Heat stress response: a complex game with chaperones and transcription factors
Department of Molecular Cell Biology, Goethe University Frankfurt, Marie Curie Str. 9, D-60439 Frankfurt/M., Germany; e-mail: [email protected]
Taken from: Molecular and Cellular Biology: from plant to human. Proceedings of EMBO lecture course 2000
Plant stress response
During evolution, the origin of terrestrial plants (about 400 million years ago) required special adaptations to rapidly changing environmental conditions (Levitt 1980). Examples for these adaptations are: -
The predominant role of the sporophyte in the life cycle of plants with the sensitive gametophyte being enclosed.
The organisation of leaves as photosynthetic organs with the active cells inside, a protective outer layer of the epidermis and cuticle and the necessary gas exchange proceeding through tightly controlled apertures (stomata).
The formation of stress resistant dormant forms (seeds) for propagation and survival of unfavourable conditions.
The development of a mechanically stabilized cormophyte allowing the generation of long-lived and very big plants with systems for long-distance nutrient and water transport.
These adaptations provided the basis for a very efficient, three-dimensional occupation by plants of all habitats outside water. Plants also became specialized to grow and propagate under extreme environmental conditions, e.g. under conditions of very low or very high temperatures, of high salt or heavy metal stress or of extreme water deficiency. Most important, however, for the remarkable success of terrestrial plants as sessile organisms was the development of their capability to rapidly respond to a multiplicity of environmental changes from which they cannot escape. Hence a network of interconnected cellular stress response systems is a prerequisite for plant survival and productivity.
The molecular cell biology of the heat stress response
A milestone for the analysis of cellular stress response systems was the pioneering work of an Italian developmental biologist F. Ritossa. After a fortuitous increase of the temperature of the incubator with the Drosophila cultures, he observed remarkable changes of the puffing, i.e. gene activity patterns of the polytene chromosomes in larval salivary glands (Ritossa 1962). Surprisingly enough, the same reprogramming of transcription was also observed after addition of chemical stressors like salicylate, 2,4-dinitrophenol and azide. About 10 years later A. Tissieres and coworkers (Tissieres et al. 1974) identified the newly formed heat stress proteins (Hsps), and McKenzie and Meselson (1977) characterized the corresponding mRNAs. Soon, the rapidly developing field included investigations of other eukaryotic organisms and bacteria. It turned out that Ritossa in fact had discovered the central parts of a general stress response system conserved throughout the living world (Nover 1991; Nover and Scharf 1997; Scharf et al. 1998).
To present an overview of 35 years of molecular cell biology research in this field, I will use a hypothetic eukaryotic cell under stress (Fig. 1). The essential elements of this model can be summarized as follows (Wu 1995; Scharf et al. 1998b; Morimoto 1998):
?? Besides heat stress (hs), a large number of chemical stressors and various states of disease cause activation of heat stress genes (top of Fig. 1).
?? Very likely the stress sensing system in cells are deviations of protein homeostasis, i.e. of the equilibrium between new synthesis, folding, intracellular targeting, biological function and degradation of proteins. Proteins are shown in two states: (i) proteins in the native state (squares) and (ii) partially denatured proteins (stars) bound to chaperones. 3
?? Heat stress proteins (Hsps) and constitutively expressed members of the 11 conserved Hsp families were characterized as molecular chaperones essential for maintainance or restoration of protein homeostasis (see part 3 of this overview). Denaturation of proteins and problems in the processing of newly formed proteins during stress are assumed to result in a decrease of the pool of free chaperones.
?? The regulatory proteins (heat stress transcription factors, Hsfs) exist as inactive proteins mostly found in the cytoplasm. Stress causes activation with oligomerization and, eventually, recompartmentation to the nucleus. Binding to the target sequences (HSE) in the promoter of hs genes triggers transcription (steps 1 and 2 of the Hsf cycle, on the left of Fig.1).
?? Hsp expression is assumed to replenish the pool of free chaperones. There is good evidence that in a kind of autorepression some of the chaperones (e.g. Hsp70, Hsp90) are involved in the second part of the Hsf cycle leading to the restoration of the inactive state (steps 3 to 5 of the Hsf cycle).
?? A peculiarity of plants is the existence of hs-induced forms of Hsfs, which may play a major part in the modulation of transcription in the course of a long-term hs response.
