yeast stress responses

YEAST STRESS RESPONSES Stefan Hohmann, Willem H. Mager

Topics in Current Genetics Vol 1 In press

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Table of Contents.................................................................................................. V 1 Introduction ........................................................................................................1 Stefan Hohmann and Willem H. Mager.............................................................1 What is stress? ..........................................................................................1 Studies of stress responses ........................................................................2 Cell proliferation and stress ......................................................................3 Aim of the stress response ........................................................................4 Phases of the stress response.....................................................................5 Sensing and signalling ..............................................................................6 Adaptation to stress...................................................................................8 Yeast as a model .......................................................................................8 2 The environmental stress response: a common yeast response to diverse environmental stresses ........................................................................................11 Audrey P. Gasch ..............................................................................................11 Abstract .......................................................................................................11 2.1 Introduction ...........................................................................................11 2.2 The environmental stress response........................................................13 2.3 Responsiveness of ESR gene expression...............................................15 2.4 Transcript levels versus protein synthesis levels ...................................18 2.5 Functions represented by genes repressed in the ESR...........................19 2.5.1 Ribosome synthesis........................................................................20 2.5.2 tRNA synthesis ..............................................................................21 2.5.3 General transcription......................................................................22 2.5.4 RNA splicing and export ...............................................................22 2.5.5 Translation .....................................................................................22 2.6 Functions represented by genes induced in the ESR .............................23 2.6.1 Carbohydrate metabolism ..............................................................23 2.6.2 Fatty acid metabolism ....................................................................26 2.6.3 Respiration.....................................................................................26 2.6.4 Oxidative stress defense.................................................................27 2.6.5 Autophagy and vacuolar functions ................................................28 2.6.6 Protein folding and degradation.....................................................29 2.6.7 Cytoskeletal reorganization ...........................................................30 2.6.8 Signaling ........................................................................................31 2.7 Functional themes in the ESR ...............................................................32 2.7.1 Differential expression of isozymes...............................................32 2.7.2 Coinduction of genes with counterproductive functions................32 2.7.3 Regulation of control steps of metabolic processes .......................34 2.8 The role of the ESR ...............................................................................34

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2.9 Regulation of ESR gene expression ...................................................... 37 2.9.1 Rap1p............................................................................................. 37 2.9.2 Chromatin remodeling ................................................................... 39 2.9.3 Regulated mRNA turnover ............................................................ 41 2.9.4 Msn2p and Msn4p ......................................................................... 43 2.9.5 Condition-specific transcriptional induction.................................. 48 2.9.6 Condition-specific cellular signaling ............................................. 50 2.9.7 Advantages of the complex regulation of ESR gene expression ... 53 2.10 Orchestration of cellular responses to stress ....................................... 54 2.11 Conclusions ......................................................................................... 56 Acknowledgements ..................................................................................... 57 References................................................................................................... 57 3 The yeast response to heat shock .................................................................... 71 Amy Trott and Kevin A. Morano .................................................................... 71 Abstract ....................................................................................................... 71 3.1 Introduction ........................................................................................... 71 3.2 The heat shock and environmental stress responses.............................. 72 3.2.1 Transcriptional regulators of heat shock gene induction ............... 72 3.2.2 Delineation of the Hsf1p and Msn2p/Msn4p heat shock regulons 73 3.2.3 The role of trehalose in thermotolerance ....................................... 76 3.2.4 Thermal stress phenotypes in yeast................................................ 77 3.3 Regulation of the heat shock factor Hsf1p ............................................ 78 3.3.1 Regulation of Hsf1p transcriptional activation .............................. 79 3.3.2 The role of phosphorylation in Hsf1p regulation........................... 81 3.3.3 Genetic and structural insights into DNA binding and regulation . 82 3.3.4 Sensing the proteome: regulation by protein chaperones .............. 84 3.3.5 Hsf1p-like proteins in yeast ........................................................... 86 3.3.6 Hsf1p and the cell cycle................................................................. 88 3.4 New directions in protein chaperone biology........................................ 91 3.4.1 Hsp90 chaperone complex subunits in yeast ................................. 91 3.4.2 Endogenous yeast Hsp90 substrates .............................................. 98 Hsf1 .................................................................................................................. 99 3.4.3 Protein chaperones and yeast prion propagation.......................... 101 3.5 Stress and aging................................................................................... 105 3.6 Conclusions ......................................................................................... 108 Acknowledgements ................................................................................... 108 References................................................................................................. 109 4 The osmotic stress response of Saccharomyces cerevisiae ........................... 121 Markus J. Tamás and Stefan Hohmann ......................................................... 121 Abstract ..................................................................................................... 121 4.1 Introduction ......................................................................................... 121 4.2 Structural and morphological effects caused by osmotic stress .......... 123 4.3 Glycerol and glycerol metabolism ...................................................... 124 4.3.1 Glycerol metabolic pathways....................................................... 125

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4.3.2 Glycerol transport ........................................................................126 4.3.3 Glycerol accumulation under osmotic stress: multiple levels of control...................................................................................................127 4.4 Transport processes affected by osmotic stress ...................................130 4.4.1 MIP channels: aquaporins and glycerol channels ........................130 4.4.2 Osmolyte uptake systems.............................................................132 4.4.3 Ion channels .................................................................................133 4.5 Perception of and response to osmotic stress: the role of signalling pathways....................................................................................................133 4.5.1 S. cerevisiae MAPK pathways.....................................................134 4.5.2 The HOG MAPK pathway in Saccharomyces cerevisiae............135 4.5.3 Control of gene expression ..........................................................141 4.5.4 The cell integrity pathway ...........................................................150 4.5.5 Skn7p: a putative link between osmosensing pathways...............156 4.5.6 Additional systems involved in osmotic stress signalling............162 4.5.7 Mechanisms of osmosensing .......................................................165 4.6 Metabolic adjustments.........................................................................167 4.7 Osmotic signalling in other yeasts: the S. pombe Sty1 pathway..........168 4.8 Conclusions .........................................................................................175 Acknowledgements ...................................................................................177 References .................................................................................................177 5 Ion homeostasis in Saccharomyces cerevisiae under NaCl stress................201 Ingrid Wadskog and Lennart Adler ...............................................................201 Abstract .....................................................................................................201 5.1 Introduction .........................................................................................201 5.2 Yeast Na+ and K+ relations ..................................................................202 5.2.1 Growth and intracellular ion levels..............................................202 5.2.2 Why is K+ but not Na+ a preferred intracellular cation? ..............203 5.2.3 Na+ toxicity ..................................................................................203 5.3 Adaptation to high concentrations of salt: role of ion transporters......204 5.3.1 The plasma membrane H+-ATPase..............................................205 5.3.2 K+ transport systems ....................................................................207 5.3.3 The Pmr2Ap/Ena1p sodium transporter.......................................208 5.3.4 The Nha1p Na+/H+ antiporter.......................................................209 5.3.5 Compartmentalization of Na+ ......................................................210 5.4 Regulation of ion homeostasis.............................................................212 5.4.1 Control at transcriptional level: ENA1 .........................................212 5.4.2 Control on protein level ...............................................................220 5.4.3 Regulation of the Trk1/2p system................................................221 5.5 Ion transporters and membrane targeting ............................................221 5.5.1 Targeting of P-type ATPases to the plasma membrane ...............222 5.5.2 Nhx1p is involved in membrane traffic out of the prevacuolar compartment .........................................................................................225 5.6 The genome-wide transcriptional response .........................................226 5.7 Conclusions .........................................................................................228

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References................................................................................................. 229 6 Oxidative stress responses in yeast ............................................................... 241 Michel B. Toledano1, Agnes Delaunay1, Benoit Biteau1, Daniel Spector1,2, Dulce Azevedo1,3 ........................................................................................... 241 Abstract ..................................................................................................... 241 6.1 Introduction ......................................................................................... 241 6.2 Effects of oxygen free radicals on biological molecules ..................... 242 6.2.1 Some concepts of free radical chemistry ..................................... 242 6.3 Biological effects of oxygen free radicals in yeast.............................. 245 6.3.1 Methods for measuring the cellular toxicity of ROS ................... 245 6.3.2 Cellular effects of ROS in S. cerevisiae....................................... 247 6.4 Antioxidant defenses and thiol redox homeostasis.............................. 251 6.4.1 Metal containing antioxidants...................................................... 251 6.4.2 Thiol redox control pathways and peroxidase systems................ 254 6.5 Adaptive oxidative stress responses .................................................... 262 6.5.1 S. cerevisiae adaptive responses to oxidative stress..................... 262 6.5.2 The genomic response underlying oxidative stress adapted states263 6.6 Control of S. cerevisiae oxidative stress responses ............................. 265 6.6.1 The Yap1 pathway....................................................................... 266 6.6.2 Skn7 as a stress response coordinator.......................................... 273 6.6.3 An H2O2-inducible Msn2/4 pathway ........................................... 274 6.5.4 Other regulators of the oxidative stress response in S. cerevisiae 275 6.7 Control of S. pombe oxidative stress responses................................... 278 6.7.1 The stress-activated MAP kinase pathway .................................. 279 6.7.2 Atf1, a bZip transcription factor substrate of Spc1/Sty1.............. 281 6.7.3 The S. pombe Yap1 homologue Pap1 .......................................... 282 6.7.4 The response regulator Prr1, a homologue of Skn7..................... 283 6.7.5 Two two-component phosphorelay systems contribute to the H2O2 response ................................................................................................ 284 6.8 Regulators of the oxidative stress response in other yeasts................. 286 6.9 Conclusions ......................................................................................... 287 Acknowledgements ................................................................................... 287 References................................................................................................. 287 7 From feast to famine; adaptation to nutrient availability in yeast............. 305 Joris Winderickx1, Inge Holsbeeks1, Ole Lagatie1, Frank Giots1, Johan Thevelein1 and Han de Winde2,3 .................................................................... 305 Abstract ..................................................................................................... 305 7.1 Introduction ......................................................................................... 306 7.2.............................................................................................................. 306 Setting the stage: limitation, starvation, and cell cycle checkpoints ......... 306 7.3 Specific responses to nutrient depletion .............................................. 309 7.3.1 Carbon Source Signalling ............................................................ 309 7.3.2 Nitrogen Source Signalling.......................................................... 326 7.3.3 Phosphor Limitation and Starvation ............................................ 333

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7.3.4 Sulphur Limitation and Starvation...............................................337 7.4 Common responses to nutrient depletion ............................................340 7.4.1 General Concepts.........................................................................340 7.4.2 Nutrient signal integration and the control of metabolism and growth...................................................................................................343 7.4.3 The FGM pathway; an integrator of responses to nutrient availability ............................................................................................345 7.4.4 Nutritional control by targets of rapamycin (Tor) proteins..........347 7.4.5 Glycogen and Trehalose metabolism...........................................350 7.4.6 Morphological differentiation as a response to nutrient limitation353 7.5 Conclusions .........................................................................................357 References .................................................................................................358 Index ...................................................................................................................387

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List of contributors

Adler, Lennart Department of Cell and Molecular Biology, Göteborg University Box 462, S-405 30 Göteborg Sweden Azevedo, Dulce Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France Biteau, Benoit Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France Delaunay, Agnes Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France De Winde, Johannes Division Bakery Ingredients, Beijerinck Laboratory, DSM Life Sciences Alexander Fleminglaan 1, NL-2613 AX Delft The Netherlands Gasch, Audrey Lawrence Berkeley National Lab 1 Cyclotron Road, Mailstop 84-355, Berkeley, CA 94720 USA Giots, Frank Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium

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Hohmann, Stefan Department of Cell and Molecular Biology, Göteborg University Box 462, S-405 30 Göteborg Sweden Holsbeeks, Inge Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium Lagatie, Ole Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium Mager, Willem Department of Biochemistry and Molecular Biology, Vrije Universiteit De Boelelaan 1083, NL-1081 HV Amsterdam The Netherlands Morano, Kevin Microbiology and Molecular Genetics, University of Texas Medical School 6431 Fannin St, JFB 1.765, Houston, TX 77030 USA Spector, Daniel Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France Tamás, Markus Department of Cell and Molecular Biology, Göteborg University Box 462, S-405 30 Göteborg Sweden Thevelein, Johan Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium Toledano, Michel Laboratoire Stress Oxydants et Cancer, LSOC/SBGM/DBJC/DSV Bat. 532, CEA-Saclay, F-91191 Gif-sur-Yvette France

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Trott, Amy Microbiology and Molecular Genetics, University of Texas Medical School 6431 Fannin St, JFB 1.765, Houston, TX 77030 Wadskog, Ingrid Department of Cell and Molecular Biology, Göteborg University Box 462, S-405 30 Göteborg Sweden Winderickx, Joris Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven Kasteelpark Arenberg 31, B-3001 Heverlee Belgium

1 Introduction Stefan Hohmann and Willem H. Mager Department of Cell and Molecular Biology/Microbiology, Göteborg University, Sweden; Department of Biochemistry and Molecular Biology, Free University of Amsterdam, The Netherlands

What is stress? All cell types, even individual cells in multi-cellular organisms, have the ability to respond to changes in environmental conditions. Such responses require a complex network of sensing and signal transduction leading to adaptations of cell growth and proliferation as well as to adjustments of the gene expression programme, metabolic activities, and other features of the cell. Environmental conditions that threaten the survival of a cell, or at least prevent it from performing optimally, are commonly referred to as cell stress. The response and adaptation mechanisms to stress are highly complex. Hence, research on stress responses, specifically in times of global gene and protein expression analyses, can easily turn into a journey through almost all aspects of cell biology. In fact, many fundamental principles of cellular and molecular biology have been discovered while studying how cells respond to stressful conditions. A well-established example illustrating that many “stress proteins” fulfill homeostatic functions are heat-shock proteins that control protein folding as chaperones. Due to the complexity of stress responses and their link to fundamental cellular functions, it is problematical to focus a volume on stress responses. Instead, we have tried to concentrate on a number of specific, well-studied stress conditions that lead to overlapping though distinct responses. Each such type of stress is treated in a specific chapter. In addition, the overlap of the responses, i.e. the responses common to all stress conditions, is the subject of a separate chapter. Finally, we have focused on a specific organism, the budding yeast Saccharomyces cerevisiae, because this organism is particularly well studied and it is an important model system in cellular and molecular biology. Another important model is the fission yeast Schizosaccharomyces pombe, which is mentioned in many of the chapters. Single-celled organisms living freely in nature, such as yeasts, face large variations in their natural environment. Rapidly acting mechanisms are crucial for the survival of these cells to sudden environmental changes and powerful adaptation mechanisms are essential to maintain their capacity to proliferate. Environmental changes may be of a physical or chemical nature: temperature, pressure, radiation,

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concentration of solutes and water, presence of certain ions, toxic chemical agents, pH and nutrient availability. In nature, yeast cells often have to cope with fluctuations in more than one such growth parameter simultaneously. Studies of stress responses Stress responses are often studied by rapidly shifting yeast cells from one condition to another, for instance, from 25°C to 42°C or from a medium without salt to 1M NaCl, and then following certain aspects of cellular behaviour such as gene expression. Dramatic shifts, commonly referred to as “sub-lethal”, may not always be relevant to the conditions which cells experience in nature. We also have to keep in mind that the term sub-lethal commonly refers to the population while large proportions of individual cells may in fact die. However, such “unphysiological”, dramatic shock treatments lead to clear and strong cellular responses that are far easier to study than gradual and slow adaptive changes. We certainly should not forget that sub-lethal shock treatments may, initially at least, cause substantial damage to the cell (and kill a large proportion) and hence probably lead to systemic responses that may not be characteristic for the adaptation to a given condition. For instance, shifting a cell rapidly to a very high NaCl concentration leads to dramatic water loss, thereby, certainly damaging many cellular components; the corresponding repair mechanisms will then be stimulated. Those are similar to the mechanisms evoked by heat shock and hence not characteristic for the way cells cope with NaCl. The specific response to NaCl stimulates transport systems to adjust ion homeostasis and to counteract the toxic effects of sodium ions. The call for caution in interpreting results from sub-lethal treatments may sound trivial, but there are many instances in literature where observations of cellular behaviour are based on analyses of heavily damaged or even dead cells. Along the same argument, it is noteworthy to mention that the severity of a stress affects the profile of a response dramatically. As discussed in some detail in chapters 2 and 4 and below, the more severe a stress the longer it takes the cell to respond, at least at the level of signal transduction and gene expression. For this reason it is highly important to study stress responses over time to capture actual responses and to monitor changes, for instance when comparing mutant and wild type strains. There are numerous examples in the literature where certain mutants were reported unable to mount a certain response. Closer inspection by time courses then revealed that the response had only shifted to a later time point. The reason for the delay in response in more heavily stressed cells is not well understood. It appears that certain rescue processes in the cell have to operate before signalling and gene expression responses are mounted. Obviously a virtue of studying yeast is the possibility to perform genetic analyses. Indeed, especially signalling pathways in stress responses are excellent examples where genetic analyses have been most instrumental not only to identify pathway components but also to order by epistasis analysis those components within a pathway and even provide information on the operation of the pathway. Throughout this volume, it will become clear that genetic analyses are a driving

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force in identifying genes/proteins important for the acquisition of tolerance to stress and for the elucidation of mechanisms controlling stress responses. Additional genes/proteins that potentially play a role in stress responses have now been identified by global gene and protein expression analysis. However, it often turns out to be difficult to demonstrate a role of a protein in a given stress response if the only known link is enhanced expression of the gene encoding that protein. The criteria in genetic analysis are that mutation and/or overexpression of the gene of interest cause a relevant stress phenotype such as diminished or enhanced growth or survival under elevated temperature or osmolarity. In fact, genetic alteration of the majority of genes identified by global expression analysis does not lead to such a scorable stress phenotype. There may be several reasons for that: functional redundancy; minor effects not detected by present methods; stimulation of compensatory pathways; indirect gene expression effects due to stimulation of a pathway not directly involved in the response to the particular stress; a gene product may be involved in adaptation but not required for growth under a certain condition; and so on. These examples illustrate that we probably have to reconsider our criteria and that we also need to perform more detailed experiments to identify phenotypes, such as cocultivation for many generations of wild type and mutant. Another approach is to return to global expression analysis also for phenotypic analyses and search for compensatory mechanisms that might be stimulated in the mutant and hence prevent a visible growth phenotype. Much work is ahead to link global expression data with cellular processes and phenotypes. An important aspect in the analysis of not only stress responses concerns cells and populations. Generally, we perform our studies on a cell culture consisting of billions of cells and for instance, the mRNA levels detected by Northern blot analysis represent the average of all those cells. Perhaps it is trivial to repeat that many of those cells may actually be dead after a sub-lethal stress treatment but apart from that, there are large variations in how individual cells behave. Analysis of stress responses in individual cells, for instance by using flow cytometry, has only recently attracted some attention. Averaging cells obviously has some statistical virtue, even though we cannot calculate the standard deviation. Especially theoretical approaches by modelling will however require some thought (or rather data) of how individual cells behave. Cell proliferation and stress Control of cell proliferation on the one hand and cellular stress responses on the other are very much interrelated and in some respect seem to be two sides of the same coin. For instance, stress treatments cause a transient arrest of the cell cycle, commonly in G1 or, under osmotic stress, also at the G2-M boundary. For instance, stress treatments cause a transient arrest of the cell cycle. Arrest commonly occurs in G1, under osmotic stress also at the G2-M boundary. Such cell cycle arrest may be needed to prevent damage during cell cycle phases in which the cell is specifically vulnerable (S and M) and allow adaptation while cells are in G1 (or

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G2). Cell cycle arrest in G1 is especially well studied for nutrient starvation (chapter 7). Glucose is the favourite carbon source of Saccharomyces and glucose depletion stimulates wide cellular responses. One aspect of the adaptation to glucose depletion is the acquisition of tolerance to a range of different stress conditions. In fact, the same is true for cells depleted or starved for any other nutrient. For this reason, nutrient responses are treated in this volume, although a future volume of Topics in Current Genetics is planned to address nutrient-stimulated cellular responses in yeast and other model systems in more detail. The mechanisms underlying starvation-induced acquisition of stress tolerance are probably linked to cell cycle-controlled stress tolerance and to the activity of protein kinase A. It is well known that G1 cells, i.e. non-proliferating yeast cells, are more stress tolerant than actively dividing cells. Moreover, there are numerous indications in the literature that nutrient availability, control of residence in the G1-phase of the cell cycle, activity of protein kinase A (cAMP-dependent protein kinase), and stress tolerance are related (in fact correlated), although the detailed mechanisms are less well understood. However, a gene expression programme controlled by the transcription factors Msn2p and Msn4p as well as accumulation of the stress protectant trehalose are central to the acquisition of stress tolerance of G1 and nutrient-starved cells. Aim of the stress response The cellular response to stress is obviously aimed at protecting cells from the detrimental effects of stress and at repairing possible damage. Protective responses of living cells have initially been identified in studies of the heat shock response (chapter 3). Cells exposed to elevated temperature increase the synthesis of heat shock proteins many of which serve as molecular chaperones. They control the conformation of other proteins or keep protein complexes in a functionally competent state. The classical heat shock response studies revealed two fundamental features: first, the response leads to acquisition of stress tolerance. Once cells have been challenged with a mild stress, they become more resistant to severe stress. For instance, the fraction of yeast cells surviving a shift to 45°C is much lower among cells that have been grown at 24°C than for cells pre-conditioned at 37°C. Acquisition of stress tolerance holds for almost all stress conditions and is considered to be one of the main purposes of the cellular stress response. Interestingly, in several cases, exposure to one type of stress has been demonstrated to lead to tolerance to other types of stress as well. This phenomenon of cross-protection suggests that different stress conditions require common cellular responses, such as adjustment of energy metabolism and production of protective proteins (such as heat shock proteins) or small protective molecules (compatible solutes such as glycerol or trehalose). The second fundamental aspect of the stress response is that underlying molecular mechanisms also play an important role in normal unstressed cells such as the heat shock proteins mentioned previously. Under heat stress conditions, at which the risk of protein unfolding or complex dissociation

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may occur, the cell will simply need more of those molecular chaperones than under ambient conditions. In addition, for other stress responses, enhancement of basal cellular activities plays an important role. The response of yeast to hyperosmolarity conditions (osmostress) is characterised, amongst others, by adjustment of glycerol metabolism and initiation of accumulation of glycerol. Glycerol is normally produced by cells as a by-product of glycolytic metabolism (and serves here as a redox-valve) and excreted to the growth medium or re-metabolised (chapter 4). Ionic stress displays features in common with hyperosmotic stress, but also evokes physiological systems commonly engaged in ion homeostasis (chapter 5). The response of yeast to oxidative stress is a reflection of the protection from oxidative damage, which is normally active in yeast cells performing oxidative ATP production in the presence of oxygen (respiration; chapter 6). Protection from oxidative damage encompasses, in conjunction with others, the action of reducing compounds produced by the cell for instance glutathione and thioredoxin as well as enzymatic activities such as superoxide dismutase or catalase. The molecular processes mentioned above are clearly part of the specific responses to a certain type of stress. As indicated above, they may, however, also be activated as an indirect effect of other stress conditions. Hyperosmotic stress, for example, has been shown to induce gene expression, which was thought to be specific for the oxidative stress response. Moreover - probably related to the cross protection phenomenon mentioned above – different stress responses share certain aspects. This significant part of the stress response is variously called the general stress response or environmental stress response of yeast (chapter 2). Phases of the stress response As cells respond to sudden stress, they do so in different phases. In the primary phase, immediate cellular changes occur as a direct consequence of stress exposure and damage, defence processes are triggered in the second phase, and finally adapted cells resume proliferation. Dependent on the type of stress, these different phases can be distinguished more or less clearly. To illustrate this situation, we again use the response to hyperosmotic shock as an example (chapter 4). When yeast cells are exposed to an increase in external osmolarity by the addition of high concentrations of salt or sugar to the medium, they immediately loose intracellular water, which leads to shrinkage of cells. This evokes immediate intracellular changes such as recruitment of water from the vacuole and collapse of the cytoskeleton (actin depolarisation). The response phase consists of an immediate, transient growth arrest at G1 or the G2/M transition of the cell cycle, reduction of transmembrane glycerol transport, triggering of the HOG MAP kinase pathway and activation of gene expression. In the adaptive phase, intracellular glycerol accumulates, cellular energy metabolism is adjusted, the cytoskeleton (actin re-polarisation) is repaired, cell wall architecture is altered, and growth resumes as soon as the critical cell size under those conditions is reached.

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The immediate cellular effect of thermo stress is the (partial) denaturation and aggregation of proteins, the disassembly of protein complexes and other cellular structures and increased fluidity of cell membranes. It is not known which proteins are particularly heat stress susceptible, but it can be predicted that denaturation will have pleiotropic effects on many cellular processes. Formation of heatdenatured proteins evokes the response phase, which induces thermo protective mechanisms. Oxidative stress directly causes oxidative damage to proteins and other macromolecules, in particular lipids and DNA. Some proteins may be more susceptible to oxidative damage than others, but also in this case, multiple direct effects on cellular functioning are likely to occur. Increase of reactive oxygen species induces the secondary response phase leading to protection and adaptation. Sensing and signalling The secondary phase of the stress response is characterised by sensing, signalling, and adjustment of gene and protein expression. The underlying mechanisms of stress sensing are major issues of investigations. In the hyperosmotic stress response, at least one plasma membrane-bound protein, Sln1p, has been shown to play an important role (chapter 4). It is part of a two-component-like sensor histidine kinase – response regulator complex and may sense changes in turgor of the cellular envelope of yeast. However, as discussed in more detail in chapter 4, many potential mechanisms of sensing osmotic changes can be envisaged. Sensing of thermo stress may be based on the recruitment of heat shock proteins by unfolded proteins in the cell (chapter 3). Such recruitment may activate the heat shock transcription factor Hsf1p, which itself interacts with heat shock proteins in its inactive state. Thus, in the heat shock response sensing and gene transcription may be directly coupled. On the other hand, heat shock also elicits the cell integrity MAP kinase pathway, which suggests that thermo stress may also be sensed at the level of the cell wall or plasma membrane. The appearance of free radicals, which trigger the oxidative stress response, most likely is sensed by redox-sensitive signalling proteins, in particular transcription factors (chapter 6). One such factor is Yap1p and possibly other members of the Yap family of transcription factors. To explain the onset of the general stress response, one would expect a critical cellular activity to be susceptible to multiple kinds of stress (chapters 2 and 7). It has been proposed that protein kinase A and Msn2p and Msn4p, the stresscontrolled transcription factors downstream of protein kinase A, might be responsible for this common effect, but conclusive evidence for this hypothesis is still missing. Maybe the “general” signal is fired - for instance at the plasma membrane - through a cellular activity that modulates protein kinase A. On the other hand, recent global expression data and analysis of the promoters of multiple-stress responsive genes have shown that in many instances those genes are controlled by different stress-specific response mechanisms converging on the promoter via different transcription factors. One possibility emerging from global expression stud-

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ies is that the genuine general stress response is restricted to adjusting cellular metabolism to stressful conditions and hence the signal may be related to the cellular energy status or the cellular proliferation potential. Clearly more work is required to distinguish between these possibilities. Subsequent to sensing of stress, responsive processes are induced. In the osmotic stress response, the HOG MAP kinase cascade is triggered as a consequence of hyperosmotic stress conditions and the cell integrity MAP kinase pathway is elicited by hypo-osmotic stress. In fact, all six MAP kinase pathways in Saccharomyces mediate events that eventually lead to morphological changes or adjustments and osmoregulation is certainly a central part of morphogenesis. Indeed an important outcome of osmostress-induced MAP kinase signalling is the adjustment of cell wall structure. Adjustment of gene expression is a central feature of the stress response of yeast (at least it has attracted most attention so far). Several stress-induced transcription factors have been identified. Transcription factors Msn2p and Msn4p are the key players in the general stress response (chapter 2). Under ambient conditions, when cells proliferate, Msn2/4p are localised in the cytoplasm. Upon stress exposure they are translocated to the nucleus where they bind to so-called STREs - general stress-responsive elements - in the promoters of a large set of genes. Cellular localisation of Msn2/4p is correlated with the cellular level of protein kinase A. Under conditions of low protein kinase A, Msn2/4p is nuclear whereas at high protein kinase A conditions, Msn2/4p are cytoplasmic. Comparison of the transcript profile of general stress-responsive genes revealed large differences. Apparently, the actual contribution of Msn2/4p-mediated transcriptional activation depends on the promoter context and interaction with other stress-specific transcription factors, as indicated above. As mentioned above, the central transcription factor in the heat shock response is Hsf1p, a trimeric protein binding to HSEs - heat shock elements - in the promoters of a large number of genes (chapter 3). Activation occurs by a conformational change, which releases the activating domain of the protein. The oxidative stress response-mediating transcription factor Yap1p is cytoplasmic and upon stress exposure is translocated to the nucleus (chapter 6). Also in this case, a large set of genes is responsive via so-called ARE’s – AP1responsive elements – in the respective promoters. Skn7p is a transcription factor controlling a set of genes overlapping with the Yap1-regulon. Skn7p is a peculiar protein: it is a response-regulator protein controlled by the Sln1p osmosensing histidine kinase and it contains a DNA-binding domain similar to the heat shock transcription factor with which it has been shown to interact. Skn7p interacts with and supports the function of different transcription factors such as Yap1p, Hsf1p, Swi4p/Swi6p (involved in cell cycle dependent gene expression), and Crz1p (mediates calcineurin-dependent calcium-induced responses). Skn7p may, therefore, be a factor involved in integrating different signals at the level of gene expression and the complete understanding of its role deserves some priority.

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Adaptation to stress The stress-responsive phase is followed by the adaptation phase, at which integration of all stress-induced cellular activities leads to resumption of growth and proliferation. After a hyperosmotic challenge, intracellular glycerol levels are increased, as a combined result of the diminished efflux and the enhanced synthesis (chapter 4). This leads to uptake of water and swelling of cells up to a certain size, which is critical for resumption of growth and division. In general, the growth rate in the presence of high external osmolarity is reduced as compared to that of cells not challenged by osmotic stress. Similarly, adaptation to hypo-osmotic stress is achieved when a new osmotic balance between intracellular medium and the environment and hence an appropriate cellular turgor has been achieved. It has been demonstrated that both hyper- and hypo-osmotic stress conditions lead to adaptations in the structure of the cell wall, which most likely reflect the need to ensure cellular integrity under the new circumstances. After ionic stress - apart from the mechanisms in common with osmotic stress – energy metabolism is adjusted in order to ensure detoxification by sodium export (chapter 5). The same holds for oxidative stress with respect to the intracellular concentration of non-enzymatic and enzymatic defence components (chapter 6). Adjustment to increased growth temperature encompasses enhanced levels of protective functions (proteins, trehalose) and stimulated energy production. Remarkably, a sudden transfer of yeast cells from 24°C to 37°C is experienced as stress, while after adaptation to the elevated temperature the growth rate is higher. Yeast as a model Saccharomyces cerevisiae is generally appreciated as a model eukaryote for fundamental and applied studies. Indeed, from both the genetic and physiological point of view, yeast is a favourite organism for molecular cell biologists. But how well are stress response mechanisms conserved in evolution? Principally, the general strategies with which cells respond and adapt to stress are well conserved but the molecular details may differ somewhat. For instance, the accumulation of compatible protective molecules is common to probably all cells but the actual compounds produced (trehalose and glycerol in yeast) may be different. However, even beyond general strategies, many molecular details are similar. Heat shock chaperones are well conserved, as is the heat-shock transcription factor, although the exact mechanisms controlling that factor may be somewhat different (chapter 3). The mechanisms to combat reactive oxygen species as well as the molecules and proteins involved are well conserved and redox-sensitive transcription factors are widespread. Finally, eukaryotic cells frequently employ MAP kinase pathways in stress responses. It appears, however, that different cells utilise those MAP kinase pathways in different ways. For instance, the S. cerevisiae HOG pathway seems to specifically mediate responses to osmotic stress while the related pathways in fission yeast and mammalian cells are well documented to also mediate protective responses to other stress conditions. Perhaps, many of these differences

1 Introduction

9

may actually turn out to be far less important when the details of the function of those pathways are better understood. In any case, the studies of yeast stress responses with budding and fission yeast are complementary and reveal a large deal of information relevant also for mammalian and plant cells. In fact, stress research has a large impact on medical issues. For instance, stress responses are related to ageing, apoptosis, cancer, and immunological responses. In addition, stress plays an important role in applied biotechnology, for example in studies aimed at improvement of the resistance of plant crops against saline conditions, heat, frost, or drought. In these fields, yeast has been widely used as a model in recent years. Industrial applications of yeast also benefit from stress research such as osmotolerance or cryotolerance of baker’s yeast, ethanol tolerance of wine yeast, or protection of foodstuffs from spoilage. Taken together, there is good reason why yeast stress responses are a highly active research field and this volume provides a snapshot of our present knowledge and sets the scene for further rapid advances in the years to come. This book is based on a previously published volume within a different series (Hohmann S, Mager WH: Yeast stress responses. In Molecular Biology Intelligence Unit; Landes Company. 1997). All chapters have been completely rewritten, some even by different authors and with different emphasis to accommodate recent developments. We would like to dedicate this volume to the memory of Helmut Ruis, Vienna, a good friend and colleague, who died prematurely in fall 2001. Helmut has contributed to the previous volume on stress responses but even more so to the study of yeast stress responses in general. In recent years, his work on stress responses had focused on the role of protein kinase A and the transcription factors Msn2p and Msn4p. His spirit and ideas have influenced our own research to a large extent and we are grateful for his many stimulating discussions and friendship.

2 The environmental stress response: a common yeast response to diverse environmental stresses Audrey P. Gasch Department of Genome Science, Lawrence Berkeley National Laboratory, USA

Abstract Unicellular organisms require specific internal conditions for optimal growth and function, however sudden changes in the external environment can perturb the internal milieu, disrupting normal processes. Therefore, cells must maintain their internal system despite fluctuations in the external surroundings. One mechanism that yeast cells use to protect the internal system from the effects of environmental variation is to initiate a common gene expression program that generally protects the cell during stressful times. This program, referred to as the environmental stress response, includes ~900 genes whose expression is stereotypically altered when yeast cells are shifted to stressful environments. The coordinated expression changes of these genes is a common feature of the responses to many different environments, however the regulation of these expression changes is gene-specific and condition-specific, indicating that initiation of the program is precisely controlled in response to each new environment. This review will focus on recent developments in defining and characterizing the genes that participate in the environmental stress response and the regulatory mechanisms that the cell utilizes to orchestrate this program.

2.1 Introduction Microorganisms must have specific and delicately balanced internal conditions for optimal growth and function. The internal milieu of the cell is maintained to promote proper operation of the cell, however fluctuations in the external environment can result in a variety of cellular perturbations that can disrupt the internal environment. These perturbations can prevent optimal enzyme activities, disrupt metabolic fluxes, destabilize cellular structures, perturb chemical gradients, etc., leading to overall instability. Thus, cells must be able to protect and maintain the

12

Audrey P. Gasch

critical features of the internal homeostasis in the face of variable external conditions. Yeast cells have evolved to be exceptionally proficient at surviving sudden and often harsh changes in their external environment. In the wild, yeast cells must contend with fluctuations in temperature, osmolarity, and acidity of their environment, the presence of radiation and toxic chemicals, and long periods of nutrient starvation. Growth under these various conditions requires maintenance of the internal system, however the cellular program required for its maintenance differs depending on the external challenges that the cell must deal with. Thus, when environmental conditions change abruptly, the cell must rapidly adjust its internal milieu to that required for growth at the new conditions. Details regarding the mechanisms that the yeast S. cerevisiae uses to adapt to new environments have been emerging over the years. Yeast cells gain cross protection against different stresses, evident by the fact that cells exposed to a mild dose of one stress become resistant to large, normally lethal doses of other stresses (for example Mitchel and Morrison 1982; Blomberg et al. 1988; Wieser et al. 1991; Flattery-O'Brien et al. 1993; Lewis et al. 1995). This observation sparked the idea that yeast cells use a general mechanism of cellular protection that is provoked when cells are exposed to stressful stimuli. Concordant with this model was the realization that a set of so-called “heat shock” genes was induced not only by temperature shock but also by other stressful environmental changes, hinting that the genes played a more general role in protecting the cell in response to stressful environments (Kurtz et al. 1986; Werner-Washburne et al. 1989; Kobayashi and McEntee 1990; Susek and Lindquist 1990). Although these observations suggested a general stress response in yeast, the role and regulation of this response remained obscure. Subsequently, it became apparent that the expression of the stress-induced genes was controlled by a common mechanism. A number of studies identified a sequence element common to the promoters of the stress-induced genes, referred to as the Stress Response Element (STRE), strongly suggesting that these genes were coregulated by a common factor (Kobayashi and McEntee 1990; Kobayashi and McEntee 1993; Marchler et al. 1993). The hypothetical STRE binding factor was proven to be either of two related zinc-finger transcription factors, Msn2p and Msn4p (Martinez-Pastor et al. 1996; Schmitt and McEntee 1996). Deletion of these factors renders cells sensitive to a variety of stressful conditions, and it was shown that Msn2p and Msn4p govern the induction of a large number of genes in response to many different stresses (Martinez-Pastor et al. 1996; Schmitt and McEntee 1996; Boy-Marcotte et al. 1998; Moskvina et al. 1998). Thus, these factors became known as the “general stress” transcription factors and were proposed to be generically activated in response to cellular stress to induce a set of genes that defend against environmental insult. However, it was noted that under certain conditions the genes identified as targets of these factors were normally induced regardless of MSN2 and MSN4 deletion, thereby hinting that the regulation of the stress response was more complicated than the initial model suggested (Schuller et al. 1994).

2 The environmental stress response

13

The recent increase in popularity of whole-genome studies is expanding our definition and understanding of yeast stress responses. Studies characterizing genomic transcript abundance and global protein synthesis levels allow the exploration of these aspects of the cellular responses of yeast cells to environmental changes. Using DNA arrays, the relative transcript levels of all genes in an organism’s genome can be rapidly quantified, and computational analysis of the resulting genomic expression data can implicate gene function and regulation while providing insights into the overall physiological response of the cell (Fodor et al. 1993; Pease et al. 1994; Shalon et al. 1996; Eisen et al. 1998; Brown and Botstein 1999). Large-scale changes in protein synthesis can be measured by twodimensional electrophoresis of pulse-labeled proteins, complementing gene expression studies and adding additional levels of detail about the protein repertoire in the cell (Blomberg 1995; Norbeck and Blomberg 1996; Godon et al. 1998; Lee et al. 1999a; Appella et al. 2000). These types of global studies have provided insights into the mechanisms that yeast use to defend themselves against environmental insult. Many of the observed cellular responses are specifically triggered to counteract features that are unique to each environment. The reader is directed to other chapters in this book that review the specialized yeast responses to a number of environmental stresses that are prevalent in nature. In addition to these specialized responses, global studies have identified the players in a common response to environmental stresses while providing insights into the complicated regulation of this cellular program. This review will focus on recent advances in defining and studying the common yeast response to stressful environments while summarizing existing literature on the genes and proteins that participate in and regulate this program.

2.2 The environmental stress response Characterization of the genomic expression programs in yeast responding to different environmental conditions revealed that a substantial fraction of each of the responses is not specific to the stimulus but instead represents a common response to all of the conditions tested. In a study conducted with colleagues, we used DNA microarrays to identify approximately 900 genes whose expression was stereotypically altered in S. cerevisiae responding to a variety of stressful environmental transitions (Gasch et al. 2000). (The complete list of the genes that participate in this response can be found at http://www-genome.stanford.edu/yeast_stress). These genes fell into two groups based on their expression patterns (Fig. 2.1): one group consisted of genes whose transcript levels increased in abundance in response to the environmental changes, and the other group was comprised of genes whose transcript levels decreased following environmental stress. (For the purposes of this review, genes whose transcript levels increase in response to environmental change will be referred to as induced, while genes whose transcript levels decrease will be referred to as repressed. It is important to note that the observed changes in transcript levels can be mediated by alterations in transcript

14

Audrey P. Gasch

Fig. 2.1. Gene expression patterns in the ESR. The average gene expression changes of the genes whose expression is induced ( ) and repressed ( ) in the ESR in response to 25oC to 37oC heat shock, 0.3 mM hydrogen peroxide, 1.5 mM diamide, 1M sorbitol, 0.02% methylmethane sulfonate, and 170 Gray of ionizing radiation. The same scale is used for all of the plots shown. Data were taken from Gasch et al. 2000 and Gasch et al. 2001. The complete list of the genes that participate in this response can be found at http://www-genome.stanford.edu/ yeast_stress

synthesis as well as transcript degradation; therefore, the terminology used here to indicate the increases or decreases in gene expression is not intended to imply any mechanism in the alteration of transcript abundance.) The two groups of genes that participate in the common gene expression response displayed nearly identical but opposite patterns of expression in response to the environmental shifts. This strongly suggests that the expression changes were coordinately regulated. A similar common gene expression response was also identified in a study by Causton et al. (2001). Since then, these gene expression changes have been observed in the cellular response to many environmental conditions, corroborating the commonality of the program (Table 2.1). Remarkably, the genes that participate in this response amount to ~14% of the currently predicted genes in the yeast genome (Ball et al. 2000; Blandin et al. 2000). Exploration of the genes involved in this response revealed that many of the induced genes are targets of Msn2p and/or Msn4p (Msn2/4p) and had already been implicated in a general stress response in yeast (Martinez-Pastor et al. 1996; Schmitt and McEntee 1996). However, characterization of the common gene expression program distinguished it from the previously described Msn2/4pdependent response in a number of ways. First, the common gene expression program encompasses nearly 900 genes and includes not only induced genes but also hundreds of genes whose expression is repressed in response to environmental changes. Second, as discussed below in a subsequent section, although many of the induced genes are regulated by Msn2/4p under certain conditions, the coordinate expression changes of these genes extends beyond Msn2/4p control. Furthermore, detailed characterization of the regulation of this response revealed that


15

it is not controlled by a general regulatory mechanism, but rather is mediated by condition-specific signaling pathways. Despite similarities to the previously defined “general” stress response, the coordinate increases and decreases in the expression of the genes in this program were referred to as the environmental stress response (ESR) (Gasch et al. 2000). For consistency, the terminology will be maintained in this review.

2.3 Responsiveness of ESR gene expression Each genomic expression program triggered by environmental change is unique to the specific features of the new conditions in terms of the genes affected and the magnitude and choreography of their expression, indicating that the cell precisely responds to the distinctive challenges of each new environment (see other chapters in this book). Nonetheless, the bulk of each genomic expression program is accounted for by the genes in the ESR. The ESR is initiated in response to a wide variety of environmental transitions, as indicated by the stereotyped alterations in expression of the genes in this response (Table 2.1). Although this program is commonly initiated in response to these diverse conditions, the precise levels and timing of the gene expression changes appear to be specific to the features of each new environment (Fig. 2.2), hinting at the sensitivity with which the program is regulated. Like the overall genomic expression responses, initiation of the ESR is often transient: immediately after the shift to a new environment, the cell responds with large changes in the expression of genes in the ESR, however over time the differences in expression usually subside, and transcript levels return to near pre-stress levels (Fig. 2.1) (Gasch et al. 2000; Causton et al. 2001). This observation is in line with previous observations of transient gene expression changes in response to stress (for example Parrou et al. 1997; Parrou et al. 1999; Rep et al. 1999). The transient changes in gene expression may help the cell to rapidly adjust the concentrations of the corresponding gene products to the levels required for growth at the new conditions (discussed further below, see Fig. 2.9). According to this model, the transient pattern of gene expression represents an adaptation phase during which the cell initiates the optimization of its internal milieu before resuming growth. An important exception to this observation is the case of nutrient starvation, in which the cells do not resume growth but enter a quiescent state until nutrients become available (see Chapter 7); consistently, the gene expression response to nutrient starvation involves large gene expression changes that are not transient but instead persist until starvation is alleviated (Gasch et al. 2000). The magnitude of the expression changes of genes in the ESR is graded to the severity of the environmental shock. Populations of cells experiencing larger doses of stress respond more strongly than cells experiencing subtle environmental changes (Gasch et al. 2000). For example, cells exposed to a 25oC to 37oC heat shock will show larger and more prolonged changes in gene expression before ad-

16

Audrey P. Gasch

Table 2.1. Environmental transitions that lead to ESR initiationa Environment Temperature Shocks Heat shock Ethanol Shock pH Extremes Acid Alkali Oxidative and Reductive Stress Hydrogen Peroxide Menadione Diamide Cadmium DTT Hyper-Osmotic Shock Sorbitol Potassium Chloride Sodium Chloride

Starvation Stationary Phase Amino Acid Starvation Nitrogen Starvation Phosphate Starvation Zinc Starvation Respiration Petite mutants Diauxic Shift Transition Nonfermentable Carbon Sources Diverse Drug Treatments Long-term Exposure to alpha Factor DNA-Damaging Agents Alkylating Agents Ionizing Radiation Double-strand Breaks a

References (Boy-Marcotte et al. 1999; Gasch et al. 2000; Causton et al. 2001) (Alexandre et al. 2001) (Causton et al. 2001) (Causton et al. 2001) (Godon et al. 1998; Gasch et al. 2000; Causton et al. 2001) (Gasch et al. 2000) (Gasch et al. 2000) (Momose and Iwahashi 2001) (Gasch et al. 2000; Travers et al. 2000) (Gasch et al. 2000; Rep et al. 2000; Causton et al. 2001) (S.M. O’Rourke and I. Herskowitz, personal communication) (Posas et al. 2000; Rep et al. 2000; Causton et al. 2001; Yale and Bohnert 2001; S.M. O’Rourke and I. Herskowitz, personal communication) (Fuge et al. 1994; Gasch et al. 2000) (Gasch et al. 2000; Natarajan et al. 2001) (Gasch et al. 2000) (Ogawa et al. 2000) (Lyons et al. 2000) (Traven et al. 2001) (Fuge et al. 1994; DeRisi et al. 1997) (Kuhn et al. 2001) (Hughes et al. 2000b) (Spellman et al. 1998) (Jelinsky and Samson 1999; Jelinsky et al. 2000; Gasch et al. 2001; Natarajan et al. 2001) (Gasch et al. 2001) (Lee et al. 2001b)

This table lists conditions that trigger initiation of the ESR, as monitored in global studies of genomic expression and translation initiation.


17

Fig. 2.2. Initiation of the ESR is often transient. The average gene expression changes of the genes whose expression is induced ( ) and repressed ( ) during ESR initiation in response to a 25oC to 37oC heat shock and a 29oC to 33oC heat shock. Data taken from Gasch et al. 2000

apting to their new steady-state expression program, relative to cells exposed to a mild temperature shift of 29oC to 33oC (Fig. 2.2). Furthermore, conditions that result in high levels of cell death usually provoke a substantial initiation of the ESR, with some of the ESR transcript levels changing more than 100-fold (A.P. Gasch and P.O. Brown, unpublished data). Thus, the ESR is initiated in response to a wi-

Fig. 2.3. Reciprocal expression of the ESR genes in response to reciprocal environmental changes. The average gene expression changes of genes whose expression is nor) and repressed ( ) during ESR initiation are shown as cells remally induced ( sponded to a 25oC to 37oC shock (A, left panel), 37oC to 25oC shock (A, right panel), a shift from YPD medium to YPD supplemented with 1M sorbitol (B, left panel), and YPD supplemented with 1M sorbitol to standard YPD medium (B, right panel)

18

Audrey P. Gasch

de range of environmental transitions, from subtle changes in conditions to lethal environmental shocks, in a manner that is graded to the severity of the environment-al stress. The ESR is not initiated in response to any environmental shift but appears to represent a response to suboptimal environments. This is evident from the genomic expression program of cells shifted back and forth between two environments. For example, when cells adapted to growth at 25oC were transferred to 37oC, they responded with large and transient changes in the expression of the ESR genes (Fig. 2.3A) (Gasch et al. 2000). In contrast, when cells adapted to 37oC were shifted to 25oC, they showed reciprocal changes in the expression of these genes: genes whose expression is normally induced during ESR initiation showed decreased expression in response to the reverse temperature shift, and genes whose expression is normally repressed in response to stressful environments became induced under these conditions. This observation indicates that initiation of the ESR is relieved when cells that are adapted to 37oC are shifted to 25oC. Furthermore, the cells immediately (within 5 minutes) adjusted their transcript levels to the final steady-state required for growth at 25oC, with no observable transient features. Thus, while a shift from 25oC to 37oC triggered initiation of the ESR, the reciprocal shift rapidly relieved the ESR gene expression differences within a very short period (Gasch et al. 2000).

2.4 Transcript levels versus protein synthesis levels The cell goes to great lengths to alter the expression of its genome, presumably to alter the abundance of the corresponding gene products. Indeed, many of the changes in ESR transcript levels correlate with changes in protein synthesis. Proteomic studies have identified proteins whose translation increases or decreases following starvation, osmotic shock, oxidative stress, and heat shock (Fuge et al. 1994; Norbeck and Blomberg 1997; Godon et al. 1998; Boy-Marcotte et al. 1999; Norbeck and Blomberg 2000). Although in each study, only a subset of these changes (10 mM) (Jamieson, 1992). In contrast, when these cells have been pre-treated during an hour with low non-toxic doses of either H2O2 (0.2-0.7 mM) or menadione (0.2-1 mM), they become resistant to much higher doses of these oxidants (Collinson and Dawes, 1992; Jamieson, 1992; Flattery-O'Brien et al., 1993). A mild heat shock (from 23oC to 37oC) can induce a protective response towards both H2O2 and menadione (Jamieson, 1992; Flattery-O'Brien et al., 1993). However, whether H2O2 pre-treatment results in cross-protection towards menadione and reciprocally, is controversial (Jamieson, 1992; Flattery-O'Brien et al., 1993). Nevertheless, in contrast to bacteria, the H2O2 and menadione responses overlap largely (Jamieson et al., 1994; Gasch et al., 2000). More recently, adaptive responses to the toxic alde-

6 Oxidative stress responses in yeast

263

hyde malondialdehyde (MDA) (Turton et al., 1997), and to linoleic acid hydroperoxide (LoaOOH) (Evans et al., 1998), both by-products of lipid peroxidation, have been demonstrated in S. cerevisiae. Here also, cross-protective effects between different peroxides are observed. However, these do not occur always, suggesting the presence of both common and oxidant-specific defense mechanisms and signal pathways. 6.5.2 The genomic response underlying oxidative stress adapted states As in bacteria, yeast oxidative stress adaptive responses require de novo protein synthesis, since they can be inhibited by cycloheximide (Collinson and Dawes, 1992; Flattery-O'Brien et al., 1993). Over the last few years, an increasing number of genes induced by H2O2, menadione, paraquat, or diamide have been identified (table 6.1). Proteomics (Godon et al., 1998) and DNA microarrays (Gasch et al., 2000; Causton et al., 2001) have now provided a genome-wide description of the gene expression program that determines the oxidative stress adapted states in S. cerevisiae. The genomic response to low doses of H2O2 (0.3-0.4 mM) is both rapid and transient, starting as early as 1 min after stress imposition and ending within 90 min. This response is also extensive, and involves a wide array of genes. More than 100 induced spots and about 50 repressed spots were identified on twodimensional maps in response to H2O2. Under similar conditions, more than 900 induced genes and about 600 repressed genes were detected by DNA microarrays mRNA profiling. Gasch (2000) and Causton (2001) showed that about 14% of the genome responded in a stereotypical manner to H2O2, menadione, diamide and to other environmental changes tested (heat shock, hyperosmotic shock, dithiothreitol, starvation). Two thirds of this stereotyped stress response, referred to as the environmental stress response (ESR; see also Chapter 2), are repressed genes involved in growth-related processes, mRNA metabolism, protein synthesis, and secretion. The other third of the ESR consist of genes involved in energy generation and storage (in particular glycogen and trehalose), ROS detoxification, cell wall modifications, protein folding and turnover, and DNA damage repair. In addition to the ESR, and specific to the H2O2 genomic response, is the super-induction of most H2O2 and O2- scavenging enzymes, most of the activities of the GSH and thioredoxin thioltransferases systems and other reductases, enzymes of the pentose phosphate pathway, and several heat shock proteins (HSP) (Godon et al., 1998; Gasch et al., 2000; see table 6.1). The gene expression programs following H2O2 and menadione treatment are largely identical, but distinct from that of diamide (Gasch et al., 2000). Genome-wide approaches are also contributing to the identification of oxidative stress regulatory pathways, also called regulons.

