JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 4352-4358
Vol. 172, No. 8
0021-9193/90/084352-07$02.00/0 Copyright © 1990, American Society for Microbiology
Thermotolerance Is Independent of Induction of the Full Spectrum of Heat Shock Proteins and of Cell Cycle Blockage in the Yeast Saccharomyces cerevisiae CHRISTINE A.
BARNES,lt
GERALD C.
JOHNSTON,'*
AND RICHARD A.
SINGER2'3
Departments of Microbiology,1 Biochemistry,2 and Medicine,3 Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 Received 7 December 1989/Accepted 7 May 1990
Cells of the yeast Saccharomyces cerevisiae are known to acquire thermotolerance in response to the stresses of starvation or heat shock. We show here through the use of cell cycle inhibitors that blockage of yeast cells in the Gl, S, or G2 phases of the mitotic cell cycle is not a stress that induces thermotolerance; arrested cells remained as sensitive to thermal killing as proliferating cells. These Gl- or S-phase-arrested cells were unimpaired in the acquisition of thermotolerance when subjected to a mild heat shock by incubation at 37°C. One cell cycle inhibitor, o-phenanthroline, did in fact cause cells to become thermotolerant but without induction of the characteristic pattern of heat shock proteins. Thermal induction of heat shock protein synthesis was unaffected; the o-phenanthroline-treated cells could still synthesize heat shock proteins upon transfer to 37°C. Use of a novel mutant conditionally defective only for the resumption of proliferation from stationary phase (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987) indicated that o-phenanthroline inhibition produces a stationary-phase arrest, a finding which is consistent with the increased thermotolerance and regulated cessation of proliferation exhibited by the inhibited cells. These findings show that the acquired thermotolerance of cells is unrelated to blockage of the mitotic cell cycle or to the rapid synthesis of the characteristic spectrum of heat shock proteins.
duction and to cell cycle position remain to be fully characterized. Here we describe further experiments that dissociate the acquisition of thermotolerance from the usual pattern of synthesis of heat shock proteins and from cell cycle position and show that even cells arrested at an unconventional cell cycle position, S phase, can acquire thermotolerance. Therefore, the stress that leads to thermotolerance is unrelated to the cell cycle or to the generally increased abundance of heat shock proteins.
The stress caused by exposure of proliferating cells of the yeast Saccharomyces cerevisiae to temperatures well above normal growth temperatures results in rapid loss of viability (34a, 35). However, yeast cells can adapt to and survive this usually lethal treatment if they are first subjected to a heat shock, a procedure in which cells are incubated at an elevated but otherwise nonlethal growth temperature (29, 34). The enhanced survival that results from the adaptation by heat-shocked cells has been termed acquired thermotolerance (18). Thermotolerance is also a characteristic of yeast cells in another stressful situation, i.e., that induced by nutrient starvation. A starved yeast cell ceases proliferation in stationary phase, expresses altered physiological properties, and becomes thermotolerant even without heat shock
MATERIALS AND METHODS Strains and culture conditions. Cultures of S. cerevisiae GR2 (MATa his6 ural) (22) were grown with gyratory shaking at 23°C in YNB defined medium (16) with added glucose (2%), amino acids (40 jig/ml), and nucleotides (20 ixg/ml). Strain MD-G3-G02, a derivative of the gcsl-J sedl-J mutant strain MAD-6 described elsewhere (10), was grown at 23°C in YM1 rich medium (16) supplemented with adenine (20 ,ug/ml) and glucose (2%). Hydroxyurea, tunicamycin, o-phenanthroline, and the yeast mating pheromone a-factor were all obtained from Sigma Chemical Co. (St. Louis, Mo.). Sinefungin was a gift from Eli Lilly & Co. (Indianapolis, Ind.). Cell concentrations were determined with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). Cell morphology was assessed microscopically; at least 200 cells were examined for each determination. Radiolabeling of protein and gel electrophoresis. Radiolabeling of protein was as described elsewhere (30). Cultures were maintained at fewer than 2 x 106 to 4 x 106 cells per ml for assessment of protein profiles. To radiolabel proteins, 1-ml samples were transferred to tubes containing 50 to 100 ,uCi of [35S]methionine and incubation with shaking was continued for 10 min at the appropriate temperature. Proteins were resolved by one-dimensional sodium dodecyl
(31).
