YEAST

VOL.

7: 367-378 (1991)

Induction of a Heat-Shock-Type Response in Saccharornyces cerevisiae Following Glucose Limitation NELLY BATAILLE, MATTHIEU REGNACQ AND HELIAN BOUCHERIE* Laboratoire de GPnPtique, UA CNRS 542, Avenue des Facultis, F-33405 Talence, France

Received 28 September 1990; revised 10 December 1990

The protein pattern of yeast cells which have arrested proliferation in response to glucose exhaustion is drastically different from that of exponentially growing cells (Boucherie, 1985). In this study, we used two-dimensional gel electrophoresis to characterize the protein events responsible for these alterations. We found that the induction of heatshock proteins is one of the major events responsible for these changes. This induction accounts for the synthesis of 18 of the 35 novel polypeptides observed in glucose-limited cells. It was shown to occur in combination with two other protein events: the derepression of carbon catabolite repressed proteins, which accounts for the synthesis of the other novel polypeptides, and an arrest of the synthesis of almost all the proteins present in exponentially growing cells. The time course of each of these events was determined by carrying out a detailed analysis of the pattern of proteins synthesized at various stages of a culture exhausting its glucose supply, and by the measurement of the rate of synthesis of individual polypeptides. The results showed in particular that the synthesis of most of the heat-shock proteins synthesized in glucose-limited cells was induced closely before glucose exhaustion, and that this synthesis was transient, climaxing by the time glucose was exhausted. Under the culture condition investigated, the entry into stationary phase associated with glucose limitation began several hours before glucose exhaustion. It was thus concluded that the observed induction of heat-shock proteins is directly related to the nutritional limitation and is independent from the arrest of cell proliferation. KEY WORDS - S. cerevisiae; heat-shock proteins;

INTRODUCTION

starvation.

and nutritional limitation. For example, hsp26 has been shown to be induced by carbon source In response to a number of environmental stresses, limitation (Kurtz et al., 1986), hspll8 by sulfur cells display an increased synthesis of a set of pro- starvation (Verma et al., 1988) and the heatteins known as heat-shock proteins (for review see inducible polyubiquitin gene, UB14, by nitrogen Lindquist and Craig, 1988).The high degree of con- as well as by carbon starvation (Finley er al., servation of these proteins among species as varied 1987). The possibility of a relationship between the as Escherichia coli, yeast, Drosophila and mammals synthesis of heat-shock proteins and nutritional suggests that they play a fundamental and important limitation is further supported by the observarole in cellular physiology. The function of heat- tion that stationary-phase cells or starved cells shock proteins, however, is unclear even if it is now are more thermoresistant than actively growing established that some of them play roles in transport cells (Schenberg-Frascino and Moustacchi, 1972). across membranes (Chirico et al., 1988; Deshaies et Indeed, thermoresistance is assumed to be the al., 1988), assembly and disassembly of macromole- consequence of increased expression of heat-shock cular complexes (Hemmingsen et al., 1988; Cheng proteins (McAlister and Finkelstein, 1980;Plesset et et al., 1989), transcription (Taylor et al., 1984), al., 1982). Recent results which demonstrate that translation (VanBogelen et al., 1983), proteolysis HsplO4 is required for induced thermotolerance are (Phillips et al., 1984) and carbon metabolism (Iida in favor of this hypothesis (Sanchez and Lindquist, and Yahara, 1985). 1990). Finally, the possibility of such a relationship In Saccharomyces cerevisiae, several results appears to be also supported by the observation indicate a relationship between heat-shock proteins that adenylate cyclase mutants which produce low CAMPlevels, constitutively synthesize certain heat*Author to whom correspondence should be addressed. 0749%503X/9 1/040367-12 $06.00 0 1991 by John Wiley & Sons Ltd

368 shock proteins (Shin et al.. 1987; Iida. 1988; Tanaka et al., 1988; Werner-Washburne et ul., 1989). Indeed, studies on cell cycle control in yeast suggest that nutrient starvation results in a low level of intracellular CAMP(Matsumoto et ul., 1985). In a previous report, we showed that glucose limitation is a particularly suitable condition for approaching questions concerning changes in protein synthesis when yeast cells undergo starvation (Boucherie, 1985). Drastic alterations in the two-dimensional pattern of protein synthesis were observed. In this study, using a modification of the basic two-dimensional gel electrophoresis technique and taking advantage of the recent progresses ofour knowledge in the two-dimensional protein map of S. cer-evisiae (Bataille et al., 1987, 1988), we report a detailed analysis of the proteins synthesized under this condition. We give evidence that a large number of proteins synthesized in response to glucose limitation correspond to heat-shockinduced polypeptides and we analyse the time course of their induction in relation to glucose exhaustion. MATERIALS A N D METHODS Yeast strain und culture conditions