264

Michel B. Toledano et al.

Table 6.1. Compilation of most of the genes induced by oxidative stress and their regulation by Yap1, Skn7 and Msn2/4 Gene

Function

Inductiona Northern blot/gene fusion-β-gal assayb

2D gelsc

DNA chipd

Regulatione Northern blot/gene fusions-β-gal assayb

2D gels c

DNA chips d

Glutathione system GSH1

Glutamate-cysteine ligase Glutathione synthetase

H, M (Stephen et al., 1995) H, TB (Sugiyama et al., 2000)

GLR1

Glutathione reductase

H, D (Grant et al., 1996)

GPX1

M (Inoue et al., 1999)

GRX1

Glutathione peroxidase Glutathione peroxidase Glutaredoxin

GRX2

Glutaredoxin

GSH2

GPX2

H, TB, CH, MV, M, D (Inoue et al., 1999) H, M, D (Luikenhuis et al., 1998) H, M, D (Luikenhuis et al., 1998)

H,D,M H,D,M H

H,D,M

Yap1 (Stephen et al., 1995); Yap1 (Sugiyama et al., 2000) Yap1 (Grant et al., 1996)

Yap1 Yap1 Yap1

Yap1

H,D,M H,D,M H

Yap1 (Inoue et al., 1999)

Yap1

H,D,M

Msn2/4

H,D,M

Thiordoxin system TRX2

Thioredoxin 2

H, TB, M, D (Kuge and Jones, 1994)

H

H,D,M

TRR1

Thioredoxin reductase 1

H, D (Morgan et al., 1997)

H

H,D,M

TSA1

Thiol peroxidase

H, D (Lee et al., 1999b; Charizanis et al., 1999)

H

H,D,M

AHP1

Thiol peroxidase

H, TB, D (Lee et al., 1999b; Lee et al.,1999a)

H

H,D,M

mTPx cTPII

Thiol peroxidase Thiol peroxidase

H, D (Park et al., 2000) H, D (Park et al., 2000)

H

H,D,M H,D,M

H (Lee et al., 1999b)

H

H,D,M

H (Jungmann et al, 1993; Marchler et al., 1993) H, P (Galiazzo and Labbe-Bois, 1993) P (Galiazzo and LabbeBois, 1993) M (Liu and Thiele, 1996)

H

H,D,M

H

H,D,M

H

H,D,M

H (Jamieson et al., 1994; Stephen et al., 1995)

H

D

H

D H,D H,D,M

Yap1/Skn7 (Kuge and Jones, 1994; Morgan et al., 1997) Yap1/Skn7 (Morgan et al., 1997) Yap1/Skn7 (Lee et al., 1999b; Charizanis et al., 1999) Yap1/Skn7 (Lee et al., 1999a)

Yap1 /skn7

Yap1

Yap1 /skn7

Yap1

Yap1 /skn7

Yap1

Yap1 /skn7 Yap1 /skn7

Yap1

Yap1/Skn7 (Lee et al., 1999b)

Yap1 /skn7

Yap1

Msn2 (Amoros et al., 2001)

Yap1 /skn7

Msn2/4

Yap1 /skn7 Yap1 /skn7

Yap1

Others antioxidants CCP1

Cytochrome-c peroxidase

CTA1 CTT1

Catalase A, peroxisomal Catalase T, cytosolic

SOD1

Superoxide dismutase

SOD2

Superoxide dismutase

CUP1

Metallothionein

H

Yap1

Heat shock proteins SSA1

HSP70 family

SSA3 SSA4 HSP12

HSP70 family HSP70 family HSP

H (Charizanis et al.,1999)

H

Yap1/Skn7 (Lee et al., 1999b); Skn7 (Raitt et al., 2000) Skn7 (Raitt et al., 2000);

Yap1 /skn7

Ms2/4


HSP26

HSP

HSP42 HSP82

HSP HSP

HSP104

HSP

H (Raitt et al., 2000; Amoros et al., 2001)

H

H,D

H (Lee et al., 1999b)

H H

H,D,M H,D

H (Schüller et al., 1994)

H

H,D,M

265

Msn2 (Charizanis et al., 1999) Skn7 (Raitt et al., 2000); Msn2 (Amoros et al., 2001) Yap1/Skn7 (Lee et al., 1999b) Skn7 (Raitt et al., 2000)

Yap1 /skn7

Drug transporters FLR1

ATP transporter

ATR1

ATP transporter

H, TB, D (Nguyên et al., 2001)

H,D,M

Yap1 (Nguyên et al., 2001)

Yap1

H,D,M

Yap1

Carbohydrates metabolism ZWF1 NTH1

G-6-P dehydrogenase Neutral trehalase

TSL1

Trehalose-6-P synthase Trehalose-6-P synthase Trehalose-6-P synthase Succinate semialdehyde dehydrogenase Aldehyde dehydrogenase

TPS1 TPS2 UGA2 ALD3 Others DDR2 UBI4 GRE1 GRE2 GRE3 PDI1

Ubiquitin Similar to dihydrofavonol reductase Aldose reductase Protein disulfide isoerase

H (Izawa et al., 1998) H (Zahringer et al., 1997)

H

H,D H,D

Yap1

H,D,M H

H,D

Yap1 Msn2/4

Yap1

H,D,M H (Coleman et al., 2001)

H,D

Msn2/4

M (Navarro-Avino et al., 1999)

H,D

Msn2/4

H (Schüller et al., 1994) H (Schüller et al., 1994) H (Garay-Arroyo and Covarrubias, 1999) H (Garay-Arroyo and Covarrubias, 1999)

H H

H,D,M H,D H,D,M

Yap1

H,D,M H

D

Yap1 Msn2/4

Yap1

a

Induction by H2O2 (H), tert-butyl hydroperoxide (TB), diamide (D), menadione (M), and paraquat (P). Gene expression assayed by: b Northern blot or promoter-β-galacosidase fusions, with the corresponding references. c Proteomics (2D gels) (Godon et al., 1998; Lee et al., 1999). d DNA microarrays (DNA chips) (Gasch et al., 2000). e Regulation by Yap1, Skn7, Msn2/4.

6.6 Control of S. cerevisiae oxidative stress responses The pathways that control the oxidative stress adaptive responses are activated by sophisticated redox sensory mechanisms that detect changes in the intracellular concentration of oxidants. Here also pioneering work has been done in E. coli and S. tiphymurium, leading to the discovery of the OxyR and SoxR transcription factors, which are the specific sensors of H2O2 and O2-, respectively (for review, see Zheng and Storz, 2000). In S. cerevisiae, the hunt for redox sensors has lead to the discovery of the Yap1 transcription factor that co-regulates, in association with Skn7, an important

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H2O2-inducible oxidative stress response regulon. Other transcription factors are involved in the oxidative stress response, in particular Msn2/4, but at the moment there is no indication that any of these pathways are directly activated by oxidants. 6.6.1 The Yap1 pathway The DNA-binding properties of Yap1 led to its initial identification Yap1 was identified as a sequence-specific DNA-binding activity, which is capable of binding to and activating the SV40 AP-1 recognition element (ARE). It was purified by virtue of its ARE-specific DNA-binding affinity as a 90 kDa protein (Harshman et al., 1988). The cloning of the YAP1 gene revealed a basic-leucine zipper (bZip)-family protein homologous to GCN4 and c-Jun. However, the Yap1 bZIP domain is located in the amino terminal part of the protein, in contrast to other AP-1 factors where it is located in the carboxyl terminus (Moye-Rowley et al., 1989). In addition, the Yap1 basic domain differs from AP-1 factors at two of the five highly conserved residues that directly contact DNA, conferring to this factor a distinctive DNA binding specificity (Fernandes et al., 1997). The identification of several natural Yap1 binding sites (Kuge and Jones, 1994; Wu and Moye-Rowley, 1994), and further analysis of the Yap1 DNA-binding properties (Fernandes et al., 1997), have established that the Yap1 recognition element (YRE) is either TTACTAA present in the promoter of TRX2 (Kuge and Jones, 1994) or TGACTAA present in the promoter of GSH1 (Wu and Moye-Rowley, 1994). Both are distinct from the Gcn4 recognition site (TGACTCA). There may be one or more other Yap1 recognition sites, since many bona fide Yap1 target promoters lack either of the known YREs, although they can bind Yap1, as shown for TSA1 (Lee et al., 1999a). As all bZip motif proteins, Yap1 binds DNA most likely as a homodimer, but this has not been yet demonstrated experimentally (Fernandes et al., 1997). Nevertheless, a detailed ultrastructural analysis of the S. pombe Yap1 homologue Pap1, showed that Pap1 binds to DNA as an homodimer. This analysis also revealed the basis for Pap1 DNA binding specificity that is distinct from that of other bZIP subfamilies (Fujii et al., 2000). Activation of Yap1 at the level of DNA binding was initially suggested (Kuge and Jones, 1994), but has not been confirmed. Yap1 is a transcriptional regulator of the oxidative, metal and drug stress responses Yap1 has a crucial role in stress tolerance that can be separated into oxidative, cadmium, and drug stress responses. These stress responses might be especially important during plant invasion as a protection against plant defense mechanisms that include both oxidative stress and a variety of fungicidal chemicals. These functions have not been immediately recognized, due to lack of phenotype of ∆yap1 under non-stress conditions (Moye-Rowley et al., 1989).


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Historically, we will start with the function of Yap1 in drug stress responses. This was recognized by the independent identification of the YAP1 locus as PDR4, SNQ3, and PAR1 in high copy plasmid screens for activities that would provide resistance to several unrelated drugs [4-nitroquinoline-N-oxide (4-NQO), Nmethyl-N’-nitro-N-nitrosoguanine (MNNG), triaziquone, sulfomethuron methyl, cycloheximide, the iron chelators o-phenanthroline, 1-nitroso-2-naphtol] (Leppert et al., 1990; Hertle et al., 1991; Hussain and Lenard, 1991; Schnell and Entian, 1991). Although phenotypes resulting from overexpression of a transcription factor do not necessarily reflect its true function, the demonstration that ∆yap1 is hypersensitive to methylglyoxal (Wu et al., 1993), 4NQO, and to a lesser extent to cycloheximide, MNNG, and sulfomethuron methyl (Hertle et al., 1991) was an indication that YAP1 might indeed have a function in some aspects of drug stress responses. This assumption has gained credence when it was shown that Yap1 is activated by several toxic chemical, including benomyl and MMS (Nguyen et al., 2001), and when several Yap1 target genes were identified as encoding the membrane-associated transporters YCF1, ATR1, and FLR1 that operate as drug-efflux pumps. Ycf1 or yeast cadmium factor, is an ATP-binding cassette (ABC) transporter that function as a GSH-conjugate pump in the detoxification of cadmium (Wemmie et al., 1994; Li et al., 1997), diazaborine (Jungwirth et al., 2000), and arsenite (Ghosh et al., 1999). Atr1 and Flr1 are MDR transporters of the major facilitator family. Atr1 is involved in resistance to 4-NQO and aminotriazole (Coleman et al., 1997) and Flr1 in the resistance to fluconazole (Alarco et al., 1997; Alarco and Raymond, 1999), the pro-oxidant drugs diamide, diethylmaleate and menadione (Nguyen et al., 2001), cerulenin (Oskouian and Saba, 1999), benomyl, and methotrexate (Broco et al., 1999; Tenreiro et al., 2001). Yap1 has an essential role in the regulation of the oxidative stress response, initially observed by Schnell (1992) based on the hypersensitivity of ∆yap1 to H2O2, t-BOOH, cumene hydroperoxide and redox cycling drugs. Kuge and Jones (1994) established this role by identifying the first Yap1 target gene, TRX2. They described its Yap1-dependent induction by H2O2, t-BOOH, diamide and diethylmaleate, owing to two YREs in the TRX2 promoter. They also showed that the ∆yap1 strain is also hypersensitive to diamide and diethylmaleate. Since then, a growing number of oxidative stress-inducible Yap1-dependent genes have been identified (see table 1). As observed by global genomic approaches, Yap1 regulates the H2O2-dependent induction of most cellular antioxidants, and activities of the GSH, thioredoxin and pentose phosphate pathways (Lee et al., 1999a; Gasch et al., 2000). This correlates with the inability of ∆yap1 to adapt to H2O2 (Stephen et al., 1995). ∆yap1 is also hypersensitive and unable to adapt to MDA (Turton et al., 1997) and to LoaOOH (Evans et al., 1998). Another important phenotype of ∆yap1 is its hypersensitivity to cadmium (Wu et al., 1993; Hirata et al., 1994). Wu and Moye-Rowley (1994) showed that Yap1 is important for cadmium tolerance because it controls the expression of GSH1, the second target of Yap1 identified. Several other Yap1-dependent cadmium inducible genes important for cadmium tolerance have since been identified. These include YCF1 (Wemmie et al., 1994), other GSH biosynthesis and sulfur amino

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acid metabolism activities (Hirata et al., 1994; Stephen and Jamieson, 1997; Takeuchi et al., 1997; Vido et al., 2001). As Yap1 is activated by cadmium, these data clearly establish that Yap1 is a regulator of a cadmium detoxification pathway, a function entangled with its function in oxidative stress and drug resistance. Co-regulation of Yap1-target genes by the two-components response regulator Skn7 The transcription factor Skn7, also critical for the peroxide stress response (see fig. 6.4), has attracted much interest because of its potential involvement in several other cellular pathways. Skn7 has similarities to the receiver domain of prokaryotic two-component systems at its C-terminus (Brown and Bussey, 1993), and to the helix-turn-helix DNA-binding domain (DBD) of the heat shock transcription factor Hsf1 at its N-terminus (Brown et al., 1994). These two domains are separated by a coiled-coil structure, also similar to the leucine zipper of Hsf1 (Morgan et al., 1995). Skn7 was identified in a genetic screen for peroxide sensitive (POS) mutants and assigned to the Yap1 pathway, based on the similar H2O2 hypersensitive phenotype of ∆skn7, ∆yap1 and ∆skn7∆yap1 (Krems et al., 1995; Krems et al., 1996). However, in contrast to ∆yap1, ∆skn7 is not sensitive, but rather more resistant to diamide and cadmium (Morgan et al., 1997; Lee et al., 1999a). Skn7 is required for the activation of TRX2 and TRR1 in response to H2O2 and binds to a specific region within the TRX2 promoter (Morgan et al., 1997; Lee et al., 1999a). Since the H2O2 induction of TRX2 and TRR1 also requires Yap1, and Yap1 and Skn7 simultaneously bind to TRX2 and TSA1 promoters (Morgan et al., 1997; Lee et al., 1999a), Yap1 and Skn7 must co-operate for gene activation. However, it is not clear whether they physically interact, nor is the DNA sequence information recognized by Skn7 clearly established (Morgan et al., 1997; Lee et al., 1999a). Skn7 is, in fact, only required for induction of about half of the Yap1 regulon, hence delineating two gene subsets, that separate antioxidants and activities of the thioredoxin pathway that are under Yap1 and Skn7 control, from activities of the GSH and pentose phosphate pathways that are only dependent on Yap1 (Lee et al., 1999a). This particular gene control explains, at least in part, the dissociated requirement of Yap1 and Skn7 in cadmium and diamide resistance. Indeed, a proteome analysis of the cadmium response showed that Skn7 is not required for induction of Yap1 targets genes (Vido et al., 2001). Moreover, Skn7 has a negative effect on this response, based on the super-induced levels of some Yap1 targets in cadmium treated ∆skn7 cells (Vido et al., 2001). The fact that a subset of the Yap1 regulon is induced by H2O2 in the absence of Skn7 suggests that Yap1 is itself regulated by H2O2 (see below). Whether Skn7 is also regulated by H2O2 is still an open question, but the Skn7 receiver domain conserved aspartate (D427) is not needed for the H2O2 response (Morgan et al., 1997). Charizanis (Charizanis et al., 1999b) directly addressed this question, and showed that in response to peroxides and elevated O2 concentration, an Skn7-Gal4-DBD fusion can activate a Gal4dependent promoter in a Yap1-independent manner. These data demonstrate that redox signals can be transmitted to Skn7 in the absence of Yap1. Yet, genes solely


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dependent of Skn7 have not been identified (Biteau and Toledano, unpublished observations). A screen for mutants that failed to induce this Gal4-Skn7dependent reporter identified ISM1 encoding a mitochondrial isoleucyl tRNA synthetase and CCP1 encoding mitochondrial cytochrome c peroxidase (Charizanis et al., 1999b; Charizanis et al., 1999a), suggesting a role for the mitochondrion in transmitting a redox signal to Skn7. Another gene, FAP7, encoding an essential nuclear protein was similarly identified (Juhnke et al., 2000). However, the exact role of these activities in the signaling to Skn7 is not known. Skn7 has been shown otherwise important for the peroxide induction of several heat shock genes (HSP12, HSP26 and HSP104), maybe through an interaction with Hsf1 (Raitt et al., 2000) (see fig. 4). Indeed, the ability of purified bacterially expressed Skn7 to bind the Hsf element (HSE) (Raitt et al., 2000) indicates that this factor might activate these HSP through their HSE. In addition, the exacerbation of the ∆skn7 peroxide hypersensitive phenotype in a strain carrying an hsf1ts mutation, and co-imunoprecipitations of Skn7 and Hsf1, suggest that Skn7 and Hsf1 might form a transcription factor important for the peroxide induction of HSP (Raitt et al., 2000). Consistent with this hypothesis, investigators using an HSF1-conditional expression system in a ∆hsf1 strain could demonstrate that the H2O2 induction of HSP26 and HSP104 is dependent upon Hsf1 and also, but to a lesser extent, upon Msn2/4 (Amoros and Estruch, 2001). However, a twodimensional gel analysis of the H2O2 response did not identify HSP12, HSP26 or HSP104 as dependent upon either Skn7 or Yap1 (Lee et al., 1999a). Although some of these data show some discrepancies, probably owing to differences in strain backgrounds, they suggest the existence of a complex cooperation between the Yap1, Skn7, Hsf1, and Msn2/4 oxidative stress pathways that is not yet really appreciated. Molecular control of Yap1 Yap1-dependent transcription and target gene expression is activated by peroxides (H2O2, t-BOOH) and by diamide (Kuge and Jones, 1994), menadione (Stephen et al., 1995; Stephen and Jamieson, 1997), the electrophiles diethylmaleate (Kuge and Jones, 1994), benomyl and MMS (Nguyen et al., 2001), and cadmium (Hirata et al., 1994; Stephen and Jamieson, 1997). These multiple Yap1-activating stress conditions spurred the quest for the Yap1 input pathway(s), and revealed an unusual mode of regulation involving stress-induced Yap1 protein modifications, which target its nuclear export and cellular redistribution (Kuge et al., 1997; Kuge et al., 1998; Yan et al., 1998; Delaunay et al., 2000). Importantly, this regulated nuclear export applies to activation by diamide, diethylmaleate, and peroxides, each apparently involving a distinct protein modification (see below) (Wemmie et al., 1997; Delaunay et al., 2000), and suggests that Yap1 is directly involved in sensing oxidative and chemical stress.

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A Yap1 Yap Klyap1 Cap1 Pap1 Ap1-like

303-CSKMNQVCGTRQCPIPKK 2247-LTAEKIDTSACQCEIDQK 265-CSKLSMACGTKSNPIPK254-CVKLNEACGTKSNPVPKF 278-CQNVSTACGSIAC----354-MGGASSSCGTSPEPLTHS

B Yap1 Yap2 Klyap1 Cap1 Pap1 Ap1-like

598-CSEIWDRITTHPKYSDIDVDGLCSELMAKAKCSER-GVV 356-CYHILEEISSLPKYSSLDIDDLCSELIIKAKCTDDCKIV 531-CSEVWDRITAHPRYSDLDIDGLCLELRTKAKCSEK-GVV 447-CSEIWDRITSHPKYTELDIDGLCNELKSKAKCSEK-GVV 501-CPKVWSKIINHPRFESFDIDDLCSKLKNKAKCSSS-GVL 500-CSKIC------VQNQEIDMDALCSDLQKKAKCSGY-GAV

Fig. 6.3. Alignment of (A) N- and (B) C-CRD regions of S. cerevisiae Yap1 and Yap2, K. lactis Klyap1, C. albicans Cap1, S. pombe Pap1 and N. crassa Ap1-like factor. Yap1 amino acid residues that are conserved in other proteins are in bold. Cysteine residues that have been shown to play a role in the regulation of Yap1 by H2O2 or diamide are underscored. Leucine residues that are essential for NES constitution are also underscored. Conserved cysteine residues are indicated in bold. C303 and C598 form a presumable disulfide bond, which activates the protein in response to H2O2 (Delaunay et al., 2000). C598, C620 and C629 form together three mutually exclusive disulfide bonds that might be the mechanism, which activates the protein in response to diamide (Kuge et al., 2001). For other references, see the text

A regulated Yap1 nuclear export. Under non stress conditions, Yap1 is restricted to the cytoplasm (Kuge et al., 1997) by virtue of rapid nuclear export by the nuclear export receptor Crm1/Xpo1 that recognizes and interacts with a noncanonical hydrophobic leucine-rich nuclear export signal (NES) in the carboxyl terminal part of Yap1 (fig. 6.3) (Kuge et al., 1998; Yan et al., 1998). This domain of approximately fifty amino acids has been named the C-terminal cysteine rich domain (CRD) as it carries three repeats of the cysteine motif CSE in overlap with the NES (Kuge et al., 1997). Upon exposure to diamide, diethylmaleate (Kuge et al., 1997) or peroxides (Coleman et al., 1999; Delaunay et al., 2000), Yap1 redistributes into the nucleus, due to loss of the interaction between Yap1 and Crm1 (Kuge et al., 1998; Yan et al., 1998). Indeed, either inactivation of Crm1, deletion of the C-terminal CRD, or substitution of leucine residues important for the NES result in a constitutive nuclear localization of Yap1 (Kuge et al., 1997; Kuge et al., 1998; Yan et al., 1998). These data demonstrate that only Yap1 nuclear export is


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regulated, but not its import. The nuclear import of Yap1 appears constitutive and it is mediated by the nuclear import receptor Pse1/Kap121 (Isoyama et al., 2001). Yap1 as a redox sensor. Alteration of the NES resulting from changes of the redox state of C-terminal cysteines was immediately envisioned as regulating the interaction between Yap1 and Crm1. This was suggested by the constitutive cytoplasmic phenotype of a Yap1 allele with substitution of all three C-terminal cysteines (Kuge et al., 1998), the enhancement of the in vitro Yap1-Crm1 interaction by reducing agents and its inhibition under oxidizing conditions (Yan et al., 1998). Yet, diamide and peroxides do not activate Yap1 in the same fashion (Wemmie et al., 1997). Indeed, the Yap1 C-terminal domain can confer by itself the nuclear redistribution of a Gal4 DBD-GFP fusion protein in response to diamide (Kuge et al., 1997) but not to peroxides (Azevedo and Toledano, unpublished observations). Activation by peroxide also requires an N-terminal domain containing the other three Yap1 cysteines, called the N-terminal CRD (fig. 6.3) (Wemmie et al., 1997; Coleman et al., 1999; Delaunay et al., 2000). It appears that activation of Yap1 by peroxides proceeds through its oxidation and the formation of a disulfide bond between N- (C303) and C-terminal (C598) cysteine residues that is not observed upon diamide treatment (Delaunay et al., 2000). Further confirmation of this model awaits the formal identification of the presumed disulfide bond of oxidized Yap1. Diamide might also lead in vivo to disulfide bond formation. In contrast to peroxides, this seems to occur within C-terminal CRD cysteines, as suggested by a mass spectrometry analysis of a bacterially expressed Cterminal CRD treated in vitro with diamide (Kuge et al., 2001). It still needs to be demonstrated that these modifications induced by diamide in vitro also occur in vivo and on the full-length protein. The kinetics of Yap1 activation/oxidation by H2O2 is very rapid peaking at about 30 min and lasting for about one hour. This suggests that Yap1 is somehow deactivated by reduction (Delaunay et al., 2000). Thioredoxin is important for this deactivation, because inactivation of the thioredoxin pathway leads to constitutive partial Yap1 activation/oxidation (Izawa et al., 1999; Delaunay et al., 2000; Carmel-Harel et al., 2001). Surprisingly, in contrast to the effect of deleting thioredoxins, inactivation of the GSH pathway does not have any effect on Yap1 (Izawa et al., 1999; Delaunay et al., 2000). Thioredoxin could catalyze the reduction of the Yap1 disulfide bond, although in vivo evidence of a Yap1thioredoxin interaction could not be obtained (Izawa et al., 1999; Delaunay et al., 2000). Alternatively, the effect of deleting thioredoxin might be indirect, promoting Yap1 oxidation (activation) through its equilibration with a more oxidized environment, or by an increase in the intracellular H2O2 concentration due to impairment of the thioredoxin-dependent peroxide scavenging capacity. Arguing against the latter idea, deletion of the thioredoxin peroxidase TSA1 gene does not lead to constitutive activation of Yap1 (Inoue et al., 1999; Delaunay et al., 2000). In addition, in ∆tsa1, Yap1 is still deactivated although the amplitude and duration of the response is slightly augmented. Surprisingly, the identification of a TSA1 loss-of-function mutation in a screen for mutations that inactivate Yap1 has suggested that Tsa1 is required for Yap1 activation by low, but not high doses of H2O2 (Ross et al., 2000). These data are intriguing and difficult to reconcile

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These data are intriguing and difficult to reconcile with the above-mentioned Yap1 phenotype in ∆tsa1 (Inoue et al., 1999; Delaunay et al., 2000) and must be further explored. In conclusion, published data have demonstrated that Yap1 is a core element of a mechanism sensing elevated intracellular H2O2 concentrations, but it still remains to be known whether peroxides directly oxidize Yap1, and how exactly Yap1 is deregulated in strains with an inactivated thioredoxin pathway. In this respect, the identification of a TSA1 loss-of-function mutation abolishing Yap1 activation, although apparently counterintuitive, suggest that the yeast peroxide sensing mechanism might be more complex than one would expect from the prokaryotic paradigm of the OxyR peroxide sensor (Zheng et al., 1998). Yap1 also senses diamide through a different redox mechanism, but here again, it is not yet known how the oxidation of Yap1 by diamide occurs. It also still needs to be understood how other chemicals and cadmium activate Yap1. Interestingly, most if not all the other chemicals known to activate Yap1, such as diethylmaleate, menadione, benomyl, MMS, and malondialdehyde, have electrophilic properties. This chemical reactivity might be the basis for their ability to activate Yap1, possibly through alteration of the NES by nucleophilic addition of C-terminal cysteine residues. This also could apply to cadmium, given its property of a weak Lewis acid to react with cysteines. Other modes of regulation of Yap1. Yap1 mutations rendering this factor to be constitutively nuclear are unable to activate gene expression in response to H2O2 and to confer resistance to this oxidant, although they are paradoxically resistant to diamide and cadmium (Takeuchi et al., 1997; Wemmie et al., 1997; Coleman et al., 1999). Therefore, although crucial, the regulated nuclear export is probably not the only level of Yap1 regulation. For instance, YAP1 mRNA levels are increased about four-fold by peroxides (Gasch et al., 2000) and MMS (Jelinsky and Samson, 1999). It is not known whether this increase in YAP1 transcript levels correspond to increased transcription or mRNA stabilization, since this transcript is a target of the nonsense-mediated mRNA decay (NMD) pathway (Ruiz-Echevarria and Peltz, 2000). Upon activation, Yap1 becomes phosphorylated, but the role of this phosphorylation awaits the identification of its cognate kinase (Delaunay et al., 2000). This kinase is not PKA, although independent data suggest that the Ras-cAMPPKA pathway has a negative effect on Yap1-dependent transcription (Fernandes et al., 1997; Charizanis et al., 1999b). The Ras-PKA YAP1 negative regulatory effect might result partly from increased Yap1 turnover and from the inhibition of Yap1 function at the level of promoter occupancy (Fernandes et al., 1997). RascAMP-dependent inhibition of Yap1, as well as of Msn2/4, is somehow important for the induction of the invasive growth of haploid cells (Stanhill et al., 1999).


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Cell swelling hypo-osmotic stress Sln1

plasma membrane Ydp1

Oxidative stress (peroxides, electrophiles)

Ssk1

Yap1

HOG pathway Osmoadaptation

Yap1

Nuc l ea

r

Yap1

Skn7

ne bra em m Yap1

Skn7

Oxidative stress genes

Crz1

Skn7

Hsf1

HSP gene expression

FKS2 (cell wall genes)

Skn7 Yap1 Skn7

Oxidative stress genes

OCH1 (cell wall genes)

CLN1,2 Mbp1 Skn7 (bud emergence)

Fig. 6.4. Model depicting the possible role of Skn7 in coordinating multiple stress and other metabolic responses, based on genetic and biochemical available data (for references, see the text)

6.6.2 Skn7 as a stress response coordinator Skn7 has potential ramifications in several other cellular pathways that suggest that one function of this regulator might be to coordinate their regulation (fig. 6.4). (i) The Skn7 gene was initially discovered as a multi-copy suppressor of the ∆kre9 β-glucan cell wall synthesis growth defect, which suggested a role in cell wall biogenesis (Brown and Bussey, 1993). The requirement of the Skn7 conserved receiver motif D427 in this function suggested that Skn7 is part of a two-component signal pathway (Brown et al., 1994). (ii) Two independent studies have since provided genetic and biochemical evidence for such a pathway, consisting of the histidine kinase Sln1, the phosphorelay Ypd1, and Skn7 D427, leading to the activation of TRX2 transcription (Ketela et al., 1998; Li et al., 1998). This pathway is not activated by H2O2, but apparently by an increase in internal osmolarity, as shown by the identification of a loss-of-function mutation of the major glycerol transporter-encoded FPS1 in a search for mutants that elevate the Sln1-Ypd1dependent Skn7 function (Tao et al., 1999). Therefore, Sln1-Ypd1-Ssk1 regulates both Skn7 and the osmostress Hog1 pathway in a reciprocal fashion, activating Skn7 while deactivating Ssk1-Hog1 when the osmotic pressure is higher intracellularly and reciprocally (Tao et al., 1999). (iii) Skn7 has also been shown to interact with the small GTPase Rho1 through a region spanning its coiled coil domain, suggesting that Rho1 could regulate Skn7 (Alberts et al., 1998). (iv) Although the

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lack of Skn7 does not lead to any apparent cell cycle defect, its overexpression is capable to bypass the essential requirement of the G1-S transcription factor SBF and MBF in a D427-dependent fashion (Morgan et al., 1995). However, the way Skn7 does so is not clear since Skn7 do not bind to SBF or MBF recognition elements. Another potential link to G1-S transcription is a genetic and in vitro biochemical interaction between Skn7 and Mbp1, a component of MBF. This interaction indicates that Skn7 and Mbp1 form a transcription factor that could be involved in the process of bud-emergence (Bouquin et al., 1999). (v) Lastly, Skn7 affects the calcineurin pathway important for cellular ion tolerance by stabilizing its downstream transcription factor Crz1, through ternary complex formation with calcineurin and Crz1 (Williams and Cyert, 2001). Interestingly, although deletion of the calcineurin gene has no effect on peroxide tolerance, it increases the peroxide hypersensitivity of ∆skn7, suggesting some cross talk between this pathway and the control of the oxidative stress response. In conclusion, Skn7 either as a downstream effects of a two-components cascade and/or through its association with four different transcription activators, Yap1, Hsf1, Mbp1, and Crz1, has been implicated in multiple stress and metabolic responses. Although, as proposed, Skn7 could function as a coordinator of these diverse responses, the exact role of Skn7 still remains elusive. 6.6.3 An H2O2-inducible Msn2/4 pathway The STRE is a regulatory element with the core sequence CCCCT initially identified as an Hsf1-independent heat shock responsive element (Kobayashi and McEntee, 1990; Wieser et al., 1991). It also mediates the response to a variety of other stress conditions (low pH, osmotic, salt stress, ethanol and H2O2 stress) and metabolic changes (nitrogen starvation and the diauxic shift) (Kobayashi and McEntee, 1993; Marchler et al., 1993; Schuller et al., 1994; Boy-Marcotte et al., 1998; for a review, see Estruch, 2000 and Chapter 2, this volume). Gene activation through the STRE, referred to as the general stress response, resembles in nature the ESR (Gasch et al., 2000; Causton et al., 2001)) and see above) and might involve as many as 186 genes, as deduced from a computer search of STREs in the yeast genome (Treger et al., 1998). Known STRE-binding factor are the partially redundant zinc finger-containing transcription factors Msn2 and Msn4 (Msn2/4) that mediate responses to most of the conditions activating the STRE (MartinezPastor et al., 1996; Schmitt and McEntee, 1996). With respect to the oxidative stress response, the ∆msn2∆msn4 double deletion strain is hypersensitive to H2O2, and has a defective induction of a STRE-lacZ reporter gene in response to H2O2 (Martinez-Pastor et al., 1996). In addition, DNA microarrays identified about 180 genes whose induction by H2O2 is affected in ∆msn2∆msn4, including CTT1, several HSP, and enzymes of carbohydrate metabolism (Gasch et al., 2000; see table 6.1). Interestingly, the target genes of Msn2/4 in the H2O2 and the other stress responses overlap but are not identical (Gasch et al., 2000). Msn2/4 identifies one pathway of the H2O2 response. However, its upstream components, as those that transmit other activating stress signals to Msn2/4, have not been characterized ex-


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cept for osmotic stress, which involves the Hog1 MAP kinase pathway (Schuller et al., 1994; Gorner et al., 1998; see Chapter 4, this volume). Superimposed on the activity of Msn2/4 is a negative control by the Ras-cAMP-Protein kinase A (PKA) pathway (Marchler et al., 1993; Martinez-Pastor et al., 1996; Boy-Marcotte et al., 1998). This effect is exerted, at least in part, by a PKA-directed phosphorylation of Msn2/4 resulting in the inhibition of its stress-induced nuclear redistribution (Gorner et al., 1998), that also affects the activation of Msn2/4 by H2O2 (Hasan et al., 2002, in press). 6.5.4 Other regulators of the oxidative stress response in S. cerevisiae Sko1 and the overlap between the oxidative and osmotic stress responses Sko1, a bZip transcription factor that binds and represses transcription from the so-called cAMP response elements (CRE), mediates osmotic stress signals downstream of the Hog1 MAP kinase pathway (Nehlin et al., 1992; Vincent and Struhl, 1992; Proft and Serrano, 1999; Garcia-Gimeno and Struhl, 2000; Pascual-Ahuir et al., 2001; Proft et al., 2001; Rep et al., 2001; see also Chapter 4, this volume). To date, this is the first direct substrate of Hog1 (Proft et al., 2001). Sko1 represses basal CRE-dependent gene expression, through the recruitment of the Tup1/Ssn6/Cyc8 general repressor complex at CREs. Upon osmotic stress, repression by Sko1-Tup1/Ssn6 is relieved in a Hog1-dependent manner (Marquez et al., 1998; Proft and Serrano, 1999; Rep et al., 2001) freeing the way to a putative CRE-specific activator (Garcia-Gimeno and Struhl, 2000; Rep et al., 2001). Alternatively, in response to osmotic stress, Sko1 might be converted from a repressor to an activator of gene expression (Rep et al., 2001). Recently, five genes, GRE2, AHP1, and GLR1, SFA1, YML131W have been identified as induced by osmotic stress in a Hog1-Sko1 dependent manner (Rep et al., 2001). Interestingly, these genes all contain both YREs and CREs and are induced by H2O2 in a Yap1dependent manner ((Rep et al., 2001); see table 6.1). An analysis of the GRE2 promoter showed that, although Sko1 and Yap1 appear to mediate the osmotic stress and H2O2 responses independently, Sko1 interferes with Yap1 promoter access and/or activity. This presumably explains the observed ∆hog1 hypersensitivity and ∆sko1 hyperresistance to H2O2 reported in this study (Rep et al., 2001). Consistent with the H2O2 hypersensitive phenotype of ∆hog1 mutant, the Sln1-Ssk1 twocomponent system upstream of Hog1 has been implicated in the tolerance to acute exposure to H2O2 (Singh, 2000). How Yap1 gets access to promoters repressed by Sko1-Tup1/Ssn6, and/or whether oxidative stress affects Sko1-Tup1/Ssn6dependent repression is an important question that warrants more studies, and could give some insights into the physiological interplay between oxidative and osmotic stress.

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Hsf1 We mentioned earlier the role of Hsf1 in the activation of Hsp26 and Hsp104 by H2O2 and its potential co-operation with Skn7 in this regulation. Using a separate experimental system, investigations by Thiele and colleagues have suggested that Hsf1 might be part of an O2- sensing pathway in yeast (Tamai et al., 1993; Tamai et al., 1994; Liu and Thiele, 1996). Analysis of the expression of metallothioneinencoded CUP1, an important O2- defense activity, showed that in addition to being heat stress inducible by Hsf1, CUP1 is also induced by elevated O2 concentrations and by menadione. CUP1 induction by oxidative stress requires Hsf1 and two non-canonical HSE elements present in the CUP1 promoter, and is critical for metallothionein-mediated resistance to menadione. The signal activating Hsf1 by menadione is O2- itself, and not the H2O2 produced by dismutation of O2-, as demonstrate by the high hyper-induction of CUP1 by menadione in a strain lacking Sod1, and by the inability of H2O2 to activate Hsf1-dependent transcription of CUP1. However, the signal could also be related to macromolecular damage caused by menadione or O2-. Interestingly, Hsf1 becomes phosphorylated upon activation by menadione with sites of phosphorylation different from those observed upon heat stress activation, suggesting that a specific signaling pathway senses and transduces the O2- signal to Hsf1. However, this pathway does not lead to activation of all Hsf1 targets, since Ssa1, Ssa3, Hsc82 and Hsc82, although potently heat-inducible by Hsf1, are not induced by menadione. Thus, Hsf1-dependent CUP1 induction by oxidative stress is promoter context specific and may depend on additional transcriptional regulatory elements. Currently, Hsf1 identifies the only known S. cerevisiae pathway that is responsive to O2-. Indeed, Yap1 is also activated by menadione, but in this case, the signal is either the H2O2 produced from dismutation of O2- and/or a direct modification by menadione on Yap1 Cterminal cysteines due to its electrophilic properties (Azevedo, unpublished observation). In conclusion, Hsf1 has certainly important functions both in the H2O2 and in the O2- stress responses through its involvements in at least two distinct S. cerevisiae signal pathways. These functions have been until now underestimated, mainly due to lack of suitable genetic system to study this essential protein. Yap2 and other Yap1 family members The S. cerevisiae genome encodes seven basic-leucine zipper (bZip) transcription factors, which are highly similar to Yap1 in the basic domain that constitute the DNA-binding surface (Fernandes et al., 1997). These eight bZip proteins form the Yap1 family (Yap1 to Yap8). They are distinct from the six other S. cerevisiae bZip proteins by their basic domain, which, as Yap1, differs at two of the five highly conserved residues that directly contact DNA. This characteristic basic domain endows this bZip family with a related DNA binding specificity. Yap2, Yap3, and Yap4 interact in vitro and in vivo with the YRE TTACTAA, but do not seem to form heterodimers with each other and with Yap1. Yap5 does not bind the YRE. The three other Yaps have not been studied. Among the seven Yap proteins, Yap2, a 50 kDa protein, is most closely related to Yap1. The two proteins have a


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high degree of homology in their C-terminal domain (see fig. 6.3), especially sharing the same cysteine residues and the amino acids that define the Yap1 NES (Kuge et al., 1997). Except for Yap1, the functions of the Yap transcription factor are not known. Although overexpression of Yap2 confers increased resistance to 1,10-phenanthrolin (Bossier et al., 1993) and to cadmium (Wu et al., 1993; Hirata et al., 1994), the lack of phenotype of ∆yap2 seems to rule out a physiological function of this factor in drug and cadmium tolerance (Hirata et al., 1994). The YAP2 pleiotropic drug resistance phenotype is probably related to the ability of Yap2 to activate transcription of some Yap1 target genes when overexpressed. Indeed, it has been shown that when overexpressed, Yap2 (Wu et al., 1993; Hirata et al., 1994), Yap3, and to a much lesser extent, Yap5 can activate transcription from a YRE (Fernandes et al., 1997). It has been suggested that Yap2, in conjunction with Yap1, is important for the H2O2 tolerance to of exponential, stationary growth phase, and respiring cells (Stephen et al., 1995). However, this finding has not been observed by others (Azevedo, unpublished results), which might be due to differences in strain backgrounds. Further work will be needed to elucidate the physiological function of Yap2 and of other Yap proteins in the stress response of S. cerevisiae. Overlap between metal metabolism and oxidative stress Iron and copper are essential for life due to their involvement as cofactors in key redox enzymes involved in respiration, ROS scavenging, and in a wide variety of other metabolic processes. However, as seen previously, they are also toxic because they can engage in Fenton chemistry, leading to OH•-mediated cellular damage. Therefore, intracellular levels of iron and copper must be adequately regulated, to satisfy their need in biochemical reactions, and to prevent accumulation to levels that would unleash their potential toxicity. This idea is best exemplified by the role of the copper sensors Ace1 (Thiele, 1988; Buchman et al., 1989) and Mac1 (Jungmann et al., 1993) two related transcription factors that coordinately regulate genes important for copper uptake and detoxification (Pena et al., 1998; Gross et al., 2000b) for a comprehensive review on this topic, see Liu and Thiele, 1997; Winge, 1999). In response to low copper conditions, Mac1 activates the expression of genes involved in high affinity copper uptake, such as Ctr1, Ctr3, and Fre1. Upon copper repletion, Mac1 is inhibited by copper-induced intramolecular interactions that inhibit both Mac1 DNA binding and transactivation activity (Jensen and Winge, 1998; Zhu et al., 1998; Serpe et al., 1999; Winge, 1999). Conversely, in response to elevate extracellular copper concentrations, Ace1 activates the expression of genes involved in the protection against copper toxicity. These genes include the metallothionein gene CUP1 (Thiele, 1988), the gene for a metallothionein-like protein Crs5 that sequesters copper ions (Culotta et al., 1994), and SOD1 (Gralla et al., 1991), whose product not only scavenges O2-, but also participate in copper detoxification by direct chelation (Culotta et al., 1995). Binding of copper by a N-terminal cysteine rich domain activates the binding of Ace1 to DNA (Thiele, 1988; Furst and Hamer, 1989; Winge, 1999). The distinction between iron utilization and toxicity appears more complex. As we

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have seen previously from the analysis of ∆sod1 strains, protection of [4Fe-4S] clusters appears more important than the potential downstream effect of iron released by their oxidation by O2-. This idea is best illustrated by the improvement of the oxygen sensitivity of the ∆sod1 mutant upon addition of iron (De Freitas et al., 2000). This indicates that some [4Fe-4S] proteins might be either essential, or are required as an antioxidant defense mechanisms. A recent analysis of the Aft1 and Aft2 transcription factors also support this idea. These two homologous transcription factors are essential for iron homeostasis. Under low iron conditions, they activate genes involved in high affinity iron uptake (Yamaguchi-Iwai et al., 1995; Blaiseau et al., 2001; Rutherford et al., 2001) such as the genes encoding the high affinity iron transporter Fet3, and the copper chaperone Atx1 involved in cellular iron utilization (for a comprehensive review on iron metabolism, see Dancis, 1998). Indeed, a strain with deletion of both AFT1 and AFT2 is iron deprived (Blaiseau et al., 2001; Rutherford et al., 2001). Interestingly this mutant has also several oxygen-dependent defects improved by addition of iron, and resembling the oxygen phenotype of ∆sod1 (Blaiseau et al., 2001). ∆aft1∆aft2 is unable to respire, apparently because of an intrinsic hypersensitivity to mitochondrial ROS, and not because of defective respiration. In addition, they are methionine and lysine auxotrophs. Apparently, this oxygen phenotype is not due to a Sod1 defect, because of the presence of wild type SOD1 transcript levels and Sod1 activity. In contrast to ∆sod1, ∆aft1∆aft2 is also hypersensitive to H2O2, and this phenotype is corrected by iron addition in the medium. Taken together, these data suggest that the oxidative stress phenotype of ∆aft1∆aft2, might be caused by a defect of some antioxidants requiring iron, and needed for the tolerance to both O2- and H2O2.

6.7 Control of S. pombe oxidative stress responses The regulation of the S. pombe oxidative stress response is more complex than in S. cerevisiae, probably also closer to metazoan environmental stress responses. Although not yet fully characterized, this response can be tentatively schematized by three distinct sensing mechanisms functioning in two distinct pathways (fig. 6.5). Two questions that have been debated (see below) are whether or not the MAP kinase Sty1/Spc1 regulates Pap1, and whether Atf1 is involved in the H2O2 response. Another unsolved question is the existence of alternate(s) pathways transmitting the H2O2 signal to Wis1-Spc1/Sty1. We will review these data starting by the Spc1/Sty1 MAPK pathway.

6 Oxidative stress responses in yeast H2O2

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H2O2

? ? Mpr1

Mak3

Mak2

Histidine kinase/ phosphorelay module

Mcs4

Wak1

MAP kinase cascade

Mak1

Win1

Wis1 H2O2

Nucle ar

me m

Sty1

an br

e

Pap1

Atf1

+

gpx1 + pyp2

+

ctt1 + sod2

?

