Metabolic phenotypes of Saccharomyces cerevisiae mutants with ...

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the tps2 mutant had significant effects on fermentation rates, ... G6P, glucose 6-phosphate; PSP, purine salvage pathway; Rib-1P, ribose 1-phosphate; T6P, trehalose 6-phosphate. ..... data confirmed that the tps1 growth and fermentation phenotype .... activation, recovery of intracellular pH and proton expulsion were not due ...
Biochem. J. (2013) 454, 227–237 (Printed in Great Britain)

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doi:10.1042/BJ20130587

Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose 6-phosphate dynamics Thomas WALTHER*†‡1 , Narjes MTIMET*, Ceren ALKIM*, Am´elie VAX*, Marie-Odile LORET*†‡, Azmat ULLAH§2 , Carlos GANCEDO, Gertien J. SMITS§ and Jean Marie FRANC¸OIS*†‡ *Universit´e de Toulouse, INSA, UPS, INP, 135 Avenue de Rangueil, F-31077 Toulouse, France, †INRA, UMR792 Ing´enierie des Syst`emes Biologiques et des Proc´ed´es, F-31400 Toulouse, France, ‡CNRS, UMR5504, F-31400 Toulouse, France, §Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, Faculty of Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands, and Instituto de Investigaciones Biomedicas “Alberto Sols”, CSIC-UAM, Madrid 28029, Spain

In Saccharomyces cerevisiae, synthesis of T6P (trehalose 6phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H + ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein

from Yarrowia lipolytica, nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H + ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism.

INTRODUCTION

[2,5]; (ii) the trehalose pathway may serve to regenerate phosphate via the consecutive action of Tps1 and T6P phosphatase (Tps2); and (iii) the protein Tps1 itself may undergo physical interactions with the hexokinases and/or sugar transporters, thereby decreasing the rate of sugar phosphorylation. The experimental evidence accumulated in several studies argues against the notion that any of the above-mentioned models (i–iii) alone can fully explain the role of Tps1 or T6P. Notably, overexpression of hexokinases insensitive to T6P does not affect growth on glucose and has only very minor effects on the short-term response to glucose exposure [6]. A mutant defective in trehalose phosphatase, TPS2, grows without significant differences to wild-type strains on glucose or fructose, arguing against the trehalose-dependent phosphate recovery hypothesis [2,7]. No experimental data is available to support the hypothesis of physical interactions of Tps1 with hexokinase or sugar transporters. Furthermore, deletion of TPS1 entails sporulation defects [1,8] and causes secretion of inositol [9], thus provoking phenotypes that appear to be completely unrelated to glucose metabolism and the function of hexokinase. Taken together, these results indicate that our understanding of Tps1/T6P function is incomplete and requires further analysis. In the present study we set out to investigate the metabolic response of mutants with altered capacities to accumulate T6P. Our main findings were: (i) a tps1 strain irreversibly converts its entire adenosine nucleotide pool into inosine upon exposure to glucose; (ii) growth on fermentable carbon sources, plasma membrane H + -ATPase activation, and fermentation rates are not affected in yeast cells having a more than 10-fold smaller T6P

The yeast Saccharomyces cerevisiae is currently the most frequently used organism for the production of bioethanol; however, despite its wide use as an industrial production organism and as a scientific model, our understanding of glycolytic regulation in this yeast is still incomplete. This inevitably limits our capacity to further improve its fermentation performance on glucose, and to rationally implement more efficient fermentation pathways for the utilization of non-naturally used sugars such as xylose and arabinose. A crucial element controlling fermentative metabolism in S. cerevisiae is the T6P (trehalose 6-phosphate) synthase, encoded by TPS1. Deletion of this gene abolishes not only production of the reserve carbohydrate trehalose, but renders the mutants incapable of growing on fermentable carbon sources such as glucose and fructose [1]. Despite leaving the chemical capacity of the cells to transform glucose into ethanol unchanged (activities of all glycolytic enzymes are present and virtually unchanged), tps1 mutants become depleted of ATP within less than 1 min upon exposure to glucose, and exhibit hyperaccumulation of phosphorylated sugars upstream of Gapdh (glyceraldehyde-3-phosphate dehydrogenase), depletion of intracellular phosphate, absence of cAMP signalling and lack of repression of gluconeogenic enzymes [1–3]. The exact mechanism by which Tps1, or its reaction product (T6P), control glycolytic function is still matter of debate. Three models exist that explain the observed phenotypes [4]: (i) T6P slows down sugar phosphorylation upon glucose addition by inhibiting hexokinase 2

Key words: fermentation, glycolysis, hexokinase, metabolic regulation, trehalose, yeast.

Abbreviations used: DW, dry weight; F16bP, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; PSP, purine salvage pathway; Rib-1P, ribose 1-phosphate; T6P, trehalose 6-phosphate. 1 To whom correspondence should be addressed (email [email protected]). 2 Present address: Department of Food Science, Government College University Faisalabad, Faisalabad, Pakistan  c The Authors Journal compilation  c 2013 Biochemical Society

228 Table 1

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List of yeast strains used in the present study

Strain

Relevant genotype

Reference

Wild-type JF1469 JF1447 tps1 Yl-TPS1 tps2 amd1 tps1 amd1 hxk2 tps1 tps2 tps1 hxk2 pgm1,2,3

