INTRODUCTION

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Exposure of cells or organisms to elevated temperatures triggers the synthesis of heat shock proteins (hsps), which help protect cells against high temperatures and a variety of other potentially toxic agents (39, 51). Many of these hsps function as molecular chaperones that help prevent the accumulation of unfolded or aggregated polypeptides (21). In growing cells, the hsps catalyze the proper folding of nascent polypeptide chains, and upon heat shock, these chaperones prevent protein aggregation and promote the refolding of damaged polypeptides (15). Another important function of certain hsps is to promote the rapid degradation of such abnormal proteins (28, 32, 47). In eukaryotes, ubiquitin and certain ubiquitin-conjugating enzymes are hsps that function in the rapid breakdown of denatured proteins (39). In addition, certain molecular chaperones have been shown to serve as cofactors in the selective degradation of abnormal polypeptides (28, 32).

The induction of heat shock response can lead to increased tolerance of cells to otherwise lethal, high temperatures. For example, when yeast cells growing at 25°C are shifted to an intermediate temperature, e.g., 37°C, to cause induction of hsps, the fraction of cells able to survive a subsequent exposure to 50°C increases markedly. This increase in thermotolerance is generally believed to require the induction of hsps (39), although this requirement has been questioned (3, 20, 48). The induction of the heat shock response can also protect cells against a variety of other toxic insults, such as ethanol and hydrogen peroxide (42, 49). In fact, in experimental animals, the exposure to 42°C to induce hsps has been shown to protect heart and brain against subsequent anoxic injury (36). Consequently, there has been appreciable medical interest in the possibility of inducing this response in patients. Because elevating body temperatures is an inconvenient and potentially dangerous procedure, the identification of pharmacological agents that could elicit this protective response would be highly desirable.

Hsps are also induced by a variety of other insults to the cell, such as ethanol, heavy metals, and oxidants (42). One common feature of these various conditions is that they damage or denature cell proteins. Other treatments that prevent the proper folding of newly synthesized proteins (e.g., incorporation of amino acid analogs) or introduction of unfolded proteins into bacterial or vertebrate cells also causes the induction of hsps (1, 22, 40). Thus, it is widely believed that the common feature of the various conditions that elicit this response is the accumulation of abnormal polypeptides in cells. Similarly, it is now well established that the accumulation of unfolded proteins in the endoplasmic reticulum (ER) signals the induction of many ER-specific molecular chaperones (8, 35).

The cells' capacity to degrade rapidly such unfolded proteins is therefore likely to be one important determinant influencing the expression of hsps. The major pathway for the selective degradation of abnormal proteins in the cytosol and nucleus is the ubiquitin-proteasome pathway (7, 18). A failure of function of this degradative system should lead to the induction of hsps. In fact, increased thermotolerance was observed in a yeast mutant in which genes encoding the ubiquitin-conjugating enzymes UBC4 and UBC5 were deleted (45).

A major goal of the present study was to test whether pharmacological agents that block proteasome function, by causing an accumulation of abnormal proteins, might increase the expression of hsps and thermotolerance. The magnitude and rapidity of such a response should depend on the extent of inhibition of protein breakdown and the frequency of production of abnormal polypeptides in normal cells. Recently, several selective inhibitors of the proteasome that can enter mammalian cells and inhibit the ubiquitin-proteasome pathway (e.g., the reversible peptide aldehydes such as MG132 or the irreversible modifiers lactacystin and clasto-lactacystin -lactone) have been identified (11, 14, 25, 41). Certain of these inhibitors also selectively block the degradation of short-lived and abnormal proteins in intact Saccharomyces cerevisiae cells (31). In yeast cells, unlike in mammalian cells, these proteasome inhibitors do not affect the breakdown of bulk of cell proteins, which are long-lived and degraded in the vacuole (31). In related studies of MDCK cells, we have recently found that exposure to proteasome inhibitors can cause an induction of hsps (4). The present study of yeast not only establishes the generality of this effect but also systematically investigated the mechanism of this response and its physiological consequences. We demonstrate here that proteasome inhibitors at concentrations that do not appear harmful, through their inhibition of protein degradation, cause an induction of hsps in yeast and concomitantly cause an increase in the cells' resistance to high temperature. However, this protection against high temperature could not be explained simply by the buildup of hsps. We present evidence that proteasome inhibitors also cause the accumulation of another thermoprotectant molecule, the disaccharide trehalose, whose content correlates with the cells' resistance to high temperature.