Prr1

+

trr1 + trx2

Fig. 6.5. Hypothetical model of the components of the S. pombe stress activated protein kinase pathway involved in the H2O2 response. Pap1 might be independent of Spc1/Sty1. The three hypothetical sensing mechanisms are indicated by an arrow. For references, see the text

6.7.1 The stress-activated MAP kinase pathway The stress-activated Spc1/Sty1 MAP kinase pathway of S. pombe, the homologue of the S. cerevisiae Hog1 and mammalian SAPK p38 and JNK pathways has

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stimulated a tremendous research interest as an important model to understand how stress signals are sensed and translated into specific stress responses in eukaryotes. Unlike the Hog1 pathway, and similar to mammalian and SAPK pathways, the function of the Spc1/Sty1 pathway is not restricted to osmotic stress. This pathway controls the initiation of sexual differentiation/conjugation and the ability to survive at stationary phase, the timing of mitotic initiation, and the cellular responses to a variety of environmental stress conditions. This includes osmotic stress, heat shock, oxidative stress, UV light, certain DNA-damaging agents, metals (cadmium and arsenite), and the protein synthesis inhibitor anisomycin (for a review see Millar, 1999; Toone and Jones, 1999); and (Millar et al., 1995; Shiozaki and Russell, 1995; Degols et al., 1996; Kato et al., 1996; Degols and Russell, 1997; Shieh et al., 1997; Toone et al., 1998; see also Chapter 4 from a perspective of osmotic stress). The S. pombe SAPK pathway consists of the core MAP kinase module Spc1/Sty1 (see fig. 6.4), also known as Phh1, which is activated by the Wis1 MAPK kinase (Millar et al., 1995; Shiozaki and Russell, 1995; Kato et al., 1996), which in turn is activated by either the MAPKK kinase Wak1 (also known as Wik1 and Wis4) (Samejima et al., 1997; Shieh et al., 1997; Shiozaki et al., 1997; Shieh et al., 1998a; Shieh et al., 1998b) or Win1 (Samejima et al., 1997; Samejima et al., 1998; Shieh et al., 1998a). Other important regulatory components of the pathway are the tyrosine phosphatases Pyp1 and Pyp2 (Millar et al., 1995; Shiozaki and Russell, 1995), and the serine/threonine phosphatases Ptc1 and Ptc3 (Nguyen and Siozaki, 1999), which modulate the activity of Spc1 through tyrosine and threonine dephosphorylation, respectively. Downstream of Spc1/Sty1 are the target transcription factors Atf1 as well as possibly Pap1. These mediate all Spc1/Sty1 stress input signals, except for the timing of mitotic initiation. Sin1 is a newly identified Spc1/Sty1 interacting protein important for osmotic and heat stress tolerance and required for Atf1 and Pap1-dependent transcription in response to heat shock, osmotic stress, and H2O2. Its exact function is not yet elucidated (Wilkinson et al., 1999). A two-component module that will be described below is located upstream of Wis4/Wak1 and Win1. Genetic and biochemical dissections of this pathway have clarified the way the different stress signals are transmitted (reviewed in Millar, 1999), although this is not yet completely understood in detail, and sometimes differently interpreted. To complicate the matter, Spc1/Sty1 undergoes an active nucleo-cytoplasmic shuttling important for its regulation (Gaits et al., 1998; Gaits and Russell, 1999), and dynamic translocation of other components of the SAPK pathway cannot be excluded presently. Inactivation of Sty1/Spc1 results in hypersensitivity to osmotic stress, heat, H2O2, UV, and other stress conditions (see above). Sty1/Spc1 is activated by these same stress conditions, and these also cause induction of Atf1 target genes in a manner dependent on both Wis1 and Sty1/Spc1 (Millar et al., 1995; Degols et al., 1996; Wilkinson et al., 1996; Degols and Russell, 1997; Shieh et al., 1997). Spc1/Sty1 activation by these stress treatments requires the activation of Wis1 by either Wak1/Wis4 or Win1 (Samejima et al., 1997; Shieh et al., 1998a; Shieh et al., 1998b). It is not clear, whether the two MEKKs have redundant roles or if Win1 has a more specific role in transducing the osmotic stress signal


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(Samejima et al., 1998). Interestingly, while osmotic stress and H2O2 activate Wis1 via upstream MEKKs (Nguyen and Siozaki, 1999), heat shock does not activate Spc1 via Wis1, but through the inhibition of Pyp1 and probably Pyp2 (Shiozaki et al., 1998). These data suggest that in S. pombe, SAPK tyrosine phosphatases serve somehow as heat stress sensors, as also possibly in mammals, and might also be sensors for arsenite (Nguyen and Siozaki, 1999; Cavigelli et al., 1996). These results (Nguyen and Siozaki, 1999) are consistent with a previous study (Samejima et al., 1997) regarding the involvement of Pyp1 in the activation of Spc1 by heat stress, but disagree with the same study about the proposed involvement of Pyp1 in activation of Spc1 by H2O2. Anyhow, H2O2 sensing clearly involves the SAPK upstream two-component phosphorelay, which is described below. The mechanism for sensing osmotic stress has yet to be uncovered in S. pombe. 6.7.2 Atf1, a bZip transcription factor substrate of Spc1/Sty1 The bZip transcription factor Atf1 is downstream of the MAPK Spc1/Sty1 pathway (Takeda et al., 1995; Kanoh et al., 1996; Shiozaki and Russell, 1996) and a direct substrate of Spc1/Sty1 (Shiozaki and Russell, 1996; Wilkinson et al., 1996). Atf1 has also a role in the function of Spc1/Sty1, because it anchors this kinase into the nucleus following stress (Gaits et al., 1998). As one of its downstream substrates, Atf1 shares with Spc1/Sty1 several of its functions. Due to their inability to activate the expression of ste11+ in response to nitrogen starvation, ∆atf1 cells are sterile and do not survive in stationary growth phase (Takeda et al., 1995; Shiozaki and Russell, 1996; Wilkinson et al., 1996). As ∆spc1 cells, ∆atf1 mutants are sensitive to osmotic stress (Shiozaki and Russell, 1996; Wilkinson et al., 1996), and unable to induce expression of gpd1+ (glyceraldehydes 3 phosphate dehydrogenase), ctt1+, and pyp2+ in response to osmotic stress. However, in contrast to ∆spc1 cells, ∆atf1 mutants do not have any G2-M cell cycle defect, and are not sensitive to heat shock (Takeda et al., 1995), cadmium and arsenite, staurosporine, cycloheximide and anisomycin (Toone et al., 1998). Also, in contrast to ∆spc1, ∆atf1 is not sensitive to UV and MMS, but paradoxically Atf1 is still activated by these agents, and induces the expression of ctt1+ in response to them (Degols and Russell, 1997). The involvement of Atf1 in the oxidative stress response is differentially interpreted (Toone et al., 1998; Nguyen et al., 2000). Based on genetic evidence, Toone (1998) has opposed the function of Atf1 in the osmotic stress response to that of Pap1 in oxidative stress and multidrug resistance. These authors proposed that each activator mediates a different aspect of the Spc1/Sty1 environmental stress input. Indeed, they observed a lack of H2O2, t-BOOH and diamide phenotypes for ∆atf1, and no osmotic stress phenotype for ∆pap1. In contrast, Nguyen (2000) observed that Atf1 and Pap1 are both required and have additive effects on the induction of ctt1 by H2O2. They also found the ∆atf1 to be more sensitive to H2O2 than ∆pap1. Consistent with these data, a promoter deletion analysis of ctt1

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showed that both Pap1 and Atf1 regulate this gene in response to H2O2 through distinct cis-acting elements (Nakagawa et al., 1998; Nakagawa et al., 1999). In contrast, induction of gpx1 by H2O2 is dependent upon Atf1 but not Pap1 (Yamada et al., 1999). Therefore, Atf1 clearly seems to mediate some aspects of the H2O2 response, but there might be a compensating effect of Pap1 when Atf1 is absent. This possibly explains the lack of H2O2 sensitivity of the ∆atf1 mutant observed by Toone (1998). In agreement with the idea of an overlapping role of Aft1 and Pap1, induction of sod2 by menadione, heat and osmotic stress is dependent upon Spc1/Sty1, but this induction is neither affected in ∆pap1, nor ∆atf1, suggesting a compensating effect of each other transcription factor (Jeong et al., 2001). This model of an overlapping role for Pap1 and Atf1 might also apply to other stress conditions that activate Spc1/Sty1, such as chemicals (MMS, cadmium, arsenite) and UV (Degols and Russell, 1997; Toone et al., 1998). 6.7.3 The S. pombe Yap1 homologue Pap1 The S. pombe bZip transcription factor Pap1 and S. cerevisiae Yap1 are structural and functional homologues (for review, see Toone et al., 2001) that share similar DNA-binding specificities, the function of a transcriptional regulator of oxidative, metal and drug responses, and a Crm1-dependent regulated nuclear export (Toone et al., 1998). Like Yap1, Pap1 was initially identified as a gene conferring multi-copy resistance to staurosporine, an alkaloid with protein kinase inhibitory activity (Toda et al., 1991; Toda et al., 1992). Strains with deletion of pap1 are hypersensitive to peroxides (H2O2, t-BOOH), diamide, staurosporine, cycloheximide, anisomycin, arsenite, and cadmium. ∆pap1 mutants are also unable to elevate expression in response to H2O2 of trx2, trr1, ctt1, as well as hba2/bfr1 and pmd1, which encode ABC transporters, and apt1, which encode a flavodoxin (Toda et al., 1992; Toone et al., 1998). In response to menadione and cumene hydroperoxide, Pap1 regulates the expression of pgr1, which encodes glutathione reductase (Lee et al., 1997). As Yap1, Pap1 redistributes from the cytoplasm to the nucleus when cells are exposed to H2O2 (0.2 mM), due to a redox-sensitive Crm1-regulated export (Adachi and Yanagida, 1989; Toda et al., 1992; Toone et al., 1998; Kudo et al., 1999). Crm1 recognizes a NES in a C-terminus CRD (amino acids 488 to 544), which is highly homologous to the Yap1 CRD, containing three cysteines residues (see fig. 3) (Kudo et al., 1999). The CRD is sufficient for the response of Pap1 to diethylmaleate (Kudo et al., 1999) but the activation of Pap1 by peroxides has not been explored. Nevertheless, it is predictable that Pap1 is part of a sensing mechanism for peroxides, electrophiles and cadmium with an overall redox regulation resembling that of Yap1. Based on genetic evidences, it has been suggested that Pap1 is also regulated by the stress-activated MAPK Sty1/Spc1/Phh1 pathway (Toone et al., 1998). Indeed, ∆sty1 and ∆pap1 mutants have the same oxidative stress and multidrug resistance phenotypes, and are unable to induce the same target genes in response to H2O2 (Toone et al., 1998). Toone (1998) proposed that Sty1 might regulate the nuclear


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redistribution of Pap1, because it is largely diminished in ∆sty1. However, the way by which this regulation would occur is puzzling. Pap1 is not a substrate of Sty1 and activation of Sty1 does not lead to Pap1 nuclear redistribution. Moreover, Shiozaki and colleagues (Nguyen et al., 2000) observed that the defective Pap1dependent H2O2 induction of ctt1 in ∆spc1 is restored upon additional deletion of atf1, ruling out a role of Spc1/Sty1 in the control of Pap1. They proposed the existence of two distinct oxidative stress pathways. This fits with the hypothetical model schematized in figure 6.5. The Pap1 pathway and the Wis-Spc1/Sty1-Atf1 pathway converge at common targets such as ctt1 (see below, section III). To explain the observed Pap1 defect in ∆spc1, they suggested that in the absence of Spc1/Sty1, Atf1 becomes a transcriptional repressor of ctt1 and of other genes. 6.7.4 The response regulator Prr1, a homologue of Skn7 Prr1 is another important regulator that might function in the Pap1 oxidative stress pathway. Prr1 is a constitutively nuclear transcription factor. It is similar to Skn7, having a C-terminal receiver domain of bacterial response regulators, containing a conserved aspartate phospho-acceptor site (D418) and a N-terminal domain similar to the HSF DNA-binding domain (Ohmiya et al., 1999). The structural similarity of Prr1 with Skn7 is further extended to functional similarities, since ∆prr1 shares some but not all the phenotypes associated with ∆pap1. Like ∆pap1, ∆prr1 mutants are hypersensitive to H2O2 and t-BOOH, but in contrast to ∆pap1 (Toone et al., 1998), ∆prr1 is not sensitive to diamide, cycloheximide, and anisomycine (Ohmiya et al., 2000). Furthermore, Prr1 regulates the H2O2 induction of trr1+ and ctt1, which are also regulated by Pap1. Therefore, by inference from the S. cerevisiae model, Prr1 may assist Pap1 in the way Skn7 assists Yap1 to induce some of its targets in response to H2O2. Mak1 might be a histidine kinase candidate upstream of Prr1, but whether Prr1 actually is downstream of a histidine kinase is unlikely. Indeed, a prr1 allele carrying a mutation of D418, the amino acid accepting the phosphate in the receiver domain, complements the H2O2 hypersensitive defect of ∆prr1 (Ohmiya et al., 2000). Prr1 appears distinct from the Spc1/Sty1Atf1 pathway, because in ∆prr1 mutants activation of Spc1 by H2O2 (Nguyen et al., 2000) and induction of the Atf1 target gene gpd1 in response to osmotic stress are not affected (Ohmiya et al., 2000). Interestingly, Prr1 has another function distinct from Pap1 but shared with Atf1. Similar to ∆atf1, ∆prr1 mutants are sterile due to their inability to induce in response to nitrogen starvation ste11, a transcription factor essential for the sexual differentiation program. Bacterially expressed, Prr1 binds to a region of ste11 resembling an HSE element, again emphasizing the similarity with Skn7.

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6.7.5 Two two-component phosphorelay systems contribute to the H2O2 response Two distinct two-component phosphorelay systems, both contributing to the H2O2 response, have been identified (Aoyama et al., 2000; Nguyen et al., 2000; Buck et al., 2001). The first one upstream of the Wak1 MEKK consists of the Mak2 and Mak3 histidine kinases, the Mpr1 histidine phosphorelay and the Mcs4 response regulator (see fig. 6.5). This one is very similar to the S. cerevisiae osmosensing Sln1-Ypd1-Ssk1 phosphorelay system that controls the Hog1 MAPK pathway (Buck et al., 2001). The second one is not as well defined. It consists of the Mak1 histidine kinase and possibly the Prr1 response regulator. Mcs4 Mcs4 contains a response regulator receiver domain with its conserved aspartate at position 412 (D412). It is structurally and functionally related to the S. cerevisiae Ssk1 response regulator that controls the redundant MEKK Ssk2/Ssk22 in response to osmotic stress (Cottarel, 1997; Shieh et al., 1997; Shiozaki et al., 1997). Mcs4 is upstream of Wak1 and Win1 MEKKs (Shieh et al., 1997; Samejima et al., 1998). Based on the phenotypes of ∆mcs4, Mcs4 initially appeared to function in the activation of Spc1/Sty1 by osmotic stress, heat, H2O2 and anisomycin (Shieh et al., 1997). However, its inactivation by substitution of D412 clearly demonstrated that Mcs4 is only required for the activation of Spc1/Sty1 in response to H2O2 (Buck et al., 2001). The differences in the phenotypes observed in ∆mcs4 and Mcs4 point mutants has been interpreted as the result of disrupting the integrity of a SAPK multi-protein complex when Mcs4 is absent, therefore affecting more than one signaling pathway. Indeed, Mcs4 physically interacts in vivo with Wak1, as shown by co-immunoprecipitation assays, demonstrating that it is an integral component of the SAPK pathway. This interaction is not affected by exposure to H2O2, and it is not yet known how Mcs4 D412 phosphorylation affects Wak1. Mcs4 was initially identified as a suppressor mutation of the mitotic catastrophe phenotype (Molz et al., 1989) arising from unchecked activation of the mitotic cyclin/CDK cdc13/cdc2 (Russell and Nurse, 1987). Mcs4 is also involved in cell cycle control. This control apparently occurs both by regulating the timing of mitosis by affecting the Spc1/Sty1 pathway and the timing of Cdc2 kinase activation by an additional Spc1/Sty1-independent pathway (Shieh et al., 1997). Mpr1/Spy1 Mpr1 is located upstream of Mcs4. It is also known as Spy1, a two-component phosphorelay with a conserved histidine (H221), structurally and functionally related to the S. cerevisiae histidine phosphorelay protein Ypd1 upstream of Ssk1 (Aoyama et al., 2000; Nguyen et al., 2000). In contrast to deletion of YPD1, which leads to lethal hyperactivation of the Hog1 pathway, Mpr1 is not essential. Also, in contrast to Ypd1, the function of Mpr1 is restricted to H2O2 signaling (Nguyen et al., 2000), which is consistent with the specific role in H2O2 signaling attributed


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to Mcs4 (Buck et al., 2001). In response to H2O2, Mpr1 affects the activation of Spc1/Sty1 through its interaction with Mcs4 (Nguyen et al., 2000). This demonstrates that Mpr1 acts as a positive regulator of the pathway, in contrast to Ypd1. Surprisingly, the ∆mpr1 mutant is not sensitive to H2O2 and can elevate the expression of ctt1 in response to this oxidant even more potently than its wild type counterpart. This is interpreted as a compensating effect exerted by Pap1 on the activation of ctt1, which can proceed independent of the activation of the Wis1Spc1 pathway (Nguyen et al., 2000). Nevertheless this attractive interpretation has not been directly addressed, for instance by testing the effect on ctt1 expression of simultaneous deletion of both mpr1 and pap1. Mpr1 probably functions to transfer a H221 high-energy phosphate to D412 of Mcs4, but the outcome of this phosphorylation on the activity of Mcs4 is not clear. Indeed, phosphorylation activates Mcs4, since Mpr1/Mcs4 interaction is dependent upon the presence of Mpr1 H221 and increases in response to H2O2. D412 is required for H2O2 signaling by Mcs4 (Buck et al., 2001). However, substitutions of Mpr1 H221 (Nguyen et al., 2000) or Mcs4 D412 (Buck et al., 2001) both increase Spc1/Sty1 phosphorylation in the absence of stress conditions suggesting that phosphorylation inhibits Mcs4. Therefore, Mcs4 might regulate the Wak1-Wis1-Spc1/Sty1 pathway negatively in unstressed conditions and positively in response to H2O2. A family of histidine kinases are peroxide sensors for the SAPK pathway So far, given the high similarity of architecture of the S. pombe SAPK and the S. cerevisiae Hog1 pathway within its MAPK and two-component modules, the existence of a S. pombe histidine kinase sensor homologous to Sln1 was expected. Analysis of the S. pombe genome revealed in fact the presence of not one, but three such proteins, Mak1, Mak2, and Mak3 (Buck et al., 2001). In contrast to the single S. cerevisiae histidine kinase Sln1, genetic evidence supports the idea that S. pombe histidine kinases are all sensing H2O2 and not changes in osmolarity. All three histidine kinases have a similar architecture consisting from the Cterminus, of a typical response regulator and histidine kinase domains, followed by a PAS/PAC domain (Buck et al., 2001). Mak1 has actually two such PAS/PAC domains. Both Mak2 and Mak3 have additionally one GAF domain followed by a domain of high homology with a new family of prokaryotic serine/threonine kinases (Av-Gay and Everett, 2000). The S. pombe histidine kinases identify two distinct phosphorelay systems, Mak2-Mak3 and Mak1 that signal H2O2 to Atf1 and Pap1-Prr1, respectively (Buck et al., 2001). Indeed, cells lacking either Mak2 or Mak3 or both kinases are unable to activate Spc1/Sty1 or Atf1 or to elevate the expression of Atf1 target genes in response to H2O2. In contrast, cells lacking Mak1 can activate the Spc1/Sty1-Atf1 pathway, but not the expression of Pap1 target genes in response to H2O2. Surprisingly reminiscent of the ∆mpr1 phenotype (see previous paragraph), ∆mak2∆mak3 have wild type sensitivity to H2O2. This wild type H2O2 phenotype could result from a compensating effect of Pap1-Prr1, as suggested by the H2O2 hypersensitivity caused by simultaneous deletion of mak2, mak3 and prr1 (Buck et al., 2001).

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None of these proteins is essential under normal conditions. It has been suggested that Mak2 and mak3 are exclusively cytoplasmic (Buck et al., 2001). Mak1, Mak2, and Mak3 are clearly required for H2O2 signaling, but whether they directly sense peroxides, and if so, how this occurs, is for the moment intriguing. Indeed, PAS/PAC and GAF domains are characteristic of diverse biological sensors, yet they have never been described as peroxide sensors. The PAS/PAC domain is found in many archaeal, bacterial, and plant proteins involved in sensing light (plant phytochromes), oxygen (FixL from Rhizobium species), and energy/redox potential (Aer, ArcB) (for a review, see Pellequer et al., 1999; Galperin et al., 2001). In metazoan, PAS domains are present in transcription factors such as Hif1, which is activated by hypoxia, as well as mouse PER and CLOCK and Drosophila SIM, all involved in the regulation of circadian rhythms (for review, see Crews and Fan, 1999). One best-studied PAS domain regulator is the oxygen sensor histidine kinase FixL that regulate nitrogen fixation in the root nodule in response to changes in O2 levels. Its PAS domain binds heme and regulates the kinase activity as a function of the availability of O2 (Gong et al., 1998). The GAF domain is also involved in sensing light, energy and/or redox potential and often coexists in the same protein with PAS/PAC domains (Aravind and Ponting, 1997; Anantharaman et al., 2001; Baliga et al., 2001). More work will be needed to investigate this interesting pathway and, in particular to establish whether peroxides are directly or indirectly sensed by these histidine kinases.

6.8 Regulators of the oxidative stress response in other yeasts In addition to S. pombe Pap1, homologues of S. cerevisiae Yap1 have been characterized in other fungi. The bZip proteins Cap1 of Candida albicans, Klyap1 of Kluyveromyces lactis, and Ap1-like factor from Neurospora crassa are structurally related to Yap1, sharing in addition to the bZip motif, a highly conserved Cterminal CRD (see fig. 6.3). However, only Cap1 and Klyap1 also possess a highly conserved N-terminal CRD with a cysteine homologous to Yap1 C303, which together with C598, is important for Yap1 activation by peroxides. In S. cerevisiae, Klyap1 and Cap1 can partially substitute for Yap1 in the resistance to H2O2 and cadmium (Alarco et al., 1997; Billard et al., 1997). When overexpressed, Klyap1 confers fluconazole resistance through the expression of FLR1 (Alarco et al., 1997). More importantly, in K. lactis deletion of KLYAP1 causes the hypersensitivity to H2O2 and cadmium, demonstrating that Klyap1 regulates the oxidative and cadmium responses (Billard et al., 1997). In C.albicans too, a strain carrying a homozygous deletion of CAP1 is hypersensitive to H2O2, cadmium, 4NQO, and phenanthrolin. This demonstrates that Cap1 regulates the oxidative, cadmium and drug resistance responses in this yeast (Alarco and Raymond, 1999; Zhang et al., 2000). The similar redox regulation of Cap1 and Yap1, predicted from their high sequence homology, has already been partially confirmed by a redox-dependent nuclear redistribution of Cap1, and by showing the importance of


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C-terminal cysteine residues in this phenomenon (Zhang et al., 2000). Interestingly, as in S. cerevisiae, truncation of the C-terminal CRD of Cap1 results in a split phenotype with hypersensitivity to H2O2 and hyperresistance to cadmium, 4NQO, phenanthrolin, as well as to diamide, brefeldin A, cerulenin, and fluconazole (Alarco and Raymond, 1999). In contrast, CAP1-deleted strains are hypersensitive to all these stress conditions.

6.9 Conclusions This review should illustrate the multitude of basic biological processes involved in the yeast oxidative stress responses and their complex regulation. As best exemplified by genome-wide analyses of gene expression, protection against ROS toxicity involves antioxidants and the thiol redox control systems, DNA repair and their cell cycle regulatory checkpoints, protein repair and degradation systems, the metabolic pathways that generate reducing power and energy, an adequate control of iron and copper metabolism, and a multitude of other basic cellular processes. Oxidative stress responses are essential for yeast during successful colonization of their plant habitat. However they appear also essential as part of broader physiological processes such as the cellular adaptation to fermentative, respiratory and invasive growth, survival under starvation conditions and during stationary phase, aging, and possibly the programmed cell death of aged yeast. Each of these multiple aspects of oxidative stress responses are evolving as specialized fields of research. Future investigations on the signals that trigger these responses and their coordinated control with other stress and metabolic responses, should further clarify the link between these seemingly disparate oxidative stress response, and further our understanding of the biological importance of oxidants in living cells.

Acknowledgements This work was supported by a grant from Association de Recherche contre le Cancer (ARC 4202), and the CEA program Toxicologie Nucleaire. We are indebted to Germain Rousselet, Frédérique Tacnet, Sophie Le Maout, and members of the Toledano’s lab for insightful discussions and review of the manuscript.

References Adachi Y, Yanagida M (1989) Higher order chromosome structure is affected by coldsensitive mutations is a Schizosaccharomyces pombe gene, crm1+, which encodes a 115-kDa protein preferentially localized to the nucleus and its periphery. J Cell Biol 108:1195

288


Alarco AM, Balan I, Talibi D, Mainville N, Raymond M (1997) AP1-mediated multidrug resistance in Saccharomyces cerevisiae requires FLR1 encoding a transporter of the major facilitator superfamily. J Biol Chem 272:19304 Alarco AM, Raymond M (1999) The bZip transcription factor Cap1p is involved in multidrug resistance and oxidative stress response in Candida albicans. J Bacteriol 181:700 Alberts AS, Bouquin N, Johnston LH, Treisman R (1998) Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein beta subunits and the yeast response regulator protein Skn7. J Biol Chem 273:8616 Alic N, Higgins VJ, Dawes IW (2001) Identification of a Saccharomyces cerevisiae gene that is required for G1 arrest in response to the lipid oxidation product linoleic acid hydroperoxide. Mol Biol Cell 12:1801 Ames B, Shigenaga M, Hagen T (1993) Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA 90:7915 Amoros M, Estruch F (2001) Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene- and stress type-dependent manner. Mol Microbiol 39:1523 Anantharaman V, Koonin EV, Aravind L (2001) Regulatory potential, phyletic distribution and evolution of ancient, intracellular small-molecule-binding domains. J Mol Biol 307:1271 Aoyama K, Mitsubayashi Y, Aiba H, Mizuno T (2000) Spy1, a histidine-containing phosphotransfer signaling protein, regulates the fission yeast cell cycle through the Mcs4 response regulator. J Bacteriol 182:4868 Aravind L, Ponting CP (1997) The GAF domain: an evolutionarily link between diverse phototransducing proteins. Trends Biochem Sci 22:458 Åslund F, Zheng M, Beckwith J, Storz G (1999) Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status. Proc Natl Acad Sci U S A 96:6161 Autor AP (1982) Biosynthesis of mitochondrial manganese superoxide dismutase in saccharomyces cerevisiae Precursor form of mitochondrial superoxide dismutase made in the cytoplasm. J Biol Chem 257:2713 Av-Gay Y, Everett M (2000) The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. Trends Microbiol 8:238 Avery AM, Avery SV (2001) Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases. J Biol Chem 276:33730 Baliga NS, Kennedy SP, Ng WV, Hood L, DasSarma S (2001) Genomic and genetic dissection of an archaeal regulon. Proc Natl Acad Sci U S A 98:2521 Benov L, Fridovich I (1998) Growth in iron-enriched medium partially compensates Escherichia coli for the lack of manganese and iron superoxide dismutase. J Biol Chem 273:10313 Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313 Bilinski T, Krawiec Z, Liczmanski A, Litwinska J (1985) Is hydroxyl radical generated by the Fenton reaction in vivo? Biochem Biophys Res Commun 130:533 Billard P, Dumond H, Bolotin-Fukuhara M (1997) Characterization of an AP-1-like transcription factor that mediates an oxidative stress response in Kluyveromyces lactis. Mol Gen Genet 257:62


289

Blaiseau PL, Lesuisse E, Camadro JM (2001) Aft2p, a novel iron-regulated transcription activator that modulates, with Aft1p, intracellular iron use and resistance to oxidative stress in yeast. J Biol Chem 276:34221 Bossier P, Fernandes L, Rocha D, Rodrigues-Pousada C (1993) Overexpression of YAP2, coding for a new yAP protein, and YAP1 in Saccharomyces cerevisiae alleviates growth inhibition caused by 1,10-phenanthroline. J Biol Chem 268:23640 Bouquin N, Johnson AL, Morgan BA, Johnston LH (1999) Association of the cell cycle transcription factor Mbp1 with the Skn7 response regulator in budding yeast. Mol Biol Cell 10:3389 Boy-Marcotte E, Perrot M, Bussereau F, Boucherie H, Jacquet M (1998) Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae. J Bacteriol 180:1044 Broach J, Deschenes R (1990) The function of RAS genes in Saccharomyces cerevisiae. Adv Canc Res 54:79 Broco N, Tenreiro S, Viegas CA, Sa-Correia I (1999) FLR1 gene (ORF YBR008c) is required for benomyl and methotrexate resistance in Saccharomyces cerevisiae and its benomyl-induced expression is dependent on pdr3 transcriptional regulator. Yeast 15:1595 Brown JL, Bussey H, Stewart RC (1994) Yeast Skn7p functions in a eukaryotic twocomponent regulatory pathway. EMBO J 13:5186 Brown LL, Bussey H (1993) SKN7, a yeast multicopy suppressor of a mutation affecting cell wall b-glucan assembly, encodes a product with domains homologous to prokaryotic two-component regulators and to heat shock transcription factors. J Bacteriol 175:6908 Buchman C, Skroch P, Welch J, Fogel S, Karin M (1989) The CUP2 gene product, regulator of yeast metallothionein expression, is a copper-activated DNA-binding protein. Mol Cell Biol 9:4091 Buck V et al. (2001) Peroxide sensors for the fission yeast stress-activated mitogenactivated protein kinase pathway. Mol Biol Cell 12:407 Cabiscol E, Piulats E, Echave P, Herrero E, Ros J (2000) Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae. J Biol Chem 275:27393 Cadet J, Berger M, Douki T, Ravanat JL (1997) Oxidative damage to DNA: formation, measurement, and biological significance. Rev Physiol Biochem Pharmacol 131:1 Carmel-Harel O, Stearman R, Gasch AP, Botstein D, Brown PO, Storz G (2001) Role of thioredoxin reductase in the Yap1p-dependent response to oxidative stress in Saccharomyces cerevisiae. Mol Microbiol 39:595 Carratore RD, Della Croce C, Simili M, Taccini E, Scavuzzo M, Sbrana S (2002) Cell cycle and morphological alterations as indicative of apoptosis promoted by UV irradiation in S cerevisiae. Mutat Res 513:183 Causton HC et al. (2001) Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell 12:323 Cavigelli M, Li WW, Lin A, Su B, Yoshioka K, Karin M (1996) The tumor promoter arsenate stimulates AP-1 activity by inhibiting a JNK phosphatase. EMBO J 15:6269 Chae H, Kim I-H, Kim K, Rhee S (1993) Cloning, sequencing, and mutation of thiolspecific antioxidant gene of Saccharomyces cerevisiae. J Biol Chem 268:16815 Chae HZ, Chung SJ, Rhee SG (1994a) Thioredoxin-dependent peroxyde reductase from yeast. J Biol Chem 269:27670

290


Chae HZ, Rhee SG (1994) A thiol-specific antioxidant and sequence homology to various proteins of unknown function. Biofactors 4:177 Chae HZ, Uhm TB, Rhee SG (1994b) Dimerization of thiol-specific antioxidant and the essential role of cysteine 47. Proc Natl Acad Sci USA 91:7022 Chang EC, Kosman DJ (1990) O2-dependent methionine auxotrophy in Cu,Zn superoxide dismutase- deficient mutants of Saccharomyces cerevisiae. J Bacteriol 172:1840 Charizanis C, Juhnke H, Krems B, Entian KD (1999a) The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7). Mol Gen Genet 262:437 Charizanis C, Juhnke H, Krems B, Entian KD (1999b) The oxidative stress response mediated via Pos9/Skn7 is negatively regulated by the Ras/PKA pathway in Saccharomyces cerevisiae. Mol Gen Genet 261:740 Chaudhuri B, Ingavale S, Bachhawat AK (1997) apd1+, a gene required for red pigment formation in ade6 mutants of Schizosaccharomyces pombe, encodes an enzyme required for glutathione biosynthesis: a role for glutathione and a glutathione-conjugate pump. Genetics 145:75 Choi HJ, Kang SW, Yang CH, Rhee SG, Ryu SE (1998) Crystal structure of a novel human peroxidase enzyme at 20 A resolution. Nat Struct Biol 5:400 Coblenz A, Wolf K (1994) The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharomyces pombe. FEMS Microbiol Rev 14:303 Coleman ST, Epping EA, Steggerda SM, Moye-Rowley WS (1999) Yap1p activates gene transcription in an oxidant-specific fashion. Mol Cell Biol 19:8302 Coleman ST, Tseng E, Moye-Rowley WS (1997) Saccharomyces cerevisiae basic regionleucine zipper protein regulatory networks converge at the ATR1 structural gene. J Biol Chem 272:23224 Collinson L, Dawes I (1992) Inducibility of the response of yeast cells to peroxide stress. J Gen Microbiol 138:329 Collinson LP, Dawes IW (1995) Isolation, characterization and overexpression of the yeast gene, GLR1, encoding glutathione reductase. gene 156:123 Corson LB, Folmer J, Strain JJ, Culotta VC, Cleveland DW (1999) Oxidative stress and iron are implicated in fragmenting vacuoles of Saccharomyces cerevisiae lacking Cu,Zn-superoxide dismutase. J Biol Chem 274:27590 Cottarel G (1997) Mcs4, a two-component system response regulator homologue, regulates the Schizosaccharomyces pombe cell cycle control. Genetics 147:1043 Crawford DR, Davis KJA (1994) Adaptative response and oxidative stress. Environ Heath Perspect 102:25 Crews ST, Fan CM (1999) Remembrance of things PAS: regulation of development by HLH-PAS proteins. Curr Opin Genet 9:580 Culotta VC (2000) Superoxide dismutase, oxidative stress, and cell metabolism. Curr Top Cell Regul 36:117 Culotta VC, Howard WR, Liu XF (1994) CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J Biol Chem 269:25295 Culotta VC, Joh HD, Lin SJ, Slekar KH, Strain J (1995) A physiological role for Saccharomyces cerevisiae copper/zinc superoxide dismutase in copper buffering. J Biol Chem 270:29991 Dancis A (1998) Genetic analysis of iron uptake in the yeast Saccharomyces cerevisiae. J Pediatr 132:S24


291

Davies KJ (1987) Protein damage and degradation by oxygen radicals I general aspects. J Biol Chem 262:9895 Davies KJ (1999) The broad spectrum of responses to oxidants in proliferating cells: a new paradigm for oxidative stress. IUBMB Life 48:41 Davies KJ, Delsignore ME (1987) Protein damage and degradation by oxygen radicals III Modification of secondary and tertiary structure. J Biol Chem 262:9908 Davies KJ, Delsignore ME, Lin SW (1987) Protein damage and degradation by oxygen radicals II Modification of amino acids. J Biol Chem 262:9902 De Freitas JM, Liba A, Meneghini R, Valentine JS, Gralla EB (2000) Yeast lacking Cu-Zn superoxide dismutase show altered iron homeostasis Role of oxidative stress in iron metabolism. J Biol Chem 275:11645 Dean RT, Fu S, Stocker R, Davies MJ (1997) Biochemistry and pathology of radicalmediated protein oxidation. Biochem J 324:1 Deere D, Shen J, Vesey G, Bell P, Bissinger P, Veal D (1998) Flow cytometry and cell sorting for yeast viability assessment and cell selection. Yeast 14:147 Degols G, Russell P (1997) Discrete roles of the Spc1 kinase and the Atf1 transcription factor in the UV response of Schizosaccharomyces pombe. Mol Cel Biol 17:3356 Degols G, Shiozaki K, Russell P (1996) Activation and regulation of the Spc1 stressactivated protein kinase in Schizosaccharomyces pombe. Mol Cel Biol 16:2870 Delaunay A, Isnard AD, Toledano MB (2000) H2O2 sensing through oxidation of the Yap1 transcription factor. EMBO J 19:5157 Demple B (1991) Regulation of bacterial oxidative stress genes. Annu Rev Genet 25:315 Demple B, Halbrook J (1983) Inducible repair of oxidative DNA damage in Escherichia coli. Nature 304:466 Draculic T, Dawes IW, Grant CM (2000) A single glutaredoxin or thioredoxin gene is essential for viability in the yeast Saccharomyces cerevisiae. Mol Microbiol 36:1167 Esterbauer H, Schaur RJ, Zollner H (1991) Chemistry and biochemistry of 4hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 11:81 Estruch F (2000) Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast. FEMS Microbiol Rev 24:469 Evans MV, Turton HE, Grant CM, Dawes IW (1998) Toxicity of linoleic acid hydroperoxide to Saccharomyces cerevisiae: involvement of a respiration-related process for maximal sensitivity and adaptive response. J Bacteriol 180:483 Fabrizio P, Pozza F, Pletcher SD, Gendron CM, Longo VD (2001) Regulation of longevity and stress resistance by Sch9 in yeast. Science 292:288 Fernandes L, Rodrigues-Pousada C, Struhl K (1997) Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions. Mol Cel Biol 17:6982 Flattery-O'Brien J, Collinson L, Dawes I (1993) Saccharomyces cerevisiae has an inducible response to menadione Which differs from that to hydrogen peroxide. J Gen Microbiol 139:501 Flattery-O'Brien JA, Dawes IW (1998) Hydrogen peroxide causes RAD9-dependent cell cycle arrest in G2 in Saccharomyces cerevisiae whereas menadione causes G1 arrest independent of RAD9 function. J Biol Chem 273:8564 Fridovich I (1995) Superoxide radical and superoxide dismutases. Annu Rev Biochem 64:97 Fujii Y, Shimizu T, Toda T, Yanagida M, Hakoshima T (2000) Structural basis for the diversity of DNA recognition by bZIP transcription factors. Nat Struct Biol 7:889

292


Furst P, Hamer D (1989) Cooperative activation of a eukaryotic transcription factor: interaction between Cu(I) and yeast ACE1 protein. Proc Natl Acad Sci U S A 86:5267 Gaits F, Degols G, Shiozaki K, Russell P (1998) Phosphorylation and association with the transcription factor Atf1 regulate localization of Spc1/Sty1 stress-activated kinase in fission yeast. Genes Dev 12:1464 Gaits F, Russell P (1999) Active nucleocytoplasmic shuttling required for function and regulation of stress-activated kinase Spc1/StyI in fission yeast. Mol Biol Cell 10:1395 Galperin MY, Nikolskaya AN, Koonin EV (2001) Novel domains of the prokaryotic twocomponent signal transduction systems. FEMS Microbiol Lett 203:11 Gan Z-R (1992) Cloning and sequencing of a gene encoding yeast thioltransferase. Biochem Biophys Res Commun 187:949 Garcia-Gimeno MA, Struhl K (2000) Aca1 and Aca2, ATF/CREB activators in Saccharomyces cerevisiae, are important for carbon source utilization but not the response to stress. Mol Cell Biol 20:4340 Gardner PR, Fridovich I (1991) Superoxide sensitivity of the Escherichia coli aconitase. J Biol Chem 266:19328 Gasch AP et al. (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11:4241 Ghosh M, Shen J, Rosen BP (1999) Pathways of As(III) detoxification in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 96:5001 Gilbert HF (1995) Thiol/disulfide exchange equilibria and disulfide bound stability. Methods Enzymol 251:8 Girard PM, Boiteux S (1997) Repair of oxidized DNA bases in the yeast Saccharomyces cerevisiae. Biochimie 79:559 Godon C et al. (1998) The H2O2 stimulon in Saccharomyces cerevisiae. J Biol Chem 273:22480 Gong W, Hao B, Mansy SS, Gonzalez G, Gilles-Gonzalez MA, Chan MK (1998) Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction. Proc Natl Acad Sci U S A 95:15177 Gorner W et al. (1998) Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev 12:586 Gralla EB, Kosman DJ (1992) Molecular genetics of superoxide dismutases in yeasts and related fungi. Adv Genet 30:251 Gralla EB, Thiele DJ, Silar P, Valentine JS (1991) ACE1, a copper-dependent transcription factor, activates expression of the yeast copper, zinc superoxide dismutase gene. Proc Natl Acad Sci U S A 88:8558 Gralla EB, Valentine JS (1991) Null mutants of Saccharomyces cerevisiae Cu,Zn superoxide dismutase: characterization and spontaneous mutation rates. J Bacteriol 173:5918 Grant CM, Collinson LP, Roe JH, Dawes IW (1996a) Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation. Mol Microbiol 21:171 Grant CM, Dawes IW (1996) Synthesis and role of glutathione in protection against oxidative stress in yeast. Redox Rep 2:223 Grant CM, MacIver FH, Dawes IW (1996b) Glutathione is an essential metabolite required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae. Curr Genet 29:511


293

Grant CM, MacIver FH, Dawes IW (1996c) Stationary-phase induction of GLR1 expression is mediated by the yAP-1 transcriptional regulatory protein in the yeast Saccharomyces cerevisiae. Mol Micro 22:739 Grant CM, MacIver FH, Dawes IW (1997a) Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine. Mol Biol Cell 8:1699 Grant CM, MacIver FH, Dawes IW (1997b) Mitochondrial function is required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae. FEBS Lett 410:219 Grant CM, Perrone G, Dawes IW (1998) Glutathione and catalase provide overlapping defenses for protection against hydrogen peroxide in the yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 253:893 Grant CM, Quinn KA, Dawes IW (1999) Differential protein S-thiolation of glyceraldehyde-3-phosphate dehydrogenase isoenzymes influences sensitivity to oxidative stress. Mol Cell Biol 19:2650 Gross A et al. (2000a) Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L). Mol Cell Biol 20:3125 Gross C, Kelleher M, Iyer VR, Brown PO, Winge DR (2000b) Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays. J Biol Chem 275:32310 Guidot DM, McCord JM, Wright RM, Repine JE (1993) Absence of electron transport (Rho 0 state) restores growth of a manganese-superoxide dismutase-deficient Saccharomyces cerevisiae in hyperoxia Evidence for electron transport as a major source of superoxide generation in vivo. J Biol Chem 268:26699 Guptasarma P, Balasubramanian D (1992) Dityrosine formation in the proteins of the eye lens. Curr Eye Res 11:1121 Halliwell B, Gutteridge JM (1984) Free radicals, lipid peroxidation, and cell damage. Lancet 2:1095 Halliwell B, Gutteridge JM (1998) Free radicals in biology and medicine. Oxford University Press, Oxford Harshman KD, Moye-Rowley WS, Parker CS (1988) Transcriptional activation by the SV40 AP-1 recognition element in yeast is mediated by a factor similar to AP-1 that is distinct from GCN4. Cell 53:321 Henzler T, Steudle E (2000) Transport and metabolic degradation of hydrogen peroxide in Chara corallina: model calculations and measurements with the pressure probe suggest transport of H(2)O(2) across water channels. J Exp Bot 51:2053 Hertle K, Haase E, Brendel M (1991) The SNQ3 gene of Saccharomyces cerevisiae confers hyper-resistance to several functionally unrelated chemicals. Curr Genet 19:429 Hillar A, Nicholls P, Switala J, Loewen PC (1994) NADPH binding and control of catalase compound II formation: comparison of bovine, yeast, and Escherichia coli enzymes. Biochem J 300:531 Hirata D, Yano K, Miyakawa T (1994) Stress-induced transcriptional activation mediated by YAP1 and YAP2 genes that encode the Jun family of transcriptional activators in Saccharomyces cerevisiae. Mol Gen Genet 242:250 Holmgren A (1985) Thioredoxin. Ann Rev Biochem 54:237 Holmgren A (1989) Thioredoxin and glutaredoxin systems. J Biol Chem 264:13963 Horiguchi H, Yurimoto H, Kato N, Sakai Y (2001) Antioxidant system within yeast peroxisome Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii. J Biol Chem 276:14279

294


Hussain M, Lenard J (1991) Characterization of PDR4, a Saccharomyces cerevisiae gene that confers pleiotropic drug resistance in high-copy number: identity with YAP1, encoding a transcriptional activator [corrected]. Gene 101:149 Imlay J, Linn S (1988) DNA damage and oxygen radical toxicity. Science 240:1302 Inoue Y, Matsuda T, Sugiyama S, Izawa S, Kimura A (1999) Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae. J Biol Chem 274:27002 Isoyama T, Murayama A, Nomoto A, Kuge S (2001) Nuclear import of the yeast AP-1-like transcription factor Yap1p is mediated by transport receptor Pse1p, and this import step is not affected by oxidative stress. J Biol Chem 276:21863 Izawa S, Inoue Y, Kimura A (1995) Oxidative stress response in yeast: effect of glutathione on adaptation to hydrogen peroxide stress in Saccharomyces cerevisiae. FEBS Lett 368:73 Izawa S, Inoue Y, Kimura A (1996) Importance of catalase in the adaptive response to hydrogen peroxide: analysis of acatalasaemic Saccharomyces cerevisiae. Biochem J 320:61 Izawa S, Maeda K, Miki T, Mano J, Inoue Y, Kimura A (1998) Importance of glucose-6phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cerevisiae. Biochem J 330:811 Izawa S, Maeda K, Sugiyama K, Mano J, Inoue Y, Kimura A (1999) Thioredoxin deficiency causes the constitutive activation of Yap1, an AP-1-like transcription factor in Saccharomyces cerevisiae. J Biol Chem 274:28459 Jacobson FS, Morgan RW, Christman MF, Ames BN (1989) An alkyl hydroperoxide reductase from Salmonella typhimurium involved in the defense of DNA against oxidative damage Purification and properties. J Biol Chem 264:1488 Jakubowski W, Bartosz G (1997) Estimation of oxidative stress in Saccharomyces cerevisae with fluorescent probes. Int J Biochem Cell Biol 29:1297 Jamieson DJ (1992) Saccharomyces cerevisiae has distinct adaptive responses to both hydrogen peroxide and menadione. J Bacteriol 174:6678 Jamieson DJ, Rivers SL, Stephen DW (1994) Analysis of Saccharomyces cerevisiae proteins induced by peroxide and superoxide stress. Microbiology 140:3277 Jelinsky SA, Samson LD (1999) Global response of Saccharomyces cerevisiae to an alkylating agent. Proc Natl Acad Sci U S A 96:1486 Jensen LT, Winge DR (1998) Identification of a copper-induced intramolecular interaction in the transcription factor Mac1 from Saccharomyces cerevisiae. EMBO J 17:5400 Jeong JH, Kwon ES, Roe JH (2001) Characterrization of the manganese-containing superoxide dismutase and its gene regulation in stress response of Schizosaccharomyces pombe. Biochem Biophys Res Commun 18:908 Jeong JS, Kwon SW, Kang SW, Rhee SG, Kim K (1999) Purification and Characterization of a Second Type Thioredoxin Peroxidase (Type II TPx) from Saccharomyces cerevisiae. Biochemistry 38:776 Juhnke H, Charizanis C, Latifi F, Krems B, Entian KD (2000) The essential protein fap7 is involved in the oxidative stress response of Saccharomyces cerevisiae. Mol Microbiol 35:936 Juhnke H, Krems B, Kotter P, Entian KD (1996) Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress. Mol Gen Genet 252:456


295

Jungmann J et al. (1993) MAC1, a nuclear regulatory protein related to Cu-dependent transcription factors is involved in Cu/Fe utilization and stress resistance in yeast. EMBO J 12:5051 Jungwirth H, Wendler F, Platzer B, Bergler H, Hogenauer G (2000) Diazaborine resistance in yeast involves the efflux pumps Ycf1p and Flr1p and is enhanced by a gain-offunction allele of gene YAP1. Eur J Biochem 267:4809 Kanoh J, Watanabe Y, Ohsugi M, Iino Y, Yamamoto M (1996) Schizosaccharomyces pombe gad7+ encodes a phosphoprotein with a bZIP domain, which is required for proper G1 arrest and gene expression under nitrogen starvation. Genes Cells 1:391 Kato TJ, Okazaki K, Murakami H, Stettler S, Fantes PA, Okayama H (1996) Stress signal, mediated by a Hog1-like MAP kinase, controls sexual development in fission yeast. FEBS Lett 378:207 Ketela T, Brown JL, Stewart RC, Bussey H (1998) Yeast Skn7p activity is modulated by the Sln1p-Ypd1p osmosensor and contributes to regulation of the HOG pathway. Mol Gen Genet 259:372 Kim K, Kim IH, Lee KY, Rhee SG, Stadtman ER (1988) The isolation and purification of a specific "protector" protein which inhibits enzyme inactivation by a thiol/Fe(III)/O2 mixed-function oxidation system. J Biol Chem 263:4704 Kobayashi N, McEntee K (1990) Evidence for a heat shock transcription factorindependent mechanism for heat shock induction of transcription in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 87:6550 Kobayashi N, McEntee K (1993) Identification of cis and trans components of a novel heat shock stress regulatory pathway in Saccharomyces cerevisiae. Mol Cell Biol 13:248 Kosower NS, Kosower EM (1995) Diamide: an oxidant probe for thiols. Methods Enzymol 251:123 Krems B, Charizanis C, Entian K-D (1995) Mutants of Saccharomyces cerevisiae sensitive to oxidative and osmotic stress. Curr Genet 27:427 Krems B, Charizanis C, Entian K-D (1996) The response regulator-like protein Pos9/Skn7 of Saccharomyces cerevisiae is involved in oxidative stress resistance. Curr Genet 29:327 Kudo N, Taoka H, Toda T, Yoshida M, Horinouchi S (1999) A novel export signal sensitive to oxidative stress in the fission yeast transcription factor Pap1. J Biol Chem 274:15151 Kuge S et al. (2001) Regulation of the yeast Yap1p nuclear export signal is mediated by redox signal-induced reversible disulfide bond formation. Mol Cell Biol 21:6139 Kuge S, Jones N (1994) YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. EMBO J 13:655 Kuge S, Jones N, Nomoto A (1997) Regulation of yAP-1 nuclear localization in response to oxidative stress. EMBO J 16:1710 Kuge S, Toda T, Lizuka N, Nomoto A (1998) Crm1 (Xpo1) dependent nuclear export of the budding yeast transcription factor yAP-1 is sensitive to oxidative stress. Genes to Cells 3:521 Lai CS, Piette LH (1978) Spin-trapping studies of hydroxyl radical production involved in lipid peroxidation. Arch Biochem Biophys 190:27 Lapinskas PJ, Cunningham KW, Liu XF, Fink GR, Culotta VC (1995) Mutations in PMR1 suppress oxidative damage in yeast cells lacking superoxide dismutase. Mol Cell Biol 15:1382

296


Laun P et al. (2001) Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis. Mol Microbiol 39:1166 Lee J, Dawes IW, Roe JH (1997) Isolation, expression, and regulation of the pgr1(+) gene encoding glutathione reductase absolutely required for the growth of Schizosaccharomyces pombe. J Biol Chem 272:23042 Lee J et al. (1999a) Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast. J Biol Chem 274:16040 Lee J, Romeo A, Kosman DJ (1996) Transcriptional remodeling and G1 arrest in dioxygen stress in Saccharomyces cerevisiae. J Biol Chem 271:24885 Lee J, Spector D, Godon C, Labarre J, Toledano MB (1999b) A new antioxidant with alkyl hydroperoxide defense properties in yeast. J Biol Chem 274:4537 Leppert G, McDevitt R, Falco SC, Van Dyk TK, Ficke MB, Golin J (1990) Cloning by gene amplification of two loci conferring multiple drug resistance in Saccharomyces. Genetics 125:13 Leroy C, Mann C, Marsolier MC (2001) Silent repair accounts for cell cycle specificity in the signaling of oxidative DNA lesions. EMBO J 20:2896 Levine RL, Moskovitz J, Stadtman ER (2000) Oxidation of methionine in proteins: roles in antioxidant defense and cellular regulation. IUBMB Life 50:301 Levine RL, Mosoni L, Berlett BS, Stadtman ER (1996) Methionine residues as endogenous antioxidants in proteins. Proc Natl Acad Sci U S A 93:15036 Levine RL, Williams JA, Stadtman ER, Shacter E (1994) Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233:346 Li S et al. (1998) The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators, SSK1p and Skn7p. EMBO J 17:6952 Li ZS, Lu YP, Zhen RG, Szczypka M, Thiele DJ, Rea PA (1997) A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathionato)cadmium. Proc Natl Acad Sci USA 94:42 Ligr M, Madeo F, Frohlich E, Hilt W, Frohlich KU, Wolf DH (1998) Mammalian Bax triggers apoptotic changes in yeast. FEBS Lett 438:61 Lin SJ, Culotta VC (1996) Suppression of oxidative damage by Saccharomyces cerevisiae ATX2, which encodes a manganese-trafficking protein that localizes to Golgi-like vesicles. Mol Cell Biol 16:6303 Lin SJ, Pufahl RA, Dancis A, O'Halloran TV, Culotta VC (1997) A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport. J Biol Chem 272:9215 Liochev SI, Fridovich I (1994) The role of O2- in the production of HO: in vitro and in vivo. Free Radic Biol Med 16:29 Liu X, Elashvili I, Gralla E, Valentine J, Lapinskas P, Culotta V (1992) Yeast lacking superoxide dismutase Isolation of genetic suppressors. J Biol Chem 267:18298 Liu X-D, Thiele DJ (1996) Oxidative stress induces heat shock factor phosphorylation and HSF-dependent activation of yeast metallothionein gene transcription. Genes Dev 10:592 Liu X-D, Thiele DJ (1997) Yeast metallothionein gene expression in response to metals and oxidative stress. METHODS: A companion in methods in enzymology 11:289 Liu XF, Culotta VC (1994) The requirement for yeast superoxide dismutase is bypassed through mutations in BSD2, a novel metal homeostasis gene. Mol Cell Biol 14:7037