CEN.PK133-5D Mat a ura3-52 Mat α ura3-52 tps1Δ::URA3- hisG Mat α ura3-52 tps1Δ::hisG Mat a ura3-52 tps1Δ::URA3- hisG Mat a ura3-52 tps1Δ::hisG pCLF-Yl-TPS1 Mat a ura3-52 tps2Δ::kanMX4 Mat a ura3-52 amd1Δ::kanMX4 Mat a ura3-52 tps1::URA3-hisG amd1Δ::kanMX4 Mat a ura3-52 hxk2Δ::kanMX4 Mat a ura3-52 tps1Δ::URA3-hisG tps2Δ::kanMX4 Mat a ura3-52 tps1Δ::URA3-hisG hxk2Δ::kanMX4 Mat a ura3-52 pgm1Δ::kanMX4 pgm2Δ::kanMX4 pgm3Δ::natMX4

[11] [13] [13] [13] The present study The present study The present study The present study The present study The present study The present study [17]

content than wild-type cells; whereas (iii) permanent hyperaccumulation of T6P entails cytosolic alkalization and strongly reduced growth rates on non-fermentable carbon sources. Taken together the results of the present study demonstrate that different regulatory processes are affected by T6P in a concentrationdependent manner, and that tight regulation of T6P levels is necessary to assure optimal growth on fermentable and nonfermentable carbon sources.

MATERIALS AND METHODS Strains and cultivation conditions

Glucose-limited growth was mimicked by cultivating the strains in a medium containing 10 g/l trehalose (Sigma), 5 g/l (NH4 )2 SO4 and 1.76 g/l YNB (yeast nitrogen base without amino acids and ammonium sulfate; Difco), as well as uracil at a final concentration of 100 mg/l to complement the ura − auxotrophy of the strains. The medium was buffered at pH 5.5 using 50 mM potassium phthalate. Cells were cultivated in Erlenmeyer flasks at 30 ◦ C, and shaken on a rotary shaker (Infors) at 200 rev./min. When yeast cultures reached a biomass concentration of 1 g DW (dry weight)/l, glucose was added at a final concentration of 10 g/l using a concentrated stock solution of 400 g/l [10]. All mutants analysed in the present study were derived from the haploid strain S. cerevisiae CEN.PK133-5D ura3-52 (referred to as wild-type) [11] (Table 1). Disruption cassettes containing the kanMX4 cassette flanked by ∼ 200 bp homologous with the upstream and downstream regions of the targeted ORF were amplified from single deletion mutants of the EUROSCARF collection [12]. They were transformed into the wild-type strain using standard genetic protocols to obtain mutant strains hxk2, amd1 and tps2. The double mutants tps1 hxk2, tps1 amd1 and tps1 tps2 were obtained by crossing and sporulation of the amd1, hxk2 and tps2 single mutants with strain JF1469 [13] which carried a deletion of TPS1. Gene disruptions were verified by PCR. Plasmid pCLF1 [14] was used for expression of the Yarrowia lipolytica Tps1 protein under the control of the Sc-PGK1 promoter. The plasmid was transformed into strain JF1477 wherein the URA3 marker gene used for the TPS1 disruption was previously excised by counterselection on 5-FOA (5-fluoro-orotic acid) [13]. The same strains were transformed with pYES2-pACT1-pHluorin [15] and cultivated on a minimal defined medium with low fluorescence [16], with no uracil supplemented, buffered at pH 5.0 with 25 mM potassium phthalate, and containing the carbon source indicated.  c The Authors Journal compilation  c 2013 Biochemical Society

The methods for metabolite extraction and quantification have been described recently [17]. Briefly, cells were separated from the culture medium by filtration, and cellular metabolism was instantaneously quenched in hot ethanol (75 %, 80 ◦ C). Processing of the samples was performed as described previously [18,19]. Metabolites were quantified using an ICS-3000 system from Dionex equipped with an automatic eluent (KOH) generator system (RFIC, Dionex), an autosampler (AS50, Dionex), a photo diode array detector (Ultimate 3000, Dionex), a conductivity detector (part of ICS-3000, Dionex) and a mass-sensitive detector (MSQ Plus, Thermo Scientific) running in ESI mode [nitrogen pressure was 90 psi (1 psi = 6.9 kPa), capillary voltage was 3.5 kV and probe temperature was 450 ◦ C]. Chromatography conditions were the same as in [17]. Intracellular phosphate was measured by a colorimetric method, as described in [20]. Assay of extracellular metabolites, ATPase activity and intracellular pH

Ethanol production rates were measured by withdrawing samples from the cultures at 5–10 min intervals during the first hour after glucose addition, and quantifying ethanol using an enzymatic assay (Roche). For enzymatic assays, cells were separated from the culture medium by filtration over a 0.45 μm polyamide membrane (Sartorius) and transferred to liquid nitrogen using a spatula. Cells were stored at − 80 ◦ C until further analysis. Membrane fractions of the cells were isolated and in vitro ATPase activity was assayed (pH 7, 2 mM ATP) as described previously [21]. Proton expulsion after glucose addition was estimated by harvesting exponentially growing trehalose cultures by centrifugation (4000 rev./min for 2 min at 30 ◦ C; Beckmann Coulter, rotor S4180), washing the cells twice in deionized water, and adjusting to a cell density of 0.4 g DW/l in deionized water. The decrease in pH following glucose addition was measured at 1–10 min intervals during the first hour following glucose addition using a pH meter. Cytosolic pH (pHi ) was determined as described previously [15]. Briefly, strains expressing the ratiometrically pH-sensitive GFP variant pHluorin [22] were precultured in Verduyn medium [16] containing galactose. For analysis, strains were inoculated into Verduyn medium containing 10 g/l trehalose as the sole carbon source, and grown to a D600 of approximately 0.4 [as measured in a Fluorostar Optima microplate reader (Isogen, BMG Labtech)]. At this time they were transferred to CELLSTAR black polystyrene clear-bottomed 96-well plates, and the pH was monitored by determination of the ratio of fluorescence emission at 510 nm upon excitation at 390 and 470 nm. This in vivo ratio was converted into a pH value based on a calibration curve of the same strain at a similar D600 , permeabilized with 100 μg/l digitonin for 10 min, washed and suspended in phosphate/citrate buffers at a range of pH values between 5.0 and 8.5. RESULTS Construction of mutant strains altered in their capacity to synthesize T6P