The strain of S. cerevi.Tiue SKQ2n was obtained from Brian Cox (Oxford University) and has the following genotype: aja; udelj + ; + /ade2; + /hisl. Cells were grown in a medium which contained (per liter) 6.7g of yeast nitrogen base without amino acids (Difco Laboratories), 12.5mg each of all amino acids that occur naturally in proteins, except methionine and cysteine, and 20 mg of adenine and uracil. Carbon sources were glucose (20 g) or potassium acetate (10 g). The medium was buffered at pH 5.8 with 10 g of succinate and 6 g of NaOH. The same medium without amino acids was used when cultures were labelled with a mixture of [3H]amino acids. In all cases, 50-ml cultures were grown in 250-ml flasks. Growth was initiated at a cell concentration of 5 x lo4 cells per ml by using cells previously grown to stationary phase in YM-I medium (Hartwell, 1967). Cells were grown with rotary shaking at 22°C and growth was monitored by observing the optical density with a KlettSummerson colorimeter (one Klett unit corresponds to 1.5 x lo5cells/ml). Under these conditions, the culture generation time was 2 h. Cultures with an optical density of 80 to 100 Klett units corresponded to mid log-phase cultures.

N. B A T A I L I . ~ M. , R ~ N A C Q AND H. BOUCHERIE

Mmsurement of cellular parameters

Total number of cells and proportion of budded cells were determined by means of a hemacytometer by using sonicated samples. The number of viable cells was determined by plating sonicated samples on YEPD plates. YEPD solid medium contained (per liter) 10 g of yeast extract, 20 g of peptone, 20 g of agar (Difco) and 20 g of glucose. Glucose and ethanol determinations

The levels of glucose (mglml) in culture supernatants were determined by using Sigma glucose kit no. 115. The levels of ethanol (mgiml) in culture supernatants were determined by using Sigma ethanol assay kit no. 332 UV. An initial 1:15 dilution of the supernatant was necessary to stay within the limits of the assay. Rudioactive Iuhelling

To measure protein accumulation, samples ( 1 ml) of culture were labelled for 10 min with 0.15 pCi of [35S]methionine (610 mCi/mmol; Amersham Corp.). The incorporations were stopped by adding 1 ml of cold 10% trichloroacetic acid. Radioactivity was determined by boiling samples for 10 min, cooling, filtering the precipitated protein on glass fiber filters (Whatman, GF/C), and counting in an NCS solubilizer (Nuclear-Chicago)-containing, toluenebased scintillation fluid. For protein analysis by two-dimensional polyacrylamide gel electrophoresis, samples ( 1 ml) were labelled for 10 min with 15 pCi of ["S]methionine (200 Ci/mmol). The incorporations were stopped by adding ice to the medium. The cells were immediately pelleted at 5000 x g for 10 min, washed once in cold distilled water, and stored at - 70°C. Preparation of'cell extracts and two-dimensional polyacrylamide gel electrophoresis

Total yeast proteins were prepared by disrupting approximately 2 x lo7 cells with glass beads as described previously (Brousse et ul., 1985). Protein analysis by two-dimensional polyacrylamide gel electrophoresis was carried out according to a modification of the procedure of O'Farrell et al. (1977). Proteins were separated in the first dimension by non-equilibrium pH gradient-gel electrophoresis in gels containing 4% acrylamide, 9.2 M-urea, 2% Nonidet P-40 and 2% ampholines (pH 3.5-10). Electrophoresis was carried out for 4 h at 400 V.

INL)U('TION OF A HEAT-SHOCK-TYPE RESPONSE

The second dimension (sodium dodecyl sulfate -gel electrophoresis) was as described by Elliott and McLaughlin (1979). Gels were 10% acrylamide, 0.1 YOsodium dodecyl sulfate, 0.375 M-Tris (pH 8.8) and were run for 3 h at 4 W per gel. In comparison to the standard two-dimensional gel electrophoresis technique (O'Farrell, 1975), where proteins were separated in the first dimension by isoelectric focusing. this technique resolves in addition basic proteins. The gels were stained overnight with Coomassie brilliant blue (0.06% in 50% methanol) and destained for 5 h in 7.5% acetic acid plus 10% methanol. The labelled gels were dried and exposed to Kodak no-screen medical X-ray film. Determination ofthe changes in the relutive rute of s~nthesisof individuul polypeptides

The relative rate of synthesis of individual polypeptides was determined by double-isotope labelling experiments in conjunction with twodimensional gel electrophoresis. Basically, the rate of synthesis for a given polypeptide at each time point of the experiment was measured as a ratio of the [''Slrnethionine incorporated into this polypeptide during a 10-min pulse to the ['Hlamino acids incorporated into the same polypeptide in reference cells. 'H-labelled proteins served as an internal standard. Each ratio was determined after two-dimensional gel electrophoresis of a mixture of the "S- and 'H-labelled proteins, by cutting out the spot corresponding to the polypeptide and counting the "S and 'H radioactivity contained in the spot. The relative rate of synthesis of a polypeptide at a given time point of the experiment was expressed as the %!'H ratio in the corresponding spot at this time point divided by the higher "Si'H ratio observed among all the conditions investigated. For this purpose, samples of test cultures were labelled for 10 min with ["S]methionine (120 pCi,! ml; 10 Ci.'mmol) at various times of the experiment. After incorporation, cells were harvested and 1.2 x 10' cells from each sample were mixed with equal amounts of a reference mixture. The reference mixture contained two different cell populations, each labelled with a mixture of ['Hlamino acids (New England Nuclear). One population corresponded to cells of a culture grown on acetate as carbon source; cells were labelled with [3H]amino acids (IOmCiiml) for 20 min when the culture reached an optical density of 100 Klett units. The other population of cells corresponded to a culture