297

Longo VD, Ellerby LM, Bredesen DE, Valentine JS, Gralla EB (1997) Human Bcl-2 reverses survival defects in yeast lacking superoxide dismutase and delays death of wildtype yeast. J Cell Biol 137:1581 Longo VD, Gralla EB, Valentine JS (1996) Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae Mitochondrial production of toxic oxygen species in vivo. J Biol Chem 271:12275 Luikenhuis S, Perrone G, Dawes IW, Grant CM (1998) The yeast Saccharomyces cerevisiae contains two glutaredoxin genes that are required for protection against reactive oxygen species. Mol Biol Cell 9:1081 Machado AK, Morgan BA, Merrill GF (1997) Thioredoxin reductase-dependent inhibition of MCB cell cycle box activity in Saccharomyces cerevisiae. J Biol Chem 272:17045 Madeo F et al. (1999) Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 145:757 Marchler G, Schuller C, Adam G, Ruis H (1993) A Saccharomyces cerevisiae UAS element controlled by protein kinase A activates transcription in response to a variety of stress conditions. EMBO J 12:1997 Maris AF, Assumpcao AL, Bonatto D, Brendel M, Henriques JA (2001) Diauxic shiftinduced stress resistance against hydroperoxides in Saccharomyces cerevisiae is not an adaptive stress response and does not depend on functional mitochondria. Curr Genet 39:137 Marquez JA, Pascual-Ahuir A, Proft M, Serrano R (1998) The Ssn6-Tup1 repressor complex of Saccharomyces cerevisiae is involved in the osmotic induction of HOGdependent and -independent genes. EMBO J 17:2543 Martinez-Pastor MT, Marchler G, C S, Marchler-Bauer A, Ruis H, Estruch F (1996) The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress-response element (STRE). EMBO J 15:227 Matsuyama S, Llopis J, Deveraux QL, Tsien RY, Reed JC (2000) Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cell Biol 2:318 Millar JB (1999) Stress-activated MAP kinase (mitogen-activated protein kinase) pathways of budding and fission yeasts. Biochem SocSymp 64:49 Millar JBA, Buck V, Wilkinson MG (1995) Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing Map kinase controlling cell size at division in fission yeast. Genes & Dev 9:2117 Molz L, Booher R, Young B, Beach D (1989) cdc2 and the regulation of mitosis: six interacting mcs genes. Genetics 122:773 Morgan BA, Banks GR, Toone WM, Raitt D, Kuge S, Johnston L (1997) The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae. EMBO J 16:1035 Morgan BA, Bouquin N, Merril GF, Johnston LH (1995) A yeast transcription factor bypassing the requirement for SBF and DSC1/MBF in budding yeast has homology to bacterial signal transduction proteins. EMBO J 14:5679 Moskovitz J, Berlett BS, Poston JM, Stadtman ER (1997) The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo. Proc Natl Acad Sci U S A 94:9585 Moskovitz J, Flescher E, Berlett BS, Azare J, Poston JM, Stadtman ER (1998) Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress. Proc Natl Acad Sci U S A 95:14071

298


Mouaheb N, Thomas D, Verdoucq L, Monfort P, Meyer Y (1998) In vivo functional discrimination between plant thioredoxins by heterologous expression in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 95:3312 Moye-Rowley WS, Harshman KD, Parker CS (1989) Yeast YAP1 encodes a novel form of the jun family of transcriptional activator proteins. Genes Dev 3:283 Muller EG (1991) Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle. J Biol Chem 266:9194 Muller EG (1994) Deoxyribonucleotides are maintained at normal levels in a yeast thioredoxin mutant defective in DNA synthesis. J Biol Chem 269:24466 Muller EG (1995) A redox-dependent function of thioredoxin is necessary to sustain a rapid rate of DNA synthesis in yeast. Arch Biochem Biophys 318:356 Muller EG (1996) A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thioredoxin for growth. Mol Biol Cell 7:1805 Nakagawa CW, Yamada K, Mutoh N (1998) Two distinct upstream regions are involved in expression of the catalase gene in Schizosaccharomyces pombe in response to oxidative stress. J Biochem (Tokyo) 123:1048 Nakagawa CW, Yamada K, Mutoh N (1999) Identification of the catalase gene promoter region involved in superinduction in Schizosaccharomyces pombe caused by cycloheximide and hydrogen peroxide. FEMS Microbiol Lett 173:373 Narasimhan ML et al. (2001) A plant defense response effector induces microbial apoptosis. Mol Cell 8:921 Nehlin JO, Carlberg M, Ronne H (1992) Yeast SKO1 gene encodes a bZIP protein that binds to the CRE motif and acts as a repressor of transcription. Nucleic Acids Res 20:5271 Nestelbacher R, Laun P, Vondrakova D, Pichova A, Schuller C, Breitenbach M (2000) The influence of oxygen toxicity on yeast mother cell-specific aging. Exp Gerontol 35:63 Netto LES, Chae HZ, Kang S-W, Rhee SG, Stadtman ER (1996) Removal of hydrogen peroxide by thiol-specific antioxidant enzyme (TSA) is involved with its antioxidant properties. J biol Chem 271:15315 Nguyen AN, Lee A, Place W, Shiozaki K (2000) Multistep phosphorelay proteins transmit oxidative stress signals to the fission yeast stress-activated protein kinase. Mol Biol Cell 11:1169 Nguyen AN, Siozaki K (1999) Heat-shock-induced activation of stress MAP kinase is regulated by threonine and tyrosine-specific phosphatases. Genes Dev 13:1653 Nguyen DT, Alarco AM, Raymond M (2001) Multiple Yap1p-binding sites mediate induction of the yeast major facilitator FLR1 gene in response to drugs, oxidants, and alkylating agents. J Biol Chem 276:1138 Nogae I, Johnston M (1990) Isolation and characterization of the ZWF1 gene of Saccharomyces cerevisiae, encoding glucose-6-phosphate dehydrogenase. Gene 96:161 Ohmiya R, Kato C, Yamada H, Aiba H, Mizuno T (1999) A fission yeast gene (prr1+) that encode a response regulator implicated in oxidative stress response. J Biochem 125:1061 Ohmiya R, Yamada H, Kato C, Aiba H, Mizuno T (2000) The Prr1 response regulator is essential for transcription of ste11+ and for sexual development in fission yeast. Mol Gen Genet 264:441 Oskouian B, Saba JD (1999) YAP1 confers resistance to the fatty acid synthase inhibitor cerulenin through the transporter Flr1p in Saccharomyces cerevisiae. Mol Gen Genet 261:346


299

Park SG, Cha MK, Jeong W, Kim IH (2000) Distinct physiological functions of thiol peroxidase isoenzymes in Saccharomyces cerevisiae. J Biol Chem 275:5723 Pascual-Ahuir A, Posas F, Serrano R, Proft M (2001) Multiple levels of control regulate the yeast cAMP-response element- binding protein repressor Sko1p in response to stress. J Biol Chem 276:37373 Pedrajas JR, Kosmidou E, Miranda-Vizuete A, Gustafsson JA, Wright AP, Spyrou G (1999) Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae. J Biol Chem 274:6366 Pedrajas JR, Miranda-Vizuete A, Javanmardy N, Gustafsson JA, Spyrou G (2000) Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity. J Biol Chem 275:16296 Pellequer JL, Brudler R, Getzoff ED (1999) Biological sensors: More than one way to sense oxygen. Curr Biol 9:416 Pena MM, Koch KA, Thiele DJ (1998) Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae. Mol Cell Biol 18:2514 Penninckx M (2000) A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses. Enzyme Microb Technol 26:737 Plummer JL, Smith BR, Sies H, Bend JR (1981) Chemical depletion of glutathione in vivo. Methods Enzymol 77:50 Prinz WA, Aslund F, Holmgren A, Beckwith J (1997) The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 272:15661 Proft M, Pascual-Ahuir A, de Nadal E, Arino J, Serrano R, Posas F (2001) Regulation of the Sko1 transcriptional repressor by the Hog1 MAP kinase in response to osmotic stress. EMBO J 20:1123 Proft M, Serrano R (1999) Repressors and upstream repressing sequences of the stressregulated ENA1 gene in Saccharomyces cerevisiae: bZIP protein Sko1p confers HOGdependent osmotic regulation. Mol Cell Biol 19:537 Prouzet-Mauleon V et al. (2001) Identification in S cerevisiae of a new stable variant of alkyl hydroperoxide reductase 1 (Ahp1) induced by oxidative stress. J Biol Chem 21:21 Raitt DC et al. (2000) The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress. Mol Biol Cell 11:2335 Rep M et al. (2001) The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage. Mol Microbiol 40:1067 Rhee SG, Kang SW, Netto LE, Seo MS, Stadtman ER (1999) A family of novel peroxidases, peroxiredoxins. Biofactors 10:207 Rodriguez-Manzaneque MT, Ros J, Cabiscol E, Sorribas A, Herrero E (1999) Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae. Mol Cell Biol 19:8180 Ross SJ, Findlay VJ, Malakasi P, Morgan BA (2000) Thioredoxin peroxidase is required for the transcriptional response to oxidative stress in budding yeast. Mol Biol Cell 11:2631 Ruis H, Hamilton B (1992) Regulation of yeast catalase genes. Molecular Biology of Free Radical Scavenging System:153 Ruiz-Echevarria MJ, Peltz SW (2000) The RNA binding protein Pub1 modulates the stability of transcripts containing upstream open reading frames. Cell 101:741

300


Russell P, Nurse P (1987) Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog. Cell 49:559 Rutherford JC, Jaron S, Ray E, Brown PO, Winge DR (2001) A second iron-regulatory system in yeast independent of Aft1p. Proc Natl Acad Sci U S A 98:14322 Samejima I, Mackie S, Fantes PA (1997) Multiple modes of activation of the stressresponsive MAP kinase pathway in fission yeast. EMBO J 16:6162 Samejima I, Mackie S, Warbrick E, Weisman R, Fantes PA (1998) The fission yeast mitotic regulator win1+ encodes a MAP kinase kinase kinase that phosphorylates and activates Wis1 MAP kinase kinase in response to high osmolarity. Mol Biol Cel 9:2325 Schmitt AP, McEntee K (1996) Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 93:5777 Schnell N, Entian KD (1991) Identification and characterization of a Saccharomyces cerevisiae gene (PAR1) conferring resistance to iron chelators. Eur J Biochem 200:487 Schuller C, Brewster JL, Alexander MR, Gustin MC, Ruis H (1994) The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the saccharomyces cerevisiae CTT1 gene. EMBO J 13:4382 Serpe M, Joshi A, Kosman DJ (1999) Structure-function analysis of the protein-binding domains of Mac1p, a copper-dependent transcriptional activator of copper uptake in Saccharomyces cerevisiae. J Biol Chem 274:29211 Sharma KG, Sharma V, Bourbouloux A, Delrot S, Bachhawat AK (2000) Glutathione depletion leads to delayed growth stasis in Saccharomyces cerevisiae: evidence of a partially overlapping role for thioredoxin. Curr Genet 38:71 Shieh J-C, Wilkinson MG, Buck V, Morgan BA, Makino K, Millar JBA (1997) The Mcs4 response regulator coordinately controls the stress-activated Wak1-Wis-1-Sty1 MAP kinase pathway and fission yeast cell cycle. Genes Dev 11:1008 Shieh J-C, Wilkinson MG, Millar JBA (1998a) The Win1 mitotic regulator is a component of the stress-activated Sty1 MAP kinase pathway in fission yeast. Mol Biol Cel 9:311 Shieh JC, Martin H, Millar JB (1998b) Evidence for a novel MAPKKK-independent pathway controlling the stress activated Sty1/Spc1 MAP kinase in fission yeast. J Cell Sci 111:2799 Shiozaki K, Russell P (1995) Cell cycle control linked to the extracellular environment by MAP kinase pathway in fission yeast. Nature 378:739 Shiozaki K, Russell P (1996) Conjugation, meiosis, and the osmotic stress response are regulated by Spc1 kinase through Atf1 transcription factor in fission yeast. Genes Dev 10:2276 Shiozaki K, Shiozaki M, Russell P (1997) Mcs4 mitotic catastrophe suppressor regulates the fission yeast cell cycle through the Wik1-Wis1-Spc1 kinase cascade. Mol Biol Cel 8:409 Singh KK (2000) The Saccharomyces cerevisiae Sln1p-Ssk1p two-component system mediates response to oxidative stress and in an oxidant-specific fashion. Free Radic Biol Med 29:1043 Slekar KH, Kosman DJ, Culotta VC (1996) The yeast copper/zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection. J Biol Chem 271:28831 Spector D, Labarre J, Toledano MB (2001) A genetic investigation of the essential role of glutathione: mutations in the proline biosynthesis pathway are the only suppressors of glutathione auxotrophy in yeast. J Biol Chem 276:7011


301

Srinivasan C, Liba A, Imlay JA, Valentine JS, Gralla EB (2000) Yeast lacking superoxide dismutase(s) show elevated levels of "free iron" as measured by whole cell electron paramagnetic resonance. J Biol Chem 275:29187 Stanhill A, Schick N, Engelberg D (1999) The yeast ras/cyclic AMP pathway induces invasive growth by suppressing the cellular stress response. Mol Cell Biol 19:7529 Stephen DW, Jamieson DJ (1996) Glutathione is an important antioxidant molecule in the yeast Saccharomyces cerevisiae. FEMS Microbiol Lett 141:207 Stephen DW, Jamieson DJ (1997) Amino acid-dependent regulation of the Saccharomyces cerevisiae GSH1 gene by hydrogen peroxide. Mol Microbiol 23:203 Stephen DW, Rivers SL, Jamieson DJ (1995) The role of the YAP1 and YAP2 genes in the regulation of the adaptive oxidative stress responses of Saccharomyces cerevisiae. Mol Microbiol 16:415 Storz G, Imlay JA (1999) Oxidative stress. Curr Opin Microbiol 2:188 Strain J et al. (1998) Suppressors of superoxide dismutase (SOD1) deficiency in Saccharomyces cerevisiae Identification of proteins predicted to mediate iron-sulfur cluster assembly. J Biol Chem 273:31138 Sturtz LA, Diekert K, Jensen LT, Lill R, Culotta VC (2001) A fraction of yeast cu,znsuperoxide dismutase and its metallochaperone, ccs, localize to the intermembrane space of mitochondria a physiological role for sod1 in guarding against mitochondrial oxidative damage. J Biol Chem 276:38084 Takeda T, Toda T, Kominami K, Kohnosu A, Yanagida M, Jones N (1995) Schizosaccharomyces pombe atf1+ encodes a transcription factor required for sexual development and entry into stationary phase. EMBO J 14:6193 Takeuchi T, Miyahara K, Hirata D, Miyakawa T (1997) Mutational analysis of Yap1 protein, an AP-1-like transcriptional activator of Saccharomyces cerevisiae. FEBS Lett 416:339 Tamai KT, Gralla EB, Ellerby LM, Valentine JS, Thiele DJ (1993) Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase. Proc Natl Acad Sci U S A 90:8013 Tamai KT, Liu X, Silar P, Sosinowski T, Thiele DJ (1994) Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways. Mol Cell Biol 14:8155 Tao W, Deschenes RJ, Fassler JS (1999) Intracellular glycerol levels modulate the activity of Sln1p, a Saccharomyces cerevisiae two-component regulator. J Biol Chem 274:360 Tenreiro S, Fernandes AR, Sa-Correia I (2001) Transcriptional activation of FLR1 gene during Saccharomyces cerevisiae adaptation to growth with benomyl: role of Yap1p and Pdr3p. Biochem Biophys Res Commun 280:216 Thevelein JM et al. (2000) Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast. Enzyme Microb Technol 26:819 Thevelein JM, de Winde JH (1999) Novel sensing mechanisms and targets for the cAMPprotein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol 33:904 Thiele DJ (1988) ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene. Mol Cell Biol 8:2745 Thomas D, Cherest H, Surdin-Kerjan Y (1991) Identification of the structural gene for glucose-6-phosphate dehydrogenase in yeast Inactivation leads to a nutritional requirement for organic sulfur. EMBO J 10:547

302


Thomas D, Surdin-Kerjan Y (1997) Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 61:503 Thomas JA, Poland B, Honzatko R (1995) Protein sulfhydryls and their role in the antioxidant function of protein S-thiolation. Arch Biochem Biophys 319:1 Toda T et al. (1992) Fission yeast pap1-dependent transcription is negatively regulated by an essential nuclear protein, crm1. Mol Cel Biol 12:5474 Toda T, Shimanuki M, Yanagida M (1991) Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases. Genes Dev 5:60 Toone WM, Jones N (1999) AP-1 transcription factors in yeast. Curr Opin Gen Dev 9:55 Toone WM, Kuge S, Samuels M, Morgan BA, Toda T, Jones N (1998) Regulation of the fission yeast transcription factor Pap1 by oxidative stress: requirement for the nuclear export factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1. Genes Dev 12:1453 Toone WM, Morgan BA, Jones N (2001) Redox control of AP-1-like factors in yeast and beyond. Oncogene 20:2336 Tran LT, Inoue Y, Kimura A (1993) Oxidative stress response in yeast: purification and some properties of a membrane-bound glutathione peroxidase from Hansenula mrakii. Biochim Biophys Acta 1164:166 Treger JM, Schmitt AP, Simon JR, McEntee K (1998) Transcriptional factor mutations reveal regulatory complexities of heat shock and newly identified stress genes in Saccharomyces cerevisiae. J Biol Chem 273:26875 Turton HE, Dawes IW, Grant CM (1997) Saccharomyces cerevisiae exhibits a yAP-1mediated adaptive response to malondialdehyde. J Bacteriol 179:1096 van Loon AP, Pesold-Hurt B, Schatz G (1986) A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen. Proc Natl Acad Sci U S A 83:3820 Verdoucq L, Vignols F, Jacquot JP, Chartier Y, Meyer Y (1999) In vivo characterization of a thioredoxin h target protein defines a new peroxiredoxin family. J Biol Chem 274:19714 Vido K, Spector D, Lagniel G, Lopez S, Toledano MB, Labarre J (2001) A proteome analysis of the cadmium response in Saccharomyces cerevisiae. J Biol Chem 276:8469 Vincent AC, Struhl K (1992) ACR1, a yeast ATF/CREB repressor. Mol Cell Biol 12:5394 Wanke V, Accorsi K, Porro D, Esposito F, Russo T, Vanoni M (1999) In budding yeast, reactive oxygen species induce both RAS-dependent and RAS-independent cell cyclespecific arrest. Mol Microbiol 32:753 Wei JP, Srinivasan C, Han H, Valentine JS, Gralla EB (2001) Evidence for a novel role of copper-zinc superoxide dismutase in zinc metabolism. J Biol Chem 276:44798 Wemmie JA, Steggerda SM, Moye-Rowley WS (1997) The Saccharomyces cerevisiae AP1 protein discriminates between oxidative stress elicited by the oxidants H2O2 and diamide. J Biol Chem 272:7809 Wemmie JA, Szczypka MS, Thiele DJ, Moye-Rowley WS (1994) Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an ATP-binding cassette transporter-encoding gene, YCF1. J Biol Chem 269:32592 Werner-Washburne M, Braun E, Johnston GC, Singer RA (1993) Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol Rev 57:383


303

Wieser R et al. (1991) Heat shock factor-independent heat control of transcription of the CTT1 Gene encoding the cytosolic catalase T of Saccharomyces cerevisiae. J Biol Chem 266:12406 Wilkinson MG et al. (1999) Sin1: an evolutionarily conserved component of the eukaryotic SAPK pathway. EMBO J 18:4210 Wilkinson MG et al. (1996) The Atf1 transcription factor is a target for the Sty1 stress activated MAP kinase pathway in fission yeast. Genes Dev 10:2289 Williams KE, Cyert MS (2001) The eukaryotic response regulator Skn7p regulates calcineurin signaling through stabilization of Crz1p. EMBO J 20:3473 Winge DR (1999) Copper-regulatory domain involved in gene expression. Adv Exp Med Biol 448:237 Wong CM, Zhou Y, Ng RW, Kung Hf H, Jin DY (2001) Cooperation of yeast peroxiredoxins Tsa1p and Tsa2p in the cellular defense against oxidative and nitrosative stress. J Biol Chem 10:10 Wu A, Wemmie JA, Edgington NP, Goebl M, Guevara JL, Moye-Rowley WS (1993) Yeast bZip proteins mediate pleiotropic drug and metal resistance. J Biol Chem 268:18850 Wu AL, Moye-Rowley WS (1994) GSH1, which encodes gamma-glutamylcysteine synthetase, is a target gene for yAP-1 transcriptional regulation. Mol Cell Biol 14:5832 Yamada K, Nakagawa CW, Mutoh N (1999) Schizosaccharomyces pombe homologue of glutathione peroxidase, which does not contain selenocysteine, is induced by several stresses and works as an antioxidant. Yeast 15:1225 Yamaguchi-Iwai Y, Dancis A, Klausner RD (1995) AFT1: a mediator of iron regulated transcriptional control in Saccharomyces cerevisiae. EMBO J 14:1231 Yan C, Lee LH, Davis LI (1998) Crm1p mediates regulated nuclear export of a yeast AP-1like transcription factor. EMBO J 17:7416 Yurkow EJ, McKenzie MA (1993) Characterization of hypoxia-dependent peroxide production in cultures of Saccharomyces cerevisiae using flow cytometry: a model for ischemic tissue destruction. Cytometry 14:287 Zhang X, De Micheli M, Coleman ST, Sanglard D, Moye-Rowley WS (2000) Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p. Mol Microbiol 36:618 Zheng M, Aslund F, Storz G (1998) Activation of the OxyR transcription factor by reversible disulfide bond formation. Science 279:1718 Zheng M, Storz G (2000) Redox sensing by prokaryotic transcription factors. Biochem Pharmacol 59:1 Zhu Z, Labbe S, Pena MM, Thiele DJ (1998) Copper differentially regulates the activity and degradation of yeast Mac1 transcription factor. J Biol Chem 273:1277

7 From feast to famine; adaptation to nutrient availability in yeast Joris Winderickx1, Inge Holsbeeks1, Ole Lagatie1, Frank Giots1, Johan Thevelein1 and Han de Winde2,3 1

Laboratorium voor Moleculaire Celbiologie, Departement Biologie, Katholieke Universiteit Leuven, 2DSM Life Sciences, Bakery Ingredients RD&T, Beijerinck Laboratory, 3 Kluyver Laboratory for Biotechnology, Technical University, Delft, The Netherlands

Abstract The study of signal transduction in microorganisms has become a major research topic in molecular and cellular biology. In this era, thorough knowledge of microbial physiology is no longer the sole and exclusive interest of academic research. It is now being acknowledged as a major importance for food, feed, and nutritional R&D. Detailed investigation of the mechanisms by which cells respond to environmental stimuli is contributing largely to both our fundamental and applied understanding of microorganisms. Baker’s yeast Saccharomyces cerevisiae has proven to be an important model organism in this respect. This budding yeast is widely used in food and feed applications and for synthesis of various useful compounds. This yeast has the remarkable capacity to thrive under a large variety of growth conditions, and can adequately adapt to rapid and profound changes in its environment. Hence, this yeast has become a fruitful model for the study of the coupling between nutrientinduced signal transduction and growth control. In this chapter, we have tried to give a broad overview of the current knowledge and insight into the mechanistic of nutrient-induced signal transduction in Saccharomyces cerevisiae. Since over the last ten years or so, this field of research has expanded significantly, the overview is necessarily multi-focused. After a general introduction on implications of yeast growth control, the Chapter is then divided into two major parts. The first part describes our current understanding of nutrient-specific response mechanisms, covering aspects of carbon, nitrogen, phosphor and sulphur signalling, and responses to both depletion and replenishment of these basic nutritional compounds. In the second part, we describe common aspects of nutrient-induced signal transduction in yeast. Overlap and crosstalk mechanisms in signalling, signal integration, and general physiological responses enable the yeast cell to efficiently react to both large and subtle variations

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in its environment. Balancing and fine-tuning of physiological responses prove to be of major importance to the organism. Detailed insight into these signal transduction cascades and networks has set the stage for future investigations where the focus will not be merely on single-file mechanisms, but more and more toward interrelations between complex signal transduction networks.

7.1 Introduction Microorganisms share an unsurpassed ability to thrive within a seemingly barren environment. Of the eukaryotic kingdom, only the species at the very bottom of the evolutionary ladder manage to make themselves a living on nothing but a simple carbon source, some elementary nitrogen-, phosphor- and sulphur compounds, and some other essential trace elements. Thus, unicellular and simple multicellular fungi are not only at the bedrock of eukaryotic offspring, but are at the bottom of many food chains as well. In this respect, baker's yeast Saccharomyces cerevisiae may be regarded as a very useful example of a cultivated unicellular fungus. This budding yeast is widely used in beer brewery, winemaking, food production, and synthesis of many useful compounds. In addition, its capacity to grow under a wide variety of culturing conditions has made this yeast a fruitful model to study various aspects of nutrient-induced metabolic phenomena and growth control. As wild yeast growing in vineyards and apple trees, Saccharomyces sp. have to cope with long periods of nutritional shortage, alternating with brief encounters of plentiful abundance. Therefore, these yeasts have developed intricate ways to both profit from and survive on very low nutrient levels and to sense a sudden abundance of nutrients and efficiently reset their metabolism and growth rate to a rich environment. When a single essential nutrient becomes limiting and eventually absent, the cellular proliferative machinery is efficiently shut down and a survival program is launched. In the absence of any one of the essential nutrients, yeast cells enter a specific, non-proliferative state known as stationary phase (WernerWashburne et al. 1993; Werner-Washburne et al. 1996), with the ultimate aim of surviving the starvation period.

7.2 Setting the stage: limitation, starvation, and cell cycle checkpoints In this chapter, we will bring together current knowledge concerning the ways in which yeast cells respond to depletion of nutrients. An important discrimination is made between nutrient limitation and nutrient starvation. Since yeast, as all microorganisms that can use a wide variety of substances as nutrient sources, decreasing availability of one substrate can, in many instances, be compensated by

7 Adaptation to nutrient availability in yeast 307

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the utilisation of another. Thus, the response to nutrient limitation often encompasses a metabolic switch from a richer to a poorer nutrient source. For this, specific signalling and metabolic pathways often have to be activated. For S. cerevisiae cells growing on rich, sugar-containing media - be it in nature or under laboratory conditions - fermentable sugar will usually be the first limiting nutrient. Subsequently, metabolism and biosynthetic capacity are reprogrammed at the socalled 'diauxic shift', for the utilisation of ethanol and acetate as carbon sources, which have accumulated during previous fermentative growth. When eventually these compounds have been used up, the cells will enter stationary phase due to carbon source starvation (Fig.7.1). In S. cerevisiae, the availability of nutrients is checked within a narrow window in the G1 phase of the cell division cycle (Fig.7.2). When the nutrient supply is sufficient, the first events introducing a new proliferation cycle will be executed (START A) (Pringle and Hartwell 1981).

Diauxic shift

Exponential growth phase< fermentation

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time Post/diauxic growth phase respiration

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Fig. 7.1. Typical culture-density profile of a fermentative batch culture of Saccharomyces cerevisiae. A schematical representation of the increase in cell number and cell density of a batch culture of Saccharomyces cerevisiae inoculated in full-complement medium with a rapidly fermentable sugar (glucose, fructose, mannose) as a carbon source. A relatively brief lag-period and a rapid, exponential fermentative growth phase are followed by a second lag-phase when the sugar has become limiting. During this 'diauxic shift’, the cells reset their metabolic capacity from fermentation to respiration and subsequently resume growth using as a carbon source the ethanol, acetate and other products of the initial fermentative growth phase. When eventually the carbon source or another essential nutrient becomes exhausted, the cells enter a resting state or stationary phase, with the ultimate goal to survive the starvation period


Fig. 7.2. The Saccharomyces cerevisiae cell cycle. Proliferating budding yeast cells experience several distinct growth phases (Nurse 1992; Pringle and Hartwell 1981); G1 (gap1), S (DNA-Synthesis), G2 (gap2) and M (Mitosis, cell division) phases each are readily definable when a mother cell starts a new round of proliferation, resulting in the release of a new daughter. The latter will continue growing in G1 until she reaches the critical cell size required to traverse START into a new proliferation cycle. The difference in size between mother and daughter cells is especially pronounced in rapidly growing, fermenting cultures. The START decision in G1 is separated in two phases; START A being the nutrient and growth checkpoint and START B being the replication and proliferation checkpoint (Pringle and Hartwell 1981). When one or more essential nutrients are depleted from the growth medium, cells will enter an off-cycle quiescent state known as G0 (Drebot et al. 1990; Werner-Washburne et al. 1993; Werner-Washburne et al. 1996). When nutrients are available, cells will resume growth at START A

Limiting amounts of a certain nutrient cause the cells to stall transiently in G1 while reprogramming metabolism for the utilisation of any available alternative nutrient. When the cells experience definite starvation for one or more nutrients, they will enter stationary phase, which is distinct from G1 in many aspects and consequently named G0 (Drebot et al. 1990; Werner-Washburne et al. 1993). It is important to note that nutrient sensing and growth regulations are confined to the G1 phase of the cell cycle in other yeast species with Schizosaccharomyces pombe being a well-characterised example (D'Souza and Heitman 2001; Nurse 1992; Woollard and Nurse 1995). Hence, the general concept of eukaryotic nutrient sensing and the signal transduction pathways and metabolic regulation routes involved may be well conserved between divergent eukaryotic species.


7.3 Specific responses to nutrient depletion 7.3.1 Carbon Source Signalling Responses operative in carbon source utilisation In order to better understand the consequences of carbon source limitation and starvation in yeast, we will first provide an overview on how different carbon sources are used by the yeast cells and how any particular carbon sources affect yeast metabolism. Baker's yeast S. cerevisiae belongs to a group of so-called facultative anaerobic yeast. These microorganisms will ferment hexose sugars like glucose and fructose under both aerobic and anaerobic growth conditions. In aerobic batch cultures, typically about 70% of the available glucose is fermented to ethanol and CO2, 20% is incorporated into biomass, 8% is used in glycerol production and only 2% will yield CO2 and H2O via oxidative phosphorylation inside the mitochondria.

Fig. 7.3. Carbon flow and intermediary metabolism in yeast. Overview of utilisation (A) and intermediary metabolism (B-next page) of diverse sugar- and non-sugar carbon sources in Saccharomyces cerevisiae. Names of enzymes and enzyme families involved in the various utilisation pathways are depicted in the usual abbreviations. In 'B', the flow of various carbon sources is into the main metabolic routes glycolysis, gluconeogenesis, pentosephosphate cycle, and Krebs cycle is depicted in more detail. For more information, please refer to several excellent reviews (De Vries and Marres 1987; Fraenkel 1982; Wills 1990)


Accordingly, glycolytic flux is high and O2 consumption is low (Oehlen et al. 1993; Oehlen et al. 1990). Saccharomyces sp. will utilise a variety of compounds as carbon and energy sources (Fig. 7.3A). Glucose and fructose enter directly into the glycolytic pathway, where ATP is obtained from substrate phosphorylation and sugars are converted into pyruvate and then to ethanol and CO2. Galactose and mannose are first converted to glucose-6-phosphate and fructose-6-phosphate, which then enter glycolysis. Di- and tri-saccharides that can be utilised as carbon sources are cleaved by specific glycosidases into their compound monosaccharides and in this way yield glucose, fructose, and galactose (Fraenkel 1982; Wills 1990). As stated above, although only a small fraction of the fermentable carbon source is initially


completely metabolised and much less ATP is produced than during respiratory growth, yeast prefer fermentative growth. This may seem wasteful. However, since the ethanol - and acetate - produced during fermentation will be metabolised in the post-diauxic growth phase (Fig. 7.1) nearly all of the available carbon source will be used eventually. In addition, and perhaps more importantly, the production of high ethanol concentrations inhibits growth of most other microorganisms, allowing S. cerevisiae to dominate in spontaneous fermentation. Nonfermentable carbon sources such as ethanol, acetate, and lactate are metabolised via respiration in the TCA cycle with ATP being produced by oxidative phosphorylation (DeVries and Marres 1988). Glycerol, which enters glycolysis after its conversion to dihydroxyacetone phosphate, also requires respiration in order to serve as an energy source, because the excess NADH formed has to be reoxidised. During respiratory growth, cells produce hexose-phosphates by gluconeogenesis for biosynthesis of macromolecules. Most steps in the gluconeogenic pathway are catalysed by enzymes that are also used in glycolysis. Hence, in order to avoid futile cycling, strict control is exerted at the level of two antagonistic enzyme pairs; fructose-1,6-bisphosphatase (Fbp1) / phosphofructokinase (Pfk1) and phospho-enolpyruvate carboxykinase (Pck1)/pyruvate kinase (Pyk1) that catalyse the two irreversible steps (Fig. 7.3B) (Alonso et al. 1984; Fraenkel 1982; Wills 1990). Carbon source-dependent growth control Because of a multiple-level regulation of metabolism by the available carbon source, yeast batch cultures grown on glucose show several well-defined growth phases (Fig. 7.1). In the first phase, characterised by rapid growth, glucose is fermented with the concomitant repression of genes required for respiratory growth. When glucose is exhausted, culture enters a short adaptive lag-phase, known as diauxic shift. During this phase, the glucose-repressed genes become derepressed and the culture adapts its metabolism for the subsequent utilisation of ethanol and other by-products of fermentation (François et al. 1987). It is important to realise that derepression of certain enzymes begins well before complete glucose depletion (Moehle and Jones 1990). Genes encoding enzymes of the gluconeogenic pathway, on the other hand, are very sensitive to glucose and remain repressed at glucose concentrations that are lower than the Km of the high affinity glucose transport system (Yin et al. 2000; Yin et al. 1996). This high sensitivity is likely to be required to avoid futile cycling and to enable a clear-cut shift from glycolysis to gluconeogenesis. Interestingly, the BCY1 transcript, which encodes the regulatory subunit of protein kinase A (PKA), shows a transient five-fold increase during the diauxic shift (Werner-Washburne et al. 1993; Werner-Washburne et al. 1991).The increased expression of BCY1 may lead to a sudden drop in activity of PKA, which may be required to reset the regulatory machinery. In this way, adaptation to respiratory growth is facilitated (Boy-Marcotte et al. 1996). Growth in the third, post-diauxic phase is much slower and ceases with the exhaustion of the available ethanol and acetate after which the culture enters stationary phase (WernerWashburne et al. 1993; Werner-Washburne et al. 1996).


S. cerevisiae prefers the utilisation of fermentable sugars with glucose as the prime substrate of choice. Even when grown on a mixture of glucose and fructose or mannose, glucose will be metabolised first (Suomalainen and Oura 1971; Wills 1990). The metabolic preference for fermentable sugar is mediated through posttranslational inactivation and degradation of several enzymes and transcriptional repression of various genes involved in the utilisation of alternative carbon sources (Gancedo 1992; Johnston and Carlson 1992a; Thevelein 1994; Trumbly 1992). At the same time, glucose induces the biosynthesis and/or activation of enzymes required for its optimal metabolisation along with several components required to accomplish a faster growth rate (Cereghino and Scheffler 1996; Muller et al. 1995b; Wills 1990). Several signalling pathways are involved in this complex regulation. Of these, the main glucose-repression pathway (Carlson 1999; Gancedo 1998; Johnston 1999; Ronne 1995) and the Ras-cAMP pathway (Thevelein et al. 2000; Thevelein and de Winde 1999) have been studied in most detail. It is now generally accepted that carbon source signalling is not restricted to metabolic adaptation responses directed towards optimal use of the available carbon source but that it affects a variety of other processes as well. Examples of these are the general stress response, i.e. STRE-controlled and PDS-controlled genes, the accumulation of reserve carbohydrates (Boy-Marcotte et al. 1998; Moskvina et al. 1998; Pedruzzi et al. 2000; Tadi et al. 1999; Thevelein 1994), expression of ribosomal protein genes (RPG) (Griffioen et al. 1996; Griffioen et al. 1994; Mager and Planta 1991; Neuman-Silberberg et al. 1995), pseudohyphal growth (Gancedo 2001; Kron 1997), and senescence and life span of yeast (Jazwinski 1996; Jazwinski 1999). Some of these topics will be discussed in section 7.4. The main catabolite repression pathway Despite its name, carbon catabolite repression pathway is a signalling cascade activated by nutrient depletion. Limitation of sugar availability stimulates the activity of the central protein kinase Snf1, which subsequently causes relief of repression and activation of the transcription of a large number of genes. Several proteins have been identified that are involved in the pathways controlling transcriptional repression or derepression of genes that encode enzymes required for the utilisation of nonfermentable carbon sources. Although catabolite repression is often presented as one mechanism, it does not affect all glucose-repressible genes in the same way or to the same extent. Increasing evidence indicates that different signal transduction routes are involved. Indeed, mutants have been identified that only affect repression of a subset of the glucose repressible genes. The pathway normally referred to as the catabolite repression pathway or main glucose repression pathway has as central components: the Mig1 transcriptional repressor complex, the Snf1 protein kinase complex, and the Glc7 protein phosphatase PP1. It has been the topic of several detailed reviews (Carlson 1999; Gancedo 1998; Johnston 1999; Ronne 1995). Glucose repression affects synthesis of many enzymes, which can be classified into three main groups (Ronne 1995). The first group contains the specific gluconeogenic enzymes fructose-1,6-bisphosphatase,


Fig. 7.4. Catabolite repression in Saccharomyces cerevisiae. Simplified schematical analysis of mediators and targets in the catabolite repression response in baker's yeast. The central glucose-dependent repressor complex Mig1/Ssn6/Tup1 exerts repression on diverse gene families, including the family-specific transcriptional activator genes CAT8 (gluconeogenic genes), GAL4 (galactose utilisation genes), and HAP4 (respiratory genes). Derepression is achieved through inhibition/inactivation of the repressor complex by a complex consisting of protein kinase Snf1 and several associated (regulatory) subunits. Under repressing conditions, Snf1 activity is inhibited by several 'upstream' mediators, including Hxk1 and Hxk2, Grr1 and protein phosphatase 1 Glc7 with associated regulatory subunits


Fbp1, and phosphoenolpyruvate carboxykinase, Pck1, apparently together with glyoxylate cycle enzymes like isocitrate lyase, Icl1. Expression of these enzymes is exquisitely sensitive to glucose (Mercado et al. 1994; Yin et al. 1996) to prevent simultaneous operation of gluconeogenesis and glycolysis. The second group is represented by the mitochondrial enzymes involved in the Krebs cycle and respiration. These are repressed because they are largely, but not fully dispensable during fermentative growth (de Winde and Grivell 1993; Grivell 1995). Finally, all proteins involved in the uptake and metabolisation of alternative carbon sources, like galactose and maltose, are repressed as the cell switches to the utilisation of glucose or fructose as a more preferred fermentable carbon source (Hu et al. 1995b; Johnston et al. 1994; Klein et al. 1996). The distinction between these three groups of genes is reflected in the different sensitivities to and the extent of repression exerted by fermentable sugars. As will be discussed below, transcriptional activation of these genes in addition to relief from catabolite repression requires different specific induction mechanisms. A general scheme of the main glucose repression pathway is presented in Fig. 7.4. The glucose-dependent transcriptional repressor Mig1 binds to specific sites in the promoter of many glucose-repressed genes (Klein et al. 1996; Lundin et al. 1994; Nehlin et al. 1991; Nehlin and Ronne 1990). It prevents transcription by recruiting the general corepressor proteins Ssn6/Cyc8 and Tup1/Cyc9 (Treitel and Carlson 1995; Vallier and Carlson 1994). The glucose-regulated subcellular localisation of Mig1 determines its role and activity. Mig1 only resides in the nucleus and hence is able to repress transcription when high concentrations of glucose are available. When cells are deprived of glucose, Mig1 translocates to the cytoplasm and Mig1controlled genes become derepressed. This glucose-regulated translocation of Mig1 is controlled through phosphorylation by the serine-threonine kinase Snf1/Cat1 (Celenza and Carlson 1984; Celenza and Carlson 1986; Entian and Zimmermann 1982). The presence of glucose inhibits the activity of the Snf1/Cat1 protein kinase, which results in under-phosphorylation and nuclear localisation of Mig1 and subsequent repression of gene expression (DeVit et al. 1997; Östling and Ronne 1998; Treitel et al. 1998). Glucose-induced inactivation of Snf1/Cat1 is mediated by an autoinhibitory interaction of the regulatory domain and the catalytic domain (Fig 7.5.). When the glucose concentration drops, this interaction is relieved and the Snf1-regulatory domain interacts with the positive regulator Snf4/Cat3 (Celenza and Carlson 1989; Jiang and Carlson 1996). The association between Snf4 and Snf1 is stabilised by three scaffolding proteins Sip1, Sip2, Sip3 and Gal83 (Jiang and Carlson 1997; Lesage et al. 1994; Vincent and Carlson 1999; Yang et al. 1994). In contrast, under glucose limiting conditions, Snf1 is activated by phosphorylation by a yet unidentified upstream kinase at Thr210 of its activation loop (Ludin et al. 1998; Wilson et al. 1996). Full activation of Snf1 in response to limiting glucose is, however, essentially dependent on the regulatory subunit Snf4 (Mc Cartney and Schmidt 2001). Yeast protein phosphatase type 1 (PP1) plays an essential role in carbon control, regulation of glycogen accumulation, sporulation, cell cycle progression, and translation (Cannon et al. 1994; Feng et al. 1991; Francisco et al. 1994; Wek et al. 1992). Specificity of PP1 towards its various substrates is governed through inter-


High glucose kinase inactive Low glucose kinase active Snf4 regulatory

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Glc7 Glc7

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Snf1

Sip1 Sip2 or Gal83

Reg1 Reg1

Fig. 7.5. Proposed model for the regulation of Snf1 kinase complex. RD: regulatory domain of Snf1 and interaction domain with Snf4. KD: Catalytic domain of Snf1. The threonine residue 210 is phosphorylated in active Snf1 kinase (Adapted from Johnston 1999)

action with specific regulatory subunits. In the glucose repression control, network PP1, encoded by GLC7, acts antagonistically to Snf1. Its glucose-control targeted, regulatory subunit Reg1/Hex2 interacts with the catalytic domain of Snf1 and recruits the catalytic subunit of PP1, Glc7, to dephosphorylate Snf1 and presumably other components of the kinase complex (Ludin et al. 1998; Sanz et al. 2000a). In this way, the autoinhibited state of the Snf1 kinase complex is reintroduced. The glucose signal most likely inhibits initial phosphorylation of Snf1 and/or stimulates Snf1-dephosphorylation through the Glc7-Reg1 protein phosphatase. Recently, the protein Sip5 was found to interact both with Snf4 and Snf1 and with Reg1 providing a novel link between the Snf1 complex and PP1 (Sanz et al. 2000b). Although Mig1 has been implicated as the central glucose-dependent transcriptional repressor, the expression of various glucose-repressed genes is not affected by deletion op MIG1 (Ronne 1995). Indeed, two additional Mig1-related proteins, Mig2 and Yer028c have recently been identified (Lutfiyya et al. 1998; Lutfiyya and Johnston 1996). Yer028c binds to Mig1-consensus binding sites, however no genes have been identified that are regulated by this factor (Lutfiyya et al. 1998). Mig2, on the other hand, indeed contributes to glucose repression of SUC2 (a gene frequently used to study glucose repression) and to repression of MIG1. Although Mig1 and Mig2 appear functionally redundant, regulation of Mig2 activity involves an entirely different mechanism. Mig2 is constitutively localised within the nucleus, both in the presence and in absence of glucose, and Snf1 is not involved


in the regulation of Mig2 activity (Lutfiyya et al. 1998). In addition to the Mig1related proteins, other proteins may contribute to full repression of glucoseresponsive genes. For instance, the repressor Sfl1 binds near the SUC2 TATA box, contributing to repression through interaction with the RNA polymerase II associated Srb/mediator complex (Song and Carlson 1998). Groups of genes involved in one metabolic process are usually under the control of gene family-specific transcriptional activators. Snf1 has been shown to relieve Mig1-mediated glucose repression of genes encoding such family-specific transcriptional activators, thereby ensuring co-ordinated regulation of their expression. Well-characterised examples are Gal4, which activates transcription of genes (GAL1, GAL2, GAL7, GAL10 and MEL1) involved in the catabolism of galactose and melibiose (Johnston et al. 1994; Lohr and Lopez 1995), MalR, the regulator that activates transcription of the maltose transporter (MALT) and maltase (MALS) (Hu et al. 1995b), Cat8, which is involved in derepression of gluconeogenic genes (Hedges et al. 1995), and Hap4, which is part of a complex regulating genes involved in respiration (de Winde and Grivell 1993; Forsburg and Guarente 1989a; Forsburg and Guarente 1989b) (Fig 7.4). In parallel to regulation via Mig1, Snf1 directly regulates through phosphorylation the activity of the transcriptional activators Cat8 and Sip4, both being specific activators of gluconeogenic genes (Hiesinger et al. 2001; Lesage et al. 1996; Rahner et al. 1996; Randez-Gil et al. 1997; Vincent and Carlson 1998; Vincent and Carlson 1999). Interestingly, the Snf1 scaffolding subunit Gal83 mediates association of Snf1 with Sip4 (Vincent and Carlson 1999). It can be concluded that the Snf1 protein kinase complex exerts glucose-responsive control at multiple points in a multi-layered regulatory cascade. The nature of the glucose signal that affects Snf1 function remains a matter of discussion. Current opinions distinguish two different models (Fig. 7.6). The first model is based on the similarity between Snf1, Snf4, and Sip proteins to subunits of the mammalian AMP-activated protein kinase (Stapleton et al. 1994; Woods et al. 1994). According to this model AMP, the AMP/ATP ratio, or the ADP/ATP ratio can act as a signal for the sensing of glucose phosphorylation and metabolic activity. The Snf1 kinase activity was reported to correlate with the AMP/ATP ratio (Wilson et al. 1996). Indeed, when cells are growing in abundance of glucose, ATP generation during glycolysis depletes AMP. This results in a low AMP/ATP ratio and subsequent inactivation of Snf1. Under glucose limitation, ATP levels are depleted resulting in high AMP/ATP ratios and activation of Snf1. Since Snf1 is not directly activated by AMP, (Stapleton et al. 1994; Woods et al. 1994) it may be controlled by an Snf1 kinase in analogy to the mammalian AMPK-kinase (Hardie and Carling 1997; Johnston 1999). Thus, the signal for glucose repression is generated during glucose metabolism. Since both hexokinase 1 (Hxk1) and glucokinase 1 (Glk1) are subject to glucose-repression themselves, this model is consistent with the predominant role of hexokinase 2 (Hxk2) in glucose phosphorylation and glucose repression (de Winde et al. 1996; Stapleton et al. 1994). In the second model, a specific regulatory role is attributed to Hxk2. This model emerged from observations that Hxk2, but not Hxk1 or Glk1, are required to maintain glucose repression during growth on glucose (de Winde et al. 1996;


Fig. 7.6. The glucose repression signal. High levels of glucose are transported into the cell and phosphorylated by the hexose kinases. Model A: The production during fermentation of ATP depletes the pool of AMP. Because AMP levels are low, Snf1 is inactive and Mig1 exerts repression. When the glucose level drops significantly, the pool of AMP is repleted; Snf1 becomes active and inactivates Mig1. Model B: During the phosphorylation process, the hexokinases shift their conformation, and a regulatory function is displayed which inactivates the Snf1 complex

Entian and Fröhlich 1984; Entian et al. 1984). Several recent observations support this model. Novel alleles of HXK2 have been isolated that exhibit distinct effects on catalytic activity and catabolite repression of SUC2 (Hohmann et al. 1999; Kraakman et al. 1999b; Mayordomo and Sanz 2001). No correlation could be found between in vitro Hxk2 activity and in vivo accumulation of sugar phosphates and catabolite repression. Hence, the role of Hxk2 in glucose repression does not seem to be restricted to its sugar phosphorylating activity. Structurefunction analysis indicates that the establishment of catabolite repression is dependent on the phosphoryl transfer reaction on the kinase, and that formation of a transmission intermediate and the concomitant conformational change of the enzyme are crucial prerequisites (Hohmann et al. 1999; Kraakman et al. 1999b; Rolland et al. 2001). Interestingly, Hxk2 appears to have a direct role in regulating the phosphorylation state of the Reg1/Hex2 regulatory subunit of PP1/Glc7. Hxk2 stimulates association of Reg1 with Glc7 and thereby enhances PP1 phosphatase activity (Sanz et al. 2000a). Moreover, a fraction of the Hxk2 pool is located in the nucleus (Randez-Gil et al. 1998a) and this nuclear localisation allows Hxk2 to associate with DNA-protein complexes that may be involved in glucose repression of SUC2 (Herrero et al. 1998). In this way, Hxk2 may be involved in transducing the glucose signal directly to the transcriptional machinery that controls expression of glucose-repressed genes. Under conditions of glucose derepression, Hxk2 is phosphorylated at Ser15 within a PKA consensus sequence (Kriegel et al. 1994; Vojtek and Fraenkel


1990). Phosphorylation of Hxk2 shifts the monomer-dimer equilibrium to the monomeric state and increases glucose affinity. However, nuclear localisation does not appear to be affected (Behlke et al. 1998; Herrero et al. 1998). In the presence of excess glucose, the Hxk2 monomer is dephosphorylated by the Glc7/Reg1 protein phosphatase (Alms et al. 1999; Randez-Gil et al. 1998b). It is tempting to conclude that phosphorylation-dephosphorylation of Hxk2 plays an important role in glucose-signalling, but results obtained thus far have often been contradictory (Herrero et al. 1998; Ma et al. 1989; Randez-Gil et al. 1998b). The previously reported glucose-stimulated protein kinase activity of Hxk2 (Herrero et al. 1989) has been shown to correlate with ATP-dependent autophosphorylation (Fernandez et al. 1988) at Ser158 (Heidrich et al. 1997; Kraakman et al. 1999b). This highly conserved residue plays a central role in the phosphoryl-transfer reaction from ATP to the 6-OH group of the sugar and hence is an important determinant of sugar binding affinity, catalytic activity, and enzyme conformation. Other glucose repression mechanisms Although PKA does not appear to be directly involved in the establishment of glucose repression (de Winde et al. 1996; Hubbard et al. 1992), it is participating in transcriptional regulation of glucose-repressible genes. PKA-dependent expression control of SUC2 is mediated through the transcriptional repressor Sko1/Acr1 (Nehlin et al. 1992; Vincent and Struhl 1992) and Sfl1 (Song and Carlson 1998). Although in the fission yeast Schizosaccharomyces pombe, glucose repression of the fructose-1,6-bisphosphatase gene FBP1 is exerted through the cAMP pathway (Hoffman and Winston 1991; Nocero et al. 1994), in Saccharomyces glucose control of the gluconeogenic genes FBP1 and PCK1 is independent of the Ras-cAMP pathway or of PKA (Yin et al. 2000; Yin et al. 1996). PKA has been implicated in regulation of expression of the yeast sugar kinase genes and in this way PKA influences catabolite repression indirectly (de Winde et al. 1996). PKA also inhibits the activity of the STRE-binding transcription factors, Msn2 and Msn4, and these are required for the expression of the Yak1 kinase (Smith et al. 1998). Yak1 is a member of the DYRK family of kinases and it has now been shown that when glucose is exhausted it accumulates in the nucleus to phosphorylate Pop2, a subunit of the Ccr4-Not complex that regulates glucose derepression (Bai et al. 1999; Hata et al. 1998; Moriya et al. 2001; Sakai et al. 1992). In addition, PKA regulates the expression of many other genes whose products function in growth control and stress resistance. Therefore, they are not directly involved in the metabolic switch (Boy-Marcotte et al. 1996; Crauwels et al. 1997b; Griffioen et al. 1996; Griffioen et al. 1994; Mager and Planta 1991; Neuman-Silberberg et al. 1995; Pernambuco et al. 1996; Tadi et al. 1999). As will be discussed below, PKA activity is transiently boosted by cAMP through the Ras-cAMP pathway after glucose addition to nonfermenting cells. During growth on glucose, high PKA activity is maintained by cAMP-independent mechanisms via the FGM pathway. Catabolite repression not only influences the rate of transcription, but for several genes, it also affects the stability of the corresponding mRNA (Yin et al. 2000; Yin et al. 1996). This has been demonstrated for iso-1 cytochrome c (CYC1)


