APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2005, p. 4531–4538 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.8.4531–4538.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 8
Activation of the Protein Kinase C1 Pathway upon Continuous Heat Stress in Saccharomyces cerevisiae Is Triggered by an Intracellular Increase in Osmolarity due to Trehalose Accumulation Femke I. C. Mensonides,1 Stanley Brul,2,3 Frans M. Klis,1 Klaas J. Hellingwerf,1 and M. Joost Teixeira de Mattos1* Swammerdam Institute of Life Sciences, Department of Molecular Microbial Physiology, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands1; Swammerdam Institute of Life Sciences, Dept. Molecular Biology & Microbial Food Safety, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands,2 and Unilever Research & Development, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands3 Received 20 October 2004/Accepted 14 March 2005
This paper reports on physiological and molecular responses of Saccharomyces cerevisiae to heat stress conditions. We observed that within a very narrow range of culture temperatures, a shift from exponential growth to growth arrest and ultimately to cell death occurred. A detailed analysis was carried out of the accumulation of trehalose and the activation of the protein kinase C1 (PKC1) (cell integrity) pathway in both glucose- and ethanol-grown cells upon temperature upshifts within this narrow range of growth temperatures. It was observed that the PKC1 pathway was hardly activated in a tps1 mutant that is unable to accumulate any trehalose. Furthermore, it was observed that an increase of the extracellular osmolarity during a continuous heat stress prevented the activation of the pathway. The results of these analyses support our hypothesis that under heat stress conditions the activation of the PKC1 pathway is triggered by an increase in intracellular osmolarity, due to the accumulation of trehalose, rather than by the increase in temperature as such. resistance, it will be imperative to integrate understanding of the separate aspects of the stress response, not only qualitatively but also quantitatively. Hence, we set out to quantify and integrate physiological and molecular responses of Saccharomyces cerevisiae to heat stress conditions. S. cerevisiae can grow within a temperature range, typically from 20°C to 42°C. Whatever sets the maximal temperature at which the organism is still able to grow is unknown, but as has been shown previously (16, 27), the transition of a population of cells being able to grow to one that only can maintain viability and subsequently to one that dies takes place within a temperature range of only a few degrees Celsius. The actual range depends on the strain used and the growth conditions. At the metabolic level, the accumulation of trehalose (␣-Dglucopyranosyl ␣-D-glucopyranoside) has been correlated with survival of cells under heat stress conditions (5). Historically, trehalose was believed to be an important storage carbohydrate in yeast. However, it is hardly present in cells that grow exponentially on glucose, and it is only produced after the diauxic shift when glucose is depleted, and it is consumed much later (12, 24). Trehalose can accumulate up to 15% of the cell dry mass during nutrient starvation and under certain stress conditions, including heat stress (2, 12). It has been shown that this disaccharide suppresses the aggregation of heat-denatured proteins, thus facilitating refolding of these proteins (22, 23). It can also protect native proteins against denaturation (23). The mechanism that triggers trehalose synthesis under stress conditions has not been fully elucidated. It has been reported that the induction of trehalose biosynthesis genes under stress conditions is dependent on the transcription factors Msn2 and Msn4 and the stress-responsive elements in these promoter sequences, although this does not explain trehalose accumula-
Microorganisms encounter a constantly changing environment in their natural habitat. It is not surprising, therefore, that they have developed mechanisms to sense and respond adequately to (potentially) threatening conditions like nutrient depletion, changes in external osmolarity, or elevated temperature. These stress responses of microorganisms have been studied for a long time, although with opposite interests. On the one hand, there is an interest in increasing the survival or performance of microorganisms under stressful conditions, for instance, of yeasts in making wine and baking (2, 17). On the other hand, stress responses are studied to kill unwanted microorganisms more effectively, for instance, during the preservation process step in the food industry (3, 13). In particular, the food industry is searching for novel “minimal processing” techniques, to produce