	MATERIALS AND METHODS

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Measurement of the synthesis of hsps. The S. cerevisiae ise1 strain used in this study, which is permeable to several proteasome inhibitors (31), is JN 284 (MAT his7 leu2 ura3 ise1; kindly provided by J. C. Wang, Harvard University). This strain was grown exponentially in methionine-free glucose minimal (SD) medium at 30°C. At different times after exposure to the proteasome inhibitors, the cells were incubated with 200 µCi of [³⁵S]methionine (Tran³⁵S-label; 1,000 Ci/mmol; ICN) for 5 or 30 min. Preparation of cell extracts and immunoprecipitation were carried out as described previously (32).

Measurement of protein breakdown in vivo. The ise1 cells grown exponentially in methionine-free SD medium were incubated with proteasome inhibitors or phenylmethylsulfonyl fluoride (PMSF) for 90 min prior to labeling. These cells were then labeled for 5 min with 100 µCi of [³⁵S]methionine. After two washes, cells were resuspended in fresh SD medium containing methionine (0.5 mg/ml) and cycloheximide (0.5 mg/ml) to prevent reincorporation of radioactive amino acids released from proteins. At different time intervals, aliquots of cells were taken and mixed with 100% trichloroacetic acid to give a final concentration of 10%. After incubation at 4°C for 1 h, the samples were centrifuged, and the radioactivity in the trichloroacetic acid-insoluble material (precipitates) was measured. The rate of protein degradation is expressed as the percentage of incorporated radioactivity that is converted into acid-soluble fragments from the cells during the chase period (means ± standard deviations [SD]).

Assay of cell resistance to heat. Thermotolerance assays were carried out as described elsewhere (44), with some modifications. Prior to heat treatment, ise1 cells growing exponentially at 30°C in SD medium were incubated with proteasome inhibitors for 2 h (except in Fig. 3C) and then shifted to 52°C for the indicated time. Cells were then diluted 200-fold and plated onto YPD medium to determine the number of viable colonies.

Extraction and assay of trehalose. Trehalose was extracted from yeast cells and assayed as described previously (29), with some modifications. Exponentially growing yeast cells were collected by centrifugation and then washed twice in cold water to remove free glucose. Cells were resuspended in 10 to 20 volumes of ice-cold water and incubated at 95°C for 20 min, and then the supernatant was collected by centrifugation. The amount of trehalose was measured by treatment of this supernatant with trehalase (20 mU/sample; Sigma Chemical Co.), which hydrolyzes trehalose to glucose. After 6 to 8 h of incubation at 37°C, the amount of glucose generated was assayed with a glucose assay kit (Sigma) containing hexokinase and glucose-6-phosphate dehydrogenase. The preexistent glucose in each sample (usually less than 5% of the amount generated by trehalase) was assayed in a parallel tube without trehalase and was subtracted from total glucose. The total amount of proteins in each sample was also measured by the Bradford method (Pierce) for the calibration. The cellular content of trehalose was expressed as the nanomoles of trehalose per microgram of cell protein.