(Zitomer et al. 1979) and phosphoenolpyruvate carboxykinase (PCK1) (Mercado et al. 1994). More recently, glucose-induced mRNA turnover has also been described for the invertase gene SUC2, genes encoding the iron protein subunit of the mitochondrial Ip protein subunit of succinate dehydrogenase, SDH1 and SDH2 (Cereghino et al. 1995; Cereghino and Scheffler 1996), and the maltase gene MAL6S (Federoff et al. 1983). mRNA turnover is likely to comprise the shortterm effect of the biphasic catabolite repression response. This initial phase requires phosphorylation of the sugar by any of the sugar kinases Hxk1, Hxk2, or Glk1 (Cereghino and Scheffler 1996; de Winde et al. 1996; Scheffler et al. 1998). The hexose kinase requirement reflects the combined activation of the Ras-cAMP pathway and the main catabolite repression pathway (Scheffler et al. 1998; Tung and Hopper 1995; Yin et al. 2000). Catabolite repression is not only induced by glucose and other rapidly fermented sugars (fructose and mannose), but also occurs to some extent with galactose (Polakis and Bartley 1965) and maltose (Eraso and Gancedo 1984). However, the actual mechanisms triggering repression with sugars other than glucose have not been studied in much detail. We have investigated fructose-induced repression and observed that both yeast hexokinase isoforms, Hxk1 and Hxk2, are required (de Winde et al. 1996). Fructose and mannose appear to generate the same signals and thus most probably affect the same targets as glucose. Repression by galactose or maltose however, only affects a subset of the glucose-repressed genes (Gancedo 1998). This nicely illustrates the above-mentioned subdivision of glucoserepressible genes in separate families that exhibit different repression sensitivities. Yeast cells growing on galactose or maltose have to repress gluconeogenic enzymes in order to avoid futile cycling, while at the same time the glucoserepressible GAL and MAL genes have to be expressed. Galactose-induced repression of the cytoplasmic NAD-dependent glutamate dehydrogenase and the mitochondrial L-lactate ferricytochrome c oxidoreductase Cyb2 is dependent on the transcriptional activator Gal4 (Lodi et al. 1991). Since GAL4 itself is glucose-repressible, this indicates that the mechanisms for galactose - and glucose - induced repression follow at least partially different signalling routes. Maltose does not activate the Ras-cAMP pathway in a wild type strain (Rolland et al. 2000), but at least partially activates the FGM-pathway (J. Winderickx, H. de Winde and M. Vanoni: unpublished data). Hence, this sugar also apparently enhances PKA activity. It remains to be established how galactose and maltose regulate expression of PKA targets that are implicated in the switch from respiratory to fermentative growth, or vice-versa. The Ras-cAMP pathway; cAMP-dependent control of PKA activity Addition of glucose or another easily fermentable sugar to yeast cells grown on poorer carbon sources triggers a rapid transient increase in the cellular cAMP concentration, often referred to as the cAMP signal (Beullens et al. 1988; Mbonyi et al. 1988; Mbonyi et al. 1990; Thevelein 1991; Thevelein et al. 1987; van der Plaat 1974). For this reason (discussed in section 7.4), cAMP has been implicated as an important second messenger in the nutrient response, accomplishing the metabolic


adaptation to fermentative growth conditions through modulation of the activity of PKA. Hence, high intracellular levels of cAMP correlate with nutrient-rich growth conditions, whereas low cAMP levels reflect nutrient limitation and poor growth conditions. The concentration of cAMP in yeast cells is controlled by the RascAMP pathway (Fig.7.7 reviewed in Thevelein et al. 2000; Thevelein and de Winde 1999). Two conditions are known to activate the pathway: (1) glucose addition to nonfermenting or stationary phase cells and (2) intracellular acidification, for example by addition of a protonophore. It has now become clear how both triggers exert their effect. Adenylate cyclase encoded by CYR1/CDC35 (Kataoka et al. 1985; Matsumoto et al. 1984), is stimulated by the G-proteins Ras1 and Ras2. They are active in the GTP-bound state and inactive in the GDP-bound state (Broek et al. 1985; Toda et al. 1985). Exchange of GDP for GTP is stimulated by Cdc25 (Broek et al. 1987; Camonis et al. 1986; Jones et al. 1991) and possibly by the related Sdc25 (Camus et al. 1994). The Ras proteins possess intrinsic GTPase

Fig. 7.7. The Saccharomyces cerevisiae Ras-cAMP signalling route and FGM pathway. When yeast cells experience an abundance of fermentable carbon source the Ras-cAMP signalling cascade is initiated at the level of hexose phosphorylation and at the level of glucose detection by the GPCR system. The signal is then relayed possibly via Cdc25activated Ras1 and Ras2 to adenylate cyclase Cdc35. Subsequently synthesised cAMP (transient cAMP signal) causes the dissociation of the regulatory subunit Bcy1 from the catalytic Tpk subunit of PKA. A sugar phosphorylation-independent signalling cascade, the FGM pathway is initiated by a complete fermentable growth medium and activates the free catalytic subunits of PKA. The FGM pathway operates via the Sch9 kinase and Pph22 phosphatase independently from and in parallel to the sugar phosphorylation-dependent Ras-cAMP cascade. Activated PKA mediates various regulatory processes, leading to adaptation to fermentative metabolism. For details, see section 7.4.3


activity that causes self-inactivation. This GTPase activity is greatly stimulated by the IRA1 and IRA2 gene products (Tanaka et al. 1990). It has been shown that intracellular acidification may activate the Ras-cAMP pathway by inhibiting both GTPases (Colombo et al. 1998). For glucose, on the other hand, it was proposed that Cdc25 and Ras would be involved in the transduction of the signal (Mbonyi et al. 1988; Munder and Küntzel 1989; Van Aelst et al. 1991). Later studies reported that Cdc25 was not the receiver of the glucose signal (Goldberg et al. 1994) and that glucose did not alter the GDP/GTP ratio bound to Ras (Colombo et al. 1998). Now again, most recent data contradict the latter observation and indicate that an increased level of Ras-GTP could, at least partially, be involved in glucoseinduced activation of the pathway (Rudoni et al. 2001). Two sensing systems transmit the sugar signal (Rolland et al. 2000). In the first sensing process, a Gprotein coupled receptor (GPCR) system specifically detects extracellular Dglucose and sucrose but no other sugars (Rolland et al. 2000). This system consists of the receptor-like protein Gpr1 and its G-protein Gpa2 (Colombo et al. 1998; Kraakman et al. 1999a; Kubler et al. 1997; Lorenz et al. 2000; Xue et al. 1998). Rgs2 stimulates GTP hydrolysis on Gpa2. Deletion of RGS2 enhances while RGS2 overexpression reduces cAMP synthesis (Versele et al. 1999). The protein shows homology to Sst2, the GTPase activating protein of Gpa1, and other members of the RGS family. The phospholipase C, Plc1, has been reported to be required for the association between Gpr1 and Gpa2. Although Plc affects pseudohyphal growth similar to Gpr1 and Gpa2 (Ansari et al. 1999) (see section 4.6), it is not required for the GPCR-mediated activation of cAMP synthesis (Lemaire, Winderickx and Thevelein, unpublished data). The second sensing process is an intracellular mechanism that requires sugar uptake and phosphorylation by the hexose kinases. This process is not glucose specific. It transmits a signal stimulated by all sugars that can be phosphorylated by the hexose kinases, i.e. fructose, mannose, and even maltose (Rolland et al. 2000; Rolland et al. 2001). Like in other eukaryotic cells, cAMP activates PKA by binding to the regulatory subunit (encoded by BCY1) (Toda et al. 1987a), thereby causing the dissociation and concomitant activation of the catalytic subunits (encoded by TPK1, TPK2, and TPK3) (Toda et al. 1987b). Two additional mechanisms strictly control the activity of the Ras-cAMP pathway during fermentative growth. First, there is a clear inverse correlation between the glucose-repressed state and the ability to generate a fermentable sugar induced cAMP signal (Beullens et al. 1988). Hence, it seems that the sugar activating system contains - or at least is influenced by - a protein that is controlled by the main catabolite repression pathway. Second, PKA activity causes strong feedback inhibition explaining the transient nature of the cAMP signal (Mbonyi et al. 1990; Nikawa et al. 1987). The low-affinity phosphodiesterase Pde1 was recently identified as the first PKA target involved in this feedback control (Ma et al. 1999). The activated catalytic subunits of PKA phosphorylate a number of enzymes and transcription factors, some of which have been identified. These targets are involved in different regulatory processes required for the shift from gluconeogenic to fermentative growth. Other known targets of PKA are involved in the


breakdown of storage carbohydrates, in stress resistance, growth control, and determination of life span (reviewed in Thevelein 1994; Thevelein et al. 2000; Thevelein and de Winde 1999). Recently, the protein kinase Rim15 was identified as an immediate downstream target of PKA. Rim15 controls different adaptation responses related to nutrient limitation (Pedruzzi et al. 2000; Reinders et al. 1998). At the level of transcription, Rim15 exerts its effect through the transcription factor Gis1. Gis1 binds to the so-called PDS or post-diauxic-shift DNA-element found in the promoter of some stress-responsive genes like SSA3 (Pedruzzi et al. 2000). Whether Rim15 is involved in the control of the activity of the STREbinding factors, Msn2 and Msn4, remains to be elucidated. Data obtained so far, however, appear to indicate that the function of these transcriptional activators is directly inhibited by PKA (Görner et al. 1998). As will be discussed in more detail in section 7.4.2, the activity of PKA is controlled also by a separate signalling route termed the Fermentable Growth Medium-induced (FGM) pathway (Crauwels et al. 1997a; Durnez et al. 1994; Hirimburegama et al. 1992; Thevelein et al. 2000; Thevelein and de Winde 1999; Winderickx et al. 1996). Activation of the FGM-pathway is not only dependent on the availability of glucose or fructose but also on the availability of all other nutrients essential for growth. In contrast to the Ras-cAMP pathway, only the presence of the sugar and not its phosphorylation is required for FGM-activation, indicating that this pathway acts as a specific nutrient sensing system (Pernambuco et al. 1996). The FGM-pathway has been shown to be independent of the cAMP level. Hence, this pathway possibly acts directly on the free catalytic subunits of PKA (Durnez et al. 1994; Hirimburegama et al. 1992). Catabolite inactivation Addition of glucose to cells growing on a non-fermentable carbon source triggers the rapid inactivation of many enzymes. This process, called catabolite inactivation (Holzer 1976) has been studied in most detail for gluconeogenic Pck1 and Fbp1 (Gancedo and Gancedo 1979). Other enzymes that are known to be subject to catabolite inactivation include malate dehydrogenase and isocitrate lyase, which are required for the glyoxylate cycle (Ordiz et al. 1995), proteins involved in the uptake and metabolism of maltose and galactose (Lucero et al. 1993; Riballo et al. 1995), and the high affinity transport system for glucose. For fructose-1,6-bisphosphatase (Fbp1), catabolite inactivation consists of two steps (Gancedo 1971; Holzer 1984). The enzyme is first reversibly inactivated by phosphorylation at a site recognised by PKA in vivo and then degraded by targeted proteolysis. Mutants deficient in adenylate cyclase do not show glucose-induced inactivation of Fbp1 (Rose et al. 1988). PKA-mediated phosphorylation is regulated by cAMP and by the level of fructose-2,6-bisphosphate via allosteric alteration of Fbp1. Inactivation through phosphorylation is, however, not required for subsequent proteolytic degradation of the enzyme, which, despite some debate (Schork et al. 1994; Schork et al. 1995), appears to occur inside the vacuole (Chiang and Schekman 1991; Chiang and Schekman 1994). Glucose-induced vacuolar targeting of Fbp1 is mediated by small vesicles, which are translocated to


the vacuole, presumably by microautophagy (Chiang et al. 1996; Hoffman and Chiang 1996). A similar two-step inactivation pattern was reported for the maltose and the galactose transporters (Chiang et al. 1996; Lucero et al. 1993; Riballo et al. 1995). More recently, it was shown that specific mutations of PKA and PKC sites in the maltose transporter, Mal61, exhibited significantly reduced rates of glucoseinduced transporter inactivation. Only for some of these mutants, this was paralleled by changes in the rate of degradation (Brondijk et al. 1998). This indicates that phosphorylation results in protein inactivation but not necessarily in an increased rate of protein degradation. In addition, two separate signalling processes appear to be involved in glucose-induced inactivation of the maltose transporter. The first process requires Rgt2 and is independent of glucose uptake and metabolism. This process stimulates transporter protein degradation but not inactivation. The second process is dependent on glucose transport and phosphorylation but not on further glucose metabolism and it stimulates both very rapid inactivation of maltose transport activity and transporter degradation (Jiang et al. 2000). Glucoseinduced degradation of the maltose and galactose transporter proteins is initiated via translocation to the vacuole by the endocytic pathway (Chiang et al. 1996; Lucero et al. 1993; Riballo et al. 1995). Finally, a reversible inactivation followed by an irreversible degradation has also been reported for isocitrate lyase, Icl1 (Lopez-Boado et al. 1988; Ordiz et al. 1995). Catabolite activation and catabolite induction Yeast cells experiencing an abundance of fermentable sugar exhibit increased expression of many genes and enhanced activity of various enzymes required for efficient transport and metabolism of the sugar. These include several glycolytic enzymes such as pyruvate decarboxylase (Pdc1), alcohol dehydrogenase I (Adh1), enolase II (Eno2), one of the isoenzymes of 6-phosphofructo-2-kinase, and pyruvate kinase (Pyk1) (Entian and Barnett 1992). The mechanism for their glucosedependent induction has been the subject of some studies, however, in most cases remains elusive. Nevertheless, an increase in the intra-cellular concentration of different glycolytic metabolites is likely to be involved in the induction of several of these genes (Boles et al. 1993; Flikweert et al. 1999; Gonçalves and Planta 1998; Muller et al. 1995b). Much attention has been focussed on transcriptional regulation of the glycolytic genes. Most of these contain in their promoters binding sites for the multifunctional transcription factors Rap1, Reb1, Abf1, and/or Gcr1 (reviewed in Chambers et al. 1995). The Gcr1 protein (Baker 1986; Uemura and Jigami 1995), together with Gcr2 (Uemura and Jigami 1992) confers specific activation of glycolytic gene expression upon addition of glucose to the growth medium, as accessory factors for Rap1 (Deminoff and Santangelo 2001). Whereas Gcr1 and Gcr2 together are essential for rapid and efficient induction of glycolytic gene transcription, Gcr1 alone is involved in upregulation of ribosomal protein gene (RPG) expression (Lopez and Baker 2000; Scott and Baker 1993; Uemura and Jigami 1992). Interestingly, binding sites for Rap1 and Abf1 are also found in the pro-


moters of various RPG’s. In parallel with the glycolytic genes, also the RPG’s are highly expressed in yeast cells growing on fermentable sugars (reviewed in Doorenbosch et al. 1992). For the RPG's, it has been shown that induction is dependent on PKA activity, but not on cAMP. Accordingly, we among others have demonstrated that induction of the RPG's is largely regulated by the FGM- and TOR-pathways, which will be discussed in more detail in sections 7.4.3 and 7.4.4 (Crauwels et al. 1997b; Griffioen et al. 1996; Griffioen et al. 1994; Pernambuco et al. 1996; Powers and Walter 1999; Winderickx et al. 1996). Glucose efficiently controls the expression of components of its transport system. Enhanced transcription of various HXT genes is regulated by glucose in a concentration-dependent manner (Fig. 7.8) (Boles and Hollenberg 1997; Kruckeberg 1996). Expression of HXT1 is induced by high glucose concentrations, whereas transcription of HXT2 and HXT4 is activated by low levels of glucose and HXT3 expression is induced independently of sugar concentration (Özcan and Johnston 1995). Induction at low-glucose levels is mediated through the transporter homologue Snf3 (Celenza et al. 1988; Özcan et al. 1996a) via activation of the glucose-signalling component Grr1 (Flick and Johnston 1991). The F-box protein Grr1 interacts in a glucosedependent manner with the Skp1 subunit of the SCF-ubiquitin protein ligase complex. The Grr1-SCF complex targets Rgt1 for degradation (Kishi et al. 1998; Li and Johnston 1997) causing inhibition of the repressor-corepressor complex Rgt1/

Fig. 7.8. Regulation of the HXT transporter gene expression by glucose. Low levels of glucose are detected by Snf3, high levels by Rgt2. Low amounts of glucose inhibit the Rgt1 repressor function and causes derepression of HXT1-4. High levels of glucose enhance the Rgt1 induction function and causes induction of HXT1. Both processes are dependent on glucose phophorylation and Grr1. High levels of glucose induce Reg1, which inhibit Snf1 and induce catabolite repression control of HXT2, HXT4, and SNF3 but at the same time, Reg1 activates a transcriptional activator, which together with Rgt1 is required for full induction of HXT1. (Adapted from Özcan et al. 1998; Özcan et al. 1996a; Özcan and Johnston 1999)


Htr1-Ssn6 and, hence activation of transcription of HXT genes (Erickson and Johnston 1994; Özcan et al. 1993; Özcan et al. 1996b; Vallier et al. 1994). Highglucose induction is mediated through another transporter-homologue, Rgt2, which is very similar to Snf3 except for the extended C-terminal region (Özcan et al. 1996a). Hence, Rgt2 and Snf3 act as receptors/glucose sensors in transmitting the signal for glucose-induced gene expression (Özcan et al. 1998). It should be emphasised that carbon source dependent control of HXT genes is not separate from, but additionally mediated through regulators that are involved in glucose repression and derepression. Ssn6 and Tup1 function as corepressors of both Rgt1 (see above) and Mig1 in regulating expression of HXT2 and HXT4 (Özcan et al. 1996a; Özcan and Johnston 1995; Özcan et al. 1996b). Consequently, Hxk2 and Reg1 are required for glucose repression of these HXT genes (Özcan and Johnston 1995), and of the high-affinity transporter genes HXT6 and HXT7 (Liang and Gaber 1996; Petit et al. 2000). HXT4 transcription is additionally controlled through the glycolytic regulators Gcr1 and Gcr2 (Türkel and Bisson 1999). This multi-partite regulation of HXT genes in Saccharomyces cerevisiae enables this yeast to efficiently adapt hexose transport affinity and capacity to sugar availability under varying growth conditions (Diderich et al. 1999). Transcriptional induction of the GAL genes (Erickson and Johnston 1994; Johnston and Carlson 1992b; Johnston et al. 1994) and MAL genes (Hu et al. 1995b; Needleman 1991; Vanoni et al. 1989) by galactose and maltose, respectively, is achieved through both relief of repression and activation by specific activators. The genes encoding the Gal4 activator (Johnston et al. 1994) and the MalR activator (Hu et al. 1995b) are for instance subject to glucose repression through Mig1. Upon derepression these activators induce transcription of their specific target genes, which themselves are direct targets of catabolite repression. A comparable mechanism holds true for induction of gluconeogenic genes through their specific activators Cat8 and Sip4 (Hedges et al. 1995; Vincent and Carlson 1998). A similar mechanism also operates when glucose is limiting or absent for induction of genes encoding proteins for mitrochondrial respiration. This effect is mediated by the Hap4 activator subunit of Hap2/3/4/5 complex (de Winde and Grivell 1993; Forsburg and Guarente 1989b; Mc Nabb et al. 1995). Glucose repression of CAT8 is exerted through Mig1, albeit not exclusively (Randez-Gil et al. 1997). Upon derepression, the Cat8 protein is activated through phosphorylation by the Snf1 kinase, as is Sip4 (Randez-Gil et al. 1997; Vincent and Carlson 1998; Vincent and Carlson 1999). This enables Cat8 and Sip4 to participate in the transcriptional activation of yeast gluconeogenic genes in response to glucose depletion (Hiesinger et al. 2001). HAP4 expression is equally controlled through the glucose repression pathway (Forsburg and Guarente 1989b; Blom and de Winde, unpublished results). Interestingly, constitutive overexpression of the Hap4 subunit of the Hap2/3/4/5 activator complex is sufficient to bypass glucose repression control of respiratory metabolism (Blom et al. 2000). Taken together, the induction of many genes requires not only relief of catabolite repression but also of additional specific induction mechanisms.


7.3.2 Nitrogen Source Signalling Nitrogen catabolite repression S. cerevisiae can utilise a wide variety of nitrogen sources. The expression of nitrogen catabolic genes is controlled in response to the quality of the nitrogen source available. The presence of preferred nitrogen sources that are readily transported and metabolised represses the expression of genes encoding transporters and catabolic enzymes necessary for the uptake and utilisation of more poorly used nitrogen sources. Thus, growth of Saccharomyces sp. on media containing ammonia, glutamine, or asparagine as nitrogen source causes nitrogen catabolite repression (NCR) of enzymes necessary for the utilisation of - amongst others proline, allantoin, or γ-aminobutyrate (see Table 7.1) (Cooper 1982; Magasanik 1992). In S. cerevisiae, nitrogen catabolite gene expression is regulated by the action of four GATA family transcription factors: Gln3 and Gat1/Nil1 are transcriptional activators, and Dal80/Uga43 and Deh1/Gzf3 are transcriptional repressors (reviewed by ter Schure et al. 2000). When nitrogen is limiting, Gln3 and Gat1 are concentrated in the nucleus where they bind GATA sequences upstream of NCRsensitive genes and activate their transcription. Conversely, in excess nitrogen, these GATA sequences are unoccupied and Gln3 and Gat1 are excluded from the nucleus. Gln3 activity is mainly repressed by intracellular glutamine whereas the Gat1 activity is repressed by intracellular glutamate (Stanbrough et al. 1995). Ure2 binds to Gln3 and Gat1, and is required for NCR-sensitive transcription to be reprTable 1. List of genes controlled by Gln3 and Dal80 and corresponding proteins Transporter genes: GAP1:general amino acid permease PUT4: proline permease UGA4:GABA permease CAN1: arginine permease DAL4: allantoin permease DAL5: allantoate permease Urea catabolic pathway genes: DUR1: urea carboxylase DUR2: allophanate hydralase

Allantoine catabolic pathway genes: DAL1: allantoinase DAL2: allantoicase DAL3: ureidoglycollate hydrogenase DAL7: malate synthetase Glutamine pathway genes: GLN1: glutamine synthase GDH1: glutamate dehydrogenase (NADPH) GDH2: glutamate dehydrogenase (NAD+)

Proline catabolic pathway genes: PUT1: proline oxidase PUT2: proline P-5-C dehydrogenase

Arginine catabolic pathway genes: CAR1: arginase CAR2: ornithine aminotransferase

Gamma-amino butyrate (GABA) catabolic pathway genes: UGA1: GABA transaminase UGA2: succinic semialdehyde dehydrogenase

Specific activators: PUT3: regulator of PUT genes DAL82: positive regulator of allophanate inducible genes DAL81: positive regulator of multiple nitrogen catabolic genes


essed and for nuclear exclusion of these transcription factors. Hence, Ure2 is known as a cytoplasmic anchor protein. In addition, it is a yeast prion precursor (Cox et al. 2000; Cunningham et al. 2000a; Cunningham et al. 2000b; Kulkarni et al. 2001; Wickner 1994). Nuclear exclusion also correlates with hyperphosphorylation of Gln3. The binding of Gln3 to Ure2 requires Tor-dependent phosphorylation of Gln3 (Beck and Hall 1999). The Tor proteins (see section 4.4) also seem to be involved in the Gat1 dependent transcription (Beck and Hall 1999). The mechanistic details of the signal transduction pathway associated with Ure2, Gln3, and Gat1, however, are not yet fully understood (Kulkarni et al. 2001). NCR- sensitive gene expression is also subjected to negative regulation by two transcriptio-

Fig. 7.9. Summary of Gln3p and Dal80p regulated nitrogen catabolite repression. Gln3p is a transcriptional activator of the expression of nitrogen catabolite genes and genes encoding amino acid permeases. Gln3p itself is repressed in rich nitrogen medium by Ure2p and active when grown in presence of only poor nitrogen sources. Dal80p functions as a repressor of the expression of nitrogen pathway and amino acid transport genes and it is mainly active in proline medium (Coffman et al. 1997). Expression of Dal80p is Gln3p dependent. Proteins other than Gln3p and Dal80p (indicated with nitrogen catabolite repression or NCR) additionally regulate transcriptional regulation of several subfamilies of amino acid biosynthesis and transporter genes. The genes under control of the transcription factors are listed in Table 7.2. Arrows denote activation, bars repression, broken lines partial repression. (Adapted from Daugherty et al. 1993; Hofman-Bang 1999)

328 Joris Winderickx et al. Table 7.2. Key steps of ammonia utilisation in yeast (adapted from Magasanik 1992).

1. NH3+ α-ketoglutarate + NADPH/H+ Gdh1 2. NH3 + glutamate + ATP Gln1 3. glutamine+α-ketoglutarate +NADH/H+ GluS 4. glutamate + NAD+ Gdh2

Æ

glutamate + NADP+

Æ

glutamine + ADP/Pi

Æ

2 glutamate + NAD+

Æ

α-ketoglutarate + NH3 + NADH/H+

Ammonia can be converted to glutamate either directly by coupling to æ-ketoglutarate by the NADPH dependent glutamate dehydrogenase Gdh1 or indirectly by conversion of ammonia and glutamine to glutamine by Gln1 or glutamine synthase and subsequently conversion of glutamine and æ-ketoglutarate to glutamine by GluS or glutamate synthase. Glutamate can be degraded to æ-ketoglutarate and ammonia by the NAD+ dependent glutamate dehydrogenase Gln2. Adapted from (Cooper 1982; Magasanik 1992; ter Schure et al. 2000)

nal GATA family repressors, Dal80 and Deh1. Deh1 acts as a repressor of nitrogen catabolite gene expression only on rich media like glutamine, aspa ragines, or ammonium (Coffman et al. 1997). Dal80 functions as a repressor of expression of many nitrogen pathway genes via the GATA-sequences found in their promoter (Cunningham and Cooper 1991). A broad survey was done to determine the uniformity of Dal80 and Gln3 regulation across the spectrum of nitrogen catabolic genes concluded that these proteins fun ction in opposition to one another in the regulation of most, but not all, NCR- sensitive genes (Daugherty et al. 1993). In some cases, Dal80 and Gln3 bind to the same GATA sequences. This suggested that they antagonise one another’s operation by competing for the same GATA binding sites upstream of the genes they regulate, i.e. Dal80 behaves like a competitive transcriptional repressor (Fig. 7.9.) (Coffman et al. 1997; Cunningham and Cooper 1991; Daugherty et al. 1993). The same relation is thought to exist for Deh1p and Gat1p (Hofman-Bang 1999). Genes known to be regulated by Gln3, Gat1, Dal80, Deh1 encode general or specific permeases and enzymes in the metabolic pathways for glutamine, glutamate, proline, urea, arginine, GABA, and allantoin (Table 7.1). Like the upstream sequences of all nitrogen, catabolite repression-sensitive genes encoding enzymes and transport proteins, the promoters of the transcriptional regulator genes GAT1, DAL80, and DEH1 themselves contain multiple GATA sequences. This arrangement allows for autogenous and cross regulation (Fig. 7.10) (Cunningham et al. 2000b; Hofman-Bang 1999). The tight regulation of proteins involved in uptake and metabolism of ammonia, glutamine, asparagine (Cooper 1982; Magasanik 1992), reflects the preference for those compounds as nitrogen sources. This preference is explained by the key steps of ammonia utilisation (Table 2). The reaction catalysed by glutamine synthase is essential in cells growing on other nitrogen sources than glutamine, including ammonia. Glutamine is required for the synthe-


sis of nucleotides and several amino acids. It is therefore not surprising that both the synthesis and the activity of glutamine synthase are tightly regulated in response to the availability of glutamine and - to a lesser extent - ammonia through the NCR regulatory network.

Fig. 7.10. Regulatory circuit of GATA-dependent transcription in S. cerevisiae. The nitrogen catabolite repression is regulated by four GATA family transcription factors: Gln3p and Gat1p are transcriptional activators and Dal80p and Deh1p act as transcriptional repressors. In addition, the promoters of Dat1p, Dal80p, and Deh1p contain multiple GATA sequences as well. These GATA sequences mediate their autogenous and cross regulation dependent on the provided nitrogen source (Hofman-Bang 1999). Arrows denote activation, bars repression, broken lines partial activation or repression. (Adapted from Cunningham et al. 2000b)


General amino acid control in amino acid biosynthesis S. cerevisiae activates a regulatory network called “general control” that provides the cell with sufficient amounts of protein precursors during amino acid starvation. Gcn4 is the transcriptional activator of gene expression in this system. Under amino acid starvation conditions, the sensor kinase Gcn2 detects uncharged tRNAs and phosphorylates and thereby activates the α-subunit of eukaryotic initiation factor 2 (eIF-2). In addition, recent reports have shown that Gcn2 requires in order to function another protein, Gcn1, bearing a region homologous to translation elongation factor 3 (Kubota et al. 2001; Kubota et al. 2000). Phosphorylated eIF-2α inhibits general translation but selectively derepresses the synthesis of the transcription factor Gcn4 at the translational level (Albrecht et al. 1998; Grundmann et al. 2001). Gcn4 is a member of the basic leucine zipper family (Ellenberger et al. 1992) and binds directly as a homodimer to a conserved regulatory region (Gcn4 target site) of its target genes (Hope and Struhl 1986; Oliphant et al. 1989). Gcn4 controls, as a transcriptional activator, a network of amino acid biosynthetic pathways (Albrecht et al. 1998). Recent microarray studies have revealed that Gcn4 is also involved in the activation of genes involved in glycogen homeostasis, genes encoding protein kinases and transcription factors were identified as target. Gcn4 target genes encoding amino acid biosynthetic enzymes contain in 84% of the cases a Gcn4 binding site, compared to a 44% occurrence among the total pool of Gcn4 targets. The targets, which lack a Gcn4 binding site in their promoter, may contain Gcn4 binding sites in the coding region of 3’ untranslated region, or they may be regulated indirectly through induction of other transcription factors by Gcn4. These results suggest that Gcn4 is a master regulator of gene expression (Natarajan et al. 2001). Amino acid sensing As shown before, S. cerevisiae regulates the expression and activity of proteins involved in amino acid uptake and utilisation. In addition to internal sensors such as those monitoring levels of uncharged tRNAs, recently Ssy1, Ptr3, and Ssy5 have been shown to be components of a plasma membrane sensor for extracellular amino acids (Fig. 7.11) (Forsberg et al. 2001a; Forsberg and Ljungdahl 2001; Klasson et al. 1999). The system exhibits high specificity to hydrophobic amino acids, e.g. leucine, in presence of ammonium (Didion et al. 1998). Ssy1 is a plasma membrane protein that belongs to the amino acid permease family. It differs, however, from the other proteins of this family by its much longer N-terminal tail, two longer extracellular loops and a lower expression level (Didion et al. 1998; Iraqui et al. 1999; Klasson et al. 1999). Besides Ssy1, the sensor also requires two peripherally plasma membrane proteins, Ptr3 and Ssy5, associated to Ssy1. Each of the components of the Ssy1-Ptr3-Ssy5 (SPS) complex appears to be equally required for the sensor function (Forsberg et al. 2001a; Forsberg and Ljungdahl 2001; Klasson et al. 1999). The SPS-signalling system is required for the expression of a set of transporter genes (AGP1, BAP2, BAP3, DIP5, GAP1,


Fig. 7.11. Integration of nitrogen catabolite repression and amino acid sensing. The nitrogen catabolite repression pathway and the Ssy1p-Ptr3-Ssy5p (SPS) amino acid sensing system interact with shared transcriptional regulators to co-ordinate gene expression in response to nitrogen availability. The interaction of the NCR components with the SPS sensing system can be either on the level of transcription factors (like Stp1p or Stp2p) and ubiquitin-mediated degradation (which is required to transmit SPS derived signals to transcriptional regulation), or directly on the level of final target genes. (Adapted from Forsberg and Ljundahl 2001)

GNP1, TAT1, TAT2 and PTR2) and an arginase (CAR1). The SPS-dependent expression of these target genes is specifically induced in response to external rather than internal amino acids (Forsberg et al. 2001a; Klasson et al. 1999). A number of downstream factors that influence the transcription of SPS-regulated genes have been identified. STP1 and STP2, two genes originally identified as factors required for pre-tRNA maturation (Wang and Hopper 1998), are required for the leucine induced induction of the permease encoded by AGP1 (Iraqui et al. 1999) and the branched chain amino acid transporters encoded by BAP2 and BAP3 (de Boer et al. 1998; de Boer et al. 2000)(Jorgenson et al. 1997). Also the non-specific transcription factor, Uga35/Dal81 and the components of the SCFGrr1 ubiquitin protein


ligase complex appear to be involved in SPS-dependent transcriptional induction of AGP1 (Bernard and André 2001a; Bernard and André 2001b; Iraqui et al. 1999). Recently, three novel genes, ASI1, ASI2, and ASI3, were identified in a screen for amino acid sensor-independent genes and their deletion causes constitutive derepression of SPS target genes (Forsberg et al. 2001b). This suggests that additional mechanisms or pathways may cross talk the main SPS-dependent pathway. One such pathway is the nitrogen catabolite repression pathway or NCR. Indeed, the NCR system co-ordinates the transcription of many nitrogen-regulated genes (Forsberg et al. 2001a). NCR monitors not only amino acids but also other nitrogen sources, and almost certainly interacts with the SPS-system by sharing transcriptional regulators. In this way, regulatory networks are created in order to fine-tune gene expression in response to nutrient availability (Forsberg et al. 2001a). Integration of carbon and nitrogen control mechanism When cells are shifted from fermentable (glucose) to non-fermentable (ethanol) carbon source, a subset of genes is induced, which are also activated by shifting cells from high-to-low-quality nitrogen source and known to be under control of the nitrogen catabolite repression pathway (Shamji et al. 2000). The GATAtranscription factors, Gln3 and Gat1, are involved in both effects but appear to contribute differently, depending on the precise nutrient input signal. Some Gln3 and Gat1 target genes are regulated in the same way by nitrogen or carbon source availability, while others are regulated in a distinct way. This is consistent with findings that the target genes of Gln3 and Gat1 are partially overlapping (Shamji et al. 2000). In this Gln3 and Gat1 regulated system, the Tor proteins, Tor1 and Tor2 (see section 7.4.4) (Helliwell et al. 1994), act as central sensors of the carbon or nitrogen source quality. Tor proteins regulate Gln3 or Gat1 localisation via the cytoplasmatic anchor protein Ure2 and the phosphatase Tap4 (Cardenas et al. 1999; Kuruvilla et al. 2001; Shamji et al. 2000). One specific example for connection points between carbon and nitrogen metabolism is the reductive amination of α-ketoglutarate by the NADPH dependent glutamate dehydrogenase, which is encoded by the GDH1 gene (Table 7.1). Probably for this reason, the regulation of GDH1 appears to be quite complex. Nitrogen-dependent transcriptional control is mediated by Gln3 and by the branchedchain amino acid biosynthesis-specific activator Leu3 (Hu et al. 1995a). In addition, carbon source-dependent transcriptional control is mediated through the Hap2/3/4/5 activator complex (Dang et al. 1996). Previously, this complex was implicated in carbon source-dependent regulation of various genes required for mitochondrial biogenesis (Forsburg and Guarente 1989b; Mc Nabb et al. 1995; Olesen and Guarente 1990; Pinkham and Guarente 1985; Pinkham et al. 1987) (reviewed in de Winde 1992; Forsburg and Guarente 1989a). This finding substantiates the already longstanding observation (Forsburg and Guarente 1989a), that yeast strains carrying hap2 or hap3 mutations display a severe defect in the utilisation of ammonium sulphate as sole nitrogen source.


Other important nitrogen- and carbon-source-dependent responses of S. cerevisiae have been described. These include cAMP-independent control of PKA activity through the FGM pathway (see section 7.4.3) and pseudohyphal differentiation (see section 7.4.6). These responses are beginning to be recognised as adaptations common to different forms of nutrient depletion (see section 7.4). 7.3.3 Phosphor Limitation and Starvation Response mechanisms involved in phosphate recruitment S. cerevisiae, like many other yeast species, preferentially utilises inorganic phosphate (Pi) as the phosphor source. However, when Pi is limiting or absent a variety of organic compounds can serve as phosphate sources, through the action of several acid phosphatases (reviewed in Johnston and Carlson 1992a; Lenburg and O'Shea 1996; Oshima 1997). All genes required for the utilisation of these poorer phosphate sources are specifically repressed by Pi (Martinez et al. 1998; Oshima 1997). Upon Pi depletion, transcription of a number of phosphate-starvation response genes, such as PHO5 (encoding a secreted acid phosphatase with broad substrate specificity) is activated by Pho4 (reviewed in Lenburg and O'Shea 1996). Pho4 binds to its target promoter regions in a co-operative manner with the auxiliary activator Pho2/Bas2/Grf10 (Barbaric et al. 1996; Vogel et al. 1989). Both Pho2 and Pho4 are indispensable for transcription of PHO5 (Barbaric et al. 1998). Pho4 has a basic helix-loop-helix (bHLH) DNA-binding motif (Berben et al. 1990) and Pho2 is a homeodomain protein (Bürglin 1988; Kaffman et al. 1994). Pho2 and Pho4 directly interact in vivo (Magbanua et al. 1997). The Pho4-interaction domain of Pho2 is similar to the Pho4-association domain of Pho80, and deletion inactivates the protein (Yang et al. 1995). The functional interaction between Pho4 and Pho2 is not depending on the presence of the negative regulator Pho80, which also interacts with Pho4 (Shao et al. 1996). In the presence of high concentrations of Pi, Pho4 is negatively regulated through binding of the specific inhibitors Pho80 and Pho85. PHO80 and PHO85 encode a cyclin and a cyclin-dependent kinase (CDK), respectively (Kaffman et al. 1994; Toh-E et al. 1988). CDK activity is not only controlled by cyclin binding but also by phosphorylation, dephosphorylation, and by binding of accessory factors and cyclin-dependent kinase inhibitors (CKI) (Morgan 1997). When Pi levels drop, Pho4 inhibition by Pho80-Pho85 is alleviated by binding of a CKI homologue, Pho81, to the Pho80 cyclin in the complex, antagonising Pho80-Pho85 kinase activity (Oshima 1982; Schneider et al. 1994). Interestingly, Pho4 is predominantly cytoplasmic in a pho81 deletion mutant strain grown in high or low phosphate conditions suggesting that the Pho4 function is regulated by phosphorylation dependent nuclear export as discussed below (O'Neill et al. 1996). This specific response to phosphate depletion is schematically depicted in Fig. 7.12 (for a recent review see Lenburg and O'Shea 1996).


Pho81 may inhibit Pho80-Pho85 by a mechanism similar to the p16INK4 family of mammalian CKI, with the exception of one interesting difference. The p16INK4 family interacts with CDK subunits, whereas Pho81 interacts primarily with the cyclin Pho80 (Ogawa et al. 1995; Schneider et al. 1994). Pho81 contains six copies of an ankyrin repeat motif, known to participate in protein-protein interactions in other regulators (Schneider et al. 1994), which are sufficient to inhibit the activity of Pho80-Pho85 in vivo and in vitro. Furthermore, Pho81 associates with Pho80-Pho85 when yeast cells are grown in either low or high phosphatecontaining media. Pho81 appears to be regulated by the critical phosphate switch. It remains unclear how Pho81 is activated or inactivated in response to Pi levels. Pho81 may be regulated by posttranslational modification in response to low Pi levels since PHO5 mRNA induction is unaffected by cycloheximide treatment (Lemire et al. 1985). Regulation of the Pho4 transcription factor occurs at two levels: subcellular localisation and phosphorylation (Komeili and O'Shea 1999). In high phosphate coInorganic phosphate

Pho81

Pho80 Pho85

Pho2

PHO2

PHO5

Pho4

PHO81

PHO84

Fig. 7.12. Transcriptional regulation of the secreted acid phosphatase gene PHO5. A high level of inorganic phosphate (Pi) positively regulates PHO85 and PHO80. PHO85, which encodes a Cdk, and PHO80, it’s associated cyclin, negatively regulate the Pho4 transcription factor. Low Pi concentration causes Pho81-mediated repression of the cyclin-CDK complex and activation of Pho4, which interacts with Pho2, induces PHO5 expression. PHO5 encodes the Pi -repressible acid phosphatase. Pho4 also activates, in co-operation with Pho2, the expression of PHO84 and PHO81, a Pi-transporter and a Cdk-inhibitor respectively.


nditions, Pho4 is phosphorylated on five serine residues by the Pho80-Pho85 complex. The phosphorylated transcription factor Pho4 is exported from the nucleus into the cytoplasm where it cannot activate transcription of PHO5 (O'Neill et al. 1996). Association of Pho4 with its nuclear import or export receptor, Pse1/Kap121 or Msn5 respectively, is regulated by Pho80-Pho85 phosphorylation (Kaffman et al. 1998a; Kaffman et al. 1998b). Accordingly, Pho80 and Pho85 are primarily in the nucleus under conditions of high phosphate. Binding of phosphorylated Pho4 to Msn5 requires the GTP-bound form of the yeast Ran homologue, Gsp1. In addition, Pho80 may repress Pho4 activity by direct interaction with and masking of its activation domain (Barbaric et al. 1998). The various serine residues of Pho4 are differentially phophorylated (Komeili and O'Shea 1999). At intermediate Pi levels, Pho4 is partially phosphorylated but this does not affect its subcellular localisation (Jeffery et al. 2001). A small but significant number of genes is activated by partially phophorylated Pho4, including PHO84, encoding the high affinity phosphate transporter (Jeffery et al. 2001). Activation of the Pho84 transporter raises intracellular phosphate levels and causes increased phosphorylation of Pho4. As a result, Pho4 is exported from the nucleus and prevented from re-entering. Subsequently, Pho84 transcription is again shut down (Kaffman et al. 1998a). Induction of PHO5 transcription upon Pi-depletion in the absence of protein synthesis suggests that Pho4 can be dephosphorylated, although a Pho4 phosphatase has not been identified yet. This feedback loop allows the cells to respond over a wide range of phosphate concentrations. Recently, it was reported that Pho2 can be phosphorylated in vitro by the CDK Cdc28 (Liu et al. 2000). A phosphate switch regulates the transcriptional activity of Pho2 and mutations in a consensus sequence (SPIK) affect the transcriptional activity of Pho2 and the interaction between Pho2 and Pho4 (Liu et al. 2000). Integration of phosphate control in general metabolism and cell proliferation In addition to its well-established role in responding to phosphate-starvation, the cyclin-dependent kinase Pho85 has been implicated in a number of other physiological processes, such as glycogen biosynthesis, actin regulation, and cell cycle progression (Moffat et al. 2000). Consistent with diverse roles for Pho85, ten genes encoding known or putative Pho85 cyclins (Pcls) have been identified, which apparently target Pho85 to specific cellular functions and substrates (Measday et al. 1994). The Pho80 subfamily cyclins (composed of Pho80, Pcl6, Pcl7, Pcl8 and Pcl10) direct Pho85 to metabolic pathways (phosphate metabolism and glycogen metabolism) while the Pcl1,2 subfamily cyclins (composed of Pcl1, Pcl2, Clg1, Pcl5 and Pcl9) are thought to associate with Pho85 to regulate cell cycle functions (Measday et al. 1997). The Pcl1.2-type Pho85 kinases were suggested to be required for G1 progression in the absence of the Cdc28 cyclins CLN1 and CLN2 (Espinoza et al. 1994; Measday et al. 1994), because deletion of PCL1 and PCL2 causes G1 arrest when CLN1 and CLN2 are deleted. Strains deleted for the entire Pcl1,2 subfamily (pcl1, pcl2, clg1, pcl5 and pcl9) have morphological defects suggesting a role in cell


morphogenesis early in the cell cycle (Lee et al. 1998; Tennyson et al. 1998). Expression of PCL1 and PCL2 is controlled by SBF (a transcription factor required for timely expression of PCL1, PCL2, CLN1 and CLN2) and peaks at START (Espinoza et al. 1994; Measday et al. 1994). In addition, the expression pattern of PCL9, the Pho85 cyclin most closely related to Pcl2, is cell cycle-regulated and dependent on a transcription factor Swi5 (Aerne et al. 1998; Measday et al. 1997; Tennyson et al. 1998). Two proteins, Rvs167 and Sic1, are known as Pcl/Pho85 substrates in accordance with a role for Pho85 in actin regulation and cell cycle progression (Lee et al. 1998; Nishizawa et al. 1998). Rvs167 plays a role in endocytosis, organisation of the actin cytoskeleton, and cell survival after starvation. Rvs167 deletion mutants, pho85 deletion mutants and strains deleted for the Pcl1,2-type Pho85 cyclins (pcl1, pcl2, clg2, pcl5 and pcl9) share the same phenotypes. Phosphorylation of Rvs167 by Pcl1,2-type Pho85 kinases appears to regulate protein-protein interactions involving Rvs167 (Colwill et al. 1999; Lee et al. 1998). An essential role for Pcl1 in the absence of Cln1,2 is the requirement of Pcl1/Pho85 kinases for degradation of Sic1, a Cln-Cdc28 inhibitor (Nishizawa et al. 1998). Deletion of PHO85 affects phosphorylation and stability of Sic1 in vivo. Cln-Cdc28 and Pcl1-Pho85 have non-redundant roles with respect to Sic1 degradation. Pcl1-Pho85 and Pcl2-Pho85 have definable although redundant roles in transition from G1 to S-phase in the yeast cell cycle. All these findings support a role for Pcl1,2-type Pho85 kinases in early cell cycle events, and suggest a regulatory cross talk in the co-ordination of nutrient availability and cell cycle progression. Additional relevance for such cross talk is provided by the fact that Gsy2, the glycogen kinase (see 7.4.5), is a substrate for a Pcl-Pho85 kinase (Huang et al. 1996a; Timblin et al. 1996). Disruption of and mutations in PHO85 exhibit impaired Gsy2 kinase activity, constitutive activity of glycogen synthase reminiscent of unphosphorylated Gsy2 and consequently, hyperaccumulation of glycogen (Huang et al. 1996a). In addition, a pho85 mutation causes increased GSY2 expression and suppresses the glycogen storage defect of snf1 mutations (Huang et al. 1996a; Timblin et al. 1996). These observations led to the hypothesis that Pho85 is a constituent of a major Gsy kinase and that certain Pho85 cyclins might target Pho85 for this role (Huang et al. 1998). In fact, deletion of both PCL8 and PCL10 causes glycogen hyperaccumulation but does not result in other phenotypic defects associated with a pho85 deletion mutant (Huang et al. 1998). Pcl10-Pho85 kinase can phosphorylate Gsy2, whereas it is not clear whether Pcl8-Pho85 is capable of directly phosphorylating Gsy2 (Huang et al. 1998). Although the phosphorylation sites on Gsy2 are only slightly different from the Pho4 consensus site, Pcl10-Pho85 phosphorylates Gsy2 much more effectively than Pho4, whereas Pho80-Pho85 kinase selectively phosphorylates Pho4 (Huang et al. 1998). Thus, physiological roles of Pho80 and Pcl10 are reflected in the substrate specificity. It appears that cyclins may contain a targeting domain required for substrate recognition and phosphorylation (Kelly et al. 1998; Schulman et al. 1998). Exactly how cyclins target a specific CDK to different substrates remains to be seen. The possibility of Pho85 functioning as a glycogen synthase kinase together with identification of Pcl10 and Gsy2 as substrate for Pho85 suggests a close link


cation of Pcl10 and Gsy2 as substrate for Pho85 suggests a close link between cell cycle regulation, nutrient availability, and glycogen accumulation. Another interesting candidate to operate in cross talk between regulatory processes is Pho2/Bas2/Grf10, especially since Cdc28 was identified as a kinase of Pho2 (Arndt et al. 1987; Liu et al. 2000; Yoshida et al. 1989). Cdc28 is an essential regulator of cell cycle progression, whereas Pho2 is required for several metabolic processes. Perhaps a new cyclin would be found to help Cdc28 co-ordinate nutritional state and cell cycle progression. Gene activation in eukaryotes is depending on co-operation between different transcription factors. Pho2 is such a pleiotrophic affector, which interacts co-operatively with gene-specific factors (Barbaric et al. 1998). Besides transcription of the PHO genes, Pho2 controls transcription of HIS4 (Arndt et al. 1987), CYC1 (Sengstag and Hinnen 1988), TRP4 (Braus et al. 1989), HO (Brazas et al. 1995) and ADE1, ADE2, ADE5,7, and ADE8 (Daignan-Fornier and Fink 1992). Together with Swi5, it binds co-operatively to a regulatory element in the HO promoter (Brazas et al. 1995) and plays a complex role in its regulation (McBride et al. 1997). Bas1 and Bas2/Pho2 are transcriptional activators identified as basal regulators of HIS4, encoding an enzyme in histidine biosynthesis (Arndt et al. 1987). Although Bas1 and Bas2 both can bind to the HIS4 promoter region simultaneously, no co-operative interactions were detected (Arndt et al. 1987). In contrast, Pho2 can bind to the TRP4 promoter together with Gcn4 in a mutually exclusive manner (Braus et al. 1989). TRP4 encodes the enzyme phophoribosyl transferase, which is central in tryptophane synthesis (Braus et al. 1989). In addition, transcriptional activation of the ADE genes requires the concerted action of Pho2/Bas2 and Bas1 and is downregulated by adenine. Pho2 stimulates both DNA binding and activation by Bas1 at the ADE5,7 promoter (Rolfes et al. 1997). The response to adenine limitation by increased expression of the purine and histidine pathway is reminiscent of the general control of amino acid biosynthesis, in which limitation for one amino acid leads to induction of several biosynthetic pathways (Hinnebusch 1988). A physiological role for cross regulation of the histidine and purine pathways in response to Pi availability is not apparent. Perhaps phosphoribosylpyrophosphate (PRPP), which is used as a substrate (histidine, tryptophane, purine and pyrimidine pathways), plays an important role in this cross talk. Trp4 catalyses a PRPP utilising step. Since Pho2/Bas2 inhibits Gcn4-dependent transcriptional activation of TRP4 when Pi levels are low, utilisation of phosphate-rich PRPP may be disadvantageous in Pi-limited cells. 7.3.4 Sulphur Limitation and Starvation Response mechanisms involved in sulphur assimilation Sulphur is a low-abundance component in biomass, since its requirement is largely restricted to synthesis of methionine and cysteine. This may explain why molecular details concerning the regulation of sulphur assimilation have only recently become available (Thomas and Surdin-Kerjan 1997). Sulphate is readily used by


Fig. 7.13. The sulphate assimilation pathway in Saccharomyces cerevisiae. Schematical depiction of intermediary sulphate metabolism in yeast. (Adapted and modified from Jones and Fink 1982; Mountain et al. 1991; Thomas and Surdin-Kerjan 1997)

Saccharomyces species as a sulphur source. Following uptake through the two high-affinity sulphate permeases, Sul1 and Sul2 (Cherest et al. 1997) sulphate is reduced to sulphide, which is immediately used in the biosynthesis of methionine and cysteine (Fig. 7.13) (Jones and Fink 1982; Mountain et al. 1991; Thomas and Surdin-Kerjan 1997). All of the genes coding for enzymes of the sulphate reduction and assimilation pathway (‘MET’ genes) are strongly repressed at the transcriptional level by S-adenosyl-methionine (AdoMet) or by methionine through its


conversion to AdoMet (Thomas et al. 1989; Thomas and Surdin-Kerjan 1997). Hence, sulphate assimilation is efficiently inhibited when methionine and/or cysteine are abundant, and sulphate depletion will not be deleterious unless methionine becomes limiting. Upon methionine depletion, transcription of the MET genes is activated by a specific activation complex consisting of the basis helix-loop-helix (b/HLH) protein Cpf1, basic leucine zipper (b/LZ) accessory protein Met28, and a METpathway specific b/LZ activator subunit Met4 (Kuras et al. 1996; Mellor et al. 1990). When intracellular methionine and, hence, AdoMet levels increase a specific F-box protein, Met30, binds to Met4 and thereby targets the associated SCFMet30 ubiquitin-protein ligase complex to the transcriptional activator subunit, which is subsequently ubiquitinated (Patton et al. 1998; Rouillon et al. 2000). Surprisingly, however, ‘flagged’ Met4 is not degraded by the 26S proteasome, but cannot associate with Met28 and Cpf1, thus preventing activation of MET genes (Kaiser et al. 2000). Met30 dependent inhibition of MET-pathway specific activation through the Met4 activator therefore largely resembles Grr1 dependent inhibition of glucose specific repression through the Rgt1 repressor. In line with a broader role for F-box proteins, also the function of the essential Met30 protein is not restricted to the sulphate assimilation pathway (see also below and Patton et al. 2000; Thomas et al. 1995). AdoMet control may also require multipartite regulatory mechanisms, since methionine repression requires the presence or biosynthesis of cysteine (Hansen and Johannesen 2000; Ono et al. 1999). How this additional control layer is implemented remains to be seen. Integration of sulphur control with carbon and phosphate utilisation The MET19/ZWF1 gene, previously implicated in the sulphate assimilation pathway encodes glucose-6-phosphate dehydrogenase (G6PDH), which is the first enzyme of the pentose phosphate route (Nogae and Johnston 1990; Thomas et al. 1991). While these authors showed that the auxotrophy for organic sulphur of met19/g6pdh mutants does not result from a depletion of NADPH due to blocked pentose phosphate pathway, a subsequent study suggested that indeed NADPH limitation might cause a block in sulphate assimilation (Hudak Slekar et al. 1996). Although, in more general terms, the exact role of the pentose phosphate cycle in cellular metabolism is still a matter of debate, this finding sheds new light on the mechanisms that control anabolic carbon and sulphur fluxes. A strict coupling can be expected between carbon flux, yielding aspartate through the citric acid cycle and sulphate assimilation, in the biosynthesis of sulphur amino acids (Fig. 7.13) (Jones and Fink 1982). Expression of G6PDH is strongly repressed by methionine and AdoMet, as are most other MET genes, while that of other enzymes of the pentose phosphate route is not (Thomas et al. 1991). Transcription of MET19 is not dependent on Met4 (Thomas and Surdin-Kerjan 1997), but repression is mediated through Met30 (Thomas et al. 1991; Thomas et al. 1995). This again indicates that the function of the Met30 repressor protein is extending beyond the sulphate assimilation pathway.