369 grown on glucose as carbon source and shifted from 22 to 36°C when the culture reached the optical density of 100 Klett units; cells were labelled with [3H]amino acids (10 mCi/ml). 20-30 min after the shift. These two populations of cells were used in order to obtain a reference mixture of 'H-labelled polypeptides containing the different classes of polypeptides considered in the experiment (polypeptides synthesized during exponential phase; carbon catabolite repressed polypeptides; heatshock-induced polypeptides). In addition, the use of these two populations ofcells offered the possibility, after electrophoresis, to easily detect most of the proteins of interest by Coomassie blue staining. Protein extracts from mixed cells were prepared as described above and individual polypeptides were resolved by two-dimensional gel electrophoresis. After electrophoresis, gels were stained, dried and submitted to autoradiography. For the purpose of orientation, radioactive ink marks were placed on the gel before exposure. Alignment of the ink spots on the autoradiogram allowed identification of spot positions for polypeptides of interest which were not detectable by Coomassie blue staining. Each of the polypeptide spots of interest was cut out, taking care not to depass the boundaries of the spot, rehydrated for 10 rnin with 10 pl ofdistilled water. and digested for I h at 60°C in 500p1 of Nuclear Chicago Solubilizer (Amersham). The radioactivity was counted using a toluene-based scintillation fluid and the data were corrected for quench. According to this procedure, the results were independent of variations in the efficiency of protein extraction that may occur from one extraction to another. They were also independent of variations in the amount of protein loaded on each gel, and of variations in the area of spot excised for determining each %/'H ratio. RESULTS Comparison of the pattern ofproteins synthesized by uctively growing cells und by glucose-limited cells To analyse changes in protein synthesis that occur in yeast cells following glucose depletion, strain SKQ2n was grown in synthetic medium containing 2% glucose. Under theseconditions, growth of cultures reaches a plateau by the time glucose is fully exhausted (Boucherie, 1985). Cultures of strain SKQ2n exponentially growing on glucose and SKQ2n cultures which have arrested growth for two hours after lucose exhaustion were labelled for 10 min with ($3) methionine. Proteins were

370

N. BATAILLE, M. REGNACQ AND H. BOUCHERIE

m

U

cn

INDUCTION OF A HEAT-SHOCK-TYPE RESPONSE

separated by two-dimensional polyacrylamidc gel electrophoresis and the autoradiogram patterns were compared. In agreement with our previous report (Boucherie, 1985). the protein pattern from glucose-limited cells (Figure IA) was found to be markedly different from that of exponentially growing cells (Figure I B). Two types ofchanges were responsible for these alterations: an arrest ofsynthesis of most of the proteins synthesized during log phase and the induction of novel polypeptides. Hence, among the 250 major polypeptides detectable on the protein map of log-phase cells (Figure lB), it was observed that the synthesis of more than 90% was arrested in glucosc-limited cells. On the other hand, of only 50 polypeptides detectable on the protein map of glucose-limited cells (Figure IA), 35 were found to correspond to novel polypeptides. Some of the major polypeptides whose synthesis was arrested in glucosc-limited cells are indicated by circles in Figure I B. The induced polypeptides are indicated by arrows in Figure 1 A and are listed in Table 1. Taking advantage of the recent identification of polypeptides of the yeast protein map (Bataille (Jt d..1987. 1988) it was possible to obtain insights on the functional significance of several of these changes. Hence, i t appeared that almost all the enzymes of the fermentative pathway so far identified on the yeast protein map (PYK, spot 16; PGK, 25; ADH. 41a and b; GLD, 45 and 47; ENO, 39 and 67: TPI. 82; PDC, 147; PGI, 152) ceased to be synthesked in glucose-limited cells. The only exception was H E X PI1 (spot 130). The corresponding polypeptide. iogetherwithpolypeptides38,165,166, 208 and 256. was among the rare polypeptides whose synthesis was markedly maintained in glucoselimited cells. I t was also possible to determine that. among the novel polypeptides synthesized in glucose-limited cells, 2 1 corresponded to polypeptides submitted to carbon catabolite repression