safe products while maintaining the functionality and quality of the unprocessed material. Elevated temperature is one of the stresses under study. In spite of extensive research, however, our knowledge of the mechanisms that are involved in the response of cells to heat stress is still far from complete. Quantitative data on physiological changes upon an increase in growth temperature would surely help to fill this gap in our understanding. Although certain aspects of the response to heat stress have been studied elaborately (see below), an integrated approach to resolve the mechanisms underlying heat stress response is lacking. To fully understand the response of microorganisms to heat stress and the resulting * Corresponding author. Mailing address: Swammerdam Institute of Life Sciences, Department of Molecular Microbial Physiology, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. Phone: 31 20 525 7066. Fax: 31 20 525 7056. E-mail:
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tion at temperatures of ⬎40°C, since transcriptional induction of stress-responsive element-controlled genes is abolished at such high temperatures (19). Another aspect of the heat stress response of yeast that has been studied elaborately is the activation of the protein kinase C1 (PKC1) pathway. This pathway is mediated by PKC1 and includes a mitogen-activated protein (MAP) kinase cascade consisting of the proteins Bck1 (as the MAP kinase kinase kinase), Mkk1 and Mkk2 (as MAP kinase kinases), and Slt2 (as the MAP kinase). It regulates the transcription factors Rlm1 and SBF (involved in regulation of transcription of a set of cell wall genes) by MAP kinase-mediated phosphorylation (for a review, see references 6 and 7). The pathway is also known as the cell integrity pathway, since it is vital for maintenance of the integrity of the cell in a range of conditions that threaten cell wall stability. For example, the pathway is activated upon both a heat shock (10) and a hypotonic shock (4). A remarkable difference between these two stimuli for activation is the time scale at which the pathway is activated. A decrease in extracellular osmolarity activates the pathway within seconds (4), whereas the pathway is only activated 10 to 30 min after a temperature increase (10). The slowness of the response to heat stress suggests that this response is a secondary effect of the temperature increase. It has been proposed that the PKC1 pathway is activated by a plasma membrane stretch (10). In addition, it has been speculated that activation of the pathway under heat stress conditions is due to an increased fluidity of the plasma membrane (10). Although evidence has been provided for the activation of the pathway by membrane stretch, no evidence has been provided for the role of membrane fluidity in the pathway activation (10). It may well be that the late activation of the cell integrity pathway upon an increase of the growth temperature is due to the accumulation of trehalose in response to the heat stress. The accumulation of trehalose, which is an osmolyte, starts immediately upon an increase in temperature (2, 9). Thus, accumulation of this compound in the cytoplasm mimics a hypotonic stress, including plasma membrane stretch, a condition known to activate the cell integrity pathway (4, 10). Indeed, it has been observed that the extreme heat sensitivity of an slt2⌬ strain, i.e., a strain impaired in the cell integrity pathway, can be remedied by osmotic stabilization (11, 25). Here, we present data that support the hypothesis that under heat stress conditions the PKC1 pathway is activated due to extensive accumulation of osmolytes in the cell, rather than to the increase in temperature itself. MATERIALS AND METHODS Strain, media, and growth conditions. The Saccharomyces cerevisiae strain X2180-1A (MAT␣ SUC2 mal mel gal2 CUP1) was used as the wild-type strain. In addition, a tps1 deletant (MATa SUC2 MAL2-8c MEL tps1::TRP1) (21) and the isogenic wild-type strain CEN-PK133-7D (MATa SUC2 MAL2-8c MEL) were used. The X2180-1A cells were maintained on YPD (1% [wt/vol] yeast extract, 2% [wt/vol] Bacto Peptone, 2% [wt/vol] glucose in distilled water) plates, and the tps1 deletant cells were maintained on YPGal (same as YPD except that 2% galactose replaces glucose) plates. Precultures were grown in a flask on a rotary shaker at 28°C in YNB medium, which consisted of 0.67% (wt/vol) YNB without amino acids (Difco) and 1% (wt/vol) of the required carbon source in 100 mM potassium phthalate at pH 5.0. The tps1 deletant and the corresponding wild type were grown on 1% galactose as a carbon source, because the mutant is unable to grow on glucose. For the actual experiments, aerobic batch cultivations were performed in