As described above, a member of the family of global transcriptional regulators (Diffley 1992), Cpf1, is involved in transcriptional regulation of the methionine biosynthetic pathway (Thomas et al. 1992). This b/HLH and leucine zipper (LZ) containing protein binds to the centromere core consensus sequence CDE1 and to diverse promoter regions, including those of several genes of the sulphate assimilation pathway (Baker and Masison 1990; Bram and Kornberg 1987; Cai and Davis 1990; Mellor et al. 1990) (for reviews see de Winde 1992; Diffley 1992). Cpf1 has been shown to be involved in transcriptional regulation of several genes (de Winde and Grivell 1992; de Winde and Grivell 1995; Mellor et al. 1990; Oechsner and Bandlow 1996; Thomas et al. 1992) probably through modulation of chromatin structure in the promoter region surrounding its CDE1 consensusbinding site (de Winde et al. 1993; Kent et al. 1994). Indeed, Cpf1 modulates gene activity through interaction with a family of chromatin-related proteins encoded by SPT21, SIN3/RPD1, RPD3, and CCR4 (McKenzie et al. 1993). CPF1 disruption mutants exhibit a severe methionine auxotrophy (Cai and Davis 1990; Mellor et al. 1990), and mRNA levels of MET25 and MET16, which contain CDE1 consensus sites in their promoter regions, are reduced. However, site directed mutagenesis of the b/HLH region revealed that DNA binding, centromere functioning, and chromatin structure modulation by Cpf1 are not related to and separable from its role in maintaining methionine prototrophy (McKenzie et al. 1993; Mellor et al. 1991). The implementation of Cpf1 in the MET-gene family specific Cpf1/Met28/Met4 activator complex implies that Cpf1 may function in transcriptional control of sulphate assimilation genes and other genes through different heteromeric protein-protein interactions.

7.4 Common responses to nutrient depletion 7.4.1 General Concepts As stated in the introduction to this Chapter, when describing and investigating the cellular responses in yeast cells to gradual depletion of nutrients, distinctions should be made between limitation and starvation for certain nutrients and between specific responses to depletion of a certain nutrient and general responses to nutritional depletion. In many instances, the decreasing availability of one substrate induces or enhances specific metabolic pathways required for the utilisation of another. When, eventually, one or more essential nutrients have been used up, cells enter stationary phase. The common signalling cascades and metabolic changes that are triggered by starvation for different nutrients are closely related to the mechanisms operating in the control of growth and proliferation. Yeast cells starved for a single essential nutrient will complete their current cell cycle and arrest in the next G1 phase at 'START A' (Pringle and Hartwell 1981) (Fig. 7.2 and 7.14). Subsequently, they will progress into an 'off-cycle', G0 or stationary phase, in which they can survive nutri-


Fig. 7.14. The Start-concept of the yeast cell division cycle. Schematical depiction of genetic cell cycle check points in Saccharomyces cerevisiae. START A, START B, and other proliferation-decision landmarks are genetically characterised by several CDC genes.2 The Cdc28 protein kinase is the central mediator of phase-to-phase transitions. Phase-specific activation of Cdc28 is controlled by G1 cyclins Cln1, Cln2, and Cln3, by S-phase cyclins Clb5 and Clb6 and by G2 cyclins, including Clb2 (see Baroni et al. 1994; Tokiwa et al. 1994 and references therein)

ent starvation much longer than when arrested elsewhere in the cell cycle. When all essential nutrients are readily available the cells will exit G0 and proceed with growth in the G1 phase to reach their 'critical' cell size and traverse START into Sphase, beginning a new round of proliferation. Yeast cells that are starved for any essential nutrient entering stationary phase display a number of specific characteristic properties. They accumulate elevated levels of the storage carbohydrates glycogen and trehalose (Lillie and Pringle 1980) while expression of STRE-controlled and PDS-controlled genes like CTT1, HSP12, and SSA3 are induced (Boy-Marcotte et al. 1998; Moskvina et al. 1998; Pedruzzi et al. 2000; Tadi et al. 1999). Transcription of ribosomal protein genes is repressed (Griffioen et al. 1996; Griffioen et al. 1994; Mager and Planta 1991; Neuman-Silberberg et al. 1995). General stress resistance, and in particular thermotolerance, is greatly enhanced (Plesset et al. 1987) and resistance to cell wall lytic enzymes is elevated (De Nobel et al. 1990) (for reviews see Thevelein 1994Werner-Washburne et al. 1993; Werner-Washburne et al. 1996). Interestingly, however, most of these characteristics are qualitatively not very different from the properties of cells growing exponentially on a non-fermentable carbon source (Fig. 7.15). This is apparently the reason why general responses to and metabolic consequences of nutrient starvation and nutritional resupplementation


Fig. 7.15. Metabolic characteristics of yeast cells growing exponentially or entering stationary phase, on fermentable or non-fermentable carbon sources. Several starvation- and stress-linked characteristics of Saccharomyces cerevisiae as indicated in the box, are only absent in cells growing exponentially on fermentable carbon sources


have not been studied intensively in yeast cells growing on a non-fermentable carbon source. This does not imply, however, that these responses do not exist. On the other hand, differences between nutrient-deprived cells and cells growing exponentially on rapidly fermentable sugars are very pronounced for all the properties mentioned above. 7.4.2 Nutrient signal integration and the control of metabolism and growth When yeast cells that are growing exponentially on a glucose-containing medium are starved for another essential nutrient like nitrogen, phosphate, or sulphate, they show in all cases the same metabolic response. The trehalose and glycogen level starts to increase, the expression of STRE-controlled genes is induced, and all characteristics of cells in stationary phase are progressively acquired (Lillie and Pringle 1980; Mager and de Kruijff 1995; Ruis and Schuller 1995; Thevelein 1994; Thevelein et al. 2000; Thevelein and de Winde 1999). These metabolic changes could simply be the logical result of the growth arrest, without being modulated themselves directly by the presence of the nutrients. However, the presence of the same characteristics in cells growing exponentially on nonfermentable carbon sources - albeit with a prolonged G1 phase - already argues against this interpretation. Like the length of G1, acquisition of these metabolic characteristics clearly can be modulated by nutrient availability alone, without being necessarily linked to growth arrest. In addition, the nutrient resupplementation response strongly argues for nutrient specific control. The most telling example in this respect is the observation that re-addition of nitrogen, phosphate or sulphate to cells starved for these nutrients, respectively, in each case causes within a few minutes a rapid, post-translational activation of trehalase which triggers trehalose mobilisation (Hirimburegama et al. 1992). This activation is not dependent on protein synthesis. Hence, this response is not a mere consequence of but rather precedes the induction of cell growth. The striking question arising from these observations is how depletion or resupplementation of nutrients so different as nitrogen, phosphate, and sulphate can trigger such similar and rapid metabolic responses. An important clue to the molecular mechanism underlying these nutrientcontrolled responses has come from studies on cAMP metabolism in yeast. Yeast mutants devoid of cAMP arrest at the same point in the cell cycle as wild type cells starved for nutrients (Matsumoto et al. 1985; Pringle and Hartwell 1981). More recently, it has been shown that cells with an overactive Ras-cAMP pathway indeed do not arrest in G1 upon nutrient starvation but rather arrest growth anywhere throughout the cell cycle (Markwardt et al. 1995). Since these cells contain only low levels of storage reserves (glycogen, trehalose, amino acids), they apparently are unable to complete their current cell cycle and therefore arrest randomly upon nutrient depletion (Kataoka et al. 1985; Tatchell 1993). In addition, a connection has been revealed between PKA activity and the critical cell size required for progression over START. Elevated activity of PKA by addition of cAMP causes repression of the G1 cyclin genes CLN1 and CLN2, resulting in a larger


critical cell size (Baroni et al. 1994; Tokiwa et al. 1994). These data would fit with a nutrient sensing function for the Ras-cAMP pathway; when constitutively activated it would be unable to sense depletion and as a result cells would progress over START, regardless of the environmental conditions. However, as will be discussed in the next section, cAMP is not the only factor determining PKA activity. Another nutrient-sensing pathway, the FGM pathway, appears to determine PKA activity independent of cAMP during fermentative growth (Thevelein et al. 2000; Thevelein and de Winde 1999). Recent data indicate that the integration of the different nutrient signals involves a complex interregulatory network with different pathways. For instance, the transcription factor Msn2 and Msn4 have been identified as targets of PKA (Estruch and Carlson 1993). PKA inhibits their activity by preventing hyperphosphorylation by yet unknown kinases. In addition, PKA prevents and reverses nuclear translocation of both proteins (Görner et al. 1998). Deletion of these two STRE-binding proteins suppresses the growth defect of PKA deficient strains, indicating that they antagonise PKA dependent growth control (Smith et al. 1998). Also the TOR pathway, another nutrient-sensing pathway, impinges on the nuclear localisation of Msn2 and Msn4. Rapamycin treatment, which inactivates the Tor kinases, leads to nuclear accumulation of Msn2 and Msn4 and the proteins fail to translocate to the nucleus in a Tor1-1 mutant. Msn2 and Msn4 control the expression of many genes involved in the acquisition of stress resistance and the accumulation of reserve carbohydrates (Boy-Marcotte et al. 1998; Mager and Ferreira 1993; Ruis and Schuller 1995; Thevelein 1996; Thevelein et al. 2000; Thevelein and de Winde 1999). In addition, Msn2 and Msn4 also control expression of the DYRK family kinase Yak1 (Smith et al. 1998). This kinase antagonises the PKA proliferation-control and PKA activity is dispensable in a yak1 deletion strain (Garret and Broach 1989; Garret et al. 1991). Most recently, Yak1 has been shown to accumulate in the nucleus when glucose is exhausted. In the nucleus, it is able to phosphorylate Pop2, one of the components of the transcriptional regulatory Ccr4-Not complexes (Bai et al. 1999; Draper et al. 1995; Hata et al. 1998; Moriya et al. 2001; Sakai et al. 1992). In this way, Yak1 presumably alters interactions or activity of the Ccr4-Not complexes which regulate expression of different genes encoding growth-controlling proteins, metabolism-controlling proteins, proteins involved in stress resistance, and proteins required for proper entry into stationary phase (Liu et al. 1998; Maillet et al. 2000; Moriya et al. 2001). The suppressive effect of a yak1 deletion depends on active Sok1 and overexpression of SOK1 suppresses the growth defect of a PKA deficient strain (Ward and Garrett 1994). The opposing regulatory connection between Yak1 and Sok1 may be explained by the recently found interaction of Sok1 with Gac1, one of the regulatory subunits that target the catalytic Glc7 subunit of PP1 (Ito et al. 2001; Venturi et al. 2000).


7.4.3 The FGM pathway; an integrator of responses to nutrient availability When a nitrogen source, phosphate, or sulphate is re-supplemented to yeast cells starved on a glucose-containing medium, a rapid activation of trehalase occurs, which leads to rapid mobilisation of trehalose (Hirimburegama et al. 1992). Similar rapid changes in activity have been described for other enzymes of glycogen and trehalose metabolism and glycolysis (Crauwels et al. 1997b; François et al. 1991; François and Parrou 2001a; François et al. 1988; Moskvina et al. 1998). These rapid changes in enzyme activity are independent of protein synthesis and hence occur at the post-translational level, presumably by protein modification. Under the same conditions, ribosomal protein gene transcription is rapidly induced and mRNA levels of STRE-controlled genes rapidly decrease (Boy-Marcotte et al. 1998; Griffioen et al. 1996; Griffioen et al. 1994; Neuman-Silberberg et al. 1995; Winderickx et al. 1996). There is good evidence that these effects are controlled by PKA (Boy-Marcotte et al. 1998; Boy-Marcotte et al. 1996; Görner et al. 1998; Tadi et al. 1999). Notably however, the effects triggered by readdition of an essential nutrient in a fermentable medium do not appear to be triggered by cAMP. Under fermentative conditions, no transient increase in the cAMP level is generated (Hirimburegama et al. 1992), and the effects mentioned are observed even in mutants lacking the regulatory subunit of PKA, Bcy1 (Cameron et al. 1988; Durnez et al. 1994). In addition, many of the above mentioned nutrient-induced effects are independent of sugar phosphorylation (Pernambuco et al. 1996). This is not compatible with an increased cAMP synthesis since sugar phosphorylation constitutes a primary requirement for the activation of the Ras-cAMP pathway (Beullens et al. 1988; Rolland et al. 2000; Rolland et al. 2001). Hence, it appears that during fermentative growth PKA is activated in a novel, non-cAMP mediated way. Since this activation is dependent on the presence of glucose or another rapidlyfermented sugar, and concomitantly on all nutrients required for growth, the pathway involved was called the ‘Fermentable Growth Medium-induced (FGM) pathway’ (Fig. 7.7) (reviewed in Thevelein 1994; Thevelein et al. 2000; Thevelein and de Winde 1999). Presumably, the pathway can be activated by re-addition of any essential nutrient to cells starved for it, because starvation leads to the acquisition of typical stationary-phase characteristics while nutrient re-addition will lead to their disappearance. According to this scheme, the role of the Ras-cAMP pathway would be confined to the transition from non-fermentable to fermentable growth conditions. The transient cAMP increase triggers a rapid dissociation of the regulatory and catalytic subunits of PKA resulting in a temporary boost in PKA activity. This PKA boost probably facilitates resetting of different regulatory mechanisms (Jiang et al. 1998). Due to feedback inhibition, the cAMP level falls back to about its basal level but at the same time the FGM pathway takes over the function as main determinant of PKA activity. At the end of fermentation, before the diauxic shift, the FGM activity also drops and an increased expression and/or modification of the regulatory subunit Bcy1 reset PKA activity level. Consequently, the cells reprogram to respiratory growth and subsequently to stationary phase (BoyMarcotte et al. 1996; Werner-Washburne et al. 1993; Werner-Washburne et al.


1996; Werner-Washburne et al. 1991). In this context, it may be worthwhile to mention that the subcellular distribution of the regulatory subunit of PKA changes in response to glucose to an exclusive nuclear localisation during fermentation (Griffioen et al. 2000; Griffioen et al. 2001). Although this mechanism could in principle result in a higher PKA activity in the cytoplasm, the nuclear translocation process is too slow and cannot account for the fast nutrient-induced responses (G. Griffioen, pers. Communication). Several data indicate that the Sch9 protein kinase could function as a regulator of PKA activity through FGM signalling. Sch9 shares sequence similarity with the catalytic subunits of PKA (Jin et al. 1995; Toda et al. 1988) and it is a functional homologue of mammalian Akt1/PKB (Geyskens et al. 2000). Overexpression of SCH9 suppresses the G1 arrest of a cdc25ras1ras2 triple deletion (Toda et al. 1988). Deletion of SCH9 causes a slow growth phenotype, which however can be rescued by overexpression of TPK1 (Toda et al. 1988) but not by deletion of YAK1 (Hartley et al. 1994). In addition, the growth defect is seriously exacerbated when SCH9 is deleted in strains defective in glucose-induced cAMP signalling like a gpr1, a gpa2, or a ras2 strain (Colombo et al. 1998; Lorenz et al. 2000) or a strain with overexpression of the Gpa2 specific GTPase, Rgs2 (Versele et al. 1999). More recently, it has been shown that Sch9 deficient strains exhibit an increased resistance to oxidants and an extended life span and these effects are suppressed by the additional deletion of RIM15, a kinase immediately downstream and negatively regulated by PKA (Fabrizio et al. 2001; Reinders et al. 1998). All these data are consistent with a function of Sch9 as positive activator of the free catalytic subunits of PKA. The role of the Sch9 kinase for FGM signalling was confirmed by the observation that glucose-repressed sch9 cells are defective for nitrogen-induced trehalase activation, nitrogen-induced upshift of ribosomal protein gene expression, and nitrogen-induced repression of STRE-controlled genes while derepressed sch9 cells are still glucose-responsive for all these parameters (Crauwels et al. 1997a). It was also observed that the positive effect of Sch9 on PKA activity is restricted to fermentative growth conditions. In derepressed cells growing on nonfermetable carbon sources, Sch9 appears to act as an inhibitor of PKA (Crauwels et al. 1997a). This carbon source dependent twist in function most probably relates to differences in phosphorylation of Sch9. This would be similar, for instance, to the positive or negative effect exerted by respectively, phosphorylated, and nonphosphorylated MAPK Kss1 on the transcription complex Ste12-Tec1 in the pathway controlling the switch to pseudohyphal growth (see 7.4.6) (Bardwell et al. 1998; Cook et al. 1996; Gancedo 2001; Madhani and Fink 1998a). More recently, the protein phosphatase PP2A was identified as another component of the FGM pathway. Overexpression in wild type cells of one subunit of the phosphatase, i.e. Pph22, affected glucose and nitrogen-induced responses and resulted in a phenotype consistent with high PKA activity. Overexpression in an sch9 deletion strain had no effect on the glucose-induced responses indicating that Sch9 is necessary to mediate the effects of PP2A in derepressed cells. In glucose-repressed cells, deletion of Sch9 by itself causes the same defect in nitrogen-induced signalling as that mediated by overexpression of Pph22 (Crauwels et al. 1997a; Sugajska et al.


2001). Possibly, Pph22 dephosphorylates sites on Sch9 that are important for kinase activity or it may interfere with a downstream component. However, this remains to be elucidated. 7.4.4 Nutritional control by targets of rapamycin (Tor) proteins Yeast cells treated with the immunosuppressive rapamycin resemble cells deprived of nutrients (Barbet et al. 1996; Heitman et al. 1991a). The intracellular receptor for rapamycin in all eukaryotes is the highly conserved FKBP12 prolyl isomerase, named Fpr1 in yeast (Heitman et al. 1991b; Koser et al. 1993). The rapamycin-FKBP12 complex specifically interacts with the conserved TOR proteins, to inhibit signalling to downstream targets (Cafferkey et al. 1993; Kunz et al. 1993). In Saccharomyces cerevisiae, two Tor proteins, Tor1 and Tor2, have been identified which are 67% identical at the amino acid level (Cafferkey et al. 1994; Helliwell et al. 1994). Because Tor inhibition elicits cellular responses resembling those triggered by starvation conditions, these proteins are thought to be mediators of nutrient-sensing pathways (Barbet et al. 1996; Schmelzle and Halln 2000). Tor proteins belong to the phosphatidylinositol kinase-related kinases (or PIKKS). However, they do not function as lipid kinases, but as Ser/Thr protein kinases (Hunter 1995; Keith and Schreiber 1995).Tor activation by nutrients results in a drastic change in cell properties, such as translation initiation, ribosome biogenesis, transcriptional repression of several genes, increased amino acid transport, and actin organisation (Schmelzle and Hall 2000). Most of them are thought to be affected through a combination of direct phosphorylation and repression of phosphatase activity (Raught et al. 2001). In the latter process, Tap42 plays a crucial role (Fig. 7.16A). It was shown that Tor phosphorylates Tap42, which is dephosphorylated by PP2A (Jiang and Broach 1999). Phosphorylation of Tap42 facilitates interaction with the catalytic subunit of PP2A and the PP2A-like phosphatase Sit4 (Dicomo and Arndt 1996; Jiang and Broach 1999). PP2A consists of the catalytic subunit Pph21/22, and two other regulatory proteins, designated A (encoded by TPD3), and B (encoded by CDC55 or RTS1) (Healy et al. 1991; Shu et al. 1997; Sneddon et al. 1990; van Zyl et al. 1992; Zabrocki et al. 2002). Binding of Tap42 to Pph21/22 results in a change in the phosphorylation state of many proteins. However, it is unlikely that binding of Tap42 results in an inhibition of the phosphatase, since pph21pph22 mutants are not rapamycin resistant. Furthermore, the amount of Tap42 in cells is significantly lower than that of Pph21/22. Rather, it was suggested that the function of Tap42 is to redirect a portion of the phosphatase activity to other substrates (Jiang and Broach 1999).Activation of Tor and subsequent redirection/inactivation of phosphatase activity results in several cellular responses (Raught et al. 2001; Rohde et al. 2001; Schmelzle and Hall 2000) (Fig. 7.16B.), one of the main being translation initiation (Barbet et al. 1996; Cafferkey et al. 1993; Heitman et al. 1991a; Kunz et al.1993). Most likely, this occurs via the initiation factor eIF4E (Barbet et al. 1996; Cosentino et al. 2000). The protein Eap1 was identified based on its ability to interact with eIF4E. Eap1 inhibits binding of the initiation factor eIF4E to the


Fig. 7.16A The Tor pathway in Saccharomyces cerevisiae. Regulation of phosphatase activity by Tor. Tor phosphorylates Tap42 that results in the binding with the phosphatases Pph21, Pph22, and Sit4. Hereby, phosphatase activity is inhibited or redirected

cap structure at the 5’ termini of mRNA’s by competing with eIF4G (the large subunit of the cap-binding complex) for binding to eIF4E. Most probably, interaction between eIF4E and Eap1 is abolished by dephosphorylation of Eap1 (Cosentino et al. 2000). Besides its role in translation initiation, Tor also controls biosynthesis by regulating ribosome biogenesis. Following rapamycin treatment, RNA polymerase II (PolII)-mediated transcription of ribosomal protein genes is severely reduced (Cardenas et al. 1999; Komeili et al. 2000; Powers and Walter 1999), as well as the PolI and PolIII-mediated rRNA and tRNA synthesis (Powers and Walter 1999; van Zyl et al. 1992; Zaragoza et al. 1998). Tor inhibition also affects transcription of specific sets of genes. This control appears to occur in a conserved manner by restricting nuclear import of several transcription factors, such as Gln3, Msn2 and Msn4, and Rtg1 and Rtg3. Gln3 is retained cytoplasmically by Ure2 (Beck and Hall 1999; Bertram et al. 2000; Cardenas et al. 1999; Hardwick et al. 1999), Rtg1 and Rtg3 by Rtg2 (Komeili et


Fig. 7.16B. The Tor pathway in Saccharomyces cerevisiae. Tor effectors in yeast. Tor inactivation blocks translation initiation through inhibition of eIF4E, ribosome biogenesis through repression of ribosomal genes (transcribed by PolII) and rRNA and tRNA sysnthesis (transcribed by PolI and PolIII) and uptake of amino acids by degradation of high affinity amino acid permeases through activation of Npr1. On the other hand, Tor inactivation causes an induction of several genes through nuclear localisation of different transcription factors (Gln3, Rtg1 and Rtg3, Msn2 and Msn4). For details, see section7.4.4. (Adapted and modified from references Jiang and Broach 1999; Rohde et al. 2001; Schmelzle and Halln 2000)

al. 2000) and Msn2 and Msn4 by the 14-3-3 proteins Bmh1 and Bmh2 (Beck and Hall 1999). Tor inhibition releases these transcription factors resulting in their nuclear translocation. Upon Tor inactivation, a severe decrease in amino acid transport is observed (Beck et al. 1999; Schmidt et al. 1998). This downregulation is caused by dephosphorylation and concomitant activation of Npr1. In turn, Npr1 phosphorylates several nitrogen-source regulated amino acid permeases. This results in the increase of low-affinity permeases Gap1 and Put4 and the ubiquitination and degradation of the high-affinity permease Tat2 (Beck et al. 1999; Schmidt et al. 1998; Vandenbol et al. 1990). Finally, Tor2 also controls actin organisation by a signalling cascade containing the Rho1 GTPase, Pkc1, and a Pkc1-activated MAP kinase cascade. Remarkably, this control is rapamycin insensitive (Helliwell et al. 1998a; Helliwell et al. 1998b; Schmidt et al. 1997; Schmidt et al. 1996; Zheng et al. 1995). Since Tor is involved in entry in stationary phase and shares several targets with the Ras-cAMP pathway (e.g. control of Msn2/4-regulated genes, ribosome


biogenesis, reserve carbohydrate metabolism), one could argue that Tor is part of the pathway. However, this is unlikely since constitutive activation of the RascAMP pathway does not cause rapamycin insensitivity (Barbet et al. 1996; François and Parrou 2001a). Whether Tor plays a role in the FGM pathway (see 7.4.3) is not clear yet, but possibly, they are interconnected via PP2A and Sch9, two main players of the FGM pathway (Crauwels et al. 1997a; Geyskens et al. 2000). It was suggested that Sch9 is a target of PP2A and in this respect Tor could interfere with the FGM pathway (Zabrocki et al. 2002). We can conclude that Tor appears to act as a master regulator of the balance between protein synthesis and degradation in response to nutrient availability (Dennis et al. 2001a; Raught et al. 2001). However, the mechanisms by which Tor activity is regulated by nutrients, remains unknown. In this respect, it was recently discovered that mammalian Tor is partly regulated by ATP availability (Dennis et al. 2001b). When ATP is low (as in the case of nutrient starvation), mTor turns on a starvation response. It is not clear at present, if a similar mechanism also operates in yeast. 7.4.5 Glycogen and Trehalose metabolism The control of reserve carbohydrate metabolism, glycogen and trehalose, may certainly serve as an example of an integrated nutrient limitation response. The intracellular concentrations of these storage carbohydrates vary with the growth phase of the yeast cell (François and Parrou 2001a; Lillie and Pringle 1980; Thevelein 1984). In cells growing on a fermentable carbon source like glucose or fructose glycogen accumulates before the fermentable sugar is exhausted, with peak concentrations occurring at the diauxic shift. In contrast, trehalose only begins to accumulate at the diauxic shift and peaks when cells enter stationary phase because of nutritional shortage. During stationary phase, trehalose is slowly degraded and its disappearance is accompanied by loss of viability (Lillie and Pringle 1980; Werner-Washburne et al. 1993; Werner-Washburne et al. 1996). Both glycogen and trehalose are synthesised from glucose-6-phosphate and UDP-glucose, however biosynthetic control and the enzymes involved are completely different (Fig.7.17). The concentration of trehalose in the yeast cell is the result of the synthetic activity of the trehalose synthase complex and the degradative activity of trehalase (Thevelein 1996). The trehalose synthase complex consists of three subunits. Trehalose-6-phosphate synthase is encoded by TPS1 (Bell et al. 1992; Cannon et al. 1994; Gonzalez et al. 1992; Van Aelst et al. 1993; Vuorio et al. 1993), and trehalose-6-phosphate phosphatase by TPS2 (De Virgilio et al. 1993). TSL1 and a related gene with high sequence similarity, TPS3, both code for the largest and probably regulatory subunit of the complex (Bell et al. 1998; Reinders et al. 1997; Vuorio et al. 1993). Trehalose-6-phosphate synthase activity is strongly enhanced by fructose-6-phosphate and inhibited by phosphate, whereas trehalose-6phosphate phosphatase activity is enhanced by phosphate (Londesborough and Vuorio 1993; Vuorio et al. 1993). The biosynthesis of trehalose is controlled via transcriptional and post-translational regulation of the subunits of the synthase co-


Fig. 7.17. Metabolism of the reserve carbohydrates glycogen and trehalose. Schematical depiction of synthesis and degradation routes for the storage carbohydrates glycogen and trehalose in Saccharomyces cerevisiae. (For abbreviated enzyme names, see paragraph 7.4.5. (Adapted from François and Parrou 2001b)

mplex. The synthase subunit is proteolytically inactivated in the presence of glucose (François et al. 1991). Transcription of the four subunit-encoding genes is coregulated by growth phase and stress conditions through cis-acting STREelements in their promoter regions (Parrou et al. 1997; Winderickx et al. 1996). The STRE-sequences have been implicated as central regulatory target sites in the control of various stress-related genes (Boy-Marcotte et al. 1998; Moskvina et al. 1998; Ruis and Schuller 1995). Interestingly, however, carbon source-dependent transcriptional control of the trehalose synthase subunit genes is not conforming to the typical STRE-mediated regulation. Although for all four genes rapid glucoseinduced disappearance of the mRNA is observed, only TSL1 is permanently repressed under fermentative growth conditions, whereas TPS1, TPS2, and TPS3 are expressed only a few-fold lower compared to post-diauxic or non-fermentative growth (Winderickx et al. 1996). This probably relates to the observation that a certain minimal level of Tps1 (and hence, a complete synthase complex?) is required even during fermentative growth (Hohmann et al. 1994; Neves et al. 1995).


Mobilisation of trehalose is achieved via hydrolysis by trehalase (Muller et al. 1995a; Thevelein 1996). In S.cerevisiae, neutral trehalase is encoded by NTH1 and presumably by a second redundant gene, NTH2. The NTH1 gene appears to express the bulk of neutral trehalase under derepressing or stress conditions (Kopp et al. 1993; Kopp et al. 1994; Nwaka et al. 1995a; Nwaka et al. 1995b). ATH1 encodes a vacuolar acid trehalase (Destruelle et al. 1995; Nwaka et al. 1996). The neutral trehalase Nth1 constitutes the main source of trehalase-degradative capacity in proliferating, stationary phase, or germinating yeast. A trehalose degrading activity has not been demonstrated for Nth2 but as for Nth1, the enzyme is required for thermotolerance (Nwaka and Holzer 1998; Nwaka et al. 1995a; Nwaka et al. 1995b). The vacuolar acid trehalase Ath1 appears to be mainly required for growth on trehalose and hence extracellular trehalose hydrolysis (Nwaka and Holzer 1998; Nwaka et al. 1996). Although some level of transcriptional regulation has been reported for NTH1 and NTH2, STRE-mediated, and Msn2/Msn4 dependent in the case of NTH1 (Nwaka et al. 1995a; Nwaka et al. 1995b; Parrou et al. 1997; Zähringer et al. 2000), trehalase activity is apparently controlled mainly by phosphorylation and dephosphorylation. Neutral trehalase activity is induced by heat shock (De Virgilio et al. 1991), by readdition of nutrients to nutrient-starved cells (Durnez et al. 1994; Hirimburegama et al. 1992), and by glucose or another fermentable sugar in glucose-depleted or post-diauxic cells (Thevelein 1991; Thevelein 1996; van der Plaat 1974). An abrupt drop in activity is observed during the diauxic shift, concomitant with the start of trehalose accumulation in postdiauxic cells (François et al. 1987). Activation of neutral trehalase is dependent on PKA (Uno et al. 1983), but cAMP is not essential as a second messenger (Durnez et al. 1994; Hirimburegama et al. 1992; Pernambuco et al. 1996) (see also section 4.3). This mode of trehalase activation has also recently been described for S. pombe, Pachysolen. Tannophilus, and Candida utilis (Carrillo et al. 1995; Soto et al. 1995a; Soto et al. 1997; Soto et al. 1995b; Soto et al. 1996) and apparently represents a conserved signalling pathway operating in the fungal nutrient response. The onset of glycogen synthesis requires the presence of self-glucosylating initiator proteins encoded by GLG1 and GLG2 (Cheng et al. 1995). Glycogen synthase is encoded by the differentially regulated GSY1 and GSY2 genes (Farkas et al. 1990; Farkas et al. 1991). The glycogen branching enzyme is encoded by GLC3 (Rowen et al. 1992; Thon et al. 1992). Glycogen catabolism is achieved by glycogen phosphorylase (GPH1) (Hwang et al. 1989) and the debranching enzyme (GDB1) (François and Parrou 2001a). In sporulating cells, glycogen breakdown is achieved by a glucoamylase (Clancy et al. 1982; Johnston and Carlson 1992a). GSY2, GLC3, and GPH1 are transcriptionally activated when the glucose concentration is decreasing (Farkas et al. 1991; Hwang et al. 1989; Rowen et al. 1992; Thon et al. 1992). Additionally, transcriptional activation under different stress conditions has been reported and shown to be dependent on the STRE-binding transcriptional activators, Msn2 and Msn4 (Parrou et al. 1997). Glycogen synthase is activated via dephosphorylation by protein phosphatase type 1 consisting of GLC7 (Cannon et al. 1994; Feng et al. 1991; Huang et al. 1996b), which is targeted to glycogen particles and regulated by the GAC1, GLC8, and REG1 gene


products (Cannon et al. 1994; François et al. 1992; Huang et al. 1996b; Ramaswarmy et al. 1998; Skroch-Stuart et al. 1994). In addition, PIG1, PIG2, and YER054c, were more recently identified as putative PP1 regulatory-targeting subunits (Cheng et al. 1997; François and Parrou 2001a; Tu et al. 1996). As mentioned before, PP1 also plays a crucial role in the control of Snf1 kinase activity and consequently catabolite repression (see 3.1.3). Protein phosphatase 2A (PP2A) also appears to be involved in the control of glycogen synthase activity but its effect on glycogen metabolism might be confined to the role in the regulation of cell growth (Clotet et al. 1995; Posas et al. 1991; Stark 1996). Pho85 and the cyclinlike subunits Pcl8 and Pcl10 are involved in the phosphorylation and inactivation of glycogen synthase (Huang et al. 1998) (see 3.3.2). Interestingly, Pho85 was identified as second site suppressor for the glycogen storage defect of cells deleted for the Snf1 kinase (Cannon et al. 1994; Huang et al. 1996a; Hubbard et al. 1992; Thompson-Jaeger et al. 1991). The Pho85 kinase complex phosphorylates the synthase at two of the three sites that have been shown to be phosphorylated in vivo (François and Parrou 2001a; Hardy and Roach 1993). The kinase involved in phosphorylation of the third site remains to be determined. Although several observations indicated the involvement of PKA in the control of glycogen synthase phosphorylation (François et al. 1988; Hardy et al. 1994; Hardy and Roach 1993), no evidence has been provided so far that glycogen synthase is a direct substrate. 7.4.6 Morphological differentiation as a response to nutrient limitation Another response affected by integration of nutrient availability is the morphogenetic switch to filamentous or pseudohyphal growth in S. cerevisiae (for a recent review see Gancedo 2001; Pan et al. 2000). Pseudohyphal growth was originally observed in heterozygous a/α diploids grown on medium containing low ammonia concentrations or proline as a sole nitrogen source. Under these conditions, the cells switched their bipolar budding pattern to a strictly polarised and elongated morphology (Gimeno et al. 1992). A similar dimorphic shift was later reported for haploid cells and this was described as invasive growth because the filaments penetrate the agar below the colony. To date, several findings indicate that the onset of pseudohyphal differentiation in S. cerevisiae is not restricted to conditions of nitrogen limitation but it appears to be a more general response to nutrient limitation (Blacketer et al. 1995a; Dickinson 1994; Dickinson 1996; Lambrechts et al. 1996b) and stress (Davenport et al. 1999; Stanhill et al. 1999; Zaragoza and Gancedo 2000) which appears to be strictly dependent on the presence of oxygen (Wright et al. 1993). Differences in susceptibility of the morphogenetic switch in response to various nutrient limitation conditions are likely due to differences in genetic background of the yeast strains under investigation (Kron 1997; Liu et al. 1996; Stanhill et al. 1999). Haploid yeast cells grown on medium containing the poor carbon source ethanol and leucine as a nitrogen source develop large hyphallike extensions (Dickinson 1994). This finding has been substantiated, since 'fusel' alcohols like isoamylalcohol, which are the natural catabolic products of several


amino acids, induce pseudohyphal growth (Dickinson 1996). Several starch degrading strains of S. cerevisiae display strong pseudohyphal differentiation and invasive growth when grown either on low ammonium medium or on medium with excess nitrogen but with maltotriose or starch as sole carbon source (Lambrechts et al. 1996b). Pseudohyphal growth is also observed when cells are plated on rich medium containing glucose and subjected to stress, e.g. heat stress or osmotic stress (Davenport et al. 1999; Stanhill et al. 1999; Zaragoza and Gancedo 2000). Although several differences between diploid pseudohyphal growth and haploid invasive growth are observed, both growth forms depend on concerted ch anges in different cellular processes like cell elongation, budding pattern and cell adhesion. Not surprisingly, several genes involved in the control of these processes have been found to influence the ability to form pseudohyphae (reviewed in Gancedo 2001; Pan et al. 2000). At present, the picture evolving is that the switch to pseudohyphal or invasive growth in diploid or haploid Saccharomyces appears to be controlled by an interplay of two different signalling pathways, i.e. the RascAMP pathway and a MAPK cascade (Fig. 7.18). A direct involvement of the Ras-cAMP pathway was suggested by the early observations that a dominant activated RAS2Val19 mutation or cAMP addition enhances pseudohyphal differentiation (Gimeno et al. 1992; Kubler et al. 1997; Lorenz and Heitman 1997) while overexpression of PDE2, encoding the most active phosphodiesterase, inhibits pseudohyphal growth (Ward et al. 1995). More recent-

Fig. 7.18. Pseudohyphal differentiation in Saccharomyces cerevisiae. Schematical representation of the MAPK cascade and Ras-cAMP components that govern the switch to pseudohyphal differentiation. For details, see paragraph 7.4.6. (Adapted from Gancedo 2001)


ly, the involvement of the cAMP pathway has been confirmed and other components such as the GPCR system, Gpr1 (Kraakman et al. 1999a; Lorenz et al. 2000; Xue et al. 1998; Yun et al. 1998) and Gpa2 (Colombo et al. 1998; Kubler et al. 1997; Lorenz and Heitman 1997; Nakafuku et al. 1988), and the Tpk subunits of PKA (Pan and Heitman 1999; Robertson and Fink 1998; Toda et al. 1987b) were described to function in the signal cascade leading to pseudohyphae formation. Interestingly, a differential role can be attributed to the three different Tpk kinases. Tpk2 induces pseudohyphal growth while Tpk1 and Tpk3 have an inhibitory effect (Pan and Heitman 1999; Robertson and Fink 1998). A two-hybrid screen identified two DNA binding proteins, Sfl1 and Mga1. Sfl1 act as repressor since its deletion enhances pseudohyphal growth (Robertson and Fink 1998). It was also reported to act as repressor of the invertase gene, SUC2 (Song and Carlson 1998). The role of Mga1 is still controversial (Lorenz and Heitman 1998b; Robertson and Fink 1998). The downstream target of Sfl1 is MUC1/FLO11 a gene encoding a cell-surface flocculin (Lambrechts et al. 1996a; Lo and Dranginis 1998). Flo8, another gene involved in flocculation (Kobayashi et al. 1996), induces expression of MUC1/FLO11 and is required for pseudohyphal growth (Liu et al. 1996). In contrast to the Sfl1 repressor, Flo8 is not strictly dependent on Tpk2 and can be activated by any catalytic PKA subunit (Gancedo 2001; Pan and Heitman 1999; Robertson and Fink 1998). Finally, another protein Msn1 (Phd2/Mss10/Fup4) has been implicated in the transcriptional regulation of MUC1/FLO11. Interestingly, Msn1 has also been isolated as an activator of SUC2 transcription (Estruch and Carlson 1990). Hence, it is possible that Msn1 is under negative control of Sfl1. Epistasis analysis has shown that it functions below Ras2 and Mep2 but independent of the MAP kinases and Ste12 (see below)(Gagiano et al. 1999b). The MAPK cascade regulating pseudohyphal growth in diploids and invasive growth in haploids shares several components with the mating pheromoneinduced pathway including Cdc42, the kinases Ste20, Ste11, Ste7, and the transcription factor Ste12 (Liu et al. 1993). The MAP kinase Fus3 is not shared and is specific for the pheromone-induced pathway. Instead, the filamentation-specific MAP kinase, Kss1, is involved. This is important to achieve specificity and to prevent inappropriate cross talk between the different MAPK cascades (Madhani and Fink 1998a; Madhani and Fink 1998b). The activation of the MAPK cascade specifically requires Ras2. Ras1 is not involved (Mosch et al. 1999; Mosch et al. 1996). Ras2 activates the guanine nucleotide exchange factor Cdc24 that in turn activates Cdc42. Activated Cdc42 interacts with and activates Ste20 by displacement of the negative regulator Hsl7 (Fujita et al. 1999; Leberer et al. 1997; Peter et al. 1996). Ste20 can now further activate the MAPK cascade that ultimately results in phophorylation of Kss1. In its unphosphorylated form, Kss1 interacts with the transcription factor Ste12 and the negative regulators Rst1/Dig1 and Rst2/Dig2 (Bardwell et al. 1998; Cook et al. 1996). When phosphorylated, Kss1 will in turn phosphorylate Ste12, Rts1/Dig1, and Rts2/Dig2. The Rts/Dig proteins dissociate (Bardwell et al. 1998) and this allows Ste12 to bind to its DNA site in the socalled FRE promoter element (Madhani and Fink 1998a; Madhani and Fink 1998b). FRE elements also contain the binding site of yet another transcription factor, Tec1 (Gavrias et al. 1996). The co-operative binding of Ste12 with Tec1 to


the FRE elements is sufficient to derepress the target genes. Thus far, only three targets have been identified; the MUC1/FLO11 gene, the PGU gene, which encodes a secreted pectin-degrading enzyme, and the G1 cyclin gene CLN1 (Gagiano et al. 1999b; Lo and Dranginis 1998; Madhani et al. 1999; Rupp et al. 1999). Since Tec1 is a filamentation specific transcription factor that associates and co-operatively binds with Ste12, it represents a transcriptional mechanism to ensure specificity of the MAPK cascade (Madhani and Fink 1998a; Madhani and Fink 1998b). Ste12 is also required for derepression mediated by Mss11 (Gagiano et al. 1999b). Whether this reflects a co-operative transcriptional regulation of Mss11 and Ste12 or an indirect effect remains to be elucidated. For instance, Mss11 could in combination with Ste12 be required for activation of the TEC1 gene (Madhani and Fink 1997). On the other hand, Mss11 is required to mediate derepression of MUC1/FLO11 by Msn1 and Flo8, two components downstream of Ras2 that act independently of Msn1 (Gagiano et al. 1999a; Rupp et al. 1999). Hence, Mss11 represents a component where the different stimuli converge, including those of the Ras-cAMP and the MAPK cascade. Several other components have been identified. The phosphatidylinositolspecific phospholipase C, Plc1, is necessary for filamentation upon nitrogen starvation. Plc1 was shown to be required for the interaction between Gpr1 and Gpa2, the two proteins that constitute the glucose- and sucrose-specific GPCR system (Ansari et al. 1999). It is therefore speculated that Plc1 would act through the RascAMP pathway. However, deletion of PLC1 does not affect cAMP signalling (Lemaire, Winderickx and Thevelein, unpublished data). The high affinity ammonium permease, Mep2, has been described as the only ammonium permease able to signal low-nitrogen to the filamentous signalling cascades (Lorenz and Heitman 1998a). As for Plc1, Mep2 was suggested to act upstream of the Ras-cAMP pathway. More recently, however, a negative role for high affinity ammonium permease, Mep1, on filamentation was described (Lorenz and Heitman 1998b). SOK2 encodes an inhibitor of pseudohyphal differentiation, exhibiting a high degree of identity in its DNA-binding domain with the DNA-binding region of Phd1 (Ward et al. 1995). Interestingly, the Sok2 protein appears to be a mediator of PKAdependent regulation of pseudohyphal growth that acts through a set of transcription factors including Phd1, Ash1, and Swi5 (Pan and Heitman 2000). As mentioned above, pseudohyphal differentiation can also be triggered on rich medium under conditions of stress (Zaragoza and Gancedo 2000). Most likely, part of these responses will involve components of the Ras-cAMP pathway, given its importance in the general stress response. On the other hand, for certain stress condition pseudohyphal differentiation could result from cross talk between MAPK cascades (Madhani and Fink 1998b). This appears to be the case for osmotic stress (Davenport et al. 1999). The membrane-spanning protein Sho1, one of the upstream sensing proteins of the high osmolarity glycerol response (HOG) pathway appears to be required to produce filaments and probably to sense low nitrogen signal (Blacketer et al. 1995a; O'Rourke and Herskowitz 1998). Several additional mutations causing constitutive pseudohyphal growth have been described in an extensive genetic analysis of the morphological differentia-


tion (Blacketer et al. 1993; Blacketer et al. 1994; Blacketer et al. 1995b). This study has thusfar revealed the involvement of a novel protein kinase Elm1 and protein phosphatase 2A (PP2A) (Blacketer et al. 1993), and of phosphoribosylpyrophosphate (PRPP) synthase, encoded by the PPS1 gene (Blacketer et al. 1994). Pps1 is a central enzyme in nitrogen metabolism and PRPP is essential for the biosynthesis of nucleotides, histidine, and tryptophane (Jones and Fink 1982; Jones 1980).

7.5 Conclusions Signal transduction mechanisms have become major research topics in molecular and cellular biology. Adequate perception of and efficient response to the environment is of vital importance to all living organisms, ranging from simple prokaryotes to multicellular eukaryotes. As the number of identified components increases and the complexity of the signal transduction pathways involved is revealed, the most striking finding is the high degree of evolutionary conservation of the underlying basic mechanisms, especially within the eukaryotic kingdom. Since baker's yeast S. cerevisiae is a relatively simple, genetically and biochemically well-characterised and an easy-to-handle eukaryote it serves as a valuable tool in the scrutiny of eukaryotic signal transduction. The way in which yeast cells respond to nutritional stimuli increasingly appears to present models for nutrient sensing and signalling in various specialised cell types of multicellular higher eukaryotes. An important new aspect appears to be the balanced coupling of extracellular nutritional detection mechanisms with intracellular sensing of the metabolic status in order to achieve responses that are more integrated. A good example of this is the dual glucose-induced signalling process for cAMP induction, which involves extracellular glucose detection via the Gpr1-Gpa2 GPCR system and glucose phosphorylation-dependent intracellular sensing via the hexose kinases. In the era where yeast research has evolved towards a genome-wide approach, a new picture emerges. Focus is shifting from single, usually parallel signal transduction cascades, to more complex interregulatory transduction networks. In such networks, parts of different cascades are being relayed and combined to create novel signalling pathways with higher flexibility, sensitivity, or robustness. A fine example is presented by the recently discovered integration of the Ras-cAMP pathway and a MAPK cascade in the control of pseudohyphal differentiation in yeast. A genome-wide, global view on signal transduction will inevitably dominate future research on the signalling networks that are operating in cell growth and nutrition.