37 1 (ccrps). These ccrps include seven mitochondria1 polypeptides (spots 2, 8, 10, 28, 38,46 and 7 9 , and HEX PI (128 and 129). Two polypeptides (38 and 208) already present in glucose-growing cells but whose levels were markedly increased in glucoselimited cells were found also to correspond to ccrps. A pattern of the proteins synthesized by logphase cells of a SKQ2n culture grown on acetate, a derepressive carbon source, is reported in Figure 1 C to give evidence of the location of these c u p s on the protein map of exponentially growing cells. Relationship of proteins synthesized by glucoselimited cells to heui-shock-inducedpolypeptides To determine whether some of the polypeptides synthesized by glucose-limited cells corresponded to heat-shock-induced polypeptides (hsps),cultures of strain SKQ2n growing exponentially on minimal medium were shifted from 22 to 36'C. Cells were pulse-labelled for 10 min with [35S]methionine.prior to the shift, and at various times (10, 20.45,60 and 120 min) following the shift. The patterns were compared after two-dimensional gel electrophoresis and autoradiography. Thirty-five polypeptides were found to be induced following the shift. These polypeptides are indicated by arrows in Figure 1 D. They have been classified in two classes, class I (31 polypeptides; thin arrows) and class I1 (four polypeptides; large arrows), depending on their presence in log-phase cells. Class I corresponds to polypeptides which were absent or barely detectable in log-phase cells (Figure I A), class I1 corresponds to polypeptides already synthesized in significant amounts prior to the shift. In agreement with previous reports (McAlister et ul., 1979; Miller et al., 1982), alteration in the levels of these polypeptides was found to be transient, climaxing in most cases 2C30 min following the shift. All these polypeptides returned to preshock levels by 2 h following the heat shock (data not

Figure I . Comparison of the two-dimensional pattern of the proteins synthesized in glucose-limited cells, glucose-growing cells. acetate-growing cells and heat-shocked cells. In all cases SKQ2n cells were pulse-labclled with ['JS]methionine for 10 min and the cxtracted proteins were fractionated by two-dimensional gel electrophoresis as described in Materials and Methods. The gels were dried and exposed at room temperature to X-ray film. (A) Glucose-limited cells. The cells were labelled 2 h after glucose exhaustion: 0.2 x lo* cpm was loaded on the first dimension gel and the exposure was for 8 days. (B) Glucose-growingcells. The cells were labelled when the culture reached the optical density of 100 Klett units (KU) (1.3 x IOhcpm; 3 days). (C) Acetate-growing cells (100 KU: 0.6 x lobcpm: X days). (D) Heat-shocked cells. The cells were grown on glucose as carbon source and shifted from 22 to 36°C when the culture reached the optical density of I 0 0 K U . Cells were labelled 20 min after the shift ( 1 . 1 x 106 cpm. 3 days). Spotscorresponding to identified polypeptides have been enumerated according to Bataille et a / .(1988). New numbers have been attributed to the polypeptides induced i n heat-shocked cells. Arrows indicate, respectively. polypeptides induced in glucose-limited cells (A), polypeptides submitted t o carbon catabolite repression (C). polypeptides induced following heat shock (D). Circles give the location of major polypeptides whose synthesis is arrested in glucose-limited cells.

372

N. BATAILLE, M. RECNACQ AND H. BOUCHERIE

Table 1. Identity and regulation class of the major polypeptides synthesized in glucose-limited cells

Spot number"

2 8 10

Identification Mit. poly.' Mit. p ~ l y . ~ Mit. poly.'

12 13 17

22 23 28 29 30 31 32 38 46 48 61 65 66 70 75 81 83 84 128 129 130 132

HSP56' HSP56' Mit. poly'

Mit. p ~ l y . ~ Mit. poly.' HSP35*

Mit. p ~ l y . ~

HEX PIh HEX PIh HEX PIl'

shown). The polypeptides corresponding to known heat-shock proteins which were previously located on the yeast protein map are indicated in Table 1. Comparison of the protein patterns from heatshocked cells (Figure ID) and glucose-limited cells (Figure I A ) gave evidence that 22 of the polypeptides synthesized in glucose-limited cells corresponded to hsp.~.Eighteen corresponded to hsps of class I and four to hsps of class 11. Evidence that these 22 polypeptides were identical to hsps was further provided by co-electrophoresis of proteins from both types of cells (data not shown). Concerning the 18 polypeptides corresponding to hsps of class I it is interesting to note that three of them corresponded to novel polypeptides of glucose-limited cells previously characterized as ccrps. This was so for spot 46, a mitochondria1 polypeptide, and for spots 139 and 140. Finally, it is also

Carbon catabolite repressed polypeptides

+ + + + + + + + + + + + + + + +

Heat-shockinduced polypeptides

+ +

+ +

+ +

+ +

of interest to note that four of the six polypeptides of log-phase cells whose synthesis was particularly well maintained after glucose exhaustion (spots 130, 165, 166 and 256) corresponded to the hsps of class 11. Time course of the changes in the pattern ofprotein synthesis associated with glucose limitation As shown in Figure 2, strain SKQ2n displayed three different growth phases during the period surrounding glucose depletion. The first phase was a transition phase, which began about 4 h before glucose exhaustion, and during which the culture progressively entered stationary phase; this phase was characterized by a progressive accumulation of cells in the unbudded part of the cell cycle (Figure 2A). The second phase was a stationary phase, whose beginning was concomitant with glucose exhaustion, which lasted about 10 h, and which