To study the impact of altered capacity to synthesize T6P on metabolic regulation, we constructed a panel of mutants affected in T6P metabolism. Strains carrying deletions of TPS1, encoding the T6P synthase, or of TPS2, encoding the T6P phosphatase, are defective in T6P synthesis or degradation respectively (Figure 1) (reviewed in [23]). Mutants with decreased phosphoglucomutase activity are

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Figure 2 T6P accumulation in strains mutated in trehalose pathway genes after exposure of respiring trehalose-grown cells to 10 g/l glucose Figure 1 Metabolic pathways implicated in trehalose synthesis and degradation

Glucose was added at t = 0 min. Values are means + − S.D. Results represent the average of at least two independent experiments. wt, wild-type.

impaired in the interconversion of G6P (glucose 6-phosphate) and G1P (glucose 1-phosphate) (Figure 1), and were previously shown to exhibit slowed-down accumulation of trehalose in response to heat shock [24]. In order to test whether T6P accumulation during the respiro-fermentative transition could be significantly decreased by impeding phosphoglucomutase function, we studied a pgm1 pgm2 pgm3 triple deletion mutant (denoted pgm1,2,3) [24,25]. The trehalose content of Y. lipolytica is very low even though this yeast expresses a functional T6P synthase [14]. To test whether T6P accumulation in S. cerevisiae could be decreased when replacing its natural T6P synthetase by Yl-Tps1 (Y. lipolytica Tps1) we constructed a tps1 mutant strain (denoted Yl-TPS1) expressing the Y. lipolytica TPS1 gene from a plasmid. The capacity of the constructed mutants to produce T6P was characterized during the respiro-fermentative transition. To mimic glucose-limited respiratory growth, the strains were cultivated in shake flasks on minimal medium containing trehalose whose Ath1-catalysed cleavage into glucose was rate-limiting for growth [10,26]. The metabolic response to the relief from glucose limitation was investigated by adding glucose at a final concentration of 10 g/l and following intracellular metabolite dynamics. In the wild-type strain, T6P concentration increased from 0.5 μmol/g DW to 8 μmol/g DW during the first 2 min after administration of glucose (Figure 2). Within the following 30 min, the T6P level slowly declined and eventually stabilized at concentrations similar to those measured before glucose addition. As reported previously, the tps1 strain was completely defective in T6P production, whereas T6P concentrations in the tps2 mutant were already high (20 μmol/g DW) under fully respiratory conditions (Figure 2) [2]. Following exposure to glucose, the T6P content of the tps2 mutant further increased by approximately the same value (∼ 8 μmol/g DW) as in wild-type cells and remained constant during the observed time span of 60 min (1 h time point not shown). T6P accumulation in the pgm1,2,3 mutant was attenuated upon exposure to glucose, reaching only one-fourth of the wild-type concentrations at 2 min, and approximately onethird after 5 and 10 min following glucose addition (Figure 2). These data demonstrate that G1P synthesis was significantly reduced and has become the rate-limiting step in T6P synthesis under these conditions. However, the results of the present study also indicate that G1P synthesis is still significant and probably catalysed by the essential proteins Sec53 [24] and/or Pcm1 [24,27]. Interestingly, the Yl-TPS1 strain accumulated only approximately one-tenth of the T6P observed in wild-type

cells. These data demonstrated the feasibility to modulate T6P accumulation in response to glucose by introducing appropriate mutations. The mutants described were used to study the impact of altered T6P accumulation on respiratory and fermentative growth, and on the metabolic regulation during the respiro-fermentative transition. Residual accumulation of T6P restores growth and fermentation on fermentable carbon sources

The rate of ethanol production upon glucose addition was not affected either in the mutant that hyperaccumulated T6P (tps2), or in the mutants that had a mild (pgm1,2,3) or strong (YlTPS1) decrease in the ability to synthesize T6P (Figure 3A). Consistently, growth on fermentable carbon sources under stable conditions (Figure 3B), and during the start-up phase immediately after glucose or fructose addition (Supplementary Figure S1 at http://www.biochemj.org/bj/454/bj4540227add.htm) was not affected in these mutant strains. Only in the tps1 mutant did the fermentation rate drop significantly to less than one-half of that observed for the other strains (Figure 3A). This residual fermentative capacity was not sufficient to confer growth on fermentable carbon sources (Figure 3B and Supplementary Figure S1). However, we noticed that after prolonged incubation times our tps1 mutant eventually started to grow on glucose but not on fructose. This observation is in agreement with results of other groups investigating tps1 mutants in the CEN.PK background (E. Boles, personal communication). We found that the ability of S. cerevisiae to produce ethanol and to grow on the fermentable carbon sources glucose and fructose is insensitive to the actual amount of accumulated T6P over a wide range of intracellular concentrations. From the fact that a strongly increased concentration of T6P did not cause a decrease in glycolytic rate, we conclude that the in vivo inhibition of hexokinase by T6P is negligible for the regulation of glycolytic flux in the presence of high glucose concentrations. Hyperaccumulation of T6P causes growth phenotype on non-fermentable carbon sources

Mutants with decreased capacity to synthesize T6P were not affected in their ability to grow under respiratory conditions (Figure 3C). In contrast, the tps2 mutant strain grew significantly slower than wild-type cells on non-fermentable carbon sources (Figure 3C). This growth phenotype was due to the unscheduled  c The Authors Journal compilation  c 2013 Biochemical Society

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Figure 3

T. Walther and others

Specific ethanol formation and growth rates of strains mutated in trehalose pathway genes

(A) Specific ethanol production rates during the first hour after exposure of respiring trehalose-grown cells to 10 g/l glucose. Specific growth rates on (B) fermentable carbon sources and (C) non-fermentable carbon sources. Values are means + − S.D. Results represent the average of at least two independent experiments. wt, wild-type.

increased accumulation of T6P as witnessed by the normal growth of the tps1 tps2 double mutant (Figure 3C) which does not produce T6P.

