Proc. Nati. Acad. Sci. USA Vol. 88, pp. 11091-11094, December 1991 Genetics
Uncoupling thermotolerance from the induction of heat shock proteins (heat-shock factor/Saccharomyces cerevisiae/stress response)
BARBARA J. SMITH* AND MICHAEL P. YAFFEt University of California, San Diego, Department of Biology, B-022, La Jolla, CA 92093
Communicated by Dan L. Lindsley, September 19, 1991
gene. Cells expressing the mutant HSF protein display temperature-sensitive defects in growth, mitochondrial protein import, cell-cycle progression, and the induction of a major hsp70 gene, SSAI (23). Here we report an analysis of the effect of the hsfl-m3 lesion on the expression of a number of hsps and a test of the ability of the mutant cells to acquire thermotolerance.
Exposure of cells to elevated temperatures ABSTRACT causes a rapid increase in the synthesis of heat shock proteins (hsps) and induces thermotolerance, the increased ability of cells to survive exposure to lethal temperatures; however, the connection between hsp induction and the acquisition of thermotolerance is unclear. hsp induction in the yeast Saccharo-
myces cerevisiae is mediated by the activation of heat-shock transcription factor, and recently we have described a mutation, hsfl-m3, in heat-shock transcription factor that prevents the factor's activation. We now demonstrate that this mutation results in a general block in heat-shock induction but does not affect the acquisition of thermotolerance. Our results indicate that high-level induction of the major hsps is not required for cells to acquire thermotolerance.
MATERIALS AND METHODS Yeast Strains and Media. Saccharomyces cerevisiae AH216 (MATa, leu2, his3) and MYY243 (MATa, leu2, his3, hsfl-m3) were described (23). These strains are isogeneic except for the hsfl-m3 lesion. Derivatives of AH216 and MYY243 containing an HSE2-4acz fusion gene integrated into the URA3 chromosomal locus were created. The HSE2lacz fusion was constructed by first annealing the -HSEcontaining synthetic oligonucleotide 5'-CTCGATCTAGAAGCTTCTAGAAGCTTCTAGA-3' to itself and then ligating the duplex DNA fragment into the unique Xho I site located upstream of the lacz gene in the plasmid pLG669Z (24). Yeast replication sequences were removed from the plasmid on an EcoRP fragment, and the yeast LEU2 gene, isolated from plasmid YEp13 (25) on a Sal I-Xho I fragment, was inserted in the Sal I site. The DNA construct was integrated into the chromosomal URA3 gene of strains AH216 and MYY243 by integrative transformation (26) after cutting the plasmid DNA at the unique Stu I site in the URA3 gene. YPD medium contained 1% yeast extract, 2% (wt/vol) Bacto Peptone, and 2% (wt/vol) glucose. SD medium (27) was prepared without asparagine and threonine. Analysis of (3-Galactosidase Activity and hsp Synthesis. ,3-Galactosidase activities were determined as described (28).in For analysis of hsp synthesis, yeast were grown at 230C SD medium to 0.5 OD6w unit. Cells were concentrated 2-fold by centrifugation and incubated for 15 min at 230C or 370C. Next, L-[4,5-3H]isoleucine (84 Ci/mmol; 1 Ci = 37 GBq) was added to 50 ,uCi/ml of cells and incubations were continued at the same temperature for 30 min. Proteins were extracted immediately as described (29), separated by SDS/PAGE, and detected by fluorography. Analysis of Thermotolerance. Cells were grown in YPD medium at 230C to 5 x 105 cells per ml or to 5 x 106 cells per ml. Cultures were divided into two portions, one of which was shifted directly to 520C, and a second portion was preincubated at 370C for 1 hr prior to exposure to 520C. Samples of cells were removed after incubation at the high temperature, diluted with ice water, and plated immediately