Both the acquired thermotolerance of heat-shocked, proliferating cells and the intrinsic thermotolerance of stationary-phase cells have been correlated with cell cycle position (31, 33) and also with the production of heat shock proteins (for a review, see reference 28). Thermotolerant yeast cells in stationary phase contain an unreplicated (Gl) complement of DNA and display elevated levels of heat shock proteins (2, 3, 32). Similarly, proliferating yeast cells subjected to a heat shock transiently arrest proliferation in Gl (23) and induce the synthesis of heat shock proteins at the same time that they acquire thermotolerance (28). More recently, a particular heat shock protein, HsplO4, has been shown to be involved in acquired thermotolerance (34a). Although it has been shown that the induction of heat shock genes does not depend on cell cycle position (1), the relationships of thermotolerance to heat shock protein pro* Corresponding author. t Present address: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
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sulfate-polyacrylamide gel electrophoresis; the gels were then stained, destained, and dried (24) prior to autoradiography. Thermotolerance assay. Cells were grown overnight at 23°C to a density of 2 x 106 to 4 x 106/ml. Portions of each culture were incubated for 1 h at 37°C or were incubated at 23°C in the presence of an inhibitor of cell proliferation or were maintained at 23°C. At intervals, 1-ml samples were incubated at 52°C for 5 min and then put on ice for a minimum of 5 min. At the titne of each 52°C heat treatment, a control sample from the same culture, but without exposure to 52°C, was also placed on ice. Both samples were then diluted with phosphate-buffered saline, spread onto YEPD solid medium (16), and incubated at 23°C for 3 to 5 days. Thermotolerance, assessed by colony formation, was expressed as percent survival (29). RESULTS Thermotolerance, either upon the stress of heat shock at 37°C (29) or in stationary phase (31), is in each case correlated with a transient or permanent arrest of cell proliferation (23, 31). To determine whether cessation of proliferation is a stress sufficient to induce thermotolerance, conditions were imposed to block cells in the Gi, S, or G2 phase of the mitotic cell cycle, and arrested cells were then assessed for thermotolerance. The temperature-sensitive cdc mutations commonly used to impose cell cycle blocks were not suitable for these experiments, since blockage of the cell cycle by these cdc mutations requires incubation at 36°C, a treatment that itself imposes a stress sufficient to induce thermotolerance (29). Therefore, to arrest cell proliferation without the interfering incubation at 36°C associated with the arrest treatment, we used cell cycle inhibitors, which allowed cells to be maintained at 23°C throughout each experiment. The inhibitors o-phenanthroline, sinefungin, and the mating pheromone a-factor were each used to bring about concerted arrest in Gl (4, 22, 26); hydroxyurea was used to arrest cells in S phase (36); and tunicamycin was used to arrest cells in G2 (39). Cell cycle arrest does not induce thermotolerance. After 5 h of incubation in the presence of the G2-specific inhibitor tunicamycin, cell division had ceased, with 85% of the population accumulated as unbudded cells (Fig. 1B). Although a high percentage of unbudded cells usually indicates arrest in Gl (17), Vai et al. (39) showed that an unbudded tunicamycin-treated cell contains a replicated content of DNA in a single nucleus, which is indicative of arrest in G2. The cells arrested in G2 by tunicamycin treatment remained sensitive to the lethal effects of 52°C treatment. Even after 12 h of incubation in the presence of tunicamycin, arrested cells were as thermosensitive as untreated, proliferating cells (Fig. 1A). In a parallel experiment, arrest of cells in S phase was brought about by the inhibitor hydroxyurea. After 5 h of exposure to hydroxyurea, more than 85% of the cells were budded (Fig. 2B), exhibiting the standard morphology of hydroxyurea-treated cells that is indicative of an S-phase block (36). S-phase-arrested cells were as sensitive as untreated cells to the lethal effects of a 5-min incubation at 52°C (Fig. 2A). Therefore, cell cycle blockage leading to arrest of proliferation either in G2 or in S phase is not a stress that induces thermotolerance. Cells treated with a-factor or sinefungin arrest proliferation in the Gl phase of the cell cycle at the regulatory step designated start (4, 26). After 12 h of incubation in the
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of sinefungin, a time at which 85% of the cells were arrested in Gi, cells remained as thermosensitive as untreated cells (Fig. 3). Cells arrested in Gi by a-factor treatment also remained thermosensitive (33) (Fig. 4). The kinetics of acquisition and loss of thermotolerance differ among different stress situations that elicit thermotolerance. For example, stationary-phase cells maintain a thermotolerant state for long periods (35), whereas heatshocked, proliferating cells are only transiently thermotolerant (29). To assess whether the cell cycle inhibitor tunicamycin, hydroxyurea, sinefungin, or a-factor could have induced only a transient thermotolerance similar to a response to heat shock, thermotolerance was assayed immediately after inhibitor addition and at intervals thereafter. None of these cell cycle inhibitors induced even a transient thermotolerance (Fig. 1 through 4). We conclude from these experiments that cell cycle blockage is not a stress that causes cells to become thermotolerpresence
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Cells arrested in the cell cycle can acquire thermotolerance. Cells arrested in Gl at start by the action of sinefungin or a-factor were transferred to 37°C, and incubation was continued for 1 h in the presence of the inhibitor. Each population of arrested cells that was heat shocked in this way acquired thermotolerance to the same level as that of proliferating cells subjected to an identical heat shock (Fig. 3A and 4A). Similarly, cells arrested in S phase by the inhibitor hydroxyurea and then transferred to 37°C and incubated for 1 h became at least 200-fold more thermotolerant than S-phase-arrested cells simply maintained at 23°C (Fig. 2A). These findings show that blockage of the cell cycle does not preclude the usual acquisition of thermotolerance upon heat shock.