References Aerne BL, Johnson AL, Toyn JH, Johnston LH (1998) Swi5 controls a novel wave of cyclin synthesis in late mitosis. Mol Biol Cell 9:945-956 Albrecht G, Mösch H-U, Hoffmann B, Reusser U, Braus GH (1998) Monitoring the Gcn4 protein-mediated response in yeast Saccharomyces cerevisiae. J Biol Chem 273:12696-12702 Alms GR, Sanz P, Carlson M, Haystead TAJ (1999) Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae:characterizing the effects of a phosphatase subunit on the yeast proteome. EMBO J 18:4157-4168 Alonso A, C. Pascual C, Herrera L, Gancedo JM, Gancedo C (1984) Metabolic imbalance in a Saccharomyces cerevisiae mutant unable to grow on fermentable hexoses. Eur J Biochem 138:407-411 Ansari K, Martin S, Farkasovsky M, Ehbrecht IM, Kuntzel H (1999) Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae. J Biol Chem 274:30052-30058 Arndt KT, Styles C, Fink GR (1987) Multiple global regulators control HIS4 transcription in yeast. Science 237:874-880 Bai Y, Salvadore C, Chiang YC, Collart MA, Liu HY, Denis CL (1999) The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4 and NOT5. Mol Cell Biol 19:6642-6651 Baker HV (1986) Glycolytic gene expression in Saccharomyces cerevisiae: nucleotide sequence of GCR1, null mutants, and evidence for expression. Mol Cell Biol 6:37743784 Baker RE, Masison DC (1990) Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1. Mol Cell Biol 10:2458-2467 Barbaric S, Münsterkötter M, Goding C, Hörz W (1998) Cooperative Pho2-Pho4 interactions at the PHO5 promoter are critical for binding of Pho4 to UASp1 and for efficient transactivation by Pho4 at UASp2. Mol Cell Biol 18:2629-2639 Barbaric S, Münsterkötter M, Svaren J, Hörz W (1996) The homeodomain protein Pho2 and the basic-helix-loop-helix protein Pho4 bind DNA cooperatively at the yeast PHO5 promoter. Nucleic Acids Res 24:4479-4486 Barbet NC, Schneider U, Helliwell SB, Stansfield I, Tuite MF, Hall MN (1996) TOR controls translation initiation and early g1 progression in yeast. Mol Biol Cell 7:25-42 Bardwell L, Cook JG, Voora D, Baggott DM, Martinez AR, Thorner J (1998) Repression of the yeast Ste12 transcription factor by direct binding of unphosphorylated Kss1 MAPK and its regulation by the Ste7 MEK. Genes Dev 12:2887-2898 Baroni MD, Monti P, Alberghina L (1994) Repression of growth-regulated G1 cyclin expression by cyclic AMP in budding yeast. Nature 371:339-342 Beck T, Hall MN (1999) The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402:689-692 Beck T, Schmidt A, Hall MN (1999) Starvation induces vacuolar targeting and degradation of the tryptophan permease in yeast. J Cell Biol 146:1227-1238 Behlke J, Heidrich K, Naumann M, Muller EC, Otto A, Reuter R, Kriegel T (1998) Hexokinase 2 from Saccharomyces cerevisiae:Regulation of oligomeric structure by in vivo phosphorylation at serine-15. Biochemistry 37:11989-11995

7 Adaptation to nutrient availability in yeast 359 Bell W, Klaassen P, Ohnacker M, Boller T, Herweijer M, Schoppink P, van der Zee P, Wiemken A (1992) Characterization of the 56 kDa subunit of the yeast trehalose-6phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation. Eur J Biochem 209:951-959 Bell W, Sun W, Hohmann S, Wera S, Reinders A, De Virgilio C, Wiemken A, Thevelein JM (1998) Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex. J Biol Chem 273:33311-33319 Berben G, Legrain M, Gilliquet V, Hilger F (1990) The yeast regulatory gene PHO4 encodes a helix-loop-helix motif. Yeast 6:451-454 Bernard F, André B (2001a) Genetic analysis of the signalling pathway activated by external amino acids in Saccharomyces cerevisiae. Mol Microbiol 41:489-502 Bernard F, André B (2001b) Ubiquitin and the SCFGrr1 ubiquitin ligase complex are involved in the signalling pathway activated by external amino acids in Saccharomyces cerevisiae. FEBS Lett 496:81-85 Bertram PG, Choi JH, Carvalho J, Ai W, Zeng C, Chan TF, Zheng XF (2000) Tripartite regulartion of Gln3p by TOR, Ure2p and phosphatases. J Biol Chem 275:35727-35733 Beullens M, Mbonyi K, Geerts L, Gladines D, Detremerie K, Jans AWH, Thevelein JM (1988) Studies on the mechanism of the glucose-induced cAMP signal in glycolysis and glucose repression mutants of the yeast Saccharomyces cerevisiae. Eur J Biochem 172:227-231 Blacketer MJ, Koehler CM, Coats SG, Meyers AM, Madaule P (1993) Regulation of dimorphism in Saccharomyces cerevisiae:involvement of the novel protein kinase homolog Elm1p and protein phospatase 2A. Mol Cell Biol 13:5567-5581 Blacketer MJ, Madaule P, Meyers AM (1994) The Saccharomyces cerevisiae mutation elm4-1 facilitates pseudohyphal differentiation and interacts with a deficiency in phosphoribosylpyrophosphate synthase activity to cause constitutive pseudohyphal growth. Mol Cell Biol 14:4671-4681 Blacketer MJ, Madaule P, Meyers AM (1995a) Mutational analysis of morphologic differentiation in Saccharomyces cerevisiae. Genetics 140:1259-1275 Blacketer MJ, Madaule P, Myers AM (1995b) Mutational analysis of morphologic differentiation in saccharomyces cerevisiae. Genetics 140:1259-1275 Blom J, Teixeira de Mattos MJ, Grivell LA (2000) Redirection of the respiro-fermentative flux distribution in Saccharomyces cerevisiae by overexpression of the transcription factor Hap4. Appl Environ Microbiol 66:1970-1973 Boles E, Heinisch J, Zimmermann FK (1993) Different signals control the activation of glycolysis in the yeast Saccharomyces cerevisiae. Yeast 9:761-770 Boles E, Hollenberg CP (1997) The molecular genetics of hexose transport in yeasts. FEMS Microbiol Rev 21:85-111 Boy-Marcotte E, Perrot M, Bussereau F, Boucherie H, Jacquet M (1998) Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae. J Bacteriol 180:1044-1052 Boy-Marcotte E, Tadi D, Perrot M, Boucherie H, Jacquet M (1996) High cAMP levels antagonize the reprogramming of gene expression that occurs at the diauxic shift in Saccharomyces cerevisiae. Microbiol 142:459-467 Bram RJ, Kornberg RD (1987) Isolation of an S. cerevisiae centromere DNA-binding protein, its human homolog and its possible role as a transcription factor. Mol Cell Biol 7:403

360 Joris Winderickx et al. Braus G, Mösch H-U, Vogel K, Hütter R (1989) Interpathway regulation of the TRP4 gene of yeast. EMBO J 8:939-945 Brazas RM, Bhoite LT, Murphy MD, Yu YX, Chen YY, Neklason DW, Stillman DJ (1995) Determining the requirements for cooperative DNA binding by Swi5p and Pho2p (Grf10p/Bas2p) at the HO promoter. J Biol Chem 270:29151-29161 Broek D, Samiy N, Fasano O, Fujiyama A, Tamanoi F, Northup J, Wigler M (1985) Differential activation of yeast adenylate cyclase by wild type and mutant Ras proteins. Cell 41:763-769 Broek D, Toda T, Michaeli T, Levin L, Birchmeier C, Zoller M, Powers S, Wigler M (1987) The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell 48:789-799 Brondijk TH, van der Rest ME, Pluim D, de Vries Y, Stingl K, Poolman B, Konings WN (1998) Catabolite inactivation of wild type and mutatnt maltose transport proteins in Saccharomyces cerevisiae. J Biol Chem 19:15352-15357 Bürglin TR (1988) The yeast regulatory gene PHO2 encodes a homeo box. Cell 53:339Cafferkey R, McLaughlin MM, Young PR, Johnson RK, Livi GP (1994) Yeast TOR (DRR) proteins:amino acid sequence alignment and identification of structural motifs. Gene 14:133-136 Cafferkey R, Young PR, McLauglin MM, Bergsma DJ, Koltin Y, Sathe GM, Faucette L, Eng WK, Johnson RK, Livi GP (1993) Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity. Mol Cell Biol 13:6012-6023 Cai MJ, Davis RW (1990) Yeast Centromere-Binding Protein CBF1, of the helix-loophelix protein family, is required for chromosome stability and methionine prototrophy. Cell 61:437-446 Cameron S, Levin L, Zoller M, Wigler M (1988) cAMP-independent control of sporulation, glycogen metabolism and heat shock resistance in S. cerevisiae. Cell 53:555-566 Camonis JH, Kalékine M, Gondré B, Garreau H, Boy-Marcotte E, Jacquet M (1986) Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae. EMBO J 5:375-380 Camus C, Boymarcotte E, Jacquet M (1994) Two subclasses of guanine exchange factor (GEF) domains revealed by comparison of activities of chimeric genes constructed from CDC25, SDC25 and BUD5 in Saccharomyces cerevisiae. Mol Gen Genet 245:167-176 Cannon JF, Pringle JR, Fiechter A, Khalil M (1994) Characterization of glycogen-deficient GLC mutants of Saccharomyces cerevisiae. Genetics 136:485-503 Cardenas ME, Cutler NS, Lorenz MC, Di Como CJ, Heitman J (1999) The Tor signaling cascade regulates gene expression in response to nutrients. Genes Dev 13:3271-3279 Carlson M (1999) Glucose repression in yeast. Curr Opin Microbiol 2:202-207 Carrillo D, Vicentesoler J, Fernandez J, Soto T, Cansado J, Gacto M (1995) Activation of cytoplasmic trehalase by cyclic-AMP-dependent and cyclic-AMP-independent signalling pathways in the yeast Candida utilis. Microbiol 141:679-686 Celenza JL, Carlson M (1984) Cloning and genetic mapping of SNF1, a gene required for expression of glucose-repressible genes in S.cerevisiae. Mol Cell Biol 4:49-53 Celenza JL, Carlson M (1986) A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science 233:1175-1180.

7 Adaptation to nutrient availability in yeast 361 Celenza JL, Carlson M (1989) Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with SNF4 protein. Mol Cell Biol 9:5034-5044 Celenza JL, Marshall-Carlson L, Carlson M (1988) The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein. Proc Natl Acad Sci USA 85:21302134 Cereghino GP, Atencio DP, Saghbini M, Beiner J, Scheffler IE (1995) Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae:Sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role. Mol Biol Cell 6:1125-1143 Cereghino GP, Scheffler IE (1996) Genetic analysis of glucose regulation in Saccharomyces cerevisiae:control of transcription versus mRNA turnover. EMBO J 15:363-374. Chambers A, Packham EA, Graham IR (1995) Control of glycolytic gene expression in the budding yeast (Saccharomyces cerevisiae). Curr Genet 29:1-9 Cheng C, Huang D, Roach PJ (1997) The PIG genes:PIG1 encodes a putative type 1 phosphatase subunit that interacts with the yeast glycogen synthase Gsy2. Yeast 13:1-8 Cheng C, Mu J, Farkas I, Huang D, Goebl MG, Roach PJ (1995) Requirement of selfglucosylating intiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae. Mol Cell Biol 15:6632-6640 Cherest H, Davidian JC, Thomas D, Benes V, Ansorge W, Surdin-Kerjan Y (1997) Molecular characterisation of two high affinity sulphate transporters in Saccharomyces cerevisiae. Genetics 145:627-635 Chiang H-L, Schekman R (1991) Regulated import and degradation of a cytosolic protein in the yeast vacuole. Nature 350:313-318 Chiang H-L, Schekman R (1994) Site of catabolite inactivation. Nature 369:284. Chiang H-L, Schekman R, Hamamoto S (1996) Selective uptake of cytosolic, peroxisomal and plasma membrane proteins by the yeast vacuole. J Biol Chem 271:9934-9941 Clancy MJ, Smith LM, Magee PT (1982) Developmental regulation of a sporulationspecific enzyme activity in Saccharomyces cerevisiae. Mol Cell Biol 2:171-178 Clotet J, Posas F, Hu GZ, Ronne H, Arino J (1995) Role of protein phosphatase 2A in the control of glycogen metabolism in yeast. Eur J Biochem 229:207-214 Coffman JA, Rai R, Loprete DM, Cunningham T, Svetlov V, Cooper TG (1997) Cross regulation of four GATA factors that control nitrogen catabolic gene expression in Saccharomyces cerevisiae. J Bacteriol 179:3416-3429 Colombo S, Ma P, Cauwenberg L, Winderickx J, Crauwels M, Teunissen A, Nauwelaers D, de Winde JH, Gorwa M-F, Colavizza D, Thevelein JM (1998) Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae. EMBO J 17:3326-3341 Colwill K, Field D, Moore L, Friesen J, Andrews B (1999) In vivo analysis of the domains of yeast Rvs167p suggests Rvs167p function is mediated through multiple protein interactions. Genetics 152:881-893 Cook JG, Bardwell L, Kron SJ, Thorner J (1996) Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae. Genes Dev 10:2831-2848 Cooper TG. (1982) Nitrogen metabolism in Saccharomyces cerevisiae. In Strathern, J.N., Jones, E.W. and Broach, J. (eds.), The Molecular Biology of the Yeast Saccharomyces:Metabolism and Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 39-99

362 Joris Winderickx et al. Cosentino GP, Schmelzle T, Haghighat A, Helliwell SB, Hall MN, Sonenberg N (2000) Eap1p, a novel eukaryotic translation initiation factor 4E-associated protein in Saccharomyces cerevisiae. Mol Cell Biol 20:4604-4613 Cox KH, Rai M, Distler JR, Daugherty JA, Coffman JA, Cooper TG (2000) Saccharomyces cerevisiae GATA sequences funtion as TATA elements during nitrogen catabolite repression and when Gln3p is excluded from the nucleus by overproduction of Ure2p. J Biol Chem 275:17611-17618 Crauwels M, Donaton MCV, Pernambuco MB, Winderickx J, de Winde JH, Thevelein JM (1997a) The Sch9 protein kinase in the yeast Saccharomyces cerevisiae controls cAPK activity and is required for nitrogen activation of the fermentable-growth-mediuminduced (FGM) pathway. Microbiol 143:2627-2637 Crauwels M, Winderickx J, de Winde JH, Thevelein JM (1997b) Identification of genes with nutrient-controlled expression by PCR-mapping in the yeast Saccharomyces cerevisiae. Yeast 13:973-984 Cunningham TS, Andhare R, Cooper TG (2000a) Nitrogen catabolite repression of DAL80 expression depends on the relative levels of Gat1p and Ure2p production in Saccharomyces cerevisiae. J Biol Chem 275:14408-14414 Cunningham TS, Cooper TG (1991) Expression of the DAL80 gene, whose product is homologous to the GATA factors and is a negative regulator of multiple nitrogen catabolic genes in Saccharomyces cerevisiae, is sensitive to nitrogen catabolite repression. Mol Cell Biol 11:6205-6215 Cunningham TS, Rai R, Cooper TG (2000b) The level of DAL80 expression downregulates GATA factor-mediated transcription in Saccharomyces cerevisiae. J Bacteriol 182:6584-6591 Daignan-Fornier B, Fink GR (1992) Coregulation of purine and histidine biosynthesis by the transcriptional activators BAS1 and BAS2. Proc Natl Acad Sci USA 89:6746-6750 Dang VD, Bohn C, Bolotinfukuhara M, Daignanfornier B (1996) The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms. J Bacteriol 178:1842-1849 Daugherty JR, Rai R, Elberry HM, Cooper TG (1993) Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3-Protein and DAL80-Protein and nitrogen catabolite repression in Saccharomyces-Cerevisiae. J Bacteriol 175:64-73 Davenport KD, Williams KE, Ullmann BD, Gustin MC (1999) Activation of the Saccharomyces cerevisiae Filamentation/invasion pathway by osmotic stress in highosmolarity glycogen pathway mutants. Genetics 153:1091-1103 de Boer M, Bebelman JP, Goncalves PM, Maat J, Van Heerikhuizen H, Planta RJ (1998) Regulation of expression of the amino acid transporter gene BAP3 in Saccharomyces cerevisiae. Mol Microbiol 30:603-613 de Boer M, Nielsen PS, Bebelman JP, Heerikhuizen H, Andersen HA, Planta RJ (2000) Stp1p, Stp2p and Abf1p are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae. Nucleic Acids Res 28:974-981 De Nobel JG, Klis FM, Priem J, Munnik T, van den Ende H (1990) The glucanase-soluble mannoproteins limit cell wall porosity in Saccharomyces cerevisiae. Yeast 6:491-499 De Virgilio C, Buerckert N, Bell W, Jeno P, Boller T, Wiemken A (1993) Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of tre-

7 Adaptation to nutrient availability in yeast 363 halose-6-phosphate and loss of trehalose-6-phosphate phosphatase activity. Eur J Biochem 212:315-323 De Virgilio C, Buerckert N, Boller T, Wiemken A (1991) A method to study the rapid phosphorylation-related modulation of neutral trehalase activity by temperature shifts in yeast. FEBS Lett 291:355-358 De Vries S, Marres CAM (1987) The mitochondrial respiratory chain of yeast. Structure and biosynthesis and the role in cellular metabolism. Biochim Biophys Acta 895:205239 de Winde JH. (1992) Global regulation of mitochondrial biogenesis in the yeast Saccharomyces cerevisiae. University of Amsterdam de Winde JH, Crauwels M, Hohmann S, Thevelein JM, Winderickx J (1996) Differential requirement of the yeast sugar kinases for sugar sensing in establishing the cataboliterepressed state. Eur J Biochem 241:633-643 de Winde JH, Grivell LA (1992) Global regulation of mitochondrial biogenesis in yeast:ABF1 and CPF1 play opposite roles in regulating expression of the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol cytochrome c oxidoreductase. Mol Cell Biol 12:2872-2883 de Winde JH, Grivell LA (1993) Global regulation of mitochondrial biogenesis in Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol 46:51-91 de Winde JH, Grivell LA (1995) Regulation of mitochondrial biogenesis in Saccharomyces cerevisiae:Intricate interplay between general and specific transcription factors in the promoter of the QCR8 gene. Eur J Biochem 233:200-208 de Winde JH, Van Leeuwen HC, Grivell LA (1993) The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease-hypersensitive region in the promoter of the QCR8 gene. Yeast 9:847-857 Deminoff SJ, Santangelo GM (2001) Rap1p requires Gcr1p and Gcr2p homodimers to activate ribosomal protein and glycolytic genes, respectively. Genetics 158:133-143 Dennis PB, Fumagalli S, Thomas G (2001a) Target of rapamycin (TOR):balancing the opposing forces of protein synthesis and degradation. Curr Opin Genet Dev 9:49-54 Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, Thomas G (2001b) Mammalian TOR:a homeostatic ATP sensor. Science 294:1102-1105 Destruelle M, Holzer H, Klionsky DJ (1995) Isolation and characterization of a novel yeast gene, ATH1, that is required for vacuolar acid trehalase activity. Yeast 11:1015-1025 DeVit MJ, Waddle JA, Johnston M (1997) Regulated nuclear translocation of the Mig1 glucose repressor. Mol Biol Cell 8:1603-1618 DeVries S, Marres CAM (1988) The mitochondrial respiratory chain of yeast structure and biosynthesis and the role in cellular metabolism. Biochim Biophys Acta 895:205-239 Dickinson JR (1994) Irreversible formation of pseudohyphae by haploid Saccharomyces cerevisiae. FEMS Microbiol Lett 119:99-104 Dickinson JR (1996) 'Fusel' alcohols induce hyphal-like extensions and pseudohyphal formation in yeasts. Microbiol 142:1391-1397 Dicomo CJ, Arndt KT (1996) Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Gene Dev 10:1904-1916 Diderich JA, Schepper M, van Hoek P, Luttik MA, van Dijken JP, Pronk JT, Klaassen P, Boelens HF, de Mattos MJ, van Dam K, Kruckeberg AL (1999) Glucose uptake kinetics and transcription of HXT genes in chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 274:15350-15359

364 Joris Winderickx et al. Didion T, Regenberg B, Jorgensen MU, KiellandBrandt MC, Andersen HA (1998) The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae. Mol Microbiol 27:643-650 Diffley JFX (1992) Global regulators of chromosome function in yeast. Anton van Leeuwenhoek International Journal for General and Molecular Microbiology 61:25-33 Doorenbosch T, Mager WH, Planta RJ (1992) Multifunctional DNA-binding proteins in yeast. Gene Exp 2:193-201 Dowell SJ, Tsang JSH, Mellor J (1992) The centromere and promoter factor-1 of yeast contains a dimerisation domain located carboxy-terminal to the bHLH domain. Anal Biochem 20:4229-4236 Draper MP, Salvadore C, Denis CL (1995) Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex. Mol Cell Biol 15:3487-3495 Drebot MA, Barnes CA, Singer RA, Johnston GC (1990) Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae. J Bacteriol 172:3584-3589 D'Souza CA, Heitman J (2001) Conserved cAMP signaling cascades regulate fungal development and virulence. FEMS Microbiol Rev 25:349-364 Durnez P, Pernambuco MB, Oris E, Arguelles JC, Mergelsberg H, Thevelein JM (1994) Activation of trehalase during growth induction by nitrogen sources in the yeast Saccharomyces cerevisiae depends on the free catalytic subunits of cAMP-dependent protein kinase, but not on functional ras proteins. Yeast 10:1049-1064 Ellenberger TE, Brandl CJ, Struhl K, Harrison SC (1992) The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha-Helices - crystal structure of the Protein-DNA complex. Cell 71:1223-1237 Entian KD, Barnett JA (1992) Regulation of sugar utilization by Saccharomyces cerevisiae. Trends Biochem Sci 17:506-510 Entian KD, Fröhlich KU (1984) Saccharomyces cerevisiae mutants provide evidence of hexokinase PII as a bifunctional enzyme with catalytic and regulatory domains for triggering carbon catabolite repression. J Bacteriol 158:29-35 Entian KD, Kopetzki E, Frœhlich KU, Mecke D (1984) Cloning of hexokinase isoenzyme PI from Saccharomyces cerevisiae. PI transformants confirm the unique role of hexokinase isoenzyme PII for glucose repression in yeasts. Mol Gen Genet 198:50-54 Entian KD, Zimmermann FK (1982) New genes involved in carbon catabolite repression and derepression in the yeast Saccharomyces cerevisiae. J Bacteriol 151:1123-1128 Eraso P, Gancedo JM (1984) Catabolite repression in yeasts is not associated with low levels of cAMP. Eur J Biochem 141:195-198 Erickson JR, Johnston M (1994) Suppressors reveal two classes of glucose repression genes in the yeast Saccharomyces cerevisiae. Genetics 136:1271-1278 Espinoza FH, Ogas J, Herskowitz I, Morgan DO (1994) Cell cycle control by a complex of the cyclin HCS26 (PCL1) and the kinase PHO85. Science 266:1388-1391 Estruch F, Carlson M (1990) Increased dosage of the MSN1 gene restores invertase expression in yeast mutants defective in the Snf1 protein kinase. Nucl Acids Res 18:69596964 Estruch F, Carlson M (1993) 2 homologous zinc finger genes identified by multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae. Mol Cell Biol 13:3872-3881 Fabrizio P, Pozza F, Pletcher SD, Gendron CM, Longo VD (2001) Regulation of longevity and stress resistance by Sch9 in yeast. Science 292:288-290

7 Adaptation to nutrient availability in yeast 365 Farkas I, Hardy TA, De Paoli-Roach AA, Roach PJ (1990) Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene. J Biol Chem 265:20879-20886 Farkas I, Hardy TA, Goebl MG, Roach PJ (1991) Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled. J Biol Chem 26:15601-15607 Federoff HJ, Eccleshall TR, Marmur J (1983) Carbon catabolite repression of maltase synthesis in Saccharomyces carlsbergensis. J Bacteriol 156:301-307 Feng Z, Wilson SE, Peng ZY, Schlender KK, Reiman EM, Trumbly RJ (1991) The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase. J Biol Chem 266:23796-23801 Fernandez R, Herrero P, Fernandez E, Fernandez T, Lopez-Boado YS, Moreno F (1988) Autophosphorylation of yeast hexokinase PII. J Gen Microbiol 134:2493-2498 Fisher F, Goding CR (1992) Single amino-acid substitutions alter helix-loop-helix protein specificity for bases flanking the core CANNTG motif. EMBO J 11:4103-4109 Flick JS, Johnston M (1991) GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats. Mol Cell Biol 11:5101-5112 Flikweert MT, Kuyper M, van Maris AJ, Kotter P, van Dijken JP, Pronk JT (1999) Steadystate and transient-state analysis of growth and metabolite production in Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity. Biotechnol Bioeng 66:42-50 Forsberg H, Gilstring F, Zargari A, Martinez P, Ljungdahl PO (2001a) The role of the yeast plasma membrane SPS nutrient sensor in the metabolic response to extracellular amino acids. Mol Microbiol 42:215-228 Forsberg H, Hammer M, Andréasson C, Molinér A, Ljungdahl PO (2001b) Suppressors of ssy1 and ptr3 null mutants define novel Amino Acid Sensor Independent (ASI) genes in Saccharomyces cerevisiae. Genetics 158:973-988 Forsberg H, Ljundahl PO (2001) Sensors of extracellular nutrients in Saccharomyces cerevisiae. Curr Genet 40:91-109 Forsberg H, Ljungdahl PO (2001) Genetic and biochemical analysis of the yeast plasma membrane Ssy1pPtr3p-Ssy5p sensor of extracellular amino acids. Mol Cell Biol 21:814-826 Forsburg SL, Guarente L (1989a) Communication between mitochondria and the nucleus in regulation of cytochrome genes in the yeast Saccharomyces cerevisiae. Ann Rev Cell Biol 5:153-180 Forsburg SL, Guarente L (1989b) Identification and characterization of HAP4:a third component of the CCAAT-bound HAP2/HAP3 heteromer. Genes Dev 3:1166-1178 Fraenkel D. (1982) Carbohydrate Metabolism. In Strathern, J.N., Jones, E.W. and Broach, J.R. (eds.), The Molecular Biology of the Yeast Saccharomyces:Metabolism and Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 1-37 Francisco L, Wang W, Chan CSM (1994) Type 1 protein phosphatase acts in opposition to Ipl1 protein kinase in regulating yeast chromosome segregation. Mol Cell Biol 14:4731-4740 François J, Neves MJ, Hers HG (1991) The control of trehalose biosynthesis in Saccharomyces cerevisiae - evidence for a catabolite inactivation and repression of trehalose-6phosphate synthase and trehalose-6-phosphate phosphatase. Yeast 7:575-587

366 Joris Winderickx et al. François J, Parrou JL (2001a) Reserve carbohydrate metabolism in the yeast Saccharomyces cerevisiae. Fems microbiology reviews 25:125-145 François J, Parrou JL (2001b) Reserve carbohydrate metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev. 25:125-145 François J, Villanueva ME, Hers HG (1988) The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers. Eur J Biochem 174:551-559 François JM, Eraso P, Gancedo C (1987) Changes in the concentration of cAMP, fructose2,6-bisphosphate and related metabolites and enzymes in Saccharomyces cerevisiae during growth on glucose. Eur J Biochem 164:369-373 François JM, Thompson-Jaeger S, Skroch JZ, U., Spevak Wea (1992) GAC1 may encode a regulatory subunit for protein phosphatase type 1 in Saccharomyces cerevisiae. EMBO J 11:87-96 Fujita A, Tonouchi A, Hiroko T, Ìnose F, Nagashima T, Satoh R, Tanaka S (1999) Hsl7p, a negative regulator of ste20p protein kinase in the Saccharomyces cerevisiae filamentous growth-signaling pathway. Proc Natl Acad Sci USA 96:8522-8527 Gagiano M, van Dyk D, Bauer FF, Lambrechts MG, Pretorius IS (1999a) Divergent regulation of the evolutionary closely related promotors of the Saccharomyces cerevisiae STA2 and MUC1 genes. J Bacteriol 181:6497-6508 Gagiano M, van Dyk D, Bauer FF, Lambrechts MG, Pretorius IS (1999b) Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2 regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Microbiol 31:103-116 Gagiano M, vanDyk D, Bauer FF, Lambrechts MG, Pretorius IS (1999c) Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Microbiol 31:103-116 Gancedo C (1971) Inactivation of fructose-1,6-bisphosphatase by glucose in yeast. J Bacteriol 107:401-405 Gancedo JM (1992) Carbon catabolite repression in yeast. Eur J Biochem 206:297-313 Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol Biol Rev 62:334361 Gancedo JM (2001) Control of pseudohyphae formation in Saccharomyces cerevisiae. FEMS Microbiol Rev 25:107-123 Gancedo JM, Gancedo C (1979) Inactivation of gluconeogenic enzymes in glycolytic mutants of Saccharomyces cerevisiae. Eur J Biochem 101:455-460 Garret S, Broach J (1989) Loss of Ras activity in Saccharomyces cerevisiae is suppressed by disruption of a new kinase gene, YAK1, whose product may act downstream of the cAMP-dependent protein kinase. Genes Dev 3:1336-1348 Garret S, Menold MM, Broach JR (1991) The Saccharomycse cerevisiae YAK1 gene encodes a protein kinase that is induced by arrest early in the cell cycle. Mol Cell Biol 11:4045-4052 Gavrias V, Andrianopoulos A, Gimeno CJ, Timberlake WE (1996) Saccharomyces cervisiae TEC1 is required for pseudohyphal growth. Mol Microbiol 19:1255-1263 Geyskens I, Shanta KHMC, Donaton MCV, Bergsma JCT, Thevelein JM, Wera S. (2000) Expression of mammalian PKB partially complements deletion of the yeast protein kinase Sch9. IOS Press

7 Adaptation to nutrient availability in yeast 367 Gimeno CJ, Ljungdahl PO, Styles CA, Fink GR (1992) Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth:regulation by starvation and RAS. Cell 68:1077-1090 Goldberg D, Segal M, Levitzki A (1994) Cdc25 is not the signal receiver for glucose induced cAMP response in S. cerevisiae. FEBS Lett 356:249-254 Gonçalves P, Planta RJ (1998) Starting uo yeast glycolysis. Trends Microbiol 6:314-319 Gonzalez MI, Stucka R, Blazquez MA, Feldmann H, Gancedo C (1992) Molecular cloning of CIF1, a yeast gene necessary for growth on glucose. Yeast 8:183-192 Görner W, Durchschlag E, Martinez-Pastor MT, Estruch F, Ammerer G, Hamilton B, Ruis H, Schüller C (1998) Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev 10:1516-1531 Gorner W, Durchschlag E, Martinez-Pastor MT, Estruch F, Ammerer G, Hamilton B, Ruis S, Schuller C (1998) Nuclear localization of the C2H2 zinc finger protein is regulated by stress and protein kinase A activity. Genes Dev 12:586-597 Griffioen G, Anghileri P, Imre E, Baroni MD, Ruis H (2000) Nutritional control of nucleocytoplasmic localization of cAMP-dependent protein kinase catalytic and regulatory subunits in Saccharomyces cerevisiae. J Biol Chem 275:1449-1456 Griffioen G, Branduardi P, Ballarini A, Anghieri P, Norbeck J, Baroni MD, Ruis H (2001) Nucleocytoplasmic distribution of budding yeast protein kinase A regulatory subunit BCY1 requires Zds1 and is regulated by Yak1-dependent phosphorylation of its targetting domain. Mol Cell Biol 21:511-523 Griffioen G, Laan RJ, Mager WH, Planta RJ (1996) Ribosomal protein gene transcription in Saccharomyces cerevisiae shows a biphasic response to nutritional changes. Microbiol 142:2279-2287 Griffioen G, Mager WH, Planta RJ (1994) Nutritional upshift response of ribosomal protein gene transcription in Saccharomyces cerevisiae. FEMS Microbiol Lett 123:137-144 Grivell LA (1995) Nucleo-mitochondrial interactions in mitochondrial gene expression. Crit Rev Biochem Molec Biol 30:121-164 Grundmann O, Mösch H-U, Braus GH (2001) Repression of GCN4 mRNA translation by nitrogen starvation in Saccharomyces cerevisiae. J Biol Chem 276:25661-25671 Hansen J, Johannesen PF (2000) Cysteine is essential for transcriptional regulation of the sulfur assimilation genes in Saccharomyces cerevisiae. Mol Gen Genet 263:535-542 Hardie DG, Carling D (1997) The AMP-activated protein kinase--fuel gauge of the mammalian cell? Eur J Biochem 246:259-273 Hardwick JS, Kuruvilla FG, Tong JK, Shamij AF, Schreiber SL (1999) Rapamycinmodulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by Tor proteins. Proc Natl Acad Sci USA 96:14866-14870 Hardy TA, Huang DQ, Roach PJ (1994) Interactions between cAMP-dependent and SNF1 protein kinases in the control of glycogen accumulation in Saccharomyces cerevisiae. J Biol Chem 269:27907-27913 Hardy TA, Roach PJ (1993) Control of yeast glycogen synthase-2 by COOH-terminal phosphorylation. J Biol Chem 268:23799-23805 Hartley AD, Ward MP, Garrett S (1994) The yak1 protein kinase of Saccharomyces cerevisiae moderates thermotolerance and inhibits growth by an Sch9 protein kinaseindependent mechanism. Genetics 136:465-474 Hata H, Mitsui H, Lui H, Bai Y, Denis CL, Shimizu Y, Sakai A (1998) Dhh1, a putative RNA helicase, associates with the general transcription factors Pop2 and Ccr4 from Saccharomyces cerevisiae. Genetics 148:571-579

368 Joris Winderickx et al. Healy AM, Zolnierowiez S, Stapleton AE, Goebl M, DePaoli-Roach AA, Pringle JR (1991) CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis:identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase. Mol Cell Biol 11:5767-5780 Hedges D, Proft M, Entian KD (1995) CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae. Mol Cell Biol 15:1915-1922 Heidrich K, Otto A, Behlke J, Rush J, Wenzel K-W, Kriegel T (1997) Autophosphorylation-inactivation site of hexokinase 2 in Saccharomyces cerevisiae. Biochemistry 36:1960-1964 Heitman J, Movva NR, Hall MN (1991a) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253:905-909 Heitman J, Movva NR, Hiestand PC, Hall MN (1991b) FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 88:1948-1952 Helliwell SB, Howald I, Barbet N, Hall MN (1998a) TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae. Genetics 148:99-112 Helliwell SB, Schmidt A, Ohya Y, Hall MN (1998b) The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin skeleton. Curr Biol 8:1211-1214 Helliwell SB, Wagner P, Kunz J, Deuterreinhard M, Henriquez R, Hall MN (1994) Tor1 and tor2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol Biol Cell 5:105-118 Herrero P, Fernandez R, Moreno F (1989) The hexokinase isoenzyme PII of Saccharomyces cerevisiae is a protein kinase. J Gen Microbiol 135:1209-1216 Herrero P, Martinez Campa C, Moreno F (1998) The hexokinase 2 protein participates in regulatory DNA-protein complexes necessary for glucose repression of the SUC2 gene in Saccharomyces cerevisiae. FEBS Lett 434:71-76 Hiesinger M, Roth S, Meissner E, Schuller HJ (2001) Contribution of Cat8 and Sip4 to the transcriptional activation of yeast gluconeogenic genes by carbon-source responsive elements. Curr Genet 39:68-76 Hinnebusch AG (1988) Mechanisms of gene regulation in the general control of aminoacid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev 52:248-273 Hirimburegama K, Durnez P, Keleman J, Oris E, Vergauwen R, Mergelsberg H, Thevelein JM (1992) Nutrient-induced activation of trehalase in nutrient-starved cells of the yeast Saccharomyces cerevisiae:cAMP is not involved as second messenger. J Gen Microbiol 138:2035-2043 Hirst K, Fisher F, Mcandrew PC, Goding CR (1994) The transcription factor, the Cdk, its cyclin and their regulator:Directing the transcriptional response to a nutritional signal. EMBO J 13:5410-5420 Hoffman CS, Winston F (1991) Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway. Genes Dev 5:561-571 Hoffman M, Chiang HL (1996) Isolation of degradation-deficient mutants defective in the targeting of fructose-1,6-bisphosphatase into the vacuole for degradation in Saccharomyces cerevisiae. Genetics 143:1555-1566 Hofman-Bang J (1999) Nitrogen catabolite repression in Saccharomyces cerevisiae. Mol Biotechnol 12:35-73 Hohmann S, Van Dijck P, Luyten K, Thevelein JM (1994) The byp1-3 allele of the Saccharomyces cerevisiae GGS1/TPS1 gene and its multi copy suppressor tRNAGLN

7 Adaptation to nutrient availability in yeast 369 (CAG):Ggs1/Tps1 protein levels restraining growth on fermentable sugars and trehalose accumulation. Curr Genet 26:295-301 Hohmann S, Winderickx J, de Winde JH, Valckx D, Cobbaert P, Luyten K, de Meirsman C, Ramos J, Thevelein JM (1999) Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2. Microbiol 145:703-714 Holzer H (1976) Catabolite inactivation in yeast. Trend Biochem Sci 1:178 Holzer H. (1984) Mechanism and function of reversible phosphorylation of fructose-1,6bisphosphatase in yeast. In Cohen, P. (ed.) Molecular aspects of cellular regulation. Elsevier, Amsterdam, Vol. 3, pp. 143-154 Hope IA, Struhl K (1986) Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell 46:885-894 Hu YM, Cooper TG, Kohlhaw GB (1995a) The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation. Mol Cell Biol 15:5257 Hu Z, Nehlin JO, Ronne H, Michels CA (1995b) MIG1-dependent and MIG1-independent glucose regulation of MAL gene expression in Saccharomyces cerevisiae. Curr Genet 28:258-266 Huang D, Farkas I, Roach PJ (1996a) Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae. Mol Cell Biol 16:4357-4365 Huang D, Moffat J, Wilson WA, Moore L, Cheng C, Roach PJ, Andrews B (1998) Cyclin partners determine Pho85 protein kinase substrate spezcificity in vitro and in vivo:control of glycogen biosynthesis by Pcl8 and Pcl10. Mol Cell Biol 18:3289-3299 Huang DQ, Chun KT, Goebl MG, Roach PJ (1996b) Genetic interactions between REG1/HEX2 and GLC7, the gene encoding the protein phosphatase type 1 catalytic subunit in Saccharomyces cerevisiae. Genetics 143:119-127 Hubbard EJA, Yang X, Carlson M (1992) Relationship of the cAMP-dependent protein kinase pathway to the SNF1 protein kinase and invertase expression in Saccharomyces cerevisiae. Genetics 130:71-80 Hudak Slekar K, Kosman DJ, Cizewski Culotta V (1996) The yeast copper/zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection. J Biol Chem 271:28831-28836 Hunter T (1995) Protein kinases and phosphatases:The yin and yang of protein phosphorylation and signaling. Cell 80:225-236 Hwang PK, Tugendreich S, Fletterick RJ (1989) Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae. Mol Cell Biol 9:16591666 Iraqui I, Vissers S, Bernard F, DeCraene JO, Boles E, Urrestarazu A, Andre B (1999) Amino acid signaling in Saccharomyces cerevisiae:a permease-like sensor of external amino acids and F-box protein Grr1p are required for transcriptional induction of the AGP1 gene, which encodes a broad-specificity amino acid permease. Mol Cell Biol 19:989-1001 Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y (2001) A comprehensive twohybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci USA 98:4569-4574 Jazwinski SM (1996) Longevity, genes, and aging. Science 273:54-59 Jazwinski SM (1999) Molecular mechanisms of yeast longevity. Trends Microbiol 7:247252

370 Joris Winderickx et al. Jeffery DA, Springer M, King DS, O'Shea EK (2001) Multi-site phosphorylation of Pho4 by the cyclin-CDK Pho80-Pho85 is semi-processive with site preference. J Mol Biol 306:997-1010 Jiang H, Medintz I, Zhang B, Michels CA (2000) Metabolic signals trigger glucose-induced inactivation of maltose permease in Saccharomyces. J Bacteriol 182:647-654 Jiang R, Carlson M (1996) Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes Dev 10:3105-3115 Jiang R, Carlson M (1997) The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex. Mol Cell Biol 17:2099-2106 Jiang Y, Broach JR (1999) Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J 18:2782-2792 Jiang Y, Davis C, Broach JR (1998) Efficient transition to growth on fermentable carbon sources in Saccharomyces cerevisiae requires signaling through the Ras pathway. EMBO J 17:6942-6951 Jin M, Fujita M, Culley BM, Apolinario E, Yamamoto M, Maundrell K, Hoffman CS (1995) sck1, a high copy number suppressor of defects in the cAMP-dependent protein kinase pathway in fission yeast, encodes a protein homologous to the Saccharomyces cerevisiae SCH9 kinase. Genetics 140:457-467 Johnston M (1999) Feasting, fasting and fermenting - glucose sensing in yeast and other cells. Trends Genet 15:29-33 Johnston M, Carlson M. (1992a) Regulation of carbon and phosphate utilization. In Jones, E.W., Pringle, J.R. and Broach, J.R. (eds.), The molecular and cellular biology of the yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Vol. 2, pp. 193-281 Johnston M, Carlson M. (1992b) Regulation of carbon and phosphate utilization. In Jones, E.W., Pringle, J.R. and Broach, J.R. (eds.), The molecular and cellular biology of the yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Vol. 2, pp. 193-281 Johnston M, Flick JS, Pexton T (1994) Multiple mechanisms provide rapid and stringent glucose repression of GAL gene expression in Saccharomyces cerevisiae. Mol Cell Biol 14:3834-3841 Jones EW, Fink GR. (1982) Regulation of amino acid and nucleotide biosynthesis in yeast. In Strathern, J.N., Jones, E.W. and Broach, J.R. (eds.), The molecular biology of the yeast Saccharomyces; metabolism and gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 181-299 Jones ME (1980) Pyrimidine nucleotide biosynthesis in animals. Genes, enzymes and regulation of UMP biosynthesis. Ann Rev Biochem 49:253-279 Jones S, Vignais M-L, Broach JR (1991) The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to RAS. Mol Cell Biol 11:2641-2646 Jorgenson MU, Gjermansen C, Andersen HA, Kielland-Brandt MC (1997) STP1, a gene involved in pre-tRNA processing in yeast, is important for amino-acid uptake and transcription of the permease gene Bap2. Curr Genet 31:241-247 Kaffman A, Herskowitz I, Tjian R, O'Shea EK (1994) Phosphorylation of the transcription factor PHO4 by a cyclin-cdk complex, PHO80-PHO85. Science 263:1153-1156 Kaffman A, Rank NM, O'Neill EM, Huang LS, O'Shea EK (1998a) The receptor Msn5 exports the phosphorylated transcription factor Pho4 out of the nucleus. Nature 396:482486

7 Adaptation to nutrient availability in yeast 371 Kaffman A, Rank NM, O'Shea EK (1998b) Phosphorylation regulates association of the transcription factor Pho4 with its import receptor Pse1/Kap121. Genes Dev 12:26732683 Kaiser P, Flick K, Wittenberg C, Reed SI (2000) Regulation of transcription by ubiquitination without proteolysis:Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4. Cell 102:303-314 Kataoka T, Broek D, Wigler M (1985) DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell 43:493-505 Keith CT, Schreiber SL (1995) PIK-related kinases:DNA repair, recombination, and cell cycle checkpoints. Science 270:50-51 Kelly BL, Wolfe KG, Roberts JM (1998) Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein. Proc Natl Acad Sci USA 95:2535-2540 Kent NA, Tsang JSH, Crowther DJ, Mellor J (1994) Chromatin structure modulation in Saccharomyces cerevisiae by Centromere and Promoter Factor 1. Mol Cell Biol 14:5229-5241 Kishi T, Seno T, Yamao F (1998) Grr1 functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skp1. Mol Gen Genet 257:143-148 Klasson H, Fink GR, Ljungdahl PO (1999) Ssy1p and Ptr3p are plasma membrane components of a yeast system that senses extracellular amino acids. Mol Cell Biol 19:54055416 Klein CJL, Olsson L, Ronnow B, Mikkelsen JD, Nielsen J (1996) Alleviation of glucose repression of maltose metabolism by MIG1 disruption in Saccharomyces cerevisiae. Appl Environ Microbiol 62:4441-4449 Kobayashi O, Suda H, Ohtani T, Sone H (1996) Molecular cloning of the dominant flocculation gene FLO8 fram Saccharomyces cerevisiae. Mol Gen Genet 251:707-715 Komeili A, O'Shea EK (1999) Roles of phosphorylation sites in regulating activity of the transcription factor Pho4. Science 284:977-980 Komeili A, Wedaman KP, O'Shea EK, Powers T (2000) Mechanism of metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors. J Cell Biol 151:863-878 Kopp M, Muller H, Holzer H (1993) Molecular analysis of the neutral trehalase gene from Saccharomyces cerevisiae. J Biol Chem 268:4766-4774 Kopp M, Nwaka S, Holzer H (1994) Corrected sequence of the yeast neutral trehalaseencoding gene (NTH1):biological implications. Gene 150:403-404 Koser PL, Eng WK, Bossard MJ, McLaughlin MM, Cafferkey R, Sathe GM, Faucette L, Levy MA, Johnson RK, Bergsma DJ (1993) The tyrosine89 residue of yeast FKBP12 is required for rapamycin binding. Gene 129:159-165 Kraakman L, Lemaire K, Ma P, Teunissen AWRH, Donaton MCV, Van Dijck P, Winderickx J, de Winde JH, Thevelein JM (1999a) A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose. Mol Microbiol 32:1002-1012 Kraakman LS, Winderickx J, Thevelein JM, de Winde JH (1999b) Structure-function analysis of yeast hexokinase:structural requirements for triggering cAMP signalling and catabolite repression. Biochem J 343:159-168 Kriegel TM, Rush J, Vojtek AB, Clifton D, Fraenkel DG (1994) In vivo posphorylation site of hexokinase 2 in Saccharomyces cerevisiae. Biochemistry 33:148-152 Kron SJ (1997) Filamentous growth in budding yeast. Trends Microbiol. 5:450-451

372 Joris Winderickx et al. Kruckeberg AL (1996) The hexose transporter family of Saccharomyces cerevisiae. Arch Microbiol 166:283-292 Kubler E, Mosch HU, Rupp S, Lisanti MP (1997) Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism. J Biol Chem 272:20321-20323 Kubota H, Ota K, Sakaki Y, Ito T (2001) Budding yeast GCN1 binds the GI domain to activate the eIF2æ kinase GCN2. J Biol Chem 276:17591-17596 Kubota H, Sakaki Y, Ito T (2000) GI domain-mediated association of the eukaryotic initation factor 2æ kinase GCN2 with its activator GCN1 is required for general amino acid control in budding yeast. J Biol Chem 275:20243-20246 Kulkarni AA, Abul-Hamd A, Rai R, El Berry H, Cooper TG (2001) Gln3p nuclear localization and interaction with Ure2p in Saccharomyces cerevisiae. J Biol Chem 276:3213632144 Kunz J, Henriquez R, Schneider U, Deuter-Reinhard M, Movva NR, Hall MN (1993) Target of rapamycin in yeast, TOR2 is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell 73:585-596 Kuras L, cherest H, Surdin-Kerjan Y, Thomas D (1996) A heteromeric complex containing the centromere binding factor 1 and two basic leucine zipper factors, Met4 and Met28, mediates the transcripton activation of yeast sulfur metabolism. EMBO J 15:25192529 Kuruvilla FG, Shamji AF, Schreiber SL (2001) Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors. Proc Natl Acad Sci USA 98:7283-7288 Lambrechts MG, Bauer FF, Marmur J, Pretorius IS (1996a) Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast. Proc Natl Acad Sci USA 93:8419-8424 Lambrechts MG, Bauer FF, Marmur J, Pretorius IS (1996b) Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast. Proc Natl Acad Sci USA USA 93:8419-8424 Leberer E, Wu C, Leeuw T, Fourest-Lieuvin A, Segall JW, Thomas DY (1997) Functional characterisation of the Cdc42p binding domain of yeast Ste20p protein kinase. EMBO J 16:83-97 Lee J, Colwill K, Aneliunas V, Tennyson C, Moore L, Ho Y, Andrews B (1998) Interaction of yeast Rvs167 and Pho85 cyclin-dependent kinase complexes may link the cell cycle to the actin cytoskeleton. Curr Biol 8:1310-1321 Lemire JM, Willcocks T, Halvorson HO, Bostian KA (1985) Regulation of repressible acid phosphatase gene transcription in Saccharomyces cerevisiae. Mol Cell Biol 5:21312141 Lenburg ME, O'Shea EK (1996) Signaling phosphate starvation. TIBS 21:383-387 Lesage P, Yang XL, Carlson M (1994) Analysis of the sip3 protein identified in a TwoHybrid screen for interaction with the snf1 protein kinase. Nucleic Acids Res 22:597603 Lesage P, Yang XL, Carlson M (1996) Yeast Snf1 protein kinase interacts with Sip4, a C-6 zinc cluster transcriptional activator:a new role for Snf1 in the glucose reponse. Mol Cell Biol 16:1921-1928 Li FN, Johnston M (1997) Grr1 of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skp1:coupling glucose sensing to gene expression and the cell cycle. EMBO J 16:5629-5638

7 Adaptation to nutrient availability in yeast 373 Liang H, Gaber RF (1996) A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6. Mol Biol Cell 7:1953-1966 Lillie SH, Pringle JR (1980) Reserve carbohydrate metabolism in Saccharomyces cerevisiae:responses to nutrient limitation. J Bacteriol 143:1384-1394 Liu C, Yang Z, Yang J, Xia Z, Ao S (2000) Regulation of the yeast transcriptional factor PHO2 activity by phosphorylation. J Biol Chem 275:31972-31978 Liu H, Styles CA, Fink GR (1996) Saccharomyces cerevisiae S288C has a mutation in FLO8, a gene requiredfor filamentous growth. Genetics 145:671-674 Liu HP, Styles CA, Fink GR (1993) Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science 262:1741-1744 Liu HY, Badarinarayana V, Audino DC, Rappsilber J, Mann M, Denis CL (1998) The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively. EMBO J 17:1096-1106 Lo W-S, Dranginis AM (1998) The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae. Mol Biol Cell 9:161-171 Lodi T, Donnini C, Ferrero I (1991) Catabolite repression by galactose in overexpressed GAL4 strains of Saccharomyces cerevisiae. J Gen Microbiol 137:1039-1044 Lohr D, Lopez J (1995) GAL4/GAL80-dependent nucleosome disruption/deposition on the upstream regions of the yeast GAL1-10 and GAL80 genes. J Biol Chem 270:2767127678 Londesborough J, Vuorio OE (1993) Purification of trehalose synthase from baker's yeast its temperature-dependent activation by fructose-6-phosphate and inhibition by phosphate. Eur J Biochem 216:841-848 Lopez MC, Baker HV (2000) Understanding the growth phenotype of the yeast gcr1 mutant in terms of global genomic expression patterns. J Bacteriol 182:4920-4978 Lopez-Boado YS, Herrero P, Fernandez T, Fernandez R, Moreno F (1988) Glucosestimulated phosphorylation of yeast isocitrate lyase in vivo. J Gen Microbiol 134:2499-2505 Lorenz MC, Heitman J (1997) Yeast pseudohyphal growth is regulated by GPA2, a G protein α homolog. EMBO J 16:7008-7018 Lorenz MC, Heitman J (1998a) The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae. EMBO J 17:1236-1247 Lorenz MC, Heitman J (1998b) Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multicopy suppressor analysis in ammonium permease mutant strains. Genetics 150:1443-1457 Lorenz MC, Pan X, Harashima T, Cardenas ME, Xue Y, Hirsch JP, Heitman J (2000) The G protein-coupled receptor Gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Genetics 154:609-622 Lucero P, Herweijer M, Lagunas R (1993) Catabolite inactivation of the yeast maltose transporter is due to proteolysis. FEBS Lett 333:165-168 Ludin K, Jiang R, Carlson M (1998) Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 95:6245-6250 Lundin M, Nehlin JO, Ronne H (1994) Importance of a flanking at-Rich region in target site recognition by the GC Box-Binding zinc finger protein mig1. Mol Cell Biol 14:1979-1985