2. Transcriptional control 2.1.
The basal transcription complex
Generally, selective transcription of eukaryotic genes requires interaction of sequence specific transcription factors with the highly complex basal transcription machinery (transcriptosome) assembled from 70 to 100 subunits around the start site of a gene (Tjian and Maniatis 1994; Ptashne and Gann 1997; Struhl and Moqtaderi 1998; Kadonaga 1998; Glass et al. 2000; Sterner and Berger 2000). The remarkable
complexity of the transcription complex is connected with its multifold functions required for efficient and tightly controlled synthesis of mRNAs (see Fig. 2 and explanations in the legend).
Heat stress transcription factor (Hsfs) as prototype of a eukaryotic activator protein
As indicated in Fig. 1, heat stress transcription factors (Hsfs) are the central control proteins of the heat stress response (Wu 1995, Nover et al. 1996; Morimoto 1998). Similar to many other proteins regulating gene activity, they have a modular structure. Despite a considerable variability in size and sequence, their basic structure and promoter recognition are conserved throughout the eukaryotic kingdom (Fig. 3). (1)
Close to the N-terminus the highly structured DNA-binding domain (DBD) is formed of a three-helical bundle (H1, H2, H3) and a four stranded antiparallel ß-sheet (ß1, ß2, ß3, ß4). The hydrophobic core of this domain ensures the precise positioning of the central helix-turn-helix motif (H2-T-H3, Fig. 3B) required for specific recognition of the heat stress promoter elements (HSE). These are repetitive patterns of palindromic binding motifs (5’-AGAAnnTTCT3’) upstream of the TATA box of hs-inducible genes (Pelham 1982; Nover 1991).
The oligomerization domain (HR-A/B region) is connected to the DNA-binding domain by a flexible linker of variable length (15 to 80 amino acid residues). A heptad pattern of hydrophobic amino acid residues in the HR-A/B region suggest a coiled-coil structure charateristic of leucine zipper type protein interaction domains (Peteranderl et al. 1999). In plants, two subfamilies of Hsf are clearly separated by peculiarities of their HR-A/B regions (Nover et al. 5
1996). Hsfs of the B-type are similar to all non-plant Hsfs (see Fig. 3). In contrast to this, Hsfs of the A-type have an extended HR-A/B region due to an insertion of 21 amino acid residues between the A and B part (see example for HsfA2 in Fig. 3B ). The significance of this coexistence of two classes of Hsfs is just beginning to emerge. (3)
The nuclear localization signal (NLS) of Hsfs are formed by bipartite clusters of basic amino acid residues (Lyck et al. 1997). These positively charged recognition motifs serve the assembly of a nuclear import complex built of the NLS receptor (importin ? ) and two other subunits, importin ß and Ran(GTP) (Mattaj and Englmeier 1998; Görlich and Kutay 1999).
Recently, the dynamically changing intracellular distribution of Hsfs between cytoplasm and nucleus was shown to depend on the balance of nuclear import and export. A C-terminal leucine-rich nuclear export signal (NES) of HsfA2 is required for the receptor-mediated export (Fig. 3B).
The C-terminal parts of the Hsfs with the activator function are the least conserved in sequence and size. Their function as transcription activators evidently resides in short activator peptide motifs (AHA motifs) chracterized by aromatic (W, F, Y), large hydrophobic (L, I, V) and acidic ( E, D) amino acid residues (see example given in Fig. 3B). Similar AHA motifs are found in the center of many other transcription factors of yeast and mammals, e.g. VP16, RelA, Sp1, Fos, Jun, Gal4, Gcn4 as well as the steroid and retinoic acid receptors (see summary and references in Nover and Scharf 1997, Döring et al. 2000). Most likely, they represent the essential sites of contacts with subunits of the basal transcription complex (Fig. 2 ).
Tjian and Maniatis (1994) proposed a model of cohesive interfaces, i.e. of interacting surfaces with a mutually corresponding pattern of aromatic/hydrophobic amino acid 6
residues between activator protein and its target protein(s). In support of this concept, mutant forms with exchanges of the aromatic and/or hydrophobic residues do not interact with components of the BTC in vitro and are deficient in reporter assays in vivo. However, this simplified model provides only partial insights into the activator function. Evidence from in vitro and in vivo experiments support the concept that in the reality of complex promoters, usually several DNA binding proteins contribute synergistically to the assembly of the transcription complex, whose composition varies with the particular activator/repressor cocktail available in a given cell or in a given situation. There is experimental evidence for the essential role of a synergistic assembly of activator and helper proteins in an enhanceosome as starting point for building a transcription complex with high processivity (Thanos and Maniatis 1995; Blau et al. 1996, Carey 1998; Ellwood et al. 1999).