373

INDUCTION OF A HEAT-SHOCK-TYPE RESPONSE

Table I . Continued

Spot numbei"

Identification

I36

Carbon catabolite rcprcsscd polypcptidcs

+ +

I39

I40 163 164

I65 I66 191

HSW6'

208

254 256

HSP89' HSP70 (SSA 1

258

+ SSA2)'

HSP70 (SSA3)'

+

Heat-shock-

induced polypeptides

+ + + + + + + + + + +

"Spot numbers correspond to the numbers in Figure I . 'Identification of polypeptides 2. 8. 10, 28, 38. 46, 75, 128. 129 and 130 has been previously reported by Bataille et ul. (1988). Mit. poly., mitochondria1 polypeptides. 'Location of HSP56 and HSP89 on the yeast protein map has been previously reported by Iida and Yahara (1984). WSP35 has been identified as corresponding to glyceraldehyde phosphate dehydrogenase (Lindquist. 1986). Apart from the fact that spot 48 corresponds to a heat-shock polypeptide with a molecular weight of 35000, spot 48 is immunodetected with antibodies directed against purified yeast glyceraldehyde phosphate dehydrogenase. and it displays a protease V8 digestion pattern similar to that of purified yeast glyceraldehyde phosphate dehydrogenase (unpublished data). I t has thus been concluded that spot 48 corresponds to HSP35. 'Identification of polypeptide 191 ascorresponding to HSP96 has been previously reported by Piper c t ul. (1988). 'Identification of the heat-shock proteins o f the HSP70 family corresponding to the SSAI. SSA2 and SSA3 acne products has been Dreviously reported by Werner-Washburne er 01. ( 1989).

corresponded to an adaptation period during which cells shift their metabolism from fermentation to respiration (Perlman and Mahler, 1974);during this phase almost all cells were arrested as unbudded cells (Figure 2A) and the rate of protein synthesis was very low, about 5 % of that of actively growing cells (Figure 2C). The final phase was a second growth phase where the ethanol produced during glucose fermentation was used as carbon source (Figure 2B). To explore further the events responsible for the alterations in the pattern of protein synthesis observed in glucose-limited cells, cells were labelled at time intervals during these three different phases (time points are indicated by arrow heads on Figure 2A). After protein extraction, protein synthesis was analysed by two-dimensional gel electrophoresis and tho rate of synthesis of individual polypeptides was determined.

Two-dimensional protein patterns from selected time points are shown in Figure 3 and the relative rates of synthesis of polypeptides representative of the main categories of proteins are reported in Figure 4. It can be seen from these data that, globally, the three events involved in the alteration of the pattern of protein synthesis, i t . the induction of hsps, the derepression of ccrps and the specific arrest of pre-existing proteins, all occurred during the period where glucose was exhausted. This is particularly clear when one considers the pattern of the proteins synthesized 1 h before glucose exhaustion (Figure 3A). This pattern is still very similar to the one of exponentially growing cells (compare Figure 3A with Figure 1 B). Concerning the synthesis of hsps, a good correlation was observed between the induction of most of these polypeptides and glucose exhaustion. Hence, most hsps were barely detectable 1 h prior to

374

N. BATAILLE, M. REGNACQ AND H. BOUCHERIE

The arrest of synthesis of pre-existing proteins and the derepression of most ccrps were also essentially initiated at the onset of glucose starvation (Figure 3B). Indeed, it was observed that, at this stage of the culture, the synthesis of almost all preexisting proteins started to be specifically reduced (see in particular spots 7 and 16, Figure 3B). In the meantime, most c u p s were derepressed. However, 25 35 45 the timing of these two events was different from that of the induction of hsps. Whereas the induction of hsps was maximum at the onset of glucose starvation, the arrest of synthesis of pre-existing proteins and the derepression of c u p s were optimum 3 h later. By this time, the synthesis of preexisting proteins was completely arrested and ccrps were maximally synthesized (Figure 3C and Figure 4A and C). 25 35 45 Although an induction concomitant with glucose exhaustion appears to be the general rule for hsps and ccrps, it should be mentioned that there are some hsps and ccrprs which escape this rule. This is so for the hsps corresponding to spots 32,33 and 48, the ccrps corresponding to spots 128 and 129, and the hspslccrps corresponding to spots 139 and 140. Figure 3A gives evidence that these polypeptides were already synthesized 1 h before glucose exhaus-8 25 35 45 TIME (hours) tion. In fact, we found that the synthesis of these polypeptides was induced approximately 10 h Figure 2. Parameters ofgrowth of strain SKQ2n in 2% glucose before the complete exhaustion of glucose, at a synthetic medium during the various growth phases occurring before and after glucose exhaustion. The culture was inoculated glucose concentration of about 1.5% (results not at an initial density of 5 x lo' cells. Sampling began when the shown). A change in growth rate (the generation number of cells reached approximately 7 x lo' cells per ml. (A) time increased from 2 to 7 h; Boucherie, 1985) and Cell parameters: ( O ) ,absorbance measured by use of a KlettSummerson spectrophotometer; (A), number of cells; ( A ) , several metabolic alterations (Lillie and Pringle, 1980; Francois et al., 1987) have been reported to viable cells; ( O ) ,percentage of budded cells. (B) Time course of glucose depletion and ethanol accumulation; (0) glucose and occur at this stage of the culture. Derepression of (m) ethanol concentrations in the growth medium. (C) Incorpor- these hsps and ccrps is probably related to these ation of ["Slmethionine were calculated per viable cell and expressed as a percentage of the incorporation of [35S]methionine changes. However, it should be mentioned that per viable cell of a SKQ2n culture exponentially growing under although the synthesis of these polypeptides was the same culture condition. Growth parameters were determined induced in advance of the synthesis of other hsps and as described in Materials and Methods. Arrow heads indicate ccrps, like these later, they were maximally synthetime points at which cells were harvested for carrying out the sized by the time glucose was exhausted (Figure 3B experiments reported in Figures 3 and 4. and Figure 4D). glucose exhaustion (Figure 3A); in contrast, they were all synthesized as major polypeptides at the onset of glucose limitation (Figure 3B). The synthesis of these proteins was transient. Quickly after glucose exhaustion, their synthesis decreased sharply so that, 3 h later, most of them were detectable only as faint spots (Figure 3C). The time dependence of the synthesis of three hsps representative of this temporal category is illustrated in Figure 4B.