Altered T6P accumulation has a strong impact on the dynamics of glycolytic metabolites during the respiro-fermentative transition

To understand the effect of changes in T6P dynamics on metabolism, we analysed the dynamic changes of phosphorylated sugars in the different mutants affected in T6P dynamics in response to glucose (Figure 4). In agreement with previous studies [1,2,10] we found that wild-type cells exposed to a sudden relief from glucose limitation exhibited a persistent increase in F16bP (fructose 1,6-bisphosphate) and of pentose 5-phosphate sugars (xylose 5-phosphate, ribose 5-phosphate and and xylulose 5-phosphate could not be separated by the applied analytical methods and were measured as a single pool denoted pent-5P). These cells transiently accumulated G6P and F6P (fructose 6phosphate) (see e.g. [10,28]), and showed a modest and transient accumulation of Rib-1P (ribose 1-phosphate) (Figures 4A–4C) [17]. As reported previously [1,29], addition of glucose to the tps1 mutant was accompanied by the hyperaccumulation of the phosphorylated sugars F16bP, G6P and F6P. G6P and F6P increased within less than 1 min after the addition of glucose to a constant concentration of ∼ 7 μmol/g DW and ∼ 2 μmol/g DW respectively, whereas F16bP continuously increased to concentrations higher than 50 μmol/g DW during the observed time span (Figures 4D and 4E). The glycolytic profile of the Yl-TPS1 strain, which has a very weak capacity to produce T6P, strongly resembled that of the tps1 mutant. A pronounced and persistent accumulation of the phosphorylated sugars G6P, F6P and F16bP was observed, reaching concentrations that were 2–3-fold higher than in wildtype cells (Figures 4G and 4H). However, contrary to the tps1  c The Authors Journal compilation  c 2013 Biochemical Society

mutant, F16bP accumulation eventually stopped in the Yl-TPS1 strain, reaching only 30 μmol/g DW (Figure 4G). The pgm1,2,3 triple mutant, which exhibited a delayed and 4fold reduced accumulation of T6P in response to glucose, behaved very similarly to wild-type cells (Figures 4J–4L). However, owing to the deletion of PGM3, which is the major phosphoribomutase in yeast [17], this mutant was defective in Rib-1P to Rib-5P conversion and, consequently, hyperaccumulated Rib-1P which was released upon glucose-induced cycling of adenine nucleotides over the PSP (purine salvage pathway) [10]. Finally, the tps2 mutant that presented permanent and increased T6P accumulation both under respiratory conditions as well as after glucose addition, transiently accumulated phosphorylated sugars G6P and F6P similarly to wild-type cells (Figure 4N). However, F16bP concentrations peaked and eventually stabilized at concentrations that corresponded to only ∼ 60 and 40 % respectively of the concentrations observed in the wild-type strain (Figure 4M). As shown in Figure 5, the concentration of free inorganic phosphate (Pi ) strongly dropped in all mutants immediately upon addition of glucose. This drop was most pronounced in the tps1 mutant, whereas the behaviour of wild-type, Yl-TPS1 and tps2 was nearly indistinguishable. The difference in Pi between tps1 and wild-type cells was up to 10 μmol/g DW during the first 5 min after the addition of glucose, but became much smaller during the following 20 min. Curiously, the decrease in Pi in the pgm1,2,3 strain was less pronounced than in all other mutants tested. The methods for metabolite extraction and phosphate quantification applied in the present study did not allow for distinguishing between cytosolic or vacuolar phosphate. Therefore it remains unclear which portion of the Pi quantified in the mutants tested was located in the cytosol and, thus, actually available as a substrate for Gapdh. However, in agreement with previous studies [1,29], the results of the present study showed that the tps1 mutant experienced the strongest decrease in Pi , making the lack of free

T6P in metabolic regulation

Figure 4

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Dynamics of phosphorylated sugars in strains mutated in trehalose pathway genes after exposure to glucose

Concentrations of F16bP (left-hand panels), G6P and F6P (middle panels), and Rib-1P and the pentose 5-phosphate pool (pent-5P) (right-hand panels). Strains were grown on trehalose and exposed to 10 g/l glucose. Glucose was added at t = 0 min. Values are means + − S.D. Results represent the average of two independent experiments. wt, wild-type.

phosphate regeneration in the tps1 mutant a probable cause of the malfunction of glycolysis and accumulation of metabolites upstream of Gapdh. The tps1 mutant irreversibly converts adenine nucleotides into inosine in response to glucose

Figure 6 compares the kinetics of adenine nucleotides, the PSP intermediates inosine and IMP, and the sum ([Sum AXP] = [ATP] + [ADP] + [AMP]) of these metabolite pools in wild-type and mutant cells following the addition of glucose to trehalose grown cultures. The transient drop of ATP in wild-type cells was not accompanied by an increase in ADP or AMP (Figure 6A) [10]. Instead, AXP nucleotides were

converted into IMP and inosine whose concentration transiently increased after the addition of glucose (Figures 6B and 6C). After the re-establishment of energetic equilibrium, these PSP metabolites were recycled into the AXP pool. The tps1 mutant exhibited a pronounced and irreversible decrease in ATP after exposure to excess glucose (Figure 6D), as shown also by others [1,3]. We now demonstrate that the tps1 strain does not only experience a decrease in ATP, but irreversibly converts the entire adenine nucleotide pool into inosine (Figures 6D and 6F). Abolishing the accumulation of inosine in tps1 cells by deleting the AMP deaminase encoding gene AMD1 [10,30] caused irreversible accumulation of AMP (Supplementary Figures S2A– S2C at http://www.biochemj.org/bj/454/bj4540227add.htm), but did not restore growth on fermentable carbon sources. These  c The Authors Journal compilation  c 2013 Biochemical Society