APPL. ENVIRON. MICROBIOL. fermentors at 28°C with a stirring rate of 700 rpm and an aeration rate of 1 volume of air per min, equal to the culture volume. The fermentors were thermostatted with water jackets. Samples were taken for determination of the number of cells, cell viability, intracellular trehalose levels, and activation of the PKC1 pathway. Heat stress. During mid-exponential growth (optical density at 600 nm, 0.5), the growth temperature was raised to 37, 39, 40, 41, 42, or 43°C without disturbing the cultures in any other way. This was done by switching the thermostat setting of 28°C to a setting (water bath) that was 5°C warmer than the desired temperature. The desired temperature was reached within 5 min, and care was taken to prevent an overshoot in temperature. Cell growth and viability assays. The cell count was carried out with a CASY counter. Culture samples were kept on ice until analysis and were appropriately diluted in CASYton just prior to counting. In all experiments, the viability of the cells was assessed by appropriately diluting culture samples in sterile 0.9% NaCl and counting CFU on YNB plates (0.67% [wt/vol] YNB, the required carbon source, and 2% [wt/vol] Bacto agar). As a carbon source, glucose (2%), galactose (2%), or ethanol (1%) was added. The colonies were counted after incubation of the plates at 28°C for 3 days. Determination of intracellular trehalose. Cells were rapidly collected on ice by the addition of 12 ml of culture to 3 ml of ice-cold water in precooled tubes. The cells were pelleted, washed once, frozen, and kept at ⫺80°C until analysis. The trehalose levels in the cells were determined essentially according to reference 18. The frozen pellets were resuspended in ice-cold water. An aliquot was used for cell counts (see above) for normalization purposes. The rest of the sample was incubated in 0.25 M Na2CO3 at 95°C for 4 h. The pH of the samples was then set at 5.2 by adding 75 l of 1 M acetic acid and 300 l of 0.2 M Na acetate (pH 5.2) to 125 l of sample. For determination of the amount of trehalose present, part of the suspension was incubated with 0.05 U/ml trehalase at 37°C overnight under constant agitation. The rest of the suspension was kept overnight at room temperature to determine the background level of glucose. The amount of glucose formed out of the trehalose present was then determined by an enzymatic assay: 200 l of the reagent {0.10 mg/ml D-glucose oxidase, 0.05 mg/ml peroxidase, and 0.50 mg/ml ABTS [2,2⬘-azinobis(3-ethylbenzthiazolinesulfonic acid)] in 0.5 Tris-HCl, pH 7.0} was added to 20 l of standard or sample in a microtiter plate. The plate was incubated for 1 h at 37°C under slow agitation, after which A415 was measured. Detection of dually phosphorylated Slt2. Cells were collected on ice by the addition of 20 ml of culture to 10 ml of ice-cold water in precooled tubes within 5 s. The cells were pelleted, washed once, frozen, and kept at ⫺80°C until analysis. Just prior to analysis, cells were lysed in 90 l cold lysis buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 150 mM NaCl, 50 mM NaF, 1 mM NaO-vanadate, 50 mM -glycerol phosphate, 5 mM Na pyrophosphate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) plus 10 l protease inhibitor cocktail for fungal cells (Sigma), by vigorous shaking with glass beads (diameter, ⬃0.5 mm) in a FastPrep cell breaker (Bio101; 25 s at level 6.0). The cell extracts were separated from cell debris and glass beads by centrifugation (10,000 ⫻ g; 15 min at 4°C). The protein concentration of the extracts was determined by using the DC Protein Assay (Bio-Rad). The appropriate amount of SDS-polyacrylamide gel electrophoresis sample buffer was added, and the samples were boiled for 5 min. The protein samples (each, 100 g) were then separated on 10% SDS-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. To detect dually phosphorylated Slt2, membranes were probed with anti-phospho-p44/42 MAP kinase antibody (New England Biolabs) at a 1:1,000 dilution overnight at 4°C. To monitor the total amount of Slt2 present in the samples, a second set of membranes was probed with Slt2 antibody (14) at a 1:2,000 dilution for 2 h at room temperature. The primary antibodies were detected with a horseradish peroxidase-conjugated anti-rabbit antibody with the ECL detection system. The blots were inspected visually. In addition, protein bands were quantified using Corel software. Quantification of the phosphorylation of Slt2 was based on two independent experiments, using the mean intensity of the bands of the total amount of Slt2 to normalize the two blots. Osmotic stabilization. The extracellular osmolarity was increased by dropwise addition of 5 M NaCl or 5 M sorbitol, up to the indicated concentration in the medium during the indicated time span. The addition of the solution was started immediately after the application of the heat stress.