Heat stress proteins as molecular chaperones
As outlined in part 1.2., all organisms respond to supraoptimal temperatures by synthesizing of a specific set of heat stress proteins (Hsp). They are needed to protect cells from heat damage, and they assist in normalization of functions during recovery (reviewed by Nover, 1991; Nover and Scharf, 1997; Feder and Hofmann 1999). Heat stress proteins can be assigned to eleven protein families conserved among bacteria, plants and animals. During the last decade more and more details of the biochemical function of Hsps have emerged. As molecular chaperones they help other proteins to maintain or regain their native conformation by stabilizing partially unfolded states (Hartl 1996; Ellis 1997, 1999; Gottesman et al. 1997; Beissinger and Buchner 1998; Bukau and Horwich 1998; Forreiter and Nover 1998; Netzer and Hartl 1998; Caplan 1999). Chaperones do not contain specific information for correct folding, but rather prevent unproductive interactions (aggregation) between non-native proteins. This type of molecular chaperones can be distinguished from other heat-induced proteins acting as direct folding catalysts like peptidyl-prolyl isomerases or protein disulfide isomerases (Schmid 1995). Three major aspects in the life cycle of a protein invoke chaperone proteins: (1) They ensure that nascent polypeptides emerging from the ribosome are kept in a folding competent state until the whole sequence information is available. (2) Since fully folded proteins cannot be translocated through membranes, chaperones are needed to maintain or create a partially unfolded form of proteins destined for the transport into mitochondria or plastids (Kubrich et al. 1995; Neupert 1997; Pfanner et al. 1997; May and Soll 1999; Braun and Schmitz 1999; ). (3) They stabilize damaged proteins generated as a result of chemical or physical stress and thus facilitate renaturation and/or degradation in the recovery period. Many data collected during the last few years indicate that members of different Hsp families act together in multisubunit 8
complexes, so-called chaperone machines (Hartl, 1996; Bukau et al., 1997; Bukau and Horwich 1998), and different chaperone complexes may interact to generate a network for protein maturation, assembly and targeting (Frydman and Höhfeld, 1997; Johnson and Craig, 1997; Forreiter and Nover1998). Although many proteins are potential substrates for chaperone machines, e.g. after stress-induced protein damage, most of them ( about 80%) fold in a chaperone-independent manner under normal conditions. It is assumed that ribosomal proteins at the surface of the ribosomes or associated proteins may help to stabilize nascent polypeptide chains.
3.2 The Hsp60 (GroE) chaperone system The prototype of a chaperone machine is the GroEL/GroES complex of the bacterium Escherichia coli (Fig. 4, for reviews see Fenton et al. 1996; Hartl, 1996; Fenton and Horwich 1997). 14 large subunits (GroEL, 56 kDa) form a hollow core structure of two heptameric rings associated back to back with each other. Each subunit is composed out of three different domains, (1) an equatorial domain interacting with the second ring and harboring an ATP binding side, (2) a flexible intermediate domain required for ATP hydrolysis and (3) an apical domain interacting with the GroES subunit (Figure 4). The apical domains of GroEL expose a set of hydrophobic amino acid residues towards the inner surface of the 50Å cavity, offering cohesive binding sides for partially unfolded proteins. This part of GroEL is the most flexible. If ATP binds to the ring occupied by the misfolded polypeptide (cis-position), the apical domain is twisted by 90° and lifted from the equatorial domain to increase the size of the inner cavity to more than 80Å. Target proteins bound to the hydrophobic surfaces may therefor undergo further mechanical unfolding. Twisting of the equatorial domain buries the hydrophobic sites of the interacting apical domains and allows binding of the GroES-heptamer. The GroES-heptamer forms a 9
domelike flexible structure with a central orifice on top covering the inner ring cavity of the GroEL subunit. Therefore the unfolded protein is entrapped and protected in the GroEL/GroES cage. Binding of GroES is a prerequisite for ATP hydrolysis by the equatorial domains of GroEL. In the last part of the cycle, ATP binding to the second ring of the GroEL heptamer (trans) allows removal of the (cis) GroES heptamer and release of the (re)folded protein, which eventually may enter into a new cycle. Since there are two identical rings of GroEL, one can easily imagine two simultaneously operating folding events in each ring coupled in their timing by binding, cleavage and release of ATP.