	RESULTS

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Induction of hsps by the inhibitor of proteasome. To test whether the inhibition of proteasome function influences the synthesis of various hsps, yeast cells growing at 30°C were exposed to [³⁵S]methionine for a 30-min pulse in the presence or absence (dimethyl sulfoxide [DMSO] control) of a potent inhibitor of proteasomes, MG132 (Cbz-LLLal). We then measured the rates of incorporation of ³⁵S into four different hsps (hsp104, hsp70, Ydj1p, and Sis1p) after isolation of each by immunoprecipitation. Because these inhibitors fail to penetrate into wild-type cells (31), we used an ise1 permeability mutant strain (38). Upon incubation with 50 µM MG132, [³⁵S]methionine incorporation into all of these hsps increased within 1 h and continued to increase linearly for 3 h (Fig. 1). Synthesis of hsp104 showed the largest relative increase (three- to fourfold) after exposure to MG132. The DnaJ homologs Ydj1p and Sis1p also showed a 2- to 3-fold increase in synthetic rates, while incorporation into hsp70 seemed to rise only 1.5- to 2-fold, perhaps because the antibody used cannot distinguish the heat-inducible species from the several constitutive species of hsp70 (Fig. 1). These findings clearly indicate steadily increasing rates of labeling of multiple hsps during a 30-min pulse of [³⁵S]methionine with longer exposure to MG132. To ensure that these findings represent increased rates of synthesis and are not complicated by changes in degradation of the labeled hsps in the presence of MG132, cells were exposed to MG132 for 2 h and then to the 5-min pulse of [³⁵S]methionine. The data shown in Fig. 1C also indicated two- to threefold more rapid labeling of hsps under these conditions. Control studies showed that MG132 did not stimulate the synthesis of these hsps in wild-type yeast, where this agent does not penetrate and thus cannot affect protein breakdown (data not shown). Therefore, these data must reflect increased rates of synthesis and cannot be explained by the inhibitor's preventing degradation of hsps (especially since hsps are rather stable proteins). Also, these findings are in accord with observation in mammalian cells, where this inhibitor causes hsp accumulation through enhanced gene expression (4, 52).

View larger version (30K): [in this window] [in a new window]   FIG. 1.   MG132 increases the synthesis of hsps in yeast cells. ise1 cells were incubated during growth at 30°C with either 50 µM MG132 (dissolved in DMSO) or 0.1% DMSO (control). (A) The rates of synthesis of several hsps (hsp104, hsp70, Ydj1p, and Sis1p) were then measured by pulse-labeling cells with 200 µCi of [35S]methionine for 30 min at different times. Shown are the times at the end of the 30-min pulse (1, 2, or 3 h after MG132 was added). After cell lysis, immunoprecipitation with antibodies against these hsps was performed with equal amounts of radioactive proteins in control or MG132-treated cells. (B) Relative induction rates of these four hsps based on data in panel A. Data shown are the mean values ± SD from three independent experiments. (C) To show that these effects of MG132 were due to enhanced synthesis of hsps, the ise1 cell were incubated with either 50 µM MG132 or 0.1% DMSO (control) for 2 h and then labeled with 200 µCi of [35S]methionine for 5 min. The synthesis of hsp70, Ydj1p, and Sis1p was measured as for panel A.

Proteasome inhibitor induces hsps without reducing cell growth. Most treatments that cause induction of hsps (e.g., incorporation amino acid analogs, puromycin, or heavy metals) are themselves harmful to cells and can rapidly reduce growth rate or viability. Surprisingly, MG132, at concentrations that caused induction of hsps, had no or very little effect on the growth of yeast at 30°C (Fig. 2). Incubation of growing cells with increasing concentrations of MG132 (20 to 100 µM) for 2 h did not significantly reduce the number of colonies on YPD plates (Fig. 2A), even though this agent caused a marked inhibition of intracellular proteolysis (Fig. 3B). Also, in liquid cultures, exposure to 50 µM MG132 did not reduce cell growth for 3 h, and by 24 h, the optical density of the treated culture was only 15 to 30% lower than that in the control (Fig. 2B). These experiments all used the ise1 strain, whose growth rate was about 50% lower than those of typical wild-type strains, e.g., W303 (data not shown), presumably because of the defect in its cell membrane (19).