RESULTS The application of a (continuous) heat stress on growing cells can be performed in many ways. We chose the method
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described (see Materials and Methods), because it disturbs the cells as little as possible, except for the actually desired heat stress. The cells are neither harvested nor transferred, thus avoiding stress due to, for example, oxygen deprivation, starvation caused by centrifugation, or a change in environment due to a transfer to fresh medium. In addition, the temperature is increased gently but in a controlled way without an overshoot, thus preventing unwanted heat stress. The disadvantage of this procedure is that it takes several minutes, but so would a method consisting of harvesting and transferring the cells to fresh medium at increased temperature. In the experiments shown here, the growth temperature of exponentially growing cells was raised from 28°C to a temperature in the range of 37°C to 43°C under well-defined conditions. In addition, control measurements at 28°C were carried out. All measurements were carried out in duplicate, and no significant differences were observed. For each experiment, a typical data set is shown. Cell growth, cell viability, and trehalose synthesis in glucose-grown cells. Previously, we showed that there is a thin line between growth, growth arrest, and loss of viability with regard to growth temperature (16) (see Fig. 1). Those previous experiments were performed under the same conditions as the experiments described here. Glucose was used as a carbon source. The growth rate of cells with glucose as a carbon source increased significantly upon increase of the growth temperature from 28°C (0.28 h⫺1) to 37°C (0.35 h⫺1) (Fig. 1A). Cell growth was unaffected at a growth temperature of 39°C, while at 41°C, growth was seriously affected. After a lag phase of approximately 2 h, the cells resumed growth but at a lower rate. An increase of the growth temperature from 28°C to 42°C resulted in a complete arrest of growth. At all these growth temperatures, cell viability remained at about 90% of the initial number of cells throughout the experiment (Fig. 1B). However, an increase of the growth temperature to 43°C resulted in a rapid and complete loss of viability (Fig. 1B). No intracellular trehalose was detected in cells growing at 28°C (Fig. 2A) (t ⫽ 18 h). However, upon all temperature upshifts analyzed, trehalose accumulation started within 10 min after the temperature increase. The degree of accumulation depended on the growth temperature after the shift (Fig. 2A). At 37°C, a temperature at which the growth rate was not impaired, the accumulation was only transient; trehalose disappeared from the cells again within 2 h. Furthermore, at temperatures up to 41°C, when the cells were just able to grow, the accumulation of trehalose increased with increasing temperature, and the subsequent rate of disappearance decreased. For conditions that did not allow for growth (42°C and 43°C), the accumulation of trehalose was less. Nevertheless, upon an increase in growth temperature trehalose accumulated rapidly, regardless of the actual stress temperature (Fig. 2A). Cell growth, cell viability, and trehalose synthesis in ethanol-grown cells. To see whether the phenomena as described above were specific for growth on glucose, the data set was extended with an analysis of the same aspects of the heat stress response during growth (or nongrowth) on the nonfermentable carbon source ethanol. Since under these conditions glycolysis is not activated for energy production, we expected a different effect of heat stress on the trehalose levels in ethanol-grown cells.