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Thermotolerance can occur without general induction of heat shock proteins. Treatment of cells with another start inhibitor, o-phenanthroline, brought about a response different from that found for the other cell cycle inhibitors discussed above. As expected (22), cells treated with ophenanthroline became arrested uniformly in the Gl interval at start, but unlike cells subjected to other treatments that caused cell cycle blockage (Fig. 1 through 4), o-phenanthroline-treated cells displayed a marked increase in thermotolerance (Fig. SA). Thermotolerance has been associated with the production of heat shock proteins in general (20, 29) and, most recently, with the production of a particular heat shock protein, HsplO4 (34a). Indeed, for most of the situations described here, thermotolerance induced by incubation at the elevated temperature of 37°C was accompanied by the synthesis of heat shock proteins (Fig. 3C and 4C). For this reason, the increased thermotolerance of o-phenanthrolinetreated cells was unexpected, because none of the inhibitors used here to cause cell cycle blockage, including o-phenanthroline (Fig. 5), increased the production of the heat shock proteins at 23°C (1) (Fig. 3C, 4C, and 5C). Even after extended exposure to o-phenanthroline, a treatment that caused a high level of thermotolerance, cells did not induce the expression of major heat shock proteins. Nevertheless, for these o-phenanthroline-treated thermotolerant cells, as for the cells arrested by the other start inhibitors (Fig. 3C and 4C), the presence of the inhibitor did not preclude the production of heat shock proteins; transfer of o-phenanthroline-arrested cells to 37°C elicited the typical pattern of o-phenanthroline protein synthesis (Fig. SC). Although particular proteins may be implicated in thermotolerance (34a), the effects of the start inhibitor o-phenanthroline show that
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FIG. 3. Thermotolerance and heat shock protein synthesis for sinefungin-treated cells. Wild-type cells proliferating at 23°C were treated with sinefungin (0.02 ,ug/ml) at time zero. At intervals, cells were removed to assess thermotolerance, morphology, and heat shock protein production. (A) Thermotolerance. Symbols: 0, incubation at 23°C; k, incubation at 37°C for 1 h. (B) Morphology of cells incubated at 23°C. (C) Heat shock protein synthesis at 23°C or after incubation at 37°C for the times indicated. The positions of the molecular weight standards are indicated on the left (in thousands), and those of major heat shock proteins are indicated on the right.
THERMOTOLERANCE IN S. CEREVISIAE
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increased synthesis of the characteristic set of heat shock proteins is not required for thermotolerance. o-Phenanthroline causes cells to enter stationary phase. Acquisition of the stationary-phase state causes thermotolerance without application of a heat shock. Therefore, we 100-
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FIG. 5. Thermotolerance and heat shock protein production for o-phenanthroline-treated cells. Wild-type cells proliferating at 23°C were treated with o-phenanthroline (20 ,tg/ml) at time zero. At intervals, cells were removed to assess thermotolerance, morphology, and heat shock protein production. (A) Thermotolerance. Symbols: 0, incubation at 23°C; U, incubation at 37°C for 1 h. (B) Morphology of cells at 23°C. (C) Heat shock protein synthesis at 23°C or after transfer to 37°C for the times indicated. Lanes 1, 7, and 12, reference patterns of proteins synthesized in untreated cells.
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phenotype, when in stationary phase, do not resume proliferation if stimulated by the presence of fresh medium under restrictive conditions, but mutant cells that are already proliferating continue to proliferate when transferred to the restrictive temperature. This novel mutant phenotype pan thus be used to distinguish a cell in stationary phase from a cell that is not proliferating for other reasons (9). When mutant cells of this type were arrested in proliferation by treatment with o-phenanthroline at a temperature that elicits the mutant phenotype, the cells became defective for the resumption of proliferation at the restrictive temperature upon transfer to fresh medium free of o-phenanthroline (Fig. 6). In contrast, mutant c,ells resumed proliferation upon transfer to inhibitor-free medium at the permissive temperature. This conditional inability of mutant cells to resume proliferation indicates that o-phenanthroline treatment brings about a stationary-phase arrest of proliferation. The stationary-phase status of o-phenanthroline-treated cells is consistent with the acquisition of thermotolerance by these arrested cells and suggests that this thermotolerance is a consequenc'e of the stationary-phase arrest caused by ophenanthroline. Mutant cells arrested in cell proliferation by hydroxyurea treatment did not become conditionally defective for the resumption of proliferation (data not shown). This cell cycle inhibition therefore does not produce a stationary-phase arrest, a finding consistent with the lack of thermotolerance of these arrested cells (Fig. 2).
Proliferation arrest and thermoto_eance. In yeast cells, thermotolerance is seen usually upon arrest of proliferation in a prereplicative state. Nutrient-deprived yeast cells become arrested in a prereplicative state and in this stationaryphase state are more thermotolerant than proliferating cells (31). Similarly, transfer of proliferating cells to 37°C and incubation for 1 h cause a transient arrest of proliferation and accumulation of Gl-phase, prerepliQative cells (23) at the same time that the cells acquire thermotolerance (29). The experiments described above show that arrest of proliferation is not a signal for the induction of thermotolerance; yeast cells arrested in Gl by a-factor or sinefungin treatment, in S phase by hydroxyurea treatment, or in G2 by tunicamycin treatment remained thermosensitive. Perhaps the arrest caused by the mating pheromone a-factor does not induce the stress-related response of thermotolerance because this arrest in Gl at start is a normal cellular response in preparation for conjugation (4). However, arrest of proliferation outside of Gl, in S phase or in G2, is not part of the usual biological repertoire of S. cerevisiae. Therefore, it was unexpected that neither hydroxyurea treatment nor tunicamycin treatment provoked the stress response of thermotolerance. It has been known for some time that a cell blocked in S phase or in G2 continues to increase in mass and becomes enlarged (21). Evidently this unbalanced growth upon blockage of the cell cycle does not perturb the physiological status of the arrested cell sufficiently to trigger the induction of thermotolerance. Heat shock and thermotolerance. Acquired thermotolerance has been correlated with the expression of heat shock proteins (27, 28). Most of the major heat shock proteins are present at only low levels in yeast cells proliferating at 23°C, but within 1 h after transfer to 37°C, as cells become thermotolerant, heat shock proteins are some of the most abundant proteins present (11, 30). Indeed, this correlation was the basis for early proposals that heat shock proteins are involved in thermotolerance. Despite this correlation between heat shock protein production and thermotolerance, appreciation of the functions of particular heat shock proteins in thermotolerance induction in yeast cells has been elusive (8, 12, 28, 32, 41). It is only recently that one heat shock protein, Hspl04, has been shown to play a role in acquisition of thermotolerance; disrption of the nonessential HSP104 gene impairs the acquisition of thermotolerance by heat-shocked mutant cells (34a). However, this impairment is obvious only after prolonged incubation at the lethal temperature of 509C; the absence of the Hspl04 protein has no effect on the survival of heat-shocked mutant cells after short-term exposure to 50°C. This short-term, Hsp104-independent thermotolerance thus reveals a second, as-yetunidentified component of thermotolerance (34a). For the purposes of the study described here, cells were assessed for thermotolerance after a short-term incubation at an elevated, lethal temperature. Therefore, our procedure evaluated the role of heat shock proteins only in this initial component of thermotolerance. Treatment with o-phenanthroline resulted in short-term thermotolerance in the absence of the usual spectrum of heat shock proteins. This inhibitor may thus prove useful in the study of this initial phase of thermotolerance.