The heat-shock response, a rapid and transient increase in the expression of a discrete set ofheat-shock genes, is a universal reaction of cells exposed to such adverse conditions as increased temperatures, anoxia, elevated levels of ethanol, and certain heavy metals (1-3). The cellular concentrations of the heat shock proteins (hsps) increase dramatically as a consequence of the activated gene expression. Elevated levels of these hsps are believed to help cells survive the deleterious effects of the environmental stress (3, 4). Temperature-induced hsp expression in yeast is mediated by the specific activation of the heat-shock transcription factor (HSF) (5-7). Transcriptional induction mediated by HSF depends on a regulatory DNA sequence, the heat-shock element (HSE), which is present in a few or multiple copies in the promoter regions of heat-shock genes (8, 9). Genetic analysis of HSF function in yeast indicates that HSF is also responsible for the low-level basal transcription of hsps under noninducing conditions, and this constitutive expression is essential for normal cellular growth (10, 11). The constitutively synthesized hsps or their cognates have been implicated as essential components for protein transport into and assembly within cytoplasmic organelles (12-18), and these and other hsps are thought to mediate protein folding, conformational changes, or subunit interactions (19, 20). The role of hsp induction in response to stress remains a mystery. A common assumption (3, 4) is that increased hsp levels serve a -protective function. This assumption largely stems from studies of induced or acquired thermotolerance in which the mild stress of a cell population results in greatly increased numbers of cells surviving a subsequent normally lethal stress (4, 21, 22). Many of the conditions that induce thermotolerance also lead to a rise in hsp levels, but the connection between this activation and the acquisition of thermotolerance remains obscure. We have described (23) a yeast strain that contains a mutant allele, hsfl-m3 (formerly called mas3), of the HSF
Abbreviations: hsp, heat shock protein; HSF, heat-shock transcription factor; HSE, heat-shock element. *Present address: The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
11091
Proc. Natl. Acad. Sci. USA 88 (1991)
Genetics: Smith and Yaffe
11092
on YPD plates. Viability was scored after 5 days of incubation at 230C.
m3
WT C H
C
H
RESULTS To further characterize the consequences of the hsfl-md mutation, the levels of (3-galactosidase produced from an HSE2-4acz fusion construct were analyzed. Enzyme activity from such a reporter gene reflects transcriptional activity mediated solely by HSF (30). In wild-type cells the levels of enzyme activity increased to a maximum after 1 hr of 23-fold over basal levels and declined to a new higher level (10-fold over the uninduced level) after 4 hr at 370C (Fig. 1). In contrast, the increase in activity in cells carrying hsfl-n3 was less than 1.5-fold after 1 hr at 3T7C with a total increase of only -3.3-fold by 4 hr: The basal level of P-galactosidase activity (Fig. 1) was similar in both mutant and wild-type cells. The lack of (3-galactosidase' induction observed with hsfl-m3 cells cannot be attributed to cell death, since the number of viable cells declined only after 6-8 hr at 370C (23). These results indicate that the mutant hsfl-n3 protein retains normal basal transcriptional activity but is defective for heat-inducible activity. The pattern of protein synthesis changes rapidly in wildtype cells exposed to elevated temperatures: the synthesis of most proteins decreases or stops, synthesis of a few proteins remains the same, and the synthesis of the classical hsps increases dramatically (31). To determine if cells carrying the hsfl-m3 mutation are also defective in the induction of endogenous heat-shock genes, we examined protein.synthesis in control and heat-shocked cultures. Striking differences in the pattern of protein synthesis from mutant and wild-type cells were apparent (Fig. 2). Whereas synthesis of the major hsps of 104 kDa, 82 kta, and 70 kDa rose substantially in wild-type cells after heat shock, these same proteins. increased only slightly or not at all in heat-shocked mutant cells (Fig. 2). The levels of certain non-heat-shock proteins that did not 'change after heat-shock treatment in wild-type cells also remained constant in mutant cells. Additionally, the decreased synthesis of 'many cellular proteins after heatshock appeared similar in both mutant and wild-type cells. The above results demonstrate that the hsfl-m3 mutation specifically affects the induction of the major hsps during heat-shock treatment. A variety of stress treatments that induce hsp production also enhance the survival of cells exposed to elevated lethal temperatures (3). To test the effect of the hsfl-m3 mutation
-hsp 104 _ _
-
-hsp 82 -hsp 70
....
AM.
,
Mk
k
w
v
i _
3_E,4 _
He
IIII~
~
IS.