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When ethanol was used as a carbon source, a different effect of the various increases in the growth temperature was seen. The growth rate remained the same upon an increase in the growth temperature from 28°C to 37°C (Fig. 1D). Virtually no growth was seen anymore, however, at a growth temperature of 39°C. At 41°C, growth was arrested completely, and the cells slowly lost their viability. Upon an increase in the growth temperature to 43°C, the cells lost their viability, but at a lower rate than observed with cells growing on glucose (Fig. 1B and C). In contrast to glucose-grown cells, in ethanol-grown cells trehalose was already present at significant levels in the cells growing at 28°C (t ⫽ 18 h) (Fig. 2B). A further temperature increase resulted in increased accumulation. The accumulation rate was higher than in glucose-grown cells and the accumulation lasted longer. Hence, much higher levels were reached. Again, the degree of accumulation depended on the actual stress temperature (Fig. 2B). At 43°C, the accumulation lasted for a shorter period (Fig. 2B). Activation of the PKC1 pathway. In the PKC1 pathway, signals are transmitted by phosphorylation of the conserved tyrosine and threonine residues of the TEY motif in the MAPK cascade proteins. Therefore, their level of phosphorylation is taken as a measure for activation of the pathway. The activation of the PKC1 pathway during heat stress was followed in time by the determination of extent of Slt2 phosphorylation using Western blotting. An antibody against the dually phosphorylated form of this protein is commercially available and its high specificity has been shown previously (15). For cells grown on either of the two carbon sources, the highest temperature at which the cells were still able to grow was used to determine the extent of activation of the PKC1 pathway. Activation of the PKC1 pathway in glucose-grown cells and ethanol-grown cells. At a growth temperature of 28°C, no phosphorylated Slt2 was detected in glucose-grown cells. Upon an increase to 41°C, phosphorylation of Slt2 started within 10 min (Fig. 3A). The maximum level of phosphorylation was reached after 1 h, while after 2 h phosphorylation subsided again to an undetectable level (Fig. 3A). This period of Slt2 activation is compatible with the period of growth arrest and trehalose accumulation under these conditions. During growth on ethanol at 28°C, no dually phosphorylated Slt2 was detected (Fig. 3B). When the growth temperature was raised to 37°C, Slt2 became phosphorylated, and this phosphorylation persisted over time, just as the accumulation of trehalose did (Fig. 3B). Trehalose synthesis and activation of the PKC1 pathway in a tps1 deletant. To further investigate a possible link between trehalose accumulation and activation of the PKC1 pathway under heat stress conditions, we studied a mutant that is unable to synthesize trehalose. Upon an increase of the growth temperature from 28°C to 37°C, the highest temperature at which the mutant was still able to grow, no trehalose was synthesized in the mutant (Fig. 4), as expected. The trehalose levels in the galactose-grown corresponding wild type (CENPK133-7D) were comparable to those in the glucose-grown X2180-1A strain (compare Fig. 2A and 4). At this stress temperature, the phosphorylation level of Slt2 was low and again transient in the wild type but significantly lower in the mutant (Fig. 5).
FIG. 1. Cell growth and cell viability as a function of time in heat-stressed S. cerevisiae cells. The growth temperature was increased at t ⫽ 18 h (arrow) after the start of growth. Growth curves at the indicated temperatures for cells grown on glucose (A) (16) or ethanol (D) and the number of viable cells as percentage of the total number of cells at each temperature for cells grown on glucose (B) or ethanol (C) are shown. Error bars in panels A and D fall within the size of the symbols. Error bars in panels B and C were omitted for the sake of clarity, but the standard deviation was ⬍10%. 4534
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FIG. 4. Trehalose synthesis in heat-stressed galactose-grown wildtype and tps1⌬ cells. Trehalose levels as a function of time in a tps1 deletant (open symbols) and an isogenic wild type (CEN-PK133-7D) (closed symbols) upon increase in the growth temperature from 28°C to 37°C are shown. The growth temperature was increased at t ⫽ 18 h (arrow) after start of growth. Error bars fall within the size of the symbols.