Studies that have not specifically resolved the initial from the delayed, HspI04-related thermotolerance have shown that thermotolerance in general can increase without an increased synthesis of all heat shock proteins (6). Moreover,
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cells that acquire thermotolerance by treatments other than heat do not always synthesize the full spectrum of heat shock proteins; in some cases only certain heat shock proteins are synthesized (for reviews, see references 7 and 25). Many individual heat shock genes are not required for thermotolerance. Cells bearing mutations in the heat shock genes HSP90 and HSC90 (28), UBI4 (13), HSP48 (H. Iida, cited in reference 28), HSP26 (32, 37), and members of the HSP70 gene family (42) nevertheless become thermotolerant after a mild heat treatment. Perhaps only a minor subset of yeast heat shock proteins functions in the acquisition of thermotolerance. Although the HsplO4 protein plays a role in thermotolerance (34a), it has been shown that thermotolerance can be acquired by yeast cells even in the presence of presumably nonfunctional heat shock proteins (15), and the acquisition of thermotolerance can occur in the absence of cytoplasmic and mitochondrial protein synthesis (29, 41). Yeast cells can also lose thermotolerance in the presence of elevated levels of the major heat shock proteins (5). Investigations of mammalian systems or Escherichia coli also suggest that thermotolerance need not be correlated with the induction of the spectrum of heat shock proteins. For example, addition of the protein synthesis inhibitor cycloheximide to rat embryonic fibroblasts does not prevent thermotolerance but does prevent heat shock protein production (43). Conversely, for E. coli cells the overexpression of heat shock proteins at non-heat-shock temperatures does not protect these cells from the effects of lethal temperatures (40). Further work is needed to resolve this issue. o-Phenanthroline induces stationary-phase arrest. We show here that o-phenanthroline brings about a stationary-phase state. The ability of o-phenanthroline to induce stationary phase suggests that this inhibitor mimics nutrient deprivation. The signal for nutrient supply, and thus for nutrient deprivation, is mediated in part by a cyclic AMP (cAMP)dependent signal transduction pathway that culminates in the activity of cAMP-dependent protein kinases (for a review, see reference 14). Disruption of the BCYJ gene, which codes for the regulatory subunit of cAMP-dependent protein kinases (38), leads to unregulated activation of protein kinases and the inability of bcyl disruption strains to respond in a regulated manner to nutrient deprivation (38). Treatment of a bcyl disruption strain with o-phenanthroline caused arrest of proliferation with cells blocked in the unbudded interval of the cell cycle (our unpublished results), which is the response of wild-type cells to o-phenanthroline treatment (22). Thus, the effects of o-phenanthroline are epistatic to those of the bcyl disruption, suggesting that o-phenanthroline brings about a stationary-phase state by affecting an activity that is regulated by the cAMP signal transduction pathway. It is not yet known whether o-phenanthroline interferes with the function of cAMP-dependent protein kinases directly or with some downstream activity. It is known, however, that o-phenanthroline, at the concentrations used here to bring about concerted blockage of start, inhibits rRNA gene expression (22) at the level of transcription initiation (40a), and also induces the general control response in S. cerevisiae (19) at the transcriptional level (40a). Although neither of these responses has been shown to play a role in the acquisition of stationary phase, the general control response is activated under starvation conditions and in this way may be a response that brings about the stationary-phase state. In any case, o-phenanthroline may prove useful in studies of stationary phase and nutrient signaling.