DISCUSSION Our two-dimensional gel analysis of the proteins synthesized in glucose-limited cells reveals two interesting features of the induction of heat-shock proteins in response to glucose limitation: (i) the fact that this induction concerns a high fraction of the entire heat-shock protein population; (ii) the fact that synthesis of these heat-shock proteins is one of the major events occurring at the level of protein

NEPHGE

t

z

0

Figure 3. Two-d~mens~onal pattern of the proteins synthesized at various stages of an SKQ2n culture before and after glucose exhaustion. In all cases, cells were pulse labelled for 10 min with ["S]methionine. (A) Proteins synthesized 1 h before glucose exhaustion (a total of0.6 x lo6cpm was loaded on the gel, and exposure was for 8 days). (B) Proteins Fynthesized when glucose was exhausted (0.3 x lo* cpm; 8 days). (C) Proteins synthesized 3 h after glucose exhaustion (0.2 x lo6cpm; 8 days). (D) Proteins synthesized 10 h after glucose exhaustion (0.3 x lo6cpm; 8 days). Symbols: arrow heads, heat-shock polypeptides; thin arrows, carbon catabolite repressed polypeptides; thick arrows, heat-shock and catabolite repressed polypeptides. Numbers are as in Figure 1.

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N. B A T A I L L ~ ,M. R ~ G N A C QAND H. BOUCHERIE

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Time ( h o u r s ) Figure 4. Relative rate of synthesis of selected polypeptides during the various growth phases of an SKQ2n culture, before and after glucose exhaustion. Polypeptides are grouped in panels A to D according to their temporal pattern of synthesis in response t o starvation: (A) polypeptides present in exponentially growing cells; ( B ) and (D) induced polypeptides corresponding to heat-shock proteins; (C) induced polypeptides corresponding to carbon catabolite repressed proteins. The relative rate of synthesis was determined asdexribed in Materials and Methods. The arrow indicates the time of complete exhaustion of glucose from the medium.

synthesis in glucose-limited cells. Our analysis shows indeed that 65% of all the heat-shock proteins detectable on our two-dimensional gels are induced in response to glucose limitation and that this induction occurs when the synthesis of almost all the proteins present in exponentially growing cells is arrested. Previously, sulfur limitation was the only condition of nutritional limitation under which the synthesis of all the heat-shock proteins had been considered (Iida and Yahara, 1984). Under this condition, it was found that cells synthesized only high molecular weight heat-shock proteins, i.e.

heat-shock proteins with a molecular weight over 45000; none of the low molecular weight heatshock proteins, those with a molecular weight around 27 000, were observed to be synthesized. The present investigation provides a rather different view of the heat-shock proteins synthesized in response to a nutritional limitation. It was observed that glucose-limited cells synthesized a high fraction of both the high molecular weight and the low molecular weight heat-shock proteins. It appears thus that the set of heat-shock proteins synthesized in response to starvation may differ markedly from one nutritional limitation to another. This difference suggests that it will be interesting to undertake a systematic analysis of the heat-shock proteins synthesized under other nutritional limitations in order to characterize the heat-shock proteins which are synthesized in response to all nutritional limitations. The present observation that heat-shock proteins are almost the only polypeptides to be synthesized when glucose is exhausted emphasizes the importance of the role that these proteins play in nutritionally limited cells. The nature of their function, however, has yet to be determined. Because nutritional limitations is associated with an arrest of cell proliferation, it is tempting to speculate that heat-shock proteins are involved in establishing the resting state. Such a function in growth and cell cycle regulation has been proposed by several authors (Plesset el ul., 1987; Shin Ct ul., 1987; Tanaka rt ul., 1988). In particular, it has been suggested that heat-shock proteins are part of the CAMP effector pathway controlling the yeast cell cycle (Shin et ul., 1987; Tanaka et ul., 1988). According to our results, it does not seem that heatshock proteins play such a role in glucose-limited cells. Indeed, under our culture conditions, we d o not observe any correlation between the entry into stationary phase and the induction of heat-shock proteins: the accumulation of cells as unbudded cells starts several hours before glucose is depleted, whereas heat-shock proteins are induced only when glucose is exhausted. I t is known that nutritional limitation is also associated with marked changes in cell metabolism. This is true in particular for glucose limitation which results in drastic changes in the machinery of carbon metabolism. Hence, after glucose exhaustion, the levels of several enzymes specifically involved in glycolytic fermentation are reduced (Entian et ul., 1984; McAlister and Holland, 1982, 1985); conversely, the synthesis of enzymes involved