_
FIG. 2. Temperature-regulated induction of endogenous HSP synthesis. Cultures of wild-type (WT) and mutant (m3) cells were grown at 230C,inminimalmediumnto midloithmicsphe. A portion ofeach culture was either kept at 230C (lane C) or shafted to 370C (lane H) for 15 mu, [3H]Isoleucine was lidded to all samp iand incubations were continued at the same temperature for 30 min. .The labeled proteins were extracted (32) and separated by elcoph resis in a 7% polyacrylamide gel in the presence. of SDS. Labeled protein bands were detected by fluorography. Putative hsps were identified by molecular weight standards electrophoresed in an adjacent lane and by comparison with published hsp patterns (32, 33). hsp70 indicates a group of several isozymes with similar molecular weights (32).
on this induced thermotolerance, cell viability was determined after exposure for various periods'to 520C, with or without a prior incubation at 370(. Pretreatment at 370( induced thermotolerance to similar extents in both mutant and wild-type cells (Fig. 3). Cultures also showed essentially identical thermotolerance curves when pretreatment was carried out at 390C (data not shown).. Additionally, the hsfl-m3 and wild-type cultures both displayed striking thermosensitivity when exposed directly to 520C, although the mutant cells died more slowly than did the wild-type cells.
DISCUSSION Our results demonstrate that the activation of HSF and the resulting induction of high levels of hsps are not required for cells to acquire thermotolerance. Such'a conclusion is contrary to the prevalent view (4, 34) that hsp induction leads to a thermotolerant state (Fig. 4A). An alternative model (Fig. 4B) is that thermotolerance results from the temperature-
40 CD
20 0
ca
10
0
12
0~~~~~~~~~~~~~~~.0
0.0
1.0
2.0
3.0
4.0
Time at 370C, hr
FIG. 1. Induction of HSE2-LacZ activity. Wild-type (solid circles) and mutant (open circles) strains containing an integrated copy of the heat-inducible HSE2-4acz gene were grown at 23°C and shifted to 37rC. Samples were removed after 0, 0.5, 1, 2, and 4. hr (as indicated), and (-galactosidase activities were determined. Values for each time point are the average of three samples, and the levels of induction obtained in three experiments varied by less than 15%.
Time at 5200, mm
FIG. 3. Thermotolerance of mutant cultures at 520C bef~re and after mild heat treatments. Wild-type cells (solid symbols) and mutant cells (open symbols) were grown in YPD medium at 23°C to 5 x 105 cells per ml. One portion (triangles) of the culture was shifted directly to 520C, and a second portion (squares) was preincubated at 3rC for 1 hr prior to exposure to 52MC. Samples were removed after 0, 5, 10, and 20 min, and. samples were analyzed for cell viability. Similar results were obtained after a 30-min preincubation at 370C or with cells grown to 5 X 101 cells per ml.
Proc. Natl. Acad. Sci. USA 88 (1991)
Genetics: Smith and Yaffe
11093
B
A
HEAT
HEAT
HSF ACTIVATION
HSF ACTIVATION
FACTOR MODIFICATION
I
HSP INDUCTION
THERMOTOLERANCE
HSP INDUCTION
THERMOTOLERANCE FIG. 4. Alternative models for the heat induction of thermotolerance. (A) Increased temperature leads to the activation of HSF, resulting in increased levels of hsps. Elevated hsp levels confer thermotolerance on cells. (B) Thermotolerance results from the heat-induced modification of one or more cellular components and is independent of hsp induction.