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ACKNOWLEDGMENTS We thank Susan Lindquist for providing information about HsplO4 prior to publication. This work was supported by the Medical Research Council of Canada and the National Cancer Institute of Canada. LITERATURE CITED 1. Barnes, C. A., R. A. Singer, and G. C. Johnston. 1987. Production of heat shock protein is independent of cell cycle blockage in the yeast Saccharomyces cerevisiae. J. Bacteriol. 169:56225625. 2. Boucherie, H. 1985. Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae. J. Bacteriol. 161:385-392. 3. Brazell, C., and T. D. Ingolia. 1984. Stimuli that induce a yeast heat shock gene fused to ,B-galactosidase. Mol. Cell. Biol. 4:2573-2579. 4. Bucking-Throm, E. W., W. Duntze, L. H. Hartwell, and T. R. Manney. 1973. Reversible arrest of haploid yeast cells at the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 76:99-110. 5. Cavicchioli, R., and K. Watson. 1986. Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins. FEBS Lett. 207:149-152. 6. Chang, E. C., D. J. Kosman, and G. R. Willsky. 1989. Arsenic oxide-induced thermotolerance in Saccharomyces cerevisiae. J. Bacteriol. 171:6349-6352. 7. Craig, E. A. 1986. The heat shock response. Crit. Rev. Biochem. 18:239-280. 8. Craig, E. A., and K. Jacobsen. 1984. Mutations of the heat inducible 70 kilodalton genes of yeast confer temperature sensitive growth. Cell 38:841-849. 9. Drebot, M. A., C. A. Barnes, R. A. Singer, and G. C. Johnston. 1990. Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae. J. Bacteriol. 172:3584-3589. 10. Drebot, M. A., G. C. Johnston, and R. A. Singer. 1987. A yeast mutant conditionally defective only for reentry into the mitotic cell cycle from stationary phase. Proc. Natl. Acad. Sci. USA 84:7948-7952. 11. Finkelstein, D. B., and S. Strausberg. 1983. Heat shock-regulated production of Escherichia coli P-galactosidase in Saccharomyces cerevisiae. Mol. Cell. Biol. 3:1625-1633. 12. Finkelstein, D. B., S. Strausberg, and L. McAlister. 1982. Alterations of transcription during heat shock of Saccharomyces cerevisiae. J. Biol. Chem. 257:8405-8411. 13. Finley, D., E. Ozkaynak, and A. Varshavsky. 1987. The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 48:1035-1048. 14. Gibbs, J. B., and M. S. Marshall. 1989. The ras oncogene-an important regulatory element in lower eucaryotic organisms. Microbiol. Rev. 53:171-185. 15. Hall, B. G. 1983. Yeast thermotolerance does not require protein synthesis. J. Bacteriol. 156:1363-1365. 16. Hartwell, L. H. 1967. Macromolecule synthesis in temperaturesensitive mutants of yeast. J. Bacteriol. 93:1662-1670. 17. Hartwell, L. H. 1974. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38:164-198. 18. Henle, K. J., and L. A. Dethlefsen. 1978. Heat fractionation and thermotolerance: a review. Cancer Res. 38:570-574. 19. Hinnebusch, A. G. 1986. The general control of amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. Crit. Rev. Biochem. 21:277-317. 20. lida, H., and I. Yahara. 1984. A heat shock-resistant mutant of Saccharomyces cerevisiae shows constitutive synthesis of two heat shock proteins and altered growth. J. Cell Biol. 99: 1441-1450. 21. Johnston, G. C., J. R. Pringle, and L. H. Hartwell. 1977. Coordination of growth with cell division in the yeast Saccharomyces cerevisiae. Exp. Cell Res. 105:79-98. 22. Johnston, G. C., and R. A. Singer. 1978. RNA synthesis and control of cell division in the yeast S. cerevisiae. Cell 14: 951-958. 23. Johnston, G. C., and R. A. Singer. 1980. Ribosomal precursor
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RNA metabolism and cell division in the yeast Saccharomyces cerevisiae. Mol. Gen. Genet. 178:357-360. 24. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (London) 227:680-685. 25. Lanks, K. W. 1986. Modulators of the eukaryotic heat shock response. Exp. Cell Res. 165:1-10. 26. Li, A. W., R. A. Singer, and G. C. Johnston. 1984. Cell cycle effects in yeast cells of the antifungal agent sinefungin. FEMS Microbiol. Lett. 23:243-247. 27. Llndqulst, S. 1986. The heat-shock response. Annu. Rev. Biochem. 55:1151-1191. 28. Lindquist, S., and E. A. Craig. 1988. The heat-shock proteins. Annu. Rev. Genet. 22:631-677. 29. McAlHster, L., and D. B. Fnkeihtdn. 1980. Heat shock proteins and thermal resistance in yeast. Biochem. Biophys. Res. Commun. 93:819-824. 30. McAlister, L., S. Strausberg, A. Kulaga, and D. B. Finkestein. 1979. Altered patterns of protein synthesis induced by heat shock of yeast. Curr. Genet. 1:63-74. 31. Paris, S., and J. R. Pringle. 1983. Saccharomyces cerevisiae: heat and glusulase sensitivities of starved cells. Ann. Microbiol. (Paris) 134B:379-385. 32. Petko, L., and S. Indqulst. 1986. Hsp26 is not required for growth at high temperatures, nor for thermotolerance, spore development, or germination. Cell 45:885-894. . 1987. 33. Plesset, J., J. R. Ludwig, B. S. Cox, and C. S. ML Effect of cell cycle position on thermotolerance in Saccharomyces cerevisiae. J. Bacteriol. 169:779-784. 34. Plesset, J., C. Palm, and C. S. McLaughlin. 1982. Induction of heat shock proteins and thermotolerance by ethanol in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 108: 1340-1345. 34a.Sanchez, Y., and S. L. Llndqulst. 1990. HSP104 required for induced thermotolerance. Science 248:1112-1115. 35. Schenberg-Frascino, A., and E. Moustacchn . 1972. Lethal and
36.