View larger version (14K): [in this window] [in a new window]   FIG. 2.   MG132 does not affect the overall growth of yeast cells. (A) After incubation with different concentrations of MG132 for 2 h, ise1 cells were diluted 200-fold and then inoculated onto YPD plates to determine the numbers of surviving colonies. (B) For measurement of cell growth in the presence of 50 µM MG132 (up to 24 h) or with 0.1% DMSO (control), we took aliquots at the indicated times and measured their optical densities at 600 nm (OD600). Data shown here are the mean values ± SD from three independent experiments.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   MG132 increases thermotolerance in yeast cells. (A) ise1 cells growing at 30°C were incubated with different concentrations of MG132 or with 0.1% DMSO (control) for 2 h and then exposed to 52°C for the indicated times. The cells were diluted 200-fold and plated onto YPD medium, and the fraction of viable cells was measured as the number of colonies formed. (B) The ability to block the breakdown of short-lived proteins by MG132 is proportional to its ability to increase thermotolerance. Degradation of cell proteins after 5-min pulse-labeling with [35S]methionine was measured in the presence or absence of MG132 as described previously (32). (C) Time course for the increase in thermotolerance by MG132. The ise1 cells were incubated at 30°C with or without 50 µM MG132 for the indicated times. After exposure to 52°C, cell survival was measured. Data are mean values ± SD from four independent experiments.

Inhibition of proteasome function induces thermotolerance. To test if exposure to proteasome inhibitors also increases the cells' resistance to high temperatures, we incubated exponentially growing ise1 cells at 30°C with different concentrations of MG132 for 2 h and then exposed them to 52°C for 5 to 20 min. To measure the number of cells still viable and able to form colonies, the cells were then diluted 200-fold and plated in medium lacking the inhibitor. After dilution of the treated cells, rates of protein breakdown should have returned to control level, since MG132 is a reversible inhibitor, and its effects on proteolysis in vivo are rapidly reversed upon removal of this inhibitor (31). After a 5-min exposure to 52°C, less than 0.1% of control cells could form colonies. However, the cells incubated with MG132 showed 5- to 100-fold-greater survival, depending on the concentration used. Similarly, after 20 min at 52°C, when less than 0.01% of control cells survived, the MG132-treated cells showed 10- to 50-fold-higher survival rates (Fig. 3A). The ise1 strain was two to three times more sensitive to killing at 52°C than typical wild-type strains (e.g., W303), presumably due to its defect in the biosynthesis of principal membrane sterol (19). Nevertheless, MG132 caused a 50- to 100-fold increase in thermotolerance, such that this mutant strain became much more resistant to high temperature than wild-type cells. These results are also consistent with the finding for mammalian cells, where this same inhibitor also increased thermotolerance dramatically (4).

View larger version (21K): [in this window] [in a new window]   FIG. 4.   The ability of different protease inhibitors to induce thermotolerance and hsps correlates with their ability to inhibit degradation of short-lived proteins. ise1 cells were incubated with MG101 (50 µM), MG132 (50 µM), lactacystin (20 µM), or -lactone (20 µM) for 2 h or PMSF (1 mM) for 2 to 24 h and exposed to 52°C for 5 min, and then cell survival was measured. In parallel, cells were pulse-labeled for 5 min with [35S]methionine to measure the degradation of short-lived proteins as described previously (32) and labeled for 30 min to measure the content of hsp104 and Ydj1p as described for Fig. 1. Data presented are the mean values + SD from three independent experiments.