dependent activation of one or several factors and is independent of HSF-mediated induction of hsps. For example, elevated temperatures might activate a heat-dependent enzyme, whose product confers thermotolerance, or heat might promote the binding of a protective protein to a thermosensitive cellular structure. Previous studies have produced conflicting conclusions concerning the relationship between hsp induction and the acquisition of thermotolerance (2). Several investigations have provided evidence that the inhibition of protein synthesis blocks the acquisition of thermotolerance (21, 35). Additionally, mutant strains ofEscherichia coli and Dictyostelium discoideum, defective for the acquisition of thermotolerance, also failed to accumulate certain h'sps during heat shock (22, 36). Other studies have suggested that protein synthesis is not required for the acquisition of thermotolerance (37-39) or demonstrated a lack of correlation between thermotolerance and high hsp levels (40, 41). In certain types of cells the acquisition of thermotolerance might indeed require the induction of one or several proteins, followed by the heat-dependent modification of these polypeptides. Such cells would require protein synthesis to acquire thermotolerance. Other cells, such as those employed in this study, might possess sufficient levels of stressprotection proteins even during growth under nonstressed conditions.'This would alleviate a requirement for protein synthesis, although a temperature-dependent modification of one or several cellular proteins would still be necessary. Such a variation between different types of cells (or even different growth conditions) might explain the conflicting results concerning the requirement of protein synthesis for the acquisition of thermotolerance. Sanchez and Lindquist (33) demonstrated that a null mutation in a specific heat-shock gene, HSP104, rendered yeast cells defective for the acquisition of thermotolerance. This finding is not inconsistent with our results, which indicate induction of hspl04 is substantially deficient in the hsfl-m3 mutant (Fig. 2). A reasonable interpretation is that constitutive levels of hspl04 are necessary for thermotolerance acquisition but that high-level induction of the protein is not. Acquired thermotolerance might result, instead, from a temperature-dependent change in the activity or structure of preexisting hspl04 and, perhaps, in the alteration of other proteins. Future studies should reveal the nature of these biochemical and cellular changes and their role in helping cells survive extreme environmental stress. We thank Peter Geiduschek, Bill Loomis, and Mike Levine for critical reading of the manuscript and their valuable suggestions. We are grateful to Farah Sogo for help with plasmid construction and
Gustavo Gonzalez for helpful discussions. This work was supported by Grant MV-419 from the American Cancer Society and by a Searle Scholarship (to M.P.Y.) from the Searle Scholars Program of the Chicago Community Trust. 1. Lindquist, S. & Craig, E. A. (1988) Annu. Rev. Genet. 22,
631-677. 2. Lindquist, S. (1986) Annu. Rev. Biochem. 55, 1151-1191. 3. Morimoto, T., Tissieres, A. & Georgopoulos, C. (1990) in Stress Proteins in Biology and Medicine, eds. Morimoto, R. I., Tissieres, A. & Georgopoulos, C. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 1-36. 4. Hahn, G. M. & Li, G. C. (1990) in Stress Proteins in Biology and Medicine, eds. Morimoto, R. I., Tissitres, A. & Georgopoulos, C. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 79-100. 5. Sorger, P. K. (1990) Cell 62, 793-805. 6. Nieto-Sotelo, J., Wiederrecht, G., Okuda, A. & Parker, C. S. (1990) Cell 62, 807-817. 7. Wu, C., Wilson, S., Walker, B., Dawid, I., Paisley, T., Zimamno, V. & Ueda, H. (1987) Science 238, 1247-1253. 8. Pelham, H. R. B. (1982) Cell 30, 517-528. 9. Pelham, H. R. B. & Bienz, M. (1982) EMBO J. 1, 1473-1477. 10. Sorger, P. K. & Pelham, H. R. B. (1988) Cell 54, 855-864. 11. Wiederrecht, G., Seto, D. & Parker, C. S. (1988) Cell 54, 841-853. 12. Deshaies, R. J., Koch, B. D. & Schekman, R. (1988) Trends Biochem. Sci. 13, 384-388. 13. Murakami, H., Pain, D. & Blobel, G. (1988) J. Cell Biol. 107, 2051-2057. 14. Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A. & Schekman, R. (1988) Nature (London) 332, 800-805. 15. Cheng, M. Y., Hartl, F.-U., Martin, R. A., Pollock, R. A., Kalousek, F., Neupert, W., Hallberg, E. M., Hallberg, R. L. & Horwich, A. L. (1989) Nature (London) 337, 620-625. 16. Vogel, J. P., Misra, L. M. & Rose, M. D. (1990) J. Cell Biol. 110, 1885-1895. 17. Altman, E., Kumamoto, C. A. & Emr, S. D. (1991) EMBO J. 10, 239-245. 18. Sadler, I., Kurihara, C. T., Rothblatt, J., Way, J. & Silver, P. (1898) J. Cell Biol. 109, 2665-2675. 19. Schlesinger, M. J. (1990) J. Biol. Chem. 265, 12111-12114. 20. Pelham, H. R. B. (1990) in Stress Proteins in Biology and Medicine, eds. Morimoto, R. I., Tissieres, A. & Georgopoulos, C. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 287-300. 21. McAlister, L. & Finkelstein, D. B. (1980) Biochem. Biophys. Res. Commun. 93, 819-824. 22. Loomis, W. F. & Wheeler, S. A. (1982) Dev. Biol. 90,412-418. 23. Smith, B. J. & Yaffe, M. P. (1991) Mol. Cell. Biol. 11, 26472655. 24. Guarente, L. & Ptashne, M. (1981) Proc. Natl. Acad. Sci. USA 78, 2199-2203. 25. Broach, J. R., Strathern, J. N. & Hicks, J. B. (1979) Gene 8,
121-133.