INDUCTION OF A HEAT-SHOCK-TYPE RESPONSE

in respiratory metabolism is derepressed, a phenomenon called carbon catabolite repression (Polakis and Bartley, 1965; Perlman and Mahler, 1974). We showed here that the induction of heat-shock proteins in glucose-limited cells occurs a t the onset of the release of carbon catabolite repression. This observation leads us to suggest that the function of heat-shock proteins in glucose-limited cells may be to help readjustment of the carbon metabolism machinery. More generally, according to this hypothesis, the function of the heat-shock proteins induced in response to nutritional limitation would be to ensure the physiological adaptation of cells to starvation conditions. This hypothesis is in agreement with the observation that several heat-shock proteins appear to be involved in the biogenesis, conformation and selective destruction of proteins (Finley et d., 1987; Rothman, 1989). We are currently testing this possibility. ACKNOWLEDGEMENTS This work was supported by grants from CNRS and the University of Bordeaux 11. REFERENCES Bataille, N., Peypouquet, M.-F. and Boucherie, H. (1987). Identification of polypeptides of the carbon metabolism machinery on the two-dimensional protein map of Saccharomyces cerevisiae. Location of 23 additional polypeptides. Yeast 3, 1 1-21. Bataille, N., Thoraval, D. and Boucherie, H. (1988). Two-dimensional gel analysis of yeast proteins: application to the study of changes in the levels of major polypeptides of Saccharomyces cerevisiae depending on the fermentable or nonfermentable nature of the carbon source. Electrophoresis 9,774780. Boucherie, H . (1985). Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae. J . Bacteriol. 161,386-392. Brousse, H., Bataille, N. and Boucherie, H. (1985) Identification of glycolytic enzymes polypeptides on the two-dimensional protein map of Saccharomyces cerevisiae and application to the study of some wine yeasts. Appl. E m . Microbiol. 50,951-957. Cheng, M. Y., Hartl, F. U., Martin, J., Pollock, R. A,, Kalousek, F.,*Neupert,W., Hallberg, E. M., Hallberg, R. L. and Horwich, A. L. (1989). Mitochondria1 heatshock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature 337, 620-625. Chirico, W. J., Watter, M. G. and Blobel, G. (1988). 70K heat-shock related proteins stimulate protein translocation into mitosomes. Nature 332,805-810.

377 Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A. and Schekman, R. (1988). A subfamily of stress proteins facilitates translocation of secretory and mitochondria1 precursor polypeptides. Nature 332, 800-805. Elliott, S. G. and McLaughlin, C. S. (1979). Synthesisand modification of proteins during cell cycle of the yeast Saccharomyces cerevisiae. J. Bacteriol. 137,1185-1 190. Entian, K. D., Frolich, K. U. and Meck, D. (1984). Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 799,181-186. Finley, D., Ozkaynak, E. and Varshavsky, A. (1987). The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation and other stresses. Cell 48,1035-1046. Franqois, J., Eraso, P. and Gancedos, C. (1987). Changes in the concentration ofcAMP, fructose 2,6-biphosphate and related metabolites and enzymes in Saccharomyces cerevisiae during growth on glucose. Eur. J . Biochem. 145,187-193. Hartwell, L. H. (1967). Macromolecule synthesis in temperature-sensitive mutants of yeast. J. Bacteriol. 93,1662-1670. Hemmingsen, S. M., Woolford, C., Van der Vies, S. M., Tilly, K., Dennis, D. T., Georgopoulos, C. P., Hendrix, R. W. and Ellis, R. (1988). Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333,330-334. Iida, H. (1988). Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5’ coding region of the adenylate cyclase gene. Mol. Cell. Biol. 8,5555-5560. Iida, H. and Yahara, I. (1984). Durable synthesis of high molecular weight heat-shock proteins in Go cells of the yeast and other eucaryotes. J. Cell. Physiol. 99, 199-207. Iida, H. and Yahara, I. (1985). Yeast heat shock protein of Mr 48,000 is an isoprotein of enolase. Nature 315, 688-690. Kurtz, S., Rossi, J., Petko, L. and Lindquist, S. (1986). An ancient developmental induction: Saccharomyces sporulation and Drosophila oogenesis. Science 231, 1 154-1 I 57. Lillie, S. H. and Pringle, J. R. (1980). Reserve carbohydrate metabolism in Saccharomyces cerevisiae: response to nutrient limitation. J . Bacteriol. 143, 1384-1394. Lindquist, S. (1986). The heat shock response. Ann. Rev. Biochem. 55,1151-1191. Lindquist, S. and Craig, E. A. (1988). The heat shock proteins. Ann. Rev. Genet. 22,631-677. Matsumoto, K., Uno, I. and Ishikawa, T. (1985). Genetic analysis of the role of CAMP in yeast. Yeast I, 15-24. McAlister, L., Strausberg, S., Kulaga, A. and Finkelstein, D. B. (1979). Altered patterns of protein synthesis induced by heat shock of yeast. Current Genetics 1, 63-74.