Dissociation of thermotolerance from hsp production. When yeast (or other) cells are preincubated at a high but not lethal temperature (e.g., 37°C), hsps are induced, and a larger fraction of cells survive a subsequent exposure to 50°C than when they are switched directly from 28 to 50°C (39). If the induction of hsps and induction of thermotolerance at 37°C are in fact signalled by the generation of abnormal proteins, these effects should be greater in the presence of proteasome inhibitors, which prevent the rapid breakdown of such proteins. To test if the protective effects of preincubation at 37°C and proteasome inhibitors are additive or synergistic, we incubated exponentially growing ise1 cells at 30°C with MG132 for 2 h and then exposed them to 37°C for another 30 min prior to the shift to 52°C for 5 to 20 min. As expected, incubation with either MG132 or 37°C increased cell survival 10- to 50-fold (Fig. 5). However, the cells pretreated with MG132 and then incubated at 37°C showed an additional 3- to 10-fold-greater survival than that induced by incubation at 37°C alone (Fig. 5). As a result of the combined treatment, 25 to 30% of cells survived exposure to 52°C for 10 min, while only 0.1% of control cells did. Even after 20 min at 52°C, 8 to 10% of cells preincubated with MG132 and 37°C survived, while less than 0.01% of control cells and 1% of those preincubated only at 37°C were viable (Fig. 5). Thus, these two stimuli are clearly synergistic in enhancing thermotolerance.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Effects of combined exposure to MG132 and 37°C on thermotolerance and the level of hsps. Exponentially growing ise1 cells were preincubated for 2 h with 50 µM MG132 or with 0.1% DMSO (control). The cultures were then divided in half, and one half was incubated at 37°C for an additional 30 min. The cells were then exposed to 52°C for 5 to 20 min, and survival was measured. In parallel, equal volumes of these cells were collected before the exposure to 52°C, and their hsp104 and Ydj1p contents were determined by Western blotting. For quantitation, the blots were incubated with 125I-protein A for 2 h and subsequently subjected to autoradiography. Data presented are the mean values ± SD from four independent experiments.

View larger version (19K): [in this window] [in a new window]   FIG. 6.   Effects of MG132 removal on thermotolerance and the level of hsps. After a 2-h incubation with 50 µM MG132, ise1 cells were washed with fresh medium to remove the inhibitor. Half of the cells were then resuspended in medium containing 50 µM MG132 (MG132), and the other were incubated with 0.1% DMSO alone (MG132 removed). These cells were incubated for an additional 2 h. At the indicated times, aliquots were taken and assayed for resistance to 52°C for 5 min. In parallel, the contents of hsp104 and Ydj1p in these aliquots were assayed by Western blotting as described for Fig. 5. Data are mean values ± SD from four independent experiments.

Rapid accumulation of trehalose upon inhibition of proteasome function. One other molecule that has been shown to accumulate during heat shock in yeast and other microorganisms is the nonreducing disaccharide trehalose (2, 10, 37). Moreover, its accumulation has been shown to correlate with cellular resistance to heat and dessication (2, 10, 37), and trehalose and hsp104 appear to have synergistic effect in protecting yeast cells from heat (13). As an attempt to identify additional mechanisms by which proteasome inhibitors promote thermotolerance, we tested whether MG132 may cause an accumulation of trehalose in yeast. Upon incubation of growing ise1 cells with 50 µM MG132 at 30°C, the level of trehalose in the cells increased markedly. After 3 h, a two- to threefold increase in its level was observed (Fig. 7A). A similar buildup of trehalose was seen when part of the culture was shifted from 30 to 37°C to induce heat shock, in accord with prior reports (10, 13). Furthermore, treatment of the cells with a highly specific and irreversible inhibitor of the proteasome, clasto-lactacystin -lactone, at a concentration (20 µM) that inhibited protein breakdown by 40 to 50% (Fig. 4) caused an increase in the cellular level of trehalose similar to that seen with MG132 (Table 2).

View larger version (18K): [in this window] [in a new window]   FIG. 7.   Effect of MG132 on the cellular content of trehalose. (A) Exponentially growing ise1 cells were treated with or without MG132 (50 µM) or incubated at 37°C. At indicated times, aliquots of cells were collected and their trehalose contents were measured. (B) After 2 h of incubation with MG132 (50 µM), the inhibitor was removed by washing cells with fresh medium. These cells were then resuspended in the medium without MG132 and further incubated for 2 h. At the given times, cells were collected and trehalose contents were measured. Data are mean values ± SD from three independent experiments.

View larger version (16K): [in this window] [in a new window]   FIG. 8.   Effect of combined exposure to MG132 and 37°C on the cellular content of trehalose. Exponentially growing ise1 cells were preincubated for 2 h with 50 µM MG132 or with 0.1% DMSO (control). The cultures were then divided in half, and one half was incubated at 37°C for an additional 30 min. After collecting cells by centrifugation, we measured the cellular level of trehalose. Data presented are the mean values ± SD from three independent experiments.