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26. Orr-Weaver, T. L., Szostak, J. W. & Rothstein, R. J. (1981) Proc. NatI. Acad. Sci. USA 78, 6354-6358. 27. Sherman, F., Fink, G. R. & Hicks, J. B. (1979) Methods in Yeast Genetics (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 28. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 29. Yaffe, M. P. (1990) Methods Enzymol. 194, 627-643. 30. Sorger, P. K. & Pelham, H. R. B. (1987) EMBO J. 6, 30353041. 31. Ingolia, T. D., Slater, M. R. & Craig, E. A. (1982) Mol. Cell. Biol. 2, 1388-1398. 32. Nicolet, C. M. & Craig, E. A. (1991) Methods Enzymol. 194, 710-717. 33. Sanchez, Y. & Lindquist, S. L. (1990) Science 248, 1112-1115.
Proc. Nadl. Acad. Sci. USA 88 (1991) 34. Meluh, P. B. & Rose, M. D. (1990) Cell 60, 1029-1041. 35. Plesofsky-Vig, N. & Brambl, R. (1985) J. Bacteriol. 162, 1083-1091. 36. Gross, C. A., Straus, D. B., Erickson, J. W. & Yura, T. (1990) in Stress Proteins in Biology and Medicine, eds. Morimoto, R. I., Tissitres, A. & Georgopoulos, C. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 167-221. 37. Hall, B. G. (1983) J. Bacteriol. 156, 1363-1365. 38. Watson, K., Dunlop, G. & Cavicchioli, R. (1984) FEBS Lett. 172, 299-302. 39. Widelitz, R. B., Magun, B. E. & Gerner, E. W. (1986) Mol. Cell. Biol. 6, 1088-1094. 40. Cavicchioli, R. & Watson, K. (1986) FEBS Lett. 207, 149-152. 41. Barnes, C. A., Johnston, G. C. & Singer, R. A. (1990) J. Bacteriol. 172, 4352-4358.
Uncoupling thermotolerance from the induction of heat shock proteins (heat-shock factor/Saccharomyces cerevisiae/stress response)
BARBARA J. SMITH* AND MICHAEL P. YAFFEt University of California, San Diego, Department of Biology, B-022, La Jolla, CA 92093
Communicated by Dan L. Lindsley, September 19, 1991
gene. Cells expressing the mutant HSF protein display temperature-sensitive defects in growth, mitochondrial protein import, cell-cycle progression, and the induction of a major hsp70 gene, SSAI (23). Here we report an analysis of the effect of the hsfl-m3 lesion on the expression of a number of hsps and a test of the ability of the mutant cells to acquire thermotolerance.
Exposure of cells to elevated temperatures ABSTRACT causes a rapid increase in the synthesis of heat shock proteins (hsps) and induces thermotolerance, the increased ability of cells to survive exposure to lethal temperatures; however, the connection between hsp induction and the acquisition of thermotolerance is unclear. hsp induction in the yeast Saccharo-
myces cerevisiae is mediated by the activation of heat-shock transcription factor, and recently we have described a mutation, hsfl-m3, in heat-shock transcription factor that prevents the factor's activation. We now demonstrate that this mutation results in a general block in heat-shock induction but does not affect the acquisition of thermotolerance. Our results indicate that high-level induction of the major hsps is not required for cells to acquire thermotolerance.