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insensitive to these changes in metabolite dynamics as witnessed by the unchanged ethanol production rates in most tested mutants. In particular, strongly decreased ATP concentrations and hyperaccumulation of phosphorylated sugars in the Yl-TPS1 strain were very similar to the tps1 mutant, yet growth and ethanol formation remained unaffected.

Activation of plasma membrane ATPase and regulation of intracellular pH in response to glucose are defective only in the tps1 mutant

Figure 5 Intracellular phosphate concentration in strains mutated in trehalose pathway genes after exposure of respiring trehalose-grown cells to 10 g/l glucose Glucose was added at t = 0 min. Values are means + − S.D. Results represent the average of two independent experiments.

data confirmed that the tps1 growth and fermentation phenotype is not due to accidental loss of AXP nucleotides, or due to the incapacity to recycle inosine, but the consequence of an energetic disequilibrium caused by the deregulation of glycolysis. Addition of glucose to the Yl-TPS1 strain also caused a rapid drop in ATP as in the tps1 mutant but, in contrast with the latter, Yl-TPS1 cells were able to re-establish part of their ATP (20 % of initial content after 30 s, 50 % at 30 min after the addition of glucose). This re-establishment was also accompanied by restoration of the adenylic energy charge {e-charge = ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP])} (Figure 7). The pgm1,2,3 triple mutant exhibited ATP dynamics very similar to wild-type cells. This indicated that a 4-fold reduction in the T6P levels had no impact on the energetic equilibrium (Figures 6J and 7). Delayed inosine recycling in the pgm1,2,3 strain (Figure 6K) can be explained by a reduced activity of the purine nucleoside phosphorylase, Pnp1, due to the inhibition by Rib-1P which hyperaccumulated under this condition in this mutant (see Figure 4L). This suggestion is based on the facts that Rib-1P is a competitive inhibitor of Pnp1 in humans [31], and that the thermodynamic equilibrium of the reaction favours inosine formation under standard conditions [32]. In favour of this explanation was the finding that a pgm1,2 double mutant did not accumulate Rib-1P and exhibited inosine recycling like in wildtype cells [17]. By extension, Rib-1P accumulation is absent in the tps1 strain (see Figure 4F) and, therefore, failure of inosine recycling in the tps1 mutant must be due to a different mechanism. Finally, tps2 cells also exhibited a transient decrease in ATP with a concomitant rise of inosine, but the changes in these metabolic pools were much less pronounced than in wild-type cells (Figures 4M–4O), suggesting that the hyperaccumulation of T6P is responsible for the altered metabolite dynamics in the tps2 mutant. Indeed, deletion of TPS1 in a tps2 mutant resulted in AXP and inosine dynamics identical with that in the tps1 mutant (results not shown). Taken together the results of the present study demonstrate that the concentration of T6P has a major impact on the dynamics of glycolytic metabolites and AXP nucleotides during the respirofermentative transition. Upon decreasing T6P concentrations more phosphorylated sugars are accumulated and ATP recovery is slowed down. Increased T6P attenuates both phosphorylated sugar accumulation and the perturbation of energetic equilibrium. However, it also became clear that glycolytic rate is largely  c The Authors Journal compilation  c 2013 Biochemical Society

Insufficient regeneration of free phosphate in the tps1 mutant was earlier proposed to be the consequence of impaired trehalose formation which releases 3 mol of phosphate per mol of trehalose produced (Figure 1). However, glycolytic flux under fully fermentative conditions is 1–2 μmol/min per mg of protein [33] which is 50–100-fold higher than the comparatively small activity of Tps1-dependent trehalose synthesis capacity (∼ 10–30 nmol/min per mg of protein [34]). In addition, Tps2 requires Pi for full activity [35]. Taking these arguments together it appears unlikely that phosphate regeneration can be assured by the trehalose pathway alone. Thus if it is limited Pi regeneration that causes the metabolic behaviour, other systems contributing to the release of phosphate must also be impaired in the tps1 mutant. Plasma membrane H + -ATPase, encoded by PMA1 [36], participates in the maintenance of near-neutral cytosolic pH by the expulsion of excess H + protons. This process is energy-driven and requires the hydrolysis of one molecule of ATP to ADP and phosphate per proton expulsed [37]. Pma1 is activated in response to glucose which increases the maximum specific activity of the enzyme, lowers its K m for ATP and changes its pH optimum [21]. Given that exposure of respiring cells to glucose causes transient acidification of the cytosol [38,39] and triggers proton expulsion [40,41], plasma membrane ATPase thus directly contributes to the regeneration of cytosolic phosphate by hydrolysing ATP. Therefore activation of Pma1, proton expulsion and intracellular pH were monitored during the respiro-fermentative transition in wild-type cells and in mutants affected in T6P metabolism. Wildtype cells responded to the exposure to glucose by a ∼ 3-fold increase in plasma membrane ATPase activity (Figure 8A). Pma1 activation was abolished in the tps1 mutant (Figure 8A), whereas this activation was not impaired in mutants with low (Yl-TPS1) or elevated (tps2) T6P (Figure 8B). Moreover, lacking H + -ATPase activation in the tps1 strain was reflected by the observations that its proton expulsion rate following glucose administration was only approximately one-half compared with all other mutants tested (Figure 8C), and that intracellular pH dropped to lower levels and recovered much slower than in the other mutants tested (Figure 9). It is of note that impaired plasma membrane ATPase activation, recovery of intracellular pH and proton expulsion were not due to decreased ATP levels in the tps1 mutant. The YlTPS1 strain exhibited a similar drop in ATP immediately after the addition of glucose as a tps1 mutant, yet displayed full activation of plasma membrane ATPase, and near wild-type proton expulsion and cytosolic pH recovery rates (Figures 8B, 8C and 9A). Since these results indicated that the plasma membrane H + ATPase activity contributed less to ATP hydrolysis and phosphate recovery in the tps1 mutant, we tested whether inhibition of plasma membrane H + -ATPase in wild-type cells during the respiro-fermentative transition could provoke a metabolic phenotype that resembles the effect of the tps1 deletion. Trehalose-grown wild-type cells were either: (i) incubated in the presence of the plasma membrane ATPase inhibitor ebselen (100 μM) for 1 min before the addition of glucose, or (ii) glucose