FIG. 2. Intracellular trehalose levels as a function of time in heatstressed S. cerevisiae cells. The growth temperature was increased at t ⫽ 18 h (arrow) after the start of growth. Intracellular trehalose levels (expressed as femtomoles per cell) were measured at various growth temperatures for cells growing on glucose (A) or ethanol (B). Error bars were omitted for the sake of clarity, but the standard deviation was ⬍5%. Note the difference in scale of both graphs.
Trehalose synthesis and activation of the PKC1 pathway and osmotic stabilization. It has been shown previously that the initial activation of the PKC1 pathway in heat-stressed cells can be prevented to a large degree by an increased extracellular osmolarity (10). Unfortunately, from these data it is not clear whether the activation is decreased altogether or merely delayed, since activation of the pathway was only determined for one time point. To counteract the increase of intracellular osmolarity due to trehalose accumulation, we gradually increased the extracellular osmolarity. Previously, we observed that the intracellular trehalose level (and Slt2 phosphorylation) rose during the first hour after the temperature increase (Fig. 2A). Therefore, we increased the extracellular osmolarity for 0.5 or 1 h after the initiation of the heat stress by adding either NaCl or sorbitol to
FIG. 3. Slt2 activation in heat-stressed S. cerevisiae cells. Detection of dually phosphorylated Slt2 and total Slt2 in glucose-grown cells at 41°C (A) and ethanol-grown cells at 37°C (B) as a function of time is shown. The numbers indicate the time in minutes after temperature increases.
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FIG. 5. Slt2 activation in heat-stressed galactose-grown wild-type and tps1⌬ cells. (A) Detection of dually phosphorylated Slt2 and total Slt2 in a tps1 deletant and its isogenic wild type (CEN-PK133-7D) upon a growth temperature increase from 28°C to 37°C. The numbers indicate the time in minutes after temperature increase. All bands of either phosphorylated Slt2 or total Slt2 are taken from the same blot. (B) Quantification of intensity of the Western blots of dually phosphorylated Slt2. The analysis is based on two independent experiments.
the growth medium. We determined the effect of these changes in extracellular osmolarity on Slt2 phosphorylation for cells that were exposed to a temperature shift to 41°C. An increase in extracellular osmolarity in a time span of a full hour resulted in a significantly lower level of Slt2 phosphorylation during this hour. The level of phosphorylation also depended on the concentration of salt or sorbitol that was added (Fig. 6). When the extracellular osmolarity was increased in a period of half an hour only, the phosphorylation of Slt2 was delayed (Fig. 6). The accumulation of trehalose did not change, compared to conditions of unchanged extracellular osmolarity and the cells retained viability (data not shown).
APPL. ENVIRON. MICROBIOL.
FIG. 6. Slt2 activation in heat-stressed S. cerevisiae cells during a gradual increase of the extracellular osmolarity upon heat stress. Detection of dually phosphorylated Slt2 and total Slt2. Numbers indicate minutes after the temperature shift. Dropwise addition of NaCl or sorbitol was started simultaneously with the temperature shift. All bands of either phosphorylated Slt2 or total Slt2 in the NaCl experiment are taken from the same blot. This is also the case for the sorbitol experiment. (1A) Phosphorylation of Slt2 in glucose-grown, heatstressed (41°C) control cells, with no addition of NaCl or sorbitol. (1B to D) Phosphorylation of Slt2 in glucose-grown, heat-stressed (41°C) cells. The extracellular osmolarity was increased by dropwise addition of NaCl to a final concentration of 0.5 M in a time span of 60 min (1B), a final concentration of 0.5 M in a time span of 30 min (1C), and a final concentration of 1 M in a time span of 60 min (1D). (2B to D) Phosphorylation of Slt2 in glucose-grown, heat-stressed (41°C) cells. The extracellular osmolarity was increased by dropwise addition of sorbitol to a final concentration of 1 M in a time span of 60 min (2B), a final concentration of 1 M in a time span of 30 min (2C), and a final concentration of 2 M in a time span of 60 min (2D). (3) Quantification of intensity of the Western blots of dually phosphorylated Slt2, based on two independent experiments.