MATERIALS AND METHODS Yeast Strains and Media. Saccharomyces cerevisiae AH216 (MATa, leu2, his3) and MYY243 (MATa, leu2, his3, hsfl-m3) were described (23). These strains are isogeneic except for the hsfl-m3 lesion. Derivatives of AH216 and MYY243 containing an HSE2-4acz fusion gene integrated into the URA3 chromosomal locus were created. The HSE2lacz fusion was constructed by first annealing the -HSEcontaining synthetic oligonucleotide 5'-CTCGATCTAGAAGCTTCTAGAAGCTTCTAGA-3' to itself and then ligating the duplex DNA fragment into the unique Xho I site located upstream of the lacz gene in the plasmid pLG669Z (24). Yeast replication sequences were removed from the plasmid on an EcoRP fragment, and the yeast LEU2 gene, isolated from plasmid YEp13 (25) on a Sal I-Xho I fragment, was inserted in the Sal I site. The DNA construct was integrated into the chromosomal URA3 gene of strains AH216 and MYY243 by integrative transformation (26) after cutting the plasmid DNA at the unique Stu I site in the URA3 gene. YPD medium contained 1% yeast extract, 2% (wt/vol) Bacto Peptone, and 2% (wt/vol) glucose. SD medium (27) was prepared without asparagine and threonine. Analysis of (3-Galactosidase Activity and hsp Synthesis. ,3-Galactosidase activities were determined as described (28).in For analysis of hsp synthesis, yeast were grown at 230C SD medium to 0.5 OD6w unit. Cells were concentrated 2-fold by centrifugation and incubated for 15 min at 230C or 370C. Next, L-[4,5-3H]isoleucine (84 Ci/mmol; 1 Ci = 37 GBq) was added to 50 ,uCi/ml of cells and incubations were continued at the same temperature for 30 min. Proteins were extracted immediately as described (29), separated by SDS/PAGE, and detected by fluorography. Analysis of Thermotolerance. Cells were grown in YPD medium at 230C to 5 x 105 cells per ml or to 5 x 106 cells per ml. Cultures were divided into two portions, one of which was shifted directly to 520C, and a second portion was preincubated at 370C for 1 hr prior to exposure to 520C. Samples of cells were removed after incubation at the high temperature, diluted with ice water, and plated immediately
The heat-shock response, a rapid and transient increase in the expression of a discrete set ofheat-shock genes, is a universal reaction of cells exposed to such adverse conditions as increased temperatures, anoxia, elevated levels of ethanol, and certain heavy metals (1-3). The cellular concentrations of the heat shock proteins (hsps) increase dramatically as a consequence of the activated gene expression. Elevated levels of these hsps are believed to help cells survive the deleterious effects of the environmental stress (3, 4). Temperature-induced hsp expression in yeast is mediated by the specific activation of the heat-shock transcription factor (HSF) (5-7). Transcriptional induction mediated by HSF depends on a regulatory DNA sequence, the heat-shock element (HSE), which is present in a few or multiple copies in the promoter regions of heat-shock genes (8, 9). Genetic analysis of HSF function in yeast indicates that HSF is also responsible for the low-level basal transcription of hsps under noninducing conditions, and this constitutive expression is essential for normal cellular growth (10, 11). The constitutively synthesized hsps or their cognates have been implicated as essential components for protein transport into and assembly within cytoplasmic organelles (12-18), and these and other hsps are thought to mediate protein folding, conformational changes, or subunit interactions (19, 20). The role of hsp induction in response to stress remains a mystery. A common assumption (3, 4) is that increased hsp levels serve a -protective function. This assumption largely stems from studies of induced or acquired thermotolerance in which the mild stress of a cell population results in greatly increased numbers of cells surviving a subsequent normally lethal stress (4, 21, 22). Many of the conditions that induce thermotolerance also lead to a rise in hsp levels, but the connection between this activation and the acquisition of thermotolerance remains obscure. We have described (23) a yeast strain that contains a mutant allele, hsfl-m3 (formerly called mas3), of the HSF
Abbreviations: hsp, heat shock protein; HSF, heat-shock transcription factor; HSE, heat-shock element. *Present address: The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
11091
Proc. Natl. Acad. Sci. USA 88 (1991)
Genetics: Smith and Yaffe
11092
on YPD plates. Viability was scored after 5 days of incubation at 230C.