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Figure 6

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Dynamics of adenine nucleotides and purine salvage pathway metabolites in strains mutated in trehalose pathway genes after exposure to glucose

Intracellular concentrations of individual adenosine nucleotides (left-hand panels), IMP and inosine (middle panels), and the sum of adenine nucleotides ([Sum AXP] = [ATP] + [ADP] + [AMP]) and the sum of AXP nucleotides and selected PSP metabolites ([Sum PSP] = [Sum AXP] + [IMP] + [inosine]) (right-hand panels). Strains were grown on trehalose and exposed to 10 g/l glucose. Glucose was added at t = 0 min. Values are means + − S.D. Results represent the average of two independent experiments. wt, wild-type.

and ebselen were administered at the same time. Whereas protocol (i) resulted in complete and irreversible loss of ATP even before the addition of glucose, no changes in ATP dynamics could be observed when following protocol (ii) (results not shown). Thus no definite answer could be provided to the question whether impaired regulation of Pma1 significantly contributes to the tps1 phenotype during the respiro-fermentative transition.

T6P concentration has an impact on the regulation of intracellular pH

During the respiro-fermentative transition, cytosolic pH in the tps2 mutant increased instantaneously, whereas all other mutants

including the wild-type experienced a significant drop in pH following glucose addition (Figure 9). In addition, the initial pH value on trehalose as well as the final pH value after the addition of glucose were ∼ 0.2 pH units higher in the tps2 mutant than in wild-type cells (Figure 9B). Therefore intracellular pH was also monitored under steady growth conditions on different carbon sources. As depicted in Figure 10, the tps2 mutant exhibited significantly higher internal pH than wild-type cells on both fermentable and non-fermentable carbon sources. In contrast, tps1 mutant cells had significantly lower intracellular pH compared with the wild-type strain on the fermentable sugars glucose and galactose (data on glucose were obtained for cells that eventually picked up growth after prolonged incubation times), and exhibited no significant deviation from wild-type behaviour on trehalose.  c The Authors Journal compilation  c 2013 Biochemical Society

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Figure 7 Energy charge in strains mutated in trehalose pathway genes after exposure of respiring trehalose-grown cells to 10 g/l glucose Glucose was added at t = 0 min. Values are means + − S.D. Results represent the average of two independent experiments.

We neither observed an increased plasma membrane H + -ATPase activity in the tps2 mutant, nor increased in vitro ATPase activity upon the addition of 5 mM T6P to the assay buffer (results not shown). These results suggest that T6P content of cells has an impact on the regulation of intracellular pH by an as yet unknown mechanism, causing alkalization of the cytosol upon T6P accumulation and acidification during growth on fermentable carbon sources when T6P is absent. DISCUSSION

T6P synthase encoded by TPS1 is required for growth on most fermentable carbon sources. This requirement has been ascribed to the observation that T6P inhibits hexokinase in vitro which

Figure 8

was suggested to prevent uncontrolled sugar phosphorylation and fatal depletion of ATP [2,4,5]. However, we found that yeast cells can tolerate an up to 10-fold decrease and a 4-fold increase in T6P without any noticeable impact on glycolytic flux or on the ability to grow on fermentable carbon sources. These results are not in favour of the hypothesis that inhibition of hexokinase is required to assure growth of S. cerevisiae on fermentable carbon sources. In addition, we found that hyperaccumulation of T6P entailed both a hitherto unnoticed growth phenotype on nonfermentable carbon sources and alkalization of the cytosol. These data provided further evidence that T6P must have additional, and probably more important, functions than the inhibition of sugar phosphorylation by hexokinase. Motivated by these findings, a detailed phenotypic analysis of mutants with altered capacity to synthesize and accumulate T6P was carried out. We showed that different physiological processes respond to the presence or absence of T6P in a concentrationdependent manner. Although growth, ethanol production, plasma membrane H + -ATPase activation and cytosolic pH largely followed wild-type behaviour when yeast cells accumulated trace amounts of T6P (see results of the Yl-TPS1 strain), such a low T6P content still caused strong accumulation of phosphorylated sugars and the pronounced perturbation of ATP levels in response to glucose. In this regard, the results of the present study differed from observations made upon expression of the bacterial T6P synthase otsA [7], and upon expression of a leaky TPS1 allele, named byp1-3 [42], in a strain carrying a deletion of the TPS1 gene. Both studies reported that growth on fermentable carbon sources and fermentation rates were not re-established even though significant amounts of T6P were produced. Given that the TPS1 deletion is causing a growth defect on glucose that depends on the strain background (E. Boles, personal communication; see also [43]), it is possible that the use of different strain backgrounds in these studies and the present study can explain the observed