DISCUSSION Cell growth and cell viability. Previously, we showed that the transition from growth to death by varying growth temperature is rather abrupt, with regard to growth temperature, when yeast is growing on glucose as a carbon and energy source (16). Here, we have shown that this transition is less abrupt in ethanol-grown cells. From the observation that growth stops at a lower temperature than growth in glucose-grown cells (Fig. 1), it could be concluded that ethanol-grown cells can only cope with less stress. However, the temperature range in which this takes place is larger. One could argue that the cells are already stressed due to the ethanol present, although at a rather low concentration, and therefore cannot cope with an additional temperature stress anyway. The larger temperature range of the transition from growth to death could then be explained along the same line of reasoning: with ethanol present in the environment, stress response mechanisms are already at work at a low level at moderate temperatures, re-
sulting in a less dramatic effect of the additional stress caused by an additional temperature increase. Accumulation of trehalose and the activation of the PKC1 pathway. Trehalose is known to accumulate under heat stress conditions in S. cerevisiae (for examples, see references 2 and 9). In the experiments shown here, accumulation of this compound was indeed initiated at all temperatures used (Fig. 2). In addition, we have shown that the actual level of accumulation in the cell depends on the stress temperature (Fig. 2), peaking at the highest temperature that still allows growth. In glucosegrown cells, the accumulation is only transient, even though the growth temperature remains high. This observation suggests that trehalose plays a role in a transient process that is only important in the phase of adaptation to the new conditions, or that its function is taken over by some other metabolite in the long run. Indeed, it has been shown previously (23) that although trehalose stabilizes proteins during heat shock, it
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also interferes with protein refolding by molecular chaperones. However, this does not explain why trehalose continues to be accumulated in ethanol-grown cells, even when the cells are still growing. It cannot be excluded that this behaviour is pathological, due to the fact that the cells are already exposed to a stressful condition. Upon an increase of the growth temperature to 41°C, a transient activation of the cell integrity pathway was seen in glucose-grown cells (Fig. 3A). Previously, it was reported that this pathway is activated as long as the temperature stress is present (10, 15), although the time spans of these studies and our current experiments differ. Indeed, one would expect that a constant cell wall remodeling is needed as long as the cause for stress on the cell wall is present. Here, we present the hypothesis that the activation of the PKC1 pathway is not caused by the heat stress itself, but by the resulting accumulation of trehalose. The increase of the trehalose concentration in the cell may mimic an environmental hypotonic stress, a condition known to immediately activate the pathway (4). The data presented here show a correlation between trehalose accumulation and activation of the pathway. In the glucose-grown cells, the pathway is only activated when the trehalose level in the cell is increasing (in the first 10 min after the temperature increase, trehalose rises to a concentration of approximately 20 mM in the cells). As soon as the accumulation stops and the level drops again, the pathway is deactivated (Fig. 2A and 3A). In contrast, in ethanol-grown cells, trehalose accumulation continues over time, and so does the pathway activation (Fig. 2B and 3B). This is in good agreement with the observation that the pathway is transiently activated upon hypo-osmotic stress (4). When the increase in intracellular osmolarity stops, the activation of the pathway stops; when the increase remains, the activation remains. If the activation of the PKC1 pathway were indeed caused by an increase in intracellular osmolarity due to trehalose accumulation, one would expect phosphorylation of Slt2 neither in the absence of trehalose synthesis (or other compounds) under heat stress conditions nor upon extracellular osmostabilization of the cells. To test this, we used a tps1 deletant, which could not accumulate any trehalose (Fig. 4). Although we chose a relatively well-growing strain, this mutant was found to be quite heat sensitive (data not shown), as was shown previously (1). Consequently, a lower stress temperature had to be used, at which trehalose accumulation and Slt2 phosphorylation were low anyway (Fig. 5). This observation of lower levels was consistent with our hypothesis, but it makes interpretation of the data on the mutant more difficult. The absence of trehalose accumulation did indeed have an effect on the activation level of the pathway (Fig. 5). Slt2 phosphorylation in the mutant was even lower than that in the wild type, although not completely absent. This partial activation could be caused by the (slight) accumulation of another compound or compounds in the cell contributing to an increase in intracellular osmolarity. It should be realized that there are numerous potential compounds, making it virtually impossible to check them all. The intracellular levels of one obvious candidate, glycerol, has been checked; these did not change compared to nonstressed cells (data not shown). Of course, one could argue that the pathway activation is decreased in the tps1 deletant because the TPS1 gene is involved in stress signaling. Indeed, the gene might
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have at least one sensory function: in addition to trehalose synthesis, it has been suggested that TPS1 plays a role in glucose sensing (26). However, others have shown that there is no decisive evidence for the TPS1 gene being involved in glucose signaling (8, 20) Although no evidence has been provided disproving the possibility of the involvement of TPS1 in stress sensing, it seems unlikely. Our hypothesis is further supported by the data on osmotic stabilization. When the effect of the accumulation of trehalose (i.e., increase in intracellular osmolarity) is counteracted by an increase in extracellular osmolarity, we observed that the activation of the PKC1 pathway was significantly suppressed on increase in growth temperature (Fig. 6). Thus, we have been able to uncouple pathway activation from heat stress, suggesting that a change in temperature is not the primary trigger for activation. As was expected, this effect depends on time and concentration of the osmotic stabilizer (Fig. 6). Only when the extracellular osmolarity is increased during the whole period that the intracellular trehalose level rises (during the first hour) is activation of the pathway almost absent. The higher the concentration of the osmotic stabilizer, the lower the activation of the signaling pathway. To exclude the possibility of a toxic effect of, in particular, these high NaCl concentrations (and thus a lowering of the level of PKC1 pathway activation), cell viability was monitored and found to be unchanged. A consequence of our hypothesis would be that the final osmolyte concentration needed to fully suppress the induction of the pathway should be osmotically equivalent to the cytosolic trehalose concentration. For a cell volume of 3 m3 with a cytosolic cell fraction of 40%, it can be calculated that 1 M NaCl or 2 M sorbitol would be counterbalanced by 11 fmol/cell trehalose. This value is higher than what we measured, but it may well be that reduced cytosolic volumes occur due to enlarged vacuoles. A proper comparison would demand a timeresolved analysis of the volumes of all cellular compartments. Furthermore, trehalose is just one of the compounds of which the intracellular concentration can increase under heat stress conditions. Our data on osmotic stabilization correlate well with previous data on the effects of altering the extracellular osmolarity (10). Although Kamada and coworkers suggest that the activation of the PKC1 pathway is caused by plasma membrane stretch under hypo-osmotic stress conditions, they only speculate on the cause of activation under heat stress conditions. Our data suggest that the pathway is activated by an increased intracellular osmolarity invoked by trehalose accumulation under heat stress conditions. Finally, it has been reported that an Slt2 deletant is heat sensitive but that this phenotype can be suppressed by osmotic stabilization (11, 25). This observation ties in nicely with our hypothesis of a link between trehalose accumulation (hence, a change in intracellular osmolarity) and Slt2 pathway activation. ACKNOWLEDGMENTS This research was supported by a grant from Unilever Research. We thank M. Molina for the gift of the anti-Slt2 antibody. REFERENCES 1. Arguelles, J. C. 1994. Heat-shock response in a yeast tps1 mutant deficient in trehalose synthesis. FEBS Lett. 350:266–270.
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