m3
WT C H
C
H
RESULTS To further characterize the consequences of the hsfl-md mutation, the levels of (3-galactosidase produced from an HSE2-4acz fusion construct were analyzed. Enzyme activity from such a reporter gene reflects transcriptional activity mediated solely by HSF (30). In wild-type cells the levels of enzyme activity increased to a maximum after 1 hr of 23-fold over basal levels and declined to a new higher level (10-fold over the uninduced level) after 4 hr at 370C (Fig. 1). In contrast, the increase in activity in cells carrying hsfl-n3 was less than 1.5-fold after 1 hr at 3T7C with a total increase of only -3.3-fold by 4 hr: The basal level of P-galactosidase activity (Fig. 1) was similar in both mutant and wild-type cells. The lack of (3-galactosidase' induction observed with hsfl-m3 cells cannot be attributed to cell death, since the number of viable cells declined only after 6-8 hr at 370C (23). These results indicate that the mutant hsfl-n3 protein retains normal basal transcriptional activity but is defective for heat-inducible activity. The pattern of protein synthesis changes rapidly in wildtype cells exposed to elevated temperatures: the synthesis of most proteins decreases or stops, synthesis of a few proteins remains the same, and the synthesis of the classical hsps increases dramatically (31). To determine if cells carrying the hsfl-m3 mutation are also defective in the induction of endogenous heat-shock genes, we examined protein.synthesis in control and heat-shocked cultures. Striking differences in the pattern of protein synthesis from mutant and wild-type cells were apparent (Fig. 2). Whereas synthesis of the major hsps of 104 kDa, 82 kta, and 70 kDa rose substantially in wild-type cells after heat shock, these same proteins. increased only slightly or not at all in heat-shocked mutant cells (Fig. 2). The levels of certain non-heat-shock proteins that did not 'change after heat-shock treatment in wild-type cells also remained constant in mutant cells. Additionally, the decreased synthesis of 'many cellular proteins after heatshock appeared similar in both mutant and wild-type cells. The above results demonstrate that the hsfl-m3 mutation specifically affects the induction of the major hsps during heat-shock treatment. A variety of stress treatments that induce hsp production also enhance the survival of cells exposed to elevated lethal temperatures (3). To test the effect of the hsfl-m3 mutation
-hsp 104 _ _
-
-hsp 82 -hsp 70
....
AM.
,
Mk
k
w
v
i _
3_E,4 _
He
IIII~
~
IS.
_
FIG. 2. Temperature-regulated induction of endogenous HSP synthesis. Cultures of wild-type (WT) and mutant (m3) cells were grown at 230C,inminimalmediumnto midloithmicsphe. A portion ofeach culture was either kept at 230C (lane C) or shafted to 370C (lane H) for 15 mu, [3H]Isoleucine was lidded to all samp iand incubations were continued at the same temperature for 30 min. .The labeled proteins were extracted (32) and separated by elcoph resis in a 7% polyacrylamide gel in the presence. of SDS. Labeled protein bands were detected by fluorography. Putative hsps were identified by molecular weight standards electrophoresed in an adjacent lane and by comparison with published hsp patterns (32, 33). hsp70 indicates a group of several isozymes with similar molecular weights (32).
on this induced thermotolerance, cell viability was determined after exposure for various periods'to 520C, with or without a prior incubation at 370(. Pretreatment at 370( induced thermotolerance to similar extents in both mutant and wild-type cells (Fig. 3). Cultures also showed essentially identical thermotolerance curves when pretreatment was carried out at 390C (data not shown).. Additionally, the hsfl-m3 and wild-type cultures both displayed striking thermosensitivity when exposed directly to 520C, although the mutant cells died more slowly than did the wild-type cells.