Regulation of plasma-membrane H + -ATPase in strains mutated in trehalose pathway genes after exposure to glucose

(A) Specific plasma-membrane ATPase activity in wild-type and tps1 strains after exposure of respiring trehalose-grown cells to 10 g/l glucose. (B) Fold change of specific ATPase activity in strains mutated in trehalose pathway genes before and 60 min after the addition of 10 g/l glucose to respiring trehalose-grown cells. (C) Specific proton expulsion rates of different mutants after exposure of respiring trehalose-grown cells to 10 g/l glucose. Values are means + − S.D. Results represent the average of at least two independent experiments. wt, wild-type.  c The Authors Journal compilation  c 2013 Biochemical Society

T6P in metabolic regulation

Figure 9

235

Cytosolic pH in different trehalose-grown strains after exposure to 10 g/l glucose

Glucose was added at t = 0 min. Values are means + − S.D. Results represent the average of at least three independent experiments.

Figure 10

Cytosolic pH during unperturbed cultivation on different carbon sources

Values are means + − S.D. Results represent the average of at least three independent experiments.

differences. Alternatively, the introduction of the Yl-Tps1 protein may have rescued an essential function that is exerted by Tps1 itself rather than by its reaction product T6P. The results of the present study do not allow excluding this possibility. However, since no experimental data are available so far that support the idea of an unknown function of the Tps1 protein, the authors prefer to ascribe the observed phenotypes to the altered dynamics of T6P in the Yl-TPS1 strain. Remarkably, our analysis revealed that tps1 mutants irreversibly converted their entire adenine nucleotide pool into inosine when exposed to glucose. Since growth on fermentable carbon sources and ATP homoeostasis were not restored in the tps1 amd1 double mutant, we conclude that the tps1 phenotype is not due the inability to recycle inosine back into AXP nucleotides, but indeed due to an energetic disequilibrium. Our observations also ruled out Rib-1P accumulation and the resulting inhibition of Pnp1 as the cause for abolished inosine recycling in the tps1 strain. We propose that depletion of cytosolic phosphate is the major cause for inosine accumulation in tps1 cells since Pnp1 requires phosphate to catalyse the conversion of inosine into hypoxanthine. This view is corroborated by the results of Boer et al. [44] who detected inosine accumulation in phosphate-starved chemostatgrown yeast cells, and our own results (also compare with [1]) which show that the tps1 strain exhibits the strongest decrease in phosphate concentration in response to glucose. The inability of tps1 mutants to maintain sufficiently high concentrations of free cytosolic phosphate to sustain Gapdh was previously proposed to be a major cause of the tps1 phenotype

[23]. Since the capacity of the trehalose pathway is too small to regenerate sufficient amounts of phosphate, we directed our attention to the regulation of the plasma membrane H + -ATPase as an alternative phosphate-regenerating system. Very early studies of the phenotypes of tps1 reported that this mutant failed to activate plasma membrane H + -ATPase in response to glucose [1]. The present study confirmed these data and refined the analysis by demonstrating that absence of H + -ATPase activation in tps1 was not due to the mere decrease in ATP (the Yl-TPS1 strain properly activated H + -ATPase despite low ATP), and that decreased in vitro H + -ATPase activity actually coincided with acidification of the cytosol. It is important to note that the drop in cytosolic pH in the tps1 mutant was not due to the accumulation of phosphorylated sugars and the concurrent release of protons. The Yl-TPS1 strain accumulated nearly as much phosphorylated sugars as the tps1 mutant, yet it did not exhibit the drastic decrease in the cytosolic pH. In addition, studies on the origin of cytosolic acidification in response to glucose revealed that proton release caused by sugar phosphorylation is by far insufficient to explain the observed cytosolic acidification [45]. Although it remains unclear which process actually causes the pH decrease during the respiro-fermentative transition [45], we demonstrate that the tps1 strain exhibits delayed and incomplete restoration of cytosolic pH under fermentative conditions. In addition, the tps2 mutant which had elevated T6P levels exhibited cytosolic alkalization under all growth conditions tested, and did not experience the transient decrease in internal pH observed in wild-type cells upon glucose exposure. These observations suggest that high T6P concentration  c The Authors Journal compilation  c 2013 Biochemical Society