The Hsp20 chaperone family and a unique cytosolic multichaperone complex of plants
Proteins of the Hsp20 chaperone family, including ? A- and ? B-crystallin, are characterized by a central domain of about 100 amino acid residues (? -crystallin domain). Size variation of the Hsp20 proteins results mainly from N-terminal and Cterminal extensions of this domain (Nover, 1991; Caspers et al., 1995; Waters et al., 1996). Plants are characterized by an unusual complexity of hs-inducible proteins belonging to the Hsp20 family (small Hsps). Different isoforms of this family are found in all cellular compartments involved in protein synthesis and/or processing, e.g. in the cytosol, the ER, chloroplasts and in mitochondria (see summaries and references in Vierling, 1991; Waters et al., 1996). All proteins of the Hsp20 family form homooligomers of variable size of 200-800 kDa corresponding to 12 to 32 subunits (Fig. 5). They exhibit chaperone activity, which does not require ATP (Horwitz, 1992; Jakob et al., 1993; Plater et al., 1996). They are able to prevent aggregation of denatured proteins in vitro and in vivo. But renaturation of proteins needs interaction
with the Hsp70 chaperone system and ATP (Forreiter et al., 1997; Ehrnsperger et al., 1997; Lee et al., 1997; Lee and Vierling, 2000). In plants oligomers of the cytosolic small Hsps undergo hs-induced assembly to form large chaperone complexes comprised of hundreds of 40 nm particles (heat stress granules, HSG; Nover et al. 1989a, 1989b). There is increasing evidence for an intriguing multiplicity of functions of these unique chaperone complexes in the plant hs response (Fig. 5). (1) The results about protection of luciferase as thermosensitive reporter for chaperone activity in vivo (Forreiter et al. 1997; Löw et al. 2000; Tripp et al., unpublished) support the concept of these complexes forming an intracellular matrix for protection of denatured proteins under hs conditions. (2) HSG complexes were shown to represent storage sites for house-keeping mRNPs, not translated during the stress period but released in the recovery period for reuse in polysomes (Nover et al. 1989a). (3) Recently, HSG were found to selectively bind one of the four tomato hs transcription factors (HsfA2), whose pattern of nuclear/cytoplasmic distribution is intimately connected with its reversible structural binding to the HSG complexes (Scharf et al., 1998a, 1998b; Bharti et al. 2000).
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Fig. 1: Model of a eukaryotic cell under stress (for further explanations seee text)
SAGA RNA Pol II
TFIID IIA TBP CTD
Srb Swi/Snf Transcription of Hsp genes
Fig. 2: Transcription machinery in eukaryotic cells The complexity of the transcription machinery made of about 100 proteins results from the multifold functions required for efficient and tightly controlled synthesis of mRNA. This includes (i) promoter recognition and initiation of transcription , (ii) RNA synthesis (elongation) and (iii) termination of transcription. RNA polymerase II with its 10 subunits, the TATA binding protein TBP with its associated proteins (TAFs) as well transcription factors TFIIA, IIB, IIE, IIF and IIE are involved. Components of the SWI/SNF, SAGA, Srb and TBP/TAF complexes are required for chromatin remodelling and reversible protein modifications, e.g. phosphorylation/dephosphorylation, acetylation/deacetylation. The control of the activity of the transcription machinery is usually brought about by regulatory factors binding upstream of the TATA box. In Fig. 2 this is exemplified for Hsf trimers making contacts to components of the basal transcription complex (arrows).
Fig. 3: Functional organization of heat stress transcription factors (Hsf) A: Survey of Hsfs from tomato (Lp), yeast (Sc), Drosophila (Dm) and human (Hs) B: Sequence details of essential functional elements of tomato HsfA2 (for further explanations see text)
7 Pi ATP
Fig. 4: The bacterial GroEL/GroES complex as model for a chaperone machine ( for explanations see text)
Hsp dimer interaction Hsp monomer
Hsp oligomer (precursor-HSG)
Heat-Stress Inactivated luciferase
Luciferase aggregate (degradation) Reactivated luciferase
40 nm particle of HSG complex
Fig. 5: Structure and function of the plant HSG complex The upper part of the figure shows the hs-dependent synthesis of Hsp17 class I and class II and their oligomerization to homododecamers. Based on the crystal structure of the related Methanococcus janaschii Hsp16.5 (Kim et al. 1998) we assume that a Hsp17 dimer formed by an extensive beta sheet of both subunits, is the basic building block of the oligomers (pictures for the hypothetic dimer interface and the oligomer were taken from Kim et al. 1998). Under hs both Hsp17(I) and Hsp17(II) assemble to high molecular weight complexes with 40 nm particles. These HSG complexes are assumed to serve as storage sites for housekeeping mRNPs and partially denatured proteins, e.g. luciferase. Dissociation of HSG complexes and renaturation of luciferase in the recovery needs interaction with the Hsp70/Hsp40 chaperone system.