DISCUSSION Our results demonstrate that the activation of HSF and the resulting induction of high levels of hsps are not required for cells to acquire thermotolerance. Such'a conclusion is contrary to the prevalent view (4, 34) that hsp induction leads to a thermotolerant state (Fig. 4A). An alternative model (Fig. 4B) is that thermotolerance results from the temperature-
40 CD
20 0
ca
10
0
12
0~~~~~~~~~~~~~~~.0
0.0
1.0
2.0
3.0
4.0
Time at 370C, hr
FIG. 1. Induction of HSE2-LacZ activity. Wild-type (solid circles) and mutant (open circles) strains containing an integrated copy of the heat-inducible HSE2-4acz gene were grown at 23°C and shifted to 37rC. Samples were removed after 0, 0.5, 1, 2, and 4. hr (as indicated), and (-galactosidase activities were determined. Values for each time point are the average of three samples, and the levels of induction obtained in three experiments varied by less than 15%.
Time at 5200, mm
FIG. 3. Thermotolerance of mutant cultures at 520C bef~re and after mild heat treatments. Wild-type cells (solid symbols) and mutant cells (open symbols) were grown in YPD medium at 23°C to 5 x 105 cells per ml. One portion (triangles) of the culture was shifted directly to 520C, and a second portion (squares) was preincubated at 3rC for 1 hr prior to exposure to 52MC. Samples were removed after 0, 5, 10, and 20 min, and. samples were analyzed for cell viability. Similar results were obtained after a 30-min preincubation at 370C or with cells grown to 5 X 101 cells per ml.
Proc. Natl. Acad. Sci. USA 88 (1991)
Genetics: Smith and Yaffe
11093
B
A
HEAT
HEAT
HSF ACTIVATION
HSF ACTIVATION
FACTOR MODIFICATION
I
HSP INDUCTION
THERMOTOLERANCE
HSP INDUCTION
THERMOTOLERANCE FIG. 4. Alternative models for the heat induction of thermotolerance. (A) Increased temperature leads to the activation of HSF, resulting in increased levels of hsps. Elevated hsp levels confer thermotolerance on cells. (B) Thermotolerance results from the heat-induced modification of one or more cellular components and is independent of hsp induction.
dependent activation of one or several factors and is independent of HSF-mediated induction of hsps. For example, elevated temperatures might activate a heat-dependent enzyme, whose product confers thermotolerance, or heat might promote the binding of a protective protein to a thermosensitive cellular structure. Previous studies have produced conflicting conclusions concerning the relationship between hsp induction and the acquisition of thermotolerance (2). Several investigations have provided evidence that the inhibition of protein synthesis blocks the acquisition of thermotolerance (21, 35). Additionally, mutant strains ofEscherichia coli and Dictyostelium discoideum, defective for the acquisition of thermotolerance, also failed to accumulate certain h'sps during heat shock (22, 36). Other studies have suggested that protein synthesis is not required for the acquisition of thermotolerance (37-39) or demonstrated a lack of correlation between thermotolerance and high hsp levels (40, 41). In certain types of cells the acquisition of thermotolerance might indeed require the induction of one or several proteins, followed by the heat-dependent modification of these polypeptides. Such cells would require protein synthesis to acquire thermotolerance. Other cells, such as those employed in this study, might possess sufficient levels of stressprotection proteins even during growth under nonstressed conditions.'This would alleviate a requirement for protein synthesis, although a temperature-dependent modification of one or several cellular proteins would still be necessary. Such a variation between different types of cells (or even different growth conditions) might explain the conflicting results concerning the requirement of protein synthesis for the acquisition of thermotolerance. Sanchez and Lindquist (33) demonstrated that a null mutation in a specific heat-shock gene, HSP104, rendered yeast cells defective for the acquisition of thermotolerance. This finding is not inconsistent with our results, which indicate induction of hspl04 is substantially deficient in the hsfl-m3 mutant (Fig. 2). A reasonable interpretation is that constitutive levels of hspl04 are necessary for thermotolerance acquisition but that high-level induction of the protein is not. Acquired thermotolerance might result, instead, from a temperature-dependent change in the activity or structure of preexisting hspl04 and, perhaps, in the alteration of other proteins. Future studies should reveal the nature of these biochemical and cellular changes and their role in helping cells survive extreme environmental stress. We thank Peter Geiduschek, Bill Loomis, and Mike Levine for critical reading of the manuscript and their valuable suggestions. We are grateful to Farah Sogo for help with plasmid construction and
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