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mutagenic effects of elevated temperature on haploid yeast. Mol. Gen. Genet. 115:243-287. Slater, M. L. 1973. Effect of reversible inhibition of deoxyribonucleic acid synthesis on the yeast cell cycle. J. Bacteriol. 113:263-270. Susek, R. E., and S. L. Llndqult. 1989. hsp26 of Saccharomyces cerevisiae is related to the superfamily of small heat shock proteins but is without a demonstrable function. Mol. Cell. Biol. 9:5265-5271. Toda, T., S. Cameron, P. Sass, M. Zoler, J. D. Scott, B. McMugen, M. Hurwitz, E. G. Krebs, and M. WIgler. 1987. Cloning and characterization of BCYI, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Mol. Cell. Biol. 7:1371-1377. Vai, M., L. Popolo, and L. Albe na. 1987. Effect of tunicamycin on cell cycle progression in budding yeast. Exp. Cell Res.
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40. VanBogelen, R. A., M. A. Acton, ad F. C. Nedhardt. 1987. Induction of the heat shock regulon does not produce thermotolerance in Escherichia coli. Genes Dev. 1:525-531. . 1989. 40a.Velnot-Drebot, L. M., R. A. Singer, and G. C. Jo rRNA transcriptional initiation is decreased by inhibitors of the yeast cell cycle control step "start." J. Biol. Chem. 264:
19528-19534.
41. Watson, K., G. Dunlop, and R. Cavcchloli. 1984. Mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast. FEBS Lett. 172:299-302. 42. Werner-Washburne, M., D. E. Stone, and E. A. Craig. 1987. Complex interactions among members of an essential subfamily of hsp7O genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 7:2568-2577. 43. WideUtz, R. B., B. E. Magun, and E. W. Gerner. 1986. Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts. Mol. Cell. Biol. 6:1088-1094.
Vol. 172, No. 8
0021-9193/90/084352-07$02.00/0 Copyright © 1990, American Society for Microbiology
Thermotolerance Is Independent of Induction of the Full Spectrum of Heat Shock Proteins and of Cell Cycle Blockage in the Yeast Saccharomyces cerevisiae CHRISTINE A.
BARNES,lt
GERALD C.
JOHNSTON,'*
AND RICHARD A.
SINGER2'3
Departments of Microbiology,1 Biochemistry,2 and Medicine,3 Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 Received 7 December 1989/Accepted 7 May 1990
Cells of the yeast Saccharomyces cerevisiae are known to acquire thermotolerance in response to the stresses of starvation or heat shock. We show here through the use of cell cycle inhibitors that blockage of yeast cells in the Gl, S, or G2 phases of the mitotic cell cycle is not a stress that induces thermotolerance; arrested cells remained as sensitive to thermal killing as proliferating cells. These Gl- or S-phase-arrested cells were unimpaired in the acquisition of thermotolerance when subjected to a mild heat shock by incubation at 37°C. One cell cycle inhibitor, o-phenanthroline, did in fact cause cells to become thermotolerant but without induction of the characteristic pattern of heat shock proteins. Thermal induction of heat shock protein synthesis was unaffected; the o-phenanthroline-treated cells could still synthesize heat shock proteins upon transfer to 37°C. Use of a novel mutant conditionally defective only for the resumption of proliferation from stationary phase (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987) indicated that o-phenanthroline inhibition produces a stationary-phase arrest, a finding which is consistent with the increased thermotolerance and regulated cessation of proliferation exhibited by the inhibited cells. These findings show that the acquired thermotolerance of cells is unrelated to blockage of the mitotic cell cycle or to the rapid synthesis of the characteristic spectrum of heat shock proteins.
duction and to cell cycle position remain to be fully characterized. Here we describe further experiments that dissociate the acquisition of thermotolerance from the usual pattern of synthesis of heat shock proteins and from cell cycle position and show that even cells arrested at an unconventional cell cycle position, S phase, can acquire thermotolerance. Therefore, the stress that leads to thermotolerance is unrelated to the cell cycle or to the generally increased abundance of heat shock proteins.
The stress caused by exposure of proliferating cells of the yeast Saccharomyces cerevisiae to temperatures well above normal growth temperatures results in rapid loss of viability (34a, 35). However, yeast cells can adapt to and survive this usually lethal treatment if they are first subjected to a heat shock, a procedure in which cells are incubated at an elevated but otherwise nonlethal growth temperature (29, 34). The enhanced survival that results from the adaptation by heat-shocked cells has been termed acquired thermotolerance (18). Thermotolerance is also a characteristic of yeast cells in another stressful situation, i.e., that induced by nutrient starvation. A starved yeast cell ceases proliferation in stationary phase, expresses altered physiological properties, and becomes thermotolerant even without heat shock
MATERIALS AND METHODS Strains and culture conditions. Cultures of S. cerevisiae GR2 (MATa his6 ural) (22) were grown with gyratory shaking at 23°C in YNB defined medium (16) with added glucose (2%), amino acids (40 jig/ml), and nucleotides (20 ixg/ml). Strain MD-G3-G02, a derivative of the gcsl-J sedl-J mutant strain MAD-6 described elsewhere (10), was grown at 23°C in YM1 rich medium (16) supplemented with adenine (20 ,ug/ml) and glucose (2%). Hydroxyurea, tunicamycin, o-phenanthroline, and the yeast mating pheromone a-factor were all obtained from Sigma Chemical Co. (St. Louis, Mo.). Sinefungin was a gift from Eli Lilly & Co. (Indianapolis, Ind.). Cell concentrations were determined with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). Cell morphology was assessed microscopically; at least 200 cells were examined for each determination. Radiolabeling of protein and gel electrophoresis. Radiolabeling of protein was as described elsewhere (30). Cultures were maintained at fewer than 2 x 106 to 4 x 106 cells per ml for assessment of protein profiles. To radiolabel proteins, 1-ml samples were transferred to tubes containing 50 to 100 ,uCi of [35S]methionine and incubation with shaking was continued for 10 min at the appropriate temperature. Proteins were resolved by one-dimensional sodium dodecyl
(31).