	DISCUSSION

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Mechanism of induction of hsps and thermotolerance. A common feature of the diverse conditions that elicit the heat shock response is that they cause damage to cell proteins. The present findings provide further strong evidence that the accumulation of such abnormal proteins signals this response (1, 17, 22, 40). We found that inhibition of proteasome function by MG132 or the -lactone, which prevents the rapid degradation of abnormal proteins, causes induction of all four hsps tested and a dramatic increase in thermotolerance. The magnitude of the increase in cell survival at 52°C was directly proportional to the degree of inhibition of protein breakdown and its duration (Fig. 3). Similar effects were seen in studies with a mutant strain in which one of the peptidase activities of the proteasome is inactivated (data not shown). Furthermore, no such effects were seen with protease inhibitor that did not block protein breakdown by the proteasome (e.g., inhibitor of the vacuolar proteases) (Fig. 4). Also, high temperatures, which should cause damage to cell proteins, and MG132, which reduces their degradation, had synergistic effects in promoting thermotolerance (Fig. 6).

Dissociation of thermotolerance from induction of hsps. Induction of hsps has been generally assumed to lead to thermotolerance, especially since most hsps are either molecular chaperones which can help prevent protein aggregation and promote refolding or components of the degradative system (e.g., ubiquitin and ubiquitin-conjugating enzymes) which help eliminate such irreversibly damaged polypeptides at high temperatures. Treatment with proteasome inhibitors, while inducing hsps, increased up to 100-fold cell survival at 52°C. Under these conditions, we also have found a marked increase in cellular resistance to other toxic insults (e.g., high concentration of ethanol or oxygen radicals) (data not shown). However, the cellular content of hsps did not correlate with thermotolerance, even though both responses appear to result from the same physiological signals. The dissociation of these two responses was most dramatic after removal of MG132 when the cells' resistance to heat and oxygen radicals fell rapidly, while hsp production continued to increase (presumably due to the stability of hsps and their mRNAs). In addition, when yeast cells were exposed to both 37°C and MG132, these stimuli had synergistic effects in increasing cell survival, even though hsp content did not increase appreciably above levels seen with either stimulus alone. Thus, some component, in addition to hsps, is necessary for tolerance to heat.

Involvement of trehalose in the induced thermotolerance. The present findings favor a simpler explanation, i.e., that the resistance to high temperatures requires a short-lived, small molecule, specifically, trehalose, which has thermoprotective effects and accumulates when protein breakdown is inhibited. Several conditions that induce the heat shock response in S. cerevisiae have been found to also cause a buildup of trehalose, in part by stimulating the expression of enzymes for trehalose biosynthesis (13, 23, 37). Furthermore, the time course of the accumulation of trehalose upon heat shock and the decline in its level following the return to the normal temperature paralleled the changes in thermotolerance (2). In the present study, we have shown that trehalose rapidly accumulates in cells, when the function of the proteasome is inhibited by treatment either with MG132 or with the specific, irreversible proteasome inhibitor, the -lactone. It is noteworthy that very similar results were obtained with these structurally unrelated types of inhibitors. MG132 is a reversible peptide aldehyde that functions as a substrate analog, and the -lactone is an irreversible inhibitor that covalently modifies 20S proteasome's active site threonine and no other cell protein (14). Together these observations confirm that the buildup of trehalose was a specific consequence of the inhibition of proteasome function in the cells. Moreover, the cellular content of this disaccharide correlated closely with changes in viability at 52°C, unlike the cells' content of hsps. Upon removal of the inhibitor and restoration of protein breakdown, the levels of trehalose fell dramatically within 30 min, as did cell resistance to high temperatures. In addition, heat shock (37°C) and MG132 had additive or synergistic effects in raising trehalose content and thermotolerance. Under these same conditions, the content of hsps did not correlate with thermotolerance.