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causes increased plasma membrane H + -ATPase activity in vivo. However, since the presence of T6P did not increase the in vitro activity of isolated H + -ATPase, and since no increased activity of H + -ATPase isolated from the tps2 mutant was found, we conclude that T6P exerts its activating effect on Pma1 by a so far unidentified mediator. In any case, these results provide evidence that proton expulsion, and thus phosphate release due to ATP hydrolysis, are impaired in the tps1 mutant. Whether or not inadequate regulation of plasma membrane H + -ATPase or cytosolic pH may provide the missing explanation for the observed phenotypes of mutants with altered T6P metabolism remains unclear. However, it should be tested whether the ATP hydrolysing capacity of plasma membrane H + -ATPase suffices to compete with ATP consumption in the first phosphorylation steps of glycolysis. The present study also refined the analysis of the metabolic phenotype of the tps2 mutant which so far received comparatively little attention. In particular, our results are at odds with a previous report which found that the tps2 mutant exhibited an increase in ATP in response to glucose and had decreased growth and fermentation rates on this sugar [2]. We observed that ethanol formation immediately following glucose administration was not affected by the high T6P concentrations found in the tps2 mutant. Since glycolytic flux was not decreased in the tps2 strain, we conclude that inhibition of hexokinase by T6P was not ratelimiting for glycolytic flux under the experimental conditions used in the present study. This is in line with arguments of Bonini et al. [7] who pointed out that glucose phosphorylation by hexokinase is competitively inhibited by T6P (K i,T6P ∼ 50– 200 μM, depending on the particular hexokinase [5]), and that an increase in intracellular glucose concentration will largely override this inhibitory effect given the concentrations of T6P commonly observed in cells. Similar to the results of the present study, this group of authors found both a significant transient decrease in ATP in response to glucose, and a transient accumulation of phosphorylated sugars that was accompanied by a decrease in free intracellular phosphate [7]. However, in constrast with that study we observed that the decrease in ATP and the increase in phosphorylated sugars were much less pronounced than in wild-type cells, indicating that increased T6P concentrations had an impact on metabolite dynamics in the early adaptation phase to glucose. More importantly, we detected a hitherto unnoticed growth phenotype of tps2 mutants on nonfermentable carbon sources which was due to the permanently increased accumulation of T6P. In addition, the tps2 mutant had a significantly higher cytosolic pH on both non-fermentable and fermentable carbon sources. These results complemented a previous study where it was found that accumulation of T6P in the tps2 mutant caused an increase in both cytochrome c and cAMP content, which depended on functional Hxk2 [46]. The observations that permanently increased T6P accumulation has an impact on respiratory chain content and impairs respiratory growth, whereas the absence of T6P renders cells incapable of grow on fermentable carbon sources makes T6P a key regulatory molecule in the co-ordination of respiro-fermentative metabolism. It is tempting to speculate that accumulation of T6P primes cells for fermentative growth, thus rendering the adaptation phase to high-glucose conditions much shorter and perturbing growth on non-fermentable carbon sources. Given that T6P inhibits SnRK1 (Snf1-related protein kinase) in plants (reviewed in [47]), it will be interesting to see whether the growth phenotype of yeast on non-fermentable sugars is due to inhibition of Snf1 and/or its partners by T6P. The metabolic target of T6P which facilitates growth and ATP homoeostasis under fermentative conditions still remains to be identified.  c The Authors Journal compilation  c 2013 Biochemical Society

AUTHOR CONTRIBUTION Thomas Walther co-ordinated and oversaw the research, carried out part of the glucose pulse experiments, and wrote the paper. Narjess Mtimet and Ceren Alkim carried out glucose pulse experiments, the plasma membrane H + -ATPase assays, growth and ethanol formation assays, and strain constructions. Am´elie Vax and Marie-Odile Loret carried out metabolome analyses using IC-MS. Azmat Ullah carried out intracellular pH measurements. Gertien Smits oversaw and interpreted intracellular pH measurements, and contributed to the writing of the paper. Carlos Gancedo contributed to data interpretation and writing of the paper. Jean Marie Franc¸ois co-ordinated the project and contributed to data interpretation and writing of the paper.

FUNDING This work was supported, in part, by the ANR (Agence Nationale de la Recherche) Blanc [grant number 05-2-42128] and by the Region Midi Pyr´en´ees (France) [grant number 09003813]. C.G. was supported by the Spanish Ministry of Science and Innovation (MICINN) [grant number BFU2010-19628-CO2-02]. A.U. thanks the HEC (Higher Education Commission) Pakistan for funding.

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Received 29 April 2013/5 June 2013; accepted 14 June 2013 Published as BJ Immediate Publication 14 June 2013, doi:10.1042/BJ20130587

 c The Authors Journal compilation  c 2013 Biochemical Society

Biochem. J. (2013) 454, 227–237 (Printed in Great Britain)

doi:10.1042/BJ20130587

SUPPLEMENTARY ONLINE DATA

Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose 6-phosphate dynamics Thomas WALTHER*†‡1 , Narjes MTIMET*, Ceren ALKIM*, Am´elie VAX*, Marie-Odile LORET*†‡, Azmat ULLAH§2 , Carlos GANCEDO, Gertien J. SMITS§ and Jean Marie FRANC¸OIS*†‡ *Universit´e de Toulouse, INSA, UPS, INP, 135 Avenue de Rangueil, F-31077 Toulouse, France, †INRA, UMR792 Ing´enierie des Syst`emes Biologiques et des Proc´ed´es, F-31400 Toulouse, France, ‡CNRS, UMR5504, F-31400 Toulouse, France, §Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, Faculty of Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands, and Instituto de Investigaciones Biomedicas “Alberto Sols”, CSIC-UAM, Madrid 28029, Spain

Figure S1

Growth of trehalose-grown strains mutated in the trehalose pathway following addition of (A) 10 g/l glucose or (B) 10 g/l fructose

Figure S2

Dynamics of adenine nucleotides and purine salvage pathway metabolites in tps1 amd1 and tps1 hxk2 strains after exposure to glucose

Intracellular concentrations of individual adenosine nucleotides (left-hand panels), IMP and inosine (middle panels), and the sum of adenine nucleotides ([Sum AXP] = [ATP] + [ADP] + [AMP]) and the sum of AXP nucleotides and selected PSP metabolites ([Sum PSP] = [Sum AXP] + [IMP] + [inosine]) (right-hand panels) in tps1 amd1 and tps1 hxk2 double mutants after exposure of respiring trehalose-grown cells to 10 g/l glucose. Glucose was added at t = 0 min. Values are means + − S.D. Results represent the average of two independent experiments. Received 29 April 2013/5 June 2013; accepted 14 June 2013 Published as BJ Immediate Publication 14 June 2013, doi:10.1042/BJ20130587

1 2

To whom correspondence should be addressed (email [email protected]). Present address: Department of Food Science, Government College University Faisalabad, Faisalabad, Pakistan  c The Authors Journal compilation  c 2013 Biochemical Society