Both the acquired thermotolerance of heat-shocked, proliferating cells and the intrinsic thermotolerance of stationary-phase cells have been correlated with cell cycle position (31, 33) and also with the production of heat shock proteins (for a review, see reference 28). Thermotolerant yeast cells in stationary phase contain an unreplicated (Gl) complement of DNA and display elevated levels of heat shock proteins (2, 3, 32). Similarly, proliferating yeast cells subjected to a heat shock transiently arrest proliferation in Gl (23) and induce the synthesis of heat shock proteins at the same time that they acquire thermotolerance (28). More recently, a particular heat shock protein, HsplO4, has been shown to be involved in acquired thermotolerance (34a). Although it has been shown that the induction of heat shock genes does not depend on cell cycle position (1), the relationships of thermotolerance to heat shock protein pro* Corresponding author. t Present address: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
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sulfate-polyacrylamide gel electrophoresis; the gels were then stained, destained, and dried (24) prior to autoradiography. Thermotolerance assay. Cells were grown overnight at 23°C to a density of 2 x 106 to 4 x 106/ml. Portions of each culture were incubated for 1 h at 37°C or were incubated at 23°C in the presence of an inhibitor of cell proliferation or were maintained at 23°C. At intervals, 1-ml samples were incubated at 52°C for 5 min and then put on ice for a minimum of 5 min. At the titne of each 52°C heat treatment, a control sample from the same culture, but without exposure to 52°C, was also placed on ice. Both samples were then diluted with phosphate-buffered saline, spread onto YEPD solid medium (16), and incubated at 23°C for 3 to 5 days. Thermotolerance, assessed by colony formation, was expressed as percent survival (29). RESULTS Thermotolerance, either upon the stress of heat shock at 37°C (29) or in stationary phase (31), is in each case correlated with a transient or permanent arrest of cell proliferation (23, 31). To determine whether cessation of proliferation is a stress sufficient to induce thermotolerance, conditions were imposed to block cells in the Gi, S, or G2 phase of the mitotic cell cycle, and arrested cells were then assessed for thermotolerance. The temperature-sensitive cdc mutations commonly used to impose cell cycle blocks were not suitable for these experiments, since blockage of the cell cycle by these cdc mutations requires incubation at 36°C, a treatment that itself imposes a stress sufficient to induce thermotolerance (29). Therefore, to arrest cell proliferation without the interfering incubation at 36°C associated with the arrest treatment, we used cell cycle inhibitors, which allowed cells to be maintained at 23°C throughout each experiment. The inhibitors o-phenanthroline, sinefungin, and the mating pheromone a-factor were each used to bring about concerted arrest in Gl (4, 22, 26); hydroxyurea was used to arrest cells in S phase (36); and tunicamycin was used to arrest cells in G2 (39). Cell cycle arrest does not induce thermotolerance. After 5 h of incubation in the presence of the G2-specific inhibitor tunicamycin, cell division had ceased, with 85% of the population accumulated as unbudded cells (Fig. 1B). Although a high percentage of unbudded cells usually indicates arrest in Gl (17), Vai et al. (39) showed that an unbudded tunicamycin-treated cell contains a replicated content of DNA in a single nucleus, which is indicative of arrest in G2. The cells arrested in G2 by tunicamycin treatment remained sensitive to the lethal effects of 52°C treatment. Even after 12 h of incubation in the presence of tunicamycin, arrested cells were as thermosensitive as untreated, proliferating cells (Fig. 1A). In a parallel experiment, arrest of cells in S phase was brought about by the inhibitor hydroxyurea. After 5 h of exposure to hydroxyurea, more than 85% of the cells were budded (Fig. 2B), exhibiting the standard morphology of hydroxyurea-treated cells that is indicative of an S-phase block (36). S-phase-arrested cells were as sensitive as untreated cells to the lethal effects of a 5-min incubation at 52°C (Fig. 2A). Therefore, cell cycle blockage leading to arrest of proliferation either in G2 or in S phase is not a stress that induces thermotolerance. Cells treated with a-factor or sinefungin arrest proliferation in the Gl phase of the cell cycle at the regulatory step designated start (4, 26). After 12 h of incubation in the
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of sinefungin, a time at which 85% of the cells were arrested in Gi, cells remained as thermosensitive as untreated cells (Fig. 3). Cells arrested in Gi by a-factor treatment also remained thermosensitive (33) (Fig. 4). The kinetics of acquisition and loss of thermotolerance differ among different stress situations that elicit thermotolerance. For example, stationary-phase cells maintain a thermotolerant state for long periods (35), whereas heatshocked, proliferating cells are only transiently thermotolerant (29). To assess whether the cell cycle inhibitor tunicamycin, hydroxyurea, sinefungin, or a-factor could have induced only a transient thermotolerance similar to a response to heat shock, thermotolerance was assayed immediately after inhibitor addition and at intervals thereafter. None of these cell cycle inhibitors induced even a transient thermotolerance (Fig. 1 through 4). We conclude from these experiments that cell cycle blockage is not a stress that causes cells to become thermotolerpresence
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Cells arrested in the cell cycle can acquire thermotolerance. Cells arrested in Gl at start by the action of sinefungin or a-factor were transferred to 37°C, and incubation was continued for 1 h in the presence of the inhibitor. Each population of arrested cells that was heat shocked in this way acquired thermotolerance to the same level as that of proliferating cells subjected to an identical heat shock (Fig. 3A and 4A). Similarly, cells arrested in S phase by the inhibitor hydroxyurea and then transferred to 37°C and incubated for 1 h became at least 200-fold more thermotolerant than S-phase-arrested cells simply maintained at 23°C (Fig. 2A). These findings show that blockage of the cell cycle does not preclude the usual acquisition of thermotolerance upon heat shock.