374 Joris Winderickx et al. Lutfiyya L, Iyer VR, DeRisi J, DeVit MJ, Brown PO, Johnston M (1998) Characterization of three related glucose repressors and genes they regulate in Saccharomyces cerevisiae. Genetics 150:1377-1391 Lutfiyya LL, Johnston M (1996) Two zinc-finger-containing repressors are responsible for glucose repression of SUC2 expression. Mol Cell Biol 16:4790-4797 Ma H, Bloom LM, Walsh CT, Botstein D (1989) The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae. Mol Cell Biol 9:5643-5649 Ma P, Wera S, Van Dijck P, Thevelein JM (1999) The PDE1 encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signalling. Mol Biol Cell 10:91-104 Madhani HD, Fink GR (1998a) The control of filamentous differentiation and virulence in fungi. Trends Cell Biol 8:348-353 Madhani HD, Fink GR (1998b) The riddle of MAP kinase signaling specificty. Trends Genet 14:151-155 Madhani HD, Galitski T, Lander ES, Fink GR (1999) Effectors of a deveopmental mitogenactivated protein kinase cascade revealed by expression signatures of signaling mutants. Proc Nat Acad Sci USA 96:12530-12535 Madhani H-D, Fink GR (1997) Combinatorial control required for the specificity of yeast MAPK signaling. Science 275:1314-1317 Magasanik B. (1992) Regulation of nitrogen utilization. In Jones, E.W., Pringle, J.R. and Broach, J. (eds.), The molecular and cellular biology of the yeast Saccharomyces:Gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 283-317 Magbanua JPV, Ogawa N, Harashima S, Oshima Y (1997) The transcriptional activators of the PHO regulon, Pho4p and Pho2p, interact directly with each other and with components of the basal transcription machinery in Saccharomyces cerevisiae. J. Biochem. (Tokyo) 121:1182-1189 Mager WH, de Kruijff AJJ (1995) Stress-induced transcriptional activation. Microbiol Rev 59:506 Mager WH, Ferreira PM (1993) Stress response of yeast. Biochem J 290:1-13 Mager WH, Planta RJ (1991) Coordinate expression of ribosomal protein genes in yeast as a function of cellular growth rate. Mol Cell Biochem 104:181-187 Maillet L, Tu C, Hong YK, Shuster EO, Collart MA (2000) The essential function of Not1 lies within the Ccr4-Not complex. J Mol Biol 303:131-143 Markwardt DD, Garrett JM, Eberhardy S, Heideman W (1995) Activation of the Ras/cyclic AMP pathway in the yeast Saccharomyces cerevisiae does not prevent G(1) arrest in response to nitrogen starvation. J Bacteriol 177:6761-6765 Martinez P, Zvyagilskaya R, Allard P, Persson BL (1998) Physiological regulation of the derepressible phosphate transporter in Saccharomyces cerevisiae. J Bacteriol 180:2253-2256 Matsumoto K, Uno I, Ishikawa T (1984) Identification of the structural gene and nonsense alleles for adenylate cyclase in Saccharomyces cerevisiae. J Bacteriol 157:277-282 Matsumoto K, Uno I, Ishikawa T (1985) Genetic analysis of the role of cAMP in yeast. Yeast 1:15-24 Mayordomo I, Sanz P (2001) Hexokinase PII:structural analysis and glucose signalling in the yeast Saccharomyces cerevisiae. Yeast 18:923-930

7 Adaptation to nutrient availability in yeast 375 Mbonyi K, Beullens M, Detremerie K, Geerts L, Thevelein JM (1988) Requirement of one functional RAS gene and inability of an oncogenic ras-variant to mediate the glucoseinduced cAMP signal in the yeast Saccharomyces cerevisiae. Mol Cell Biol 8:30513057 Mbonyi K, Van Aelst L, Argüelles JC, Jans AWH, Thevelein JM (1990) Glucose-induced hyperaccumulation of cyclic AMP and defective glucose repression in yeast strains with reduced activity of cyclic AMP-dependent protein kinase. Mol Cell Biol 10:45184523 Mc Cartney RR, Schmidt MC (2001) Regulation of Snf1 kinase:Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit. J Biol Chem 276:36460-36466 Mc Nabb DS, Xing YY, Guarente L (1995) Cloning of yeast HAP5:A novel subunit of a heterotrimeric complex required for CCAAT binding. Gene Develop 9:47-58 McBride HJ, Brazas RM, Yu Y, Nasmyth K, Stillman DJ (1997) Long-range interactions at the HO promoter. Mol Cell Biol 17:2669-2678 McKenzie EA, Kent NA, Dowell SJ, Moreno F, Bird LE, Mellor J (1993) The centromere and promoter factor-1, CPF1, of Saccharomyces cerevisiae modulates gene activity through a family of factors including SPT21, RPD1 (SIN3), RPD3 and CCR4. Mol Gen Genet 240:374-386 Measday V, Moore L, Ogas J, Tyers M, Andrews B (1994) The PCL2 (ORFD)-PHO85 cyclin-dependent kinase complex:a cell cycle regulator in yeast. Science 266:1391-1395 Measday V, Moore L, Retnakaran R, Lee J, Donoviel M, Neiman AM, Andrews B (1997) A family of cyclin-like proteins that interact with the Pho85 cyclin-dependent kinase. Mol Cell Biol 17:1212-1223 Mellor J, Jiang W, Funk M, Ratjen J, Barnes CA, Hinz T, Hegemann JH, Philippsen P (1990) CPF1, a yeast protein which functions in centromeres and promoters. EMBO J 12:4017-4026 Mellor J, Rathjen J, Jiang W, Dowell SJ (1991) DNA binding of CPF1 is required for optimal centromere function but not for maintaining methionine prototrophy in yeast. Nucleic Acids Res 19:2961-2969 Mercado JJ, Smith R, Sagliocco FA, Brown AJP, Gancedo JM (1994) The levels of yeast gluconeogenic mRNAs respond to environmental factors. Eur J Biochem 224:473-481 Moehle, Jones (1990) Consequences of growth media, gene copy number and regulatory mutations on the expression of the PRB1 gene of Saccharomyces cerevisiae. Genetics 124:39-55 Moffat J, Huang D, Andrews B (2000) Functions of Pho85 cyclin-dependent kinases in budding yeast. Prog Cell Cycle Res 4:97-106 Morgan DO (1997) Cyclin-dependent kinases:engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 13:261-291 Moriya H, Shimizu Y, Omori A, Iwahita S, Katoh M, Sakai A (2001) Yak1p, a Dyrk family kinase, translocates to the nucleus and phosphorylates yeast Pop2 in response to a glucose signal. Genes Dev 15:1217-1228 Mosch HU, Kubler E, Krappmann S, Fink GR, Braus GH (1999) Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP pathways during invasive growth of Saccharomyces cerevisiae. Mol Biol Cell 10:1325-1335 Mosch HU, Roberts RL, Fink GR (1996) Ras2 signals via the Cdc42/Ste20/mitogenactivated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 93:5352-5356

376 Joris Winderickx et al. Moskvina E, Schuller C, Maurer CTC, Mager WH, Ruis H (1998) A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements. Yeast 14:1041-1050 Mountain HA, Byström HS, Tang Larsen J, Korch C (1991) Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae. Yeast 7:781-803 Muller J, Boller T, Wiemken A (1995a) Trehalose and trehalase in plants:recent developments. Plant Sci 112:1-9 Muller S, Boles E, May M, Zimmermann FK (1995b) Different internal metabolites trigger the induction of glycolytic gene expression in Saccharomyces cerevisiae. J Bacteriol 177:4517-4519 Munder T, Küntzel H (1989) Glucose-induced cAMP signaling in Saccharomyces cerevisiae is mediated by the CDC25 protein. FEBS Lett 242:341-345 Nakafuku M, Obara T, Kaibuchi K, Miyajima I, Miyajima A, Itoh H, Nakamura S, Arai K, Matsumoto K, Kaziro Y (1988) Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein :studies on its structure and possible functions. Proc Natl Acad Sci USA 85:1374-1378 Natarajan K, Meyer MR, Jackson BM, Slade D, Roberts C, Hinnebusch AG, Marton MJ (2001) Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast. Mol Cell Biol 21:4347-4368 Needleman R (1991) Control of maltase synthesis. Mol Microbiol 5:2079-2084 Nehlin JO, Carlberg M, Ronne H (1991) Control of yeast GAL genes by MIG1 repressor:a transcriptional cascade in the glucose response. EMBO J 10:3373-3377 Nehlin JO, Carlberg M, Ronne H (1992) Yeast SKO1 gene encodes a bZIP protein that binds to the CRE motif and acts as a repressor of transcription. Nucleic Acids Res 20:5271-5278 Nehlin JO, Ronne H (1990) Yeast MIG1 repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. EMBO J 9:2891-2898 Neuman-Silberberg FS, Bhattacharya S, Broach JR (1995) Nutrient availability and the RAS/cyclic AMP pathway both induce expression of ribosomal protein genes in Saccharomyces cerevisiae but by different mechanisms. Mol Cell Biol 15:3187-3196 Neves MJ, Hohmann S, Bell W, Dumortier F, Luyten K, Ramos J, Cobbaert P, Dekoning W, Kaneva Z, Thevelein JM (1995) Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis. Curr Genet 27:110-122 Nikawa J, Cameron S, Toda T, Ferguson KW, Wigler M (1987) Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae. Genes Dev 1:931-937 Nishizawa M, Kawasumi M, Fujino M, Toh-e A (1998) Phosphorylation of sic1, a cyclindependent kinase (Cdk) inhibitor, by Cdk including Pho85 kinase is required for its prompt degradation. Mol Cell Biol 9:2393-2405 Nocero M, Isshiki T, Yamamoto M, Hoffman CS (1994) Glucose repression of fbp1 transcription in Schizosaccharomyces pombe is partially regulated by adenylate cyclase activation by a G protein alpha subunit encoded by gpa2 (git8). Genetics 138:39-45 Nogae I, Johnston M (1990) Isolation and characterization of the ZWF1 gene of Saccharomyces cerevisiae, encoding glucose-6-phosphate dehydrogenase. Gene 96:161-169 Nurse P (1992) Eukaryotic Cell-Cycle Control. Biochem Soc Trans 20:239-242 Nwaka S, Holzer H (1998) Molecular biology of trehalose and the trehalases in the yeast Saccharomyces cerevisiae. Progr Nucl Acid Res Molec Biol 58:197-237

7 Adaptation to nutrient availability in yeast 377 Nwaka S, Kopp M, Holzer H (1995a) Expression and function of the trehalase genes NTH1 and YBR0106 in Saccharomyces cerevisiae. J Biol Chem 270:10193-10198 Nwaka S, Mechler B, Destruelle M, Holzer H (1995b) Phenotypic features of trehalase mutants in Saccharomyces cevevisiae. FEBS Lett 360:286-290 Nwaka S, Mechler B, Holzer H (1996) Deletion of the ATH1 gene in Saccharomyces cerevisiae prevents growth on trehalose. FEBS Lett 386:235-238 O'Connell KF, Baker RE (1992) Possible cross-regulation of phosphate and sulphate metabolism in Saccharomyces cerevisiae. Genetics 132:63-73 Oechsner U, Bandlow W (1996) Interactions of the yeast centromere and promoter factor, Cpf1p, with the cytochrome c(1) upstream region and functional implications on regulated gene expression. Nucleic Acids Res 24:2395-2403 Oehlen LJWM, Scholte ME, de Koning W, van Dam K (1993) Inactivation of the CDC25 gene product in Saccharomyces cerevisiae leads to a decrease in glycolytic activity which is independent of cAMP levels. J Gen Microbiol 139:2091-2100 Oehlen LJWM, van Doorn J, Scholte ME, Postma PW, van Dam K (1990) Changes in the incorporation of carbon derived from glucose into cellular pools during the cell cycle of Saccharomyces cerevisiae. J Gen Microbiol 136:413-418 Ogawa N, Noguchi K, Sawai H, Yamashita Y, Yompakdee C, Oshima Y (1995) Functional domains of Pho81p, an inhibitor of Pho85p protein kinase, in the transduction pathway of Pi signals in Saccharomyces cerevisiae. Mol Cell Biol 15:997-1004 Olesen JT, Guarente L (1990) The HAP2 subunit of yeast CCAAT transcriptional activator contains adjacent domains for subunit association and DNA recognition:model for the HAP2/3/4 complex. Genes Dev 4:1714-1729 Oliphant AR, Brandl CJ, Struhl K (1989) Defining the sequence specificity of DNAbinding proteins by selecting binding sites from random-sequence oligonucleotides:analysis of yeast GCN4 protein. Mol Cell Biol 9:2944-2949 O'Neill EM, Kaffman A, Jolly ER, O'Shea EK (1996) Regulation of PHO4 nuclear localization by the PHO80-PHO85 cyclin-CDK complex. Science 271:209-212 Ono BI, Hazu T, Yoshida S, Kawato T, Shinoda S, Brzvwczy J, Paszawski A (1999) Cysteine biosynthesis in Saccharomyces cerevisiae:a new outlook on the pathway and regulation. Yeast 15:1365-1375 Ordiz I, Herrero P, Rodicio R, Moreno F (1995) Glucose-induced inactivation of isocitrate lyase in Saccharomyces cerevisiae is mediated by an internal decapeptide sequence. FEBS Lett 367:219-222 O'Roucke SM, Herskowitz I (1998) The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev 12:2874-2886 Oshima Y. (1982) Regulatory circuits for gene expression:the metabolism of galactose and phosphate. In Strathern, J.N., Jones, E.W. and Broach, J.R. (eds.), The molecular biology of the yeast Saccharomyces; metabolism and gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 159-180 Oshima Y (1997) The phosphatase system in Saccharomyces cerevisiae. Genes Genet Syst 72:323-334 Östling J, Ronne H (1998) Negative control of the Mig1p repressor by Snf1p-dependent phosphorylation in the absence of glucose. Eur J Biochem 252:162-168 Özcan S, Dover J, Johnston M (1998) Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae. EMBO J 17:2566-2573

378 Joris Winderickx et al. Özcan S, Dover J, Rosenwald AG, Wölfl S, Johnston M (1996a) Two glucose transporters in S. cerevisiae are glucose sensors that generate a signal for induction of gene expression. Proc Natl Acad Sci USA 93:12428-12432 Özcan S, Freidel K, Leuker A, Ciriacy M (1993) Glucose uptake and catabolite repression in dominant HTR1 mutants of Saccharomyces cerevisiae. J Bacteriol 175:5520-5528 Özcan S, Johnston M (1995) Three different regulatory mechanisms enable yeast hexose transporter (HXT) genes to be induced by different levels of glucose. Mol Cell Biol 15:1564-1572 Özcan S, Johnston M (1999) Function and regulation of yeast hexose transporters. Microbiol Mol Biol Rev 63:554-569 Özcan S, Leong T, Johnston M (1996b) Rgt1p of Saccharomyces cerevisiae, a key regulator of glucose-induced genes, is both an activator and a repressor of transcription. Mol Cell Biol 16:6419-6426 Pan X, Harashima T, Heitman J (2000) Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae. Curr Opin Microbiol 3:567-572 Pan X, Heitman J (2000) Sok2 regulates yeast pseudohyphal differentiation via a transcriptional factor cascade that regulates cell-cell adhesion. Mol Cell Biol 20:8364-8372 Pan XW, Heitman J (1999) Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Cell Biol 19:4874-4887 Parrou JL, Teste MA, François J (1997) Effects of various types of stress on metabolism of reserve carbohydrates in Saccharomyces cerevisiae:genetic evidence for a stressinduced recycling of glycogen and trehalose. Microbiol 143:1891-1900 Patton EE, Peyraud C, Rouillon A, Surdin-Kerjan Y, Tyers M, Thomas D (2000) SCF(met30)-mediated control of the transcriptional activator Met4 is required for the G(1)-S transition. EMBO J 19:1613-1624 Patton EE, Willems AR, Sa D, Kuras L, Thomas D, Craig KL, Teyers M (1998) Cdc53 is a scaffold protein for multiple Cdc34/Skp1/F-box protein complex that regulate cell division and methionine biosynthesis in yeast. Genes Dev 12:692-705 Pedruzzi I, Burckert N, Egger P, De Virgilio C (2000) Saccharomyces cerevisiae Ras/cAMP pathway controls post-diauxic shift element-dependent transcription through the zinc finger protein Gis1. EMBO J 19:2569-2579 Pernambuco MB, Winderickx J, Crauwels M, Griffioen G, Mager WH, Thevelein JM (1996) Glucose-triggered signalling in Saccharomyces cerevisiae:different requirements for sugar phosphorylation between cells grown on glucose and those grown an non-fermentable carbon sources. Microbiol 142:1775-1782 Peter M, Neiman AM, Park H-OvL, M., Herskowitz I (1996) Functional analysis of the interaction between the small GTP binding Cdc42 and the Ste20 protein kinase in yeast. EMBO J 15:7046-7059 Petit T, Diderich JA, Kruckeberg AL, Gançedo C, van Dam K (2000) Hexokinase regulates kinetics of glucose transport and expression of genes encoding hexose transporters in Saccharomyces cerevisiae. J. Bacteriol 182:6815-6818 Pinkham JL, Guarente L (1985) Cloning and molecular analysis of the HAP2 locus:a global regulator of respiratory genes in S. cerevisiae. Mol Cell Biol 5:3410-3416 Pinkham JL, Olesen JT, Guarente LP (1987) Sequence and nuclear localisation of the S.cerevisiae HAP2 protein, a transcriptional activator. Mol Cell Biol 7:578-585 Plesset J, Ludwig J, Cox B, McLaughlin C (1987) Effect of cell cycle position on thermotolerance in Saccharomyces cerevisiae. J Bacteriol 169:779-784

7 Adaptation to nutrient availability in yeast 379 Polakis ES, Bartley W (1965) Changes in the enzyme activities of Saccharomyces cerevisiae during aerobic growth on different carbon sources. Biochem J 97:284Posas F, Clotet J, Arino J (1991) Saccharomyces cerevisiae gene SIT4 is involved in the control of glycogen metabolism. FEBS Lett 279:341-345 Powers T, Walter P (1999) Regulation of ribosome biogenesis by the rapamycinsensitiveTOR-signaling pathway in Saccharomyces cerevisiae. Mol Biol Cell 10:9871000 Pringle JR, Hartwell LH. (1981) The Saccharomyces cerevisiae cell cycle. In Strathern, J.N., Jones, E.W. and Broach, J.R. (eds.), The molecular biology of the yeast Saccharomyces cerevisiae; Life cycle and inheritance. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 97-142 Rahner A, Scholer A, Martens E, Gollwitzer B, Schuller HJ (1996) Dual influence of the yeast Cat1p (Snf1p) protein kinase on carbon source-dependent transcriptional activation of gluconeogenic genes by the regulatory gene CAT8. Nucleic Acids Res 24:2331-2337 Ramaswarmy NT, Li L, Khalil M, Cannon JF (1998) Regulation of yeast glycogen metabolism and sporulation by Glc7 protein phosphatase. Genetics 149:57-72 Randez-Gil F, Bojunga N, Proft M, Entian KD (1997) Glucose derepression of gluconeogenic enzymes in Saccharomyces cerevisiae correlates with phosphorylation of the gene activator Cat8p. Mol Cell Biol 17:2502-2510 Randez-Gil F, Herrero P, Sanz P, Prieto JA, Moreno F (1998a) Hexokinase PII has a double cytosolic-nuclear localisation in Saccharomyces cerevisiae. FEBS Lett 425:475-478 Randez-Gil F, Sanz P, Entian KD, Prieto JA (1998b) Carbon source-dependent phosphorylation of hexokinase PII and its role in the glucose-signaling response in yeast. Mol Cell Biol 18:2940-2948 Raught B, A.C. G, Sonenberg N (2001) The target of rapamycin (TOR) proteins. Proc Natl Acad Sci USA USA 98:7039-7044 Reinders A, Burckert N, Boller T, Wiemken A, De Virgilio C (1998) Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase. Genes Dev 12:2943-2955 Reinders A, Burckert N, Hohmann S, Thevelein JM, Boller T, Wiemken A, DeVirgilio C (1997) Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock. Mol Microbiol 24:687-695 Riballo E, Herweijer M, Wolf DH, Lagunas R (1995) Catabolite inactivation of the yeast maltose transporter occurs in the vacuole after internalization by endocytosis. J Bacteriol 177:5622-5627 Robertson LS, Fink GR (1998) The three yeast A kinases have specific signaling functions in pseudohyphal growth. Proc Natl Acad Sci USA 95:13783-13787 Rohde J, Heitman J, Cardenas ME (2001) The TOR kinases link nutrient sensing to cell growth. J Biol Chem 276:398-403 Rolfes RJ, Zhang F, Hinnebusch AG (1997) The transcriptional activators Bas1, Bas2, and Abf1 bind positive regulatory sites as the critical elements for adenine regulation of ADE5,7. J Biol Chem 272:13343-13354 Rolland F, de Winde JH, Lemaire K, Boles E, Thevelein JM, Winderickx J (2000) Glucoseinduced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process. Mol Microbiol 38:348-358

380 Joris Winderickx et al. Rolland F, Wanke V, Cauwenberg L, Ma P, Boles E, Vanoni M, de Winde JH, Thevelein JM, Winderickx J (2001) The role of hexose transport and phosphorylation in cAMP signalling in the yeast Saccharomyces cerevisiae. FEMS Yeast Res 1403:1-13 Ronne H (1995) Glucose repression in fungi. Trends Genet 11:12-17 Rose M, Entian KD, Hoffmann L, Vogel RF (1988) Irreversible inactivation of Saccharomyces cerevisiae fructose-1,6-bisphosphatase independent of protein phosphorylation at Ser11. FEBS Lett 241:55-59. Rouillon A, Barbey R, Patton EE, Tyers M, Thomas D (2000) Feedback-regulated degradation of the transcriptional activator Met4 is triggered by th SVCF(Met30) complex. EMBO J 19:282-294 Rowen DW, Meinke M, LaPorte DC (1992) GLC3 and GHA1 of Saccharomyces cerevisiae are allelic and encode the glycogen branching enzyme. Mol Cell Biol 12:2-29 Rudoni S, Colombo S, Coccetti P, Martegani E (2001) Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae. Biochim Biophys Acta 1538:181-189 Ruis H, Schuller C (1995) Stress signaling in yeast. Bioessays 17:959-965 Rupp S, Summers E, Lo HJ, Madhani H, Fink GR (1999) MAP kinase and cAMP filamentation signaling pathways converge on the unusual large promotor of the yeast FLO11 gene. EMBO J 18:1257-1269 Sakai A, Chibazakura T, Shimizu Y, Hishinuma F (1992) Molecular analysis of POP2 gene, a gene required for glucose-derepression of gene expression in Saccharomyces cerevisiae. Nucl Acids Res 20:6227-6233 Sanz P, Alms GR, Haystead TA, Carlson M (2000a) Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase. Mol Cell Biol 20:13211328 Sanz P, Ludin K, Carlson M (2000b) Sip5 interacts with both the Reg1/Glc7 protein phosphatase and the Snf1 protein kinase of Saccharomyces cerevisiae. Genetics 154:99-107 Scheffler IE, delaCruz BJ, Prieto S (1998) Control of mRNA turnover as a mechaism of glucose repression in Saccharomyces cerevisiae. Int J Biochem Cell Biol 30:11751193 Schmelzle T, Hall MN (2000) TOR, a central controller of cell growth. Cell 103:253-262 Schmidt A, Beck T, Koller A, Kunz J, Hall MN (1998) The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. EMBO J 17:6924-6931 Schmidt A, Bickle M, Beck T, Hall MN (1997) The yeast phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the exchange factor ROM2. Cell 88:531542 Schmidt A, Kunz J, Hall MN (1996) TOR2 is required for organization of the actin cytoskeleton in yeast. Proc Natl Acad Sci USA 93:13780-13785 Schneider KR, Smith RL, O'Shea EK (1994) Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81. Science 266:122-126 Schork SM, Bee G, Thumm M, Wolf DH (1994) Catabolite inactivation of fructose-1,6bisphosphatase in yeast is mediated by the proteasome. FEBS Lett 349:270-274 Schork SM, Thumm M, Wolf DH (1995) Catabolite inactivation of fructose-1,6bisphosphatase of saccharomyces cerevisiae - degradation occurs via the ubiquitin pathway. J Biol Chem 270:26446-26450

7 Adaptation to nutrient availability in yeast 381 Schulman BA, Lindstrom DL, Harlow E (1998) Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. Proc Natl Acad Sci USA 95:10453-10458 Scott EW, Baker HV (1993) Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the High-Level expression of the glycolytic gene TPI. Mol Cell Biol 13:543-550 Sengstag C, Hinnen A (1988) A 28-bp segment of the S.cerevisiae PHO5 upstream activator sequence confers phosphate control to the CYC1-lacZ gene fusion. Cell 67:223Shamji AF, Kuruvilla FG, Schreiber SL (2000) Partioning the transcriptional program induced by rapamycin among the effectors of Tor proteins. Curr Biol 10:1574-1581 Shao D, Creasy CL, Bergmane LW (1996) Interaction of Saccharomyces cerevisiae Pho2 with Pho4 increases the accessibility of the activation domain of Pho4. Mol Gen Genet 251:358-364 Shu YM, Yang HF, Hallberg E, Hallberg R (1997) Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A. Mol Cell Biol 17:3242-3253 Skroch-Stuart J, Frederick DL, Varner CM, Tatchell K (1994) The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit. Mol Cell Biol 14:896-905 Smith A, Ward MP, Garrett S (1998) Yeast PKA represses Msn2p/Msn4p-dependent gene expression to regulate growth, stress response and glycogen accumulation. EMBO J 17:3556-3564 Sneddon AA, Cohen PT, Stark MJ (1990) Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes. EMBO J 9:4339-4346 Song W, Carlson M (1998) Srb mediator proteins interact functionally and physically with transcriptional reprssor Sfl1. EMBO J 17:5757-5765 Soto T, Fernandez J, Cansado J, Vicentesoler J, Gacto M (1995a) Glucose-induced, cyclicAMP-independent signalling pathway for activation of neutral trehalase in the fission yeast Schizosaccharomyces pombe. Microbiol 141:2665-2671 Soto T, Fernandez J, Cansado J, VicenteSoler J, Gacto M (1997) Protein kinase Sck1 is involved in trehalase activation by glucose and nitrogen source in the fission yeast Schizosaccharomyces pombe. Microbiol 143:2457-2463 Soto T, Fernandez J, Vicentesoler J, Cansado J, Gacto M (1995b) Activation of neutral trehalase by glucose and nitrogen source in Schizosaccharomyces pombe strains deficient in cAMP-dependent protein kinase activity. FEBS Lett 367:263-266 Soto T, Fernandez J, Vicentesoler J, Cansado J, Gacto M (1996) Posttranslational regulatory control of trehalase induced by nutrients, metabolic inhibitors and physical agents in Pachysolen tannophilus. Fungal Genet Biol 20:143-151 Stanbrough M, Rowen DW, Magasanik B (1995) The role of the GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated genes. Proc Natl Acad Sci USA 92:9450-9454 Stanhill A, Schick N, Engelberg D (1999) The yeast Ras2/cyclic AMP pathway induces invasive growth by suppressing the cellular stress response. Mol Cell Biol 19:7529-7538 Stapleton D, Gao GA, Michell BJ, Widmer J, Mitchelhill K, Teh T, House CM, Witters LA, Kemp BE (1994) Mammalian 5'-AMP-activated protein kinase non-catalytic subunits are homologs of proteins that interact with yeast Snf1 protein kinase. J Biol Chem 269:29343-29346

382 Joris Winderickx et al. Stark MJR (1996) yeast protein serine/threonine phosphatases:multiple roles and diverse regulation. Yeast 12:1647-1675 Sugajska E, Swiatek W, Zabrocki P, Geyskens I, Thevelein JM, Zolnierowicz S, Wera S (2001) Multiple effects of protein phosphatase 2A on nutrient-induced signalling in the yeast Saccharomyces cerevisiae. Mol Microbiol 40:1020-1026 Suomalainen H, Oura E. (1971) Yeast nutrition and solute uptake. In Rose, A.H. and Harrison, J.H. (eds.), The Yeasts. Academic Press, N.Y., New York, Vol. 2 Tadi D, Hasan RN, Bussereau F, Boy-Marcotte E, Jacquet M (1999) Selection of genes repressed by cAMP that are induced by nutritional limitation in Saccharomyces cerevisiae. Yeast 15:1733-1745 Tanaka K, Nakafuku M, Satoh T, Marshall MS, Gibbs JB, Matsumoto K, Kaziro Y, Toh-e A (1990) S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell 60:803-807 Tatchell K. (1993) RAS genes in the budding yeast Saccharomyces cerevisiae. In Kurjan, J. and Taylor, B.J. (eds.), Signal Transduction. Prokaryotic and Simple Eukaryotic Systems. Academic Press, San Diego, pp. 147-188 Tennyson CN, Lee J, Andrews BJ (1998) A role for the Pcl9-Pho85 cyclin-cdk complex at the M/G1 boundary in Saccharomyces cerevisiae. Mol Microbiol 28:69-79 ter Schure EG, van Riel NAW, Verrips (2000) The role of ammonia metabolism in nitrogen catabolite repression in Saccharomyces cerevisiae. FEMS Microbiol Rev 24:67-83 Thevelein JM (1984) Regulation of trehalose mobilization in fungi. Microbiol Rev 48:4259 Thevelein JM (1991) Fermentable sugars and intracellular acidification as specific activators of the Ras-adenylate cyclase signalling pathway in yeast:the relationship to nutrient-induced cell cycle control. Mol Microbiol 5:1301-1307 Thevelein JM (1994) Signal transduction in yeast. Yeast 10:1753-1790 Thevelein JM. (1996) Regulation of trehalose metabolism and its relevance to cell growth and function. In Brambl/Marzluf (ed.) The Mycota III; Biochemistry and molecular biology. Springer verlag, Berlin-Heidelberg, pp. 395-420 Thevelein JM, Beullens M, Honshoven F, Hoebeeck G, Detremerie K, Griewel B, den Hollander JA, Jans AWH (1987) Regulation of the cAMP level in the yeast Saccharomyces cerevisiae:the glucose-induced cAMP signal is not mediated by a transient drop in the intracellular pH. J Gen Microbiol 133:2197-2205 Thevelein JM, Cauwenberg L, Colombo S, de Winde JH, Donaton M, Dumortier F, Kraakman L, Lemaire K, Ma P, Nauwelaers D, Rolland F, Teunissen A, Van Dijck P, Versele M, Wera S, Winderickx J (2000) Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance and growth in yeast. Enzyme Microbial Technol 26:819-825 Thevelein JM, de Winde JH (1999) Novel sensing mechanisms and targets for the cAMP protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol 32:1002-1012 Thomas D, Cherest H, Surdin-Kerjan Y (1989) Elements involved in S-adenosyl methionine-mediated regulation of the Saccharomyces cerevisiae MET25 gene. Mol Cell Biol 9:3292-3298 Thomas D, Cherest H, Surdin-Kerjan Y (1991) Identification of the structural gene for glucose-6-phosphate dehydrogenase in yeast. Inactivation leads to a nutritional requirement for organic sulfur. EMBO J 10:547-553

7 Adaptation to nutrient availability in yeast 383 Thomas D, Jaquemin I, Surdin-Kerjan Y (1992) MET4, a leucine zipper protein, and centromere-binding factor 1 are both required for transcriptional activation of sulfur metabolism in Saccharomyces cerevisiae. Mol Cell Biol 12:1719-1727 Thomas D, Kuras L, Barbey R, Cherest H, Blaiseau PL, Surdin-Kerjan Y (1995) Met30p, a yeast transcriptional inhibitor that responds to S-adenosylmethionine, is an essential protein with WD40 repeats. Mol Cell Biol 15:6526-6534 Thomas D, Surdin-Kerjan Y (1997) Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 61:503-532 Thompson-Jaeger S, Francois J, Gaughran JP, Tatchell K (1991) Deletion of SNF1 affects the nutrient response of yeast and resembles mutations which activate the adenylate cyclase pathway. Genetics 129:697-706 Thon VJ, Vigneron-Lesens C, Marianne-Pepin T, Montreuil J, decq A (1992) Coordinate regulation of glycogen metabolsm in the yeast Saccharomyces cerevisiae:induction of glycogen branching enzyme. J Biol Chem 267:15224-15228 Timblin BK, Tatchell K, Bergman LW (1996) Deletion of the gene encoding the cyclindependent protein kinase Pho85 alters glycogen metabolism in Saccharomyces cerevisiae. Genetics 143:57-66 Toda T, Cameron S, Sass P, Wigler M (1988) SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to cAMPdependent protein kinase catalytic subunits. Genes Dev 2:517-527 Toda T, Cameron S, Sass P, Zoller M, Scott JD, McBullen B, Hurwitz M, Krebs EG, Wigler M (1987a) Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Mol Cell Biol 7:1371-1377 Toda T, Cameron S, Sass P, Zoller M, Wigler M (1987b) Three different genes in Saccharomyces cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell 50:277-287 Toda T, Uno I, Ishikawa T, Powers S, Kataoka T, Broek D, Cameron S, Broach J, Matsumoto K, Wigler M (1985) In yeast, Ras proteins are controlling elements of adenylate cyclase. Cell 40:27-36 Toh-E A, Tanaka K, Uesono Y, Wickner R (1988) PHO85, a negative regulator of the PHO system, is a homolog of the protein kinase gene, CDC28, of Saccaromyces cerevisiae. Mol Gen Genet 214:162-164 Tokiwa G, Tyers M, Volpe T, Futcher B (1994) Inhibition of G1 cyclin activity by the Ras/cAMP pathway in yeast. Nature 371:342-345 Treitel MA, Carlson M (1995) Repression by SSN6-TUP1 is directed by MIG1, a repressor activator protein. Proc Natl Acad Sci USA 92:3132-3136 Treitel MA, Kuchin S, Carlson M (1998) Snf1 Protein Kinase Regulates Phosphorylation of the Mig1 Repressor in Saccharomyces cerevisiae. Mol Cell Biol 18:6373-6380 Trumbly R (1992) Glucose repression in the yeast Saccharomyces cerevisiae. Mol Microbiol 6:15-21 Tu JL, Song WJ, Carlson M (1996) Protein phosphatase type 1 interacts with proteins required for meiosis and other cellular processes in Saccharomyces cerevisiae. Mol Cell Biol 16:4199-4206 Tung KS, Hopper AK (1995) The glucose repression and RAS-cAMP signal transduction pathways of Saccharomyces cerevisiae each affect RNA processing and the synthesis of a reporter protein. Mol Gen Genet 247:48-54

384 Joris Winderickx et al. Türkel S, Bisson LF (1999) Transcription of the HXT4 gene is regulated by Gcr1 and Gcr2 in the yeast Saccharomyces cerevisiae. Yeast 15:1045-1057 Uemura H, Jigami Y (1992) Role of GCR2 in transcriptional activation of yeast glycolytic genes. Mol Cell Biol 12:3834-3842 Uemura H, Jigami Y (1995) Mutations in GCR1, a transcriptional activator of Saccharomyces cerevisiae glycolytic genes, function as suppressors of gcr2 mutations. Genetics 139:511-521 Uno I, Matsumoto K, Adachi K, Ishikawa T (1983) Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast. J Biol Chem 258:10867-10872 Vallier LG, Carlson M (1994) Synergistic release from glucose repression by mig1 and SSN mutations in saccharomyces cerevisiae. Genetics 137:49-54 Vallier LG, Coons D, Bisson LF, Carlson M (1994) Altered regulatory responses to glucose are associated with a glucose transport defect in grr1 mutants of saccharomyces cerevisiae. Genetics 136:1279-1285 Van Aelst L, Hohmann S, Bulaya B, de Koning W, Sierkstra L, Neves MJ, Luyten K, Alijo R, Ramos J, Coccetti P, Martegani E, de Magalhaes-Rocha NM, Brandao RL, Van Dijck P, Vanhalewyn M, Durnez P, Jans AWH, Thevelein JM (1993) Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae. Mol Microbiol 8:927-943 Van Aelst L, Jans AWH, Thevelein JM (1991) Involvement of the CDC25 gene product in the signal transmission pathway of the glucose-induced RAS-mediated cAMP signal in the yeast Saccharomyces cerevisiae. J Gen Microbiol 137:341-349 van der Plaat JB (1974) Cyclic 3',5'-adenosine monophosphate stimulates trehalose degradation in baker's yeast. Biochem. Biophys. Res. Commun. 56:580-587 van Zyl W, Huang W, Sneddon AA, Stark M, Carnier S, Werer M, Marck C, Sentenac A, Broach JR (1992) Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae. Mol Cell Biol 12:4946-4959 Vandenbol M, Jauniaux J-C, Grenson M (1990) The Saccharomyces cerevisiae NPR1 gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue. Mol Gen Genet 222:393-399 Vanoni M, Sollitti P, Goldenthal M, Marmur J (1989) structure and regulation of the multigene family controlling maltose fermentation in budding yeast. Progress Nucl Acid Res Mol Biol 37:281-322 Venturi GM, Bloecher A, WilliamsHart T, Tatchell K (2000) Genetic interactions between GLC7, PPZ1 and PPZ2 in Saccharomyces cerevisiae. Genetics 155:69-83 Versele M, de Winde JH, Thevelein JM (1999) A novel regulator of G-protein signalling in yeast, Rgs2, downregulates glucose-activation of the cAMP pathway through direct inhibition of Gpa2. EMBO J 18:5577-5591 Vincent AC, Struhl K (1992) ACR1, a yeast ATF/CREB repressor. Mol Cell Biol 12:59345405 Vincent O, Carlson M (1998) Sip4, a Snf1 kinase dependent transcriptional activator binds to the carbon source-responsive elements of gluconeogenic genes. EMBO J 17:70027008 Vincent O, Carlson M (1999) Gal83 mediates the interaction of the Snf1 kinase complex with the transcription activator Sip4. EMBO J 18:6672-6681

7 Adaptation to nutrient availability in yeast 385 Vogel K, Hörz W, Hinnen A (1989) The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions. Mol Cell Biol 9:2050-2057 Vojtek AB, Fraenkel DG (1990) Phosphorylation of yeast hexokinases. Eur J Biochem 190:371-375 Vuorio OE, Kalkkinen N, Londesborough J (1993) Cloning of two related genes encoding the 56-kDa and 123-kDa subunits of trehalose synthase from the yeast Saccharomyces cerevisiae. Eur J Biochem 216:849-861 Wang SS, Hopper AK (1998) Isolation of a yeast gene involved in species-specific pretRNA processing. Mol Cell Biol 8:5140-5149 Ward MP, Garrett S (1994) Suppression of a yeast cyclic AMP-dependent protein kinase defect by overexpression of SOK1, a yeast gene exhibiting sequence similarity to a developmentally regulated mouse gene. Mol Cell Biol 14:5619-5627 Ward MP, Gimeno CJ, Fink GR, Garrett S (1995) SOK2 may regulate cyclic AMPdependent protein kinase-stimulated growth and pseudohyphal development by repressing transcription. Mol Cell Biol 15:6854-6863 Wek RC, Cannon JF, Dever TE, Hinnebusch AG (1992) Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2alpha kinase GCN2. Mol Cell Biol 12:5700-5710 Werner-Washburne M, Braun E, Johnston GC, Singer RA (1993) Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol Rev 57:383-401 Werner-Washburne M, Braun EL, Crawford ME, Peck VM (1996) Stationary phase in Saccharomyces cerevisiae. Mol Microbiol 19:1159-1166 Werner-Washburne M, Brown D, Braun E (1991) Bcy1, the regulatory subunit of cAMPdependent protein kinase in yeast, is differentially modified in response to the physiological status of the cell. J Biol Chem 266:19704-19709 Wickner RB (1994) [ure3] as an altered ure2 protein - evidence for a prion analog in Saccharomyces cerevisiae. Science 264:566-569 Wills C (1990) Regulation of sugar and ethanol metabolism in Saccharomyces cerevisiae. Crit Rev Biochem Mol Biol 25:245-280 Wilson WA, Hawley SA, Hardie DG (1996) Glucose repression/derepression in budding yeast:SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP:ATP ratio. Curr Biol 6:1426-1434 Winderickx J, de Winde JH, Crauwels M, Hino A, Hohmann S, Van Dijck P, Thevelein JM (1996) Regulation of genes encoding subunits of the trehalose synthase complex in Saccharomyces cerevisiae:novel variations of STRE-mediated transcription control ? Mol Gen Genet 252:470-482 Woods A, Munday MR, Scott J, Yang X, Carlson M, Carling D (1994) Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo. J Biol Chem 269:19509-19515 Woollard A, Nurse P (1995) G(1) regulation and checkpoints operating around START in fission yeast. Bioessays 17:481-490 Wright RM, Repine T, Repine JE (1993) Reversible pseudohyphal growth in haploid Saccharomyces cerevisiae is an aerobic process. Curr Genet 23:388-391 Xue Y, Batlle M, Hirsch JP (1998) GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p G subunit and functions in a Ras-independent pathway. EMBO J 17:1996-2007

386 Joris Winderickx et al. Yang J, Wu JS, Ao SZ (1995) Analysis of activation activity of yeast PHO2, PHO4 protein and their interaction. Acta Biochim Biophy Sin 27:165-172 Yang XL, Jiang R, Carlson M (1994) A family of proteins containing a conserved domain that mediates interaction with the yeast SNF1 protein kinase complex. EMBO J 13:5878-5886 Yin Z, Hatton L, Brown AJ (2000) Differential post-transcriptional regulation of yeast mRNAs in response to high and low glucose concentrations. Mol Microbiol 35:553565 Yin ZK, Smith RJ, Brown AJP (1996) Multiple signalling pathways trigger the exquisite sensitivity of yeast gluconeogenic mRNAs to glucose. Mol Microbiol 20:751-764 Yoshida K, Ogawa N, Oshima Y (1989) Function of the PHO regulatory genes for repressible acid phosphatase synthesis in S. cerevisiae. Mol Gen Genet 217:40-46 Yun CW, Tamaki H, Nakayama R, Yamamoto K, Kumagai H (1998) Gpr1p, a putative Gprotein coupled receptor, regulates glucose-dependent cellular cAMP level in yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 252:29-33 Zabrocki P, Van Hoof C, Goris J, Thevelein JM, Winderickx J, Wera S (2002) Protein phosphatase 2A on track for nutrient-induced signalling in yeast. Mol Microbiol 43:835-842 Zähringer H, Thevelein JM, Nwaka S (2000) Induction of neutral trehalase Nth1 by heat and osmotic stress is controlled by STRE elements and Msn2/Msn4 transcription factors:variations of PKA effect during stress and growth. Mol Microbiol 35:397-406 Zaragoza D, Ghavidel A, Heitman J, Schultz MC (1998) Rapamycin induces the G0 program of transcriptional repression in yeast by interfering with the TOR signaling pathway. Mol Cell Biol 18:4463-4470 Zaragoza O, Gancedo JM (2000) Pseudohyphal growth is induced in Saccharomyces cerevisiae by a combination of stress and cAMP signalling. Ant Leeuwenhoek 78:187-194 Zheng XF, Florentino D, Chen J, Crabtree GR, Schreiber SL (1995) TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin. Cell 82:121-130 Zitomer RS, Montgomery DL, Nichols DL, Hall BD (1979) Transcriptional regulation of the yeast cytochrome c gene. Proc Natl Acad Sci USA 76:3627-3631

Index

actin cytoskeleton, 123, 155, 169, 178 actin organisation, 349 activation domain, 72, 80, 88 aging, 71, 108 alkoxyl radical, 244 alkyl hydroperoxidase, 260 AMP/ATP ratio, 317 AMP-dependent protein kinase, 170 apoptosis, 249 aquaporin, 131 ATF/CREB family, 145 ATP hydrolysis, 95 ATP turnover, 95 autophagy, 23, 29, 55 betaine, 168 bZIP domain, 145 calcineurin, 154, 160 calcofluor white, 154 calmodulin, 83, 102 Candida boidinii, 260 carbohydrate metabolism, 23, 33 carboxypeptidase Y, 226 Casein kinase, 219 catabolite inactivation, 323 catalases, 254 cell cycle, 19, 31, 37, 53 cell cycle progression, 315, 337 cell expansion, 150, 158, 169 cell integrity pathway, 130, 150 cell polarity, 123, 155, 163, 169 cell shrinking, 124, 139, 161 cell wall, 20, 54 cell wall maintenance, 158 cell wall metabolism, 145, 158 chaperonin, 20 chromatin, 31, 41, 49, 56 circadian rhythms, 287 coiled-coil domain, 85, 88 cross protection, 12, 37 cyclin-dependent protein kinase, 157 cyclophilin, 97 cyclosporin A, 97 cytochrome c, 28 cytoskeleton, 30, 54

Debaryomyces hansenii, 202 diauxic shift, 307, 311, 348 DNA arrays, 13 DNA microarrays, 75 DYRK family, 319, 346 endoplasmic reticulum, 77, 94 ergosterol, 123, 131 eukaryotic initiation factor, 332 exocyst, 225 fatty acid metabolism, 23, 27 feedback, 33 Fenton reaction, 242 filamentous growth, 159 flow cytometry, 246 fructose-2,6-bisphosphate, 27, 34 GAF domain, 286 GATA family, 327, 330 general stress response, 12, 22, 45 glucan synthase, 216 gluconeogenesis, 35, 310 glutathione peroxidases, 254, 261 glutathione synthase, 255 glutathionylation, 250 glycogen, 24, 34, 35, 55, 124 glycogen breakdown, 355 glycogen phosphorylase, 25 glycogen synthase, 25 glycogen synthesis, 355 glycolysis, 24, 35, 41, 250, 257, 310, 347 glycolytic flux, 24, 27 G-protein coupled receptor, 322 growth arrest, 19 GTPase, 274, 321, 348, 352 GTP-binding protein, 154 Haber-Weiss reaction, 242 heat shock transcription factor, 72 helix-turn-helix, 72, 84, 268 histidine kinase, 139, 160, 168, 178, 274, 284 histone deacetylase, 43, 97 histone deacetylation, 214

388 histones, 41 HO promoter, 339 isoamylalcohol, 356 K+ homeostasis, 208 Krebs cycle, 310, 315 leucine zipper, 266, 277 life-span, 247 linoleic acid, 246, 263 lipid metabolism, 123 luciferase, 77 maltase, 317 MAP kinase, 54 MAP kinase pathway, 54 mechanosensitive channels, 123 meiosis, 43 metabolic remodeling, 71, 76 metabolite transport, 23 metallothionein, 252, 276 methionine sulfoxide reductase, 261 microtubule, 96 MIP channels, 131 mitochondria, 27 mitochondrial, 19, 23, 30, 36 mitochondrial biogenesis, 334 mRNA turnover, 22, 43, 44, 57, 320 Na+ compartmentalization, 204, 211 Na+ toxicity, 203, 219 nuclear import receptor, 271 nuclear translocation, 46, 141, 165, 176 nucleo-cytoplasmic shuttling, 281 nucleolus, 22 nucleotide biosynthesis, 20 osmotolerance, 142, 170 oxygen-derived reactive species, 242 PAS/PAC domain, 286 pectin-degrading enzyme, 358 pentose phosphate, 24, 35 pentose phosphate pathway, 253, 264 pentose-phosphate cycle, 310 peroxisomal, 33, 36 peroxisome, 27 peroxyl radical, 244

pheromone response pathway, 135, 141, 149 phosphatidylinositol, 134, 154, 167 phosphatidylinositol kinase, 349 phosphatidyl-inositol signalling, 179 phosphodiesterase, 323, 356 phospholipase C, 322, 358 phospholipid biosynthesis, 124 phosphorelay system, 134, 139, 145, 160, 175 phosphotyrosine phosphatase, 176 Pichia, 202 plasma membrane sensor, 332 polyadenylation, 43 post-diauxic growth phase, 311 post-diauxic phase, 312 PP1 phosphatase, 318 prevacuolar compartment, 211, 212, 226 prion, 71, 93, 103 proline biosynthesis, 258 prolyl isomerase, 349 protein carbonylation, 249 protein glycosylation, 20 protein Kinase C, 21, 54 pseudohyphae, 356 pseudohyphal development, 135, 150, 163, 171 pseudohyphal growth, 312, 348, 356 rapamycin, 21, 45, 349 Ras-cAMP pathway, 312, 319, 346 receiver domain, 268, 284 redox homeostasis, 251 regulatory sequences, 49 replication, 20 respiration, 26, 55 response regulator, 130, 145, 155, 173 ribosomal protein genes, 312, 325, 347 ribosomal proteins, 21 secretion, 20, 54 secretory pathway, 223 senescence, 312 serine/threonine phosphatases, 149 silencing, 22, 39 SNARE, 225 Spc1/Sty1 MAP kinase, 280 sphingolipids, 225 spindle pole, 92 spontaneous fermentation, 311 sporulation, 251

Index stationary phase, 306, 312, 321, 342 steroid hormone, 93, 100 storage carbohydrates, 323 stretch-activated channels, 133 Sty1 pathway, 164, 171 sugar kinases, 320 thermotolerance, 71, 76, 84 thioredoxin peroxidases, 254, 259 TOR pathway, 154, 170, 346 translation, 16, 23, 44, 54 translation elongation factor, 332 trehalase, 24, 34, 345 trehalose, 24, 35 trehalose breakdown, 77 trehalose synthase, 24, 35 two-dimensional electrophoresis, 13 tyrosine kinase, 95

389

tyrosine phosphatases, 142, 149, 174, 179, 281 tyrosine phosphorylation, 83, 101 ubiquitin, 31 vacuolar, 23, 29, 34 vacuolar membrane, 205, 212, 217 vacuolar targeting, 324 vacuole, 134, 167 virulence, 172 Yap1 family, 277 zinc metabolism, 252 zinc-finger, 166 Zygosaccharomyces rouxii, 202