378 McAlister, L. and Finkelstein, D. B. (1980). Heat-shock proteins and thermotolerance in yeast. Biochem. Biophys. Rex Commun. 93,819-824. McAlister, L. and Holland, M. J. (1982). Targested dclction of a yeast enolase structural gene: identification and isolation of yeast enolase isoenzymes. J. Bid. Chem. 257,7 18 1--7188. McAlister, L. and Holland, M. J. (1985). Differential cxpression of the three yeast glyceraldchyde-3phosphate dehydrogenase genes. J. Biol. Chem. 260, 15019-15027. Miller, M . J.. Xuong, N.-H. and Geiduschek, P. (1982). Quantitative analysis of the heat shock response of Succhuromyces cerevisiae. J . Bacteriol. 151, 3 1 I--327. O'Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007402 I . O'Farrcll. P. Z., Goodman, H. M. and O'Farrell, P. H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1 142. Perlman, D. and Mahler, H. R. (1974). Derepression of mitochondria and their enzymes in yeast: regulatory aspect. Arch. Biochem. Biophj3.s. 162,248-27 I . Phillips, T. A., Van Bogelen. R. A. and Neidhardt, F. C. (1984). Lon gene product of Escherichia coli is a heatshock protein. J. Bacteriol. 159,283-287. Piper, P. W., Curran. B., Davies, M. W., Hirst, K., Lockheart, A. and Seward, K. (1988). Catabolite control of the elevation of PGK mRNA levels by heat shock in Saccharomyces cerevisiae. Mol. Microhiol. 2, 353 -36 I . Plcssct, J.. Palm, C. and McLaughlin, C. S. (1982). Induction of heat-shock proteins and therrnotolerance by ethanol in Saccharomyces cerevisiae. Biochem. Biophjs. Res. Commun. 108, I34Ck1345. Plesset, J., Ludwig, J. R.. Cox, B. S. and McLaughlin, C. S. (1987). Effect of cell cycle position on thermotolerance in Succharon~ycescerevisiae. J. Bacteriol. 169, 779-784.

N. BATAILLE, M. REGNACQ A N D H.BOUCHERIE

Polakis, E. S. and Bartley, W. (1965). Changes in enzyme activities of Succharomyces cerevisiae during aerobic growth on different carbon sources. Biochem. J. 97, 284-297. Rothman, J . E. (1989). Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell59,591-601. Sanchez, Y. and Lindquist, S. L. (1990). HSP104 required for induced thermotolerance. Science 248, 1 1 12-1 I 14. Schenberg-Frascino, A. and Moustacchi, E. (1972). Lethal and mutagenic effects of elevated temperature on haploid yeast. I. Variation in sensitivity during the cell cycle. Mol. G m . Genet. 115,243--257. Shin, D.-Y., Matsumoto, K., Iida, H., IJno, I. and Ishikawa, T. (1987). Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation. Mol. Cell. Biol. 7,244250. Tanaka, K., Matsumoto, K. and Toh-e, A. (1988). Dual regulation of the expression of the polyubiquitin gene by cyclic AMP and heat shock in yeast. EMBO J. 7, 495-502. Taylor, W. E., Straus, D. B., Grossman, A. D., Burton, Z. F., Gross, C. A. and Burgess, R. (1984). Transcription from a heat-inducible promoter causes heat-shock regulation of the sigma subunit of E. coli RNA polymerase. Cell 38,371 .38 I . VanBogelen, R. A., Vaughn. V. and Neidhardt, F. C. (1983). Gene for heat-inducible lysyl-t RNA synthetase (IysV) maps near cadA in Esclierichia coli. J. Bucteriol. 153,1066-1068. Verma, R.. Iida, H. and Pardee, A. B. (1988). Identification of a novel stress-inducible glycoprotein in Saccharomyces cerevisiae. J. Biol. Chem. 263, 8569-8575. Werner-Washburne. M., Becker, J., Kosic-Smithers and Craig, E. A. (1989). Yeast Hsp70 RNA levels vary in response to the physiological status of the cell. J. Bacteriol. 171,2680-2688.