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Thermotolerance can occur without general induction of heat shock proteins. Treatment of cells with another start inhibitor, o-phenanthroline, brought about a response different from that found for the other cell cycle inhibitors discussed above. As expected (22), cells treated with ophenanthroline became arrested uniformly in the Gl interval at start, but unlike cells subjected to other treatments that caused cell cycle blockage (Fig. 1 through 4), o-phenanthroline-treated cells displayed a marked increase in thermotolerance (Fig. SA). Thermotolerance has been associated with the production of heat shock proteins in general (20, 29) and, most recently, with the production of a particular heat shock protein, HsplO4 (34a). Indeed, for most of the situations described here, thermotolerance induced by incubation at the elevated temperature of 37°C was accompanied by the synthesis of heat shock proteins (Fig. 3C and 4C). For this reason, the increased thermotolerance of o-phenanthrolinetreated cells was unexpected, because none of the inhibitors used here to cause cell cycle blockage, including o-phenanthroline (Fig. 5), increased the production of the heat shock proteins at 23°C (1) (Fig. 3C, 4C, and 5C). Even after extended exposure to o-phenanthroline, a treatment that caused a high level of thermotolerance, cells did not induce the expression of major heat shock proteins. Nevertheless, for these o-phenanthroline-treated thermotolerant cells, as for the cells arrested by the other start inhibitors (Fig. 3C and 4C), the presence of the inhibitor did not preclude the production of heat shock proteins; transfer of o-phenanthroline-arrested cells to 37°C elicited the typical pattern of o-phenanthroline protein synthesis (Fig. SC). Although particular proteins may be implicated in thermotolerance (34a), the effects of the start inhibitor o-phenanthroline show that
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increased synthesis of the characteristic set of heat shock proteins is not required for thermotolerance. o-Phenanthroline causes cells to enter stationary phase. Acquisition of the stationary-phase state causes thermotolerance without application of a heat shock. Therefore, we 100-
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phenotype, when in stationary phase, do not resume proliferation if stimulated by the presence of fresh medium under restrictive conditions, but mutant cells that are already proliferating continue to proliferate when transferred to the restrictive temperature. This novel mutant phenotype pan thus be used to distinguish a cell in stationary phase from a cell that is not proliferating for other reasons (9). When mutant cells of this type were arrested in proliferation by treatment with o-phenanthroline at a temperature that elicits the mutant phenotype, the cells became defective for the resumption of proliferation at the restrictive temperature upon transfer to fresh medium free of o-phenanthroline (Fig. 6). In contrast, mutant c,ells resumed proliferation upon transfer to inhibitor-free medium at the permissive temperature. This conditional inability of mutant cells to resume proliferation indicates that o-phenanthroline treatment brings about a stationary-phase arrest of proliferation. The stationary-phase status of o-phenanthroline-treated cells is consistent with the acquisition of thermotolerance by these arrested cells and suggests that this thermotolerance is a consequenc'e of the stationary-phase arrest caused by ophenanthroline. Mutant cells arrested in cell proliferation by hydroxyurea treatment did not become conditionally defective for the resumption of proliferation (data not shown). This cell cycle inhibition therefore does not produce a stationary-phase arrest, a finding consistent with the lack of thermotolerance of these arrested cells (Fig. 2).
Proliferation arrest and thermoto_eance. In yeast cells, thermotolerance is seen usually upon arrest of proliferation in a prereplicative state. Nutrient-deprived yeast cells become arrested in a prereplicative state and in this stationaryphase state are more thermotolerant than proliferating cells (31). Similarly, transfer of proliferating cells to 37°C and incubation for 1 h cause a transient arrest of proliferation and accumulation of Gl-phase, prerepliQative cells (23) at the same time that the cells acquire thermotolerance (29). The experiments described above show that arrest of proliferation is not a signal for the induction of thermotolerance; yeast cells arrested in Gl by a-factor or sinefungin treatment, in S phase by hydroxyurea treatment, or in G2 by tunicamycin treatment remained thermosensitive. Perhaps the arrest caused by the mating pheromone a-factor does not induce the stress-related response of thermotolerance because this arrest in Gl at start is a normal cellular response in preparation for conjugation (4). However, arrest of proliferation outside of Gl, in S phase or in G2, is not part of the usual biological repertoire of S. cerevisiae. Therefore, it was unexpected that neither hydroxyurea treatment nor tunicamycin treatment provoked the stress response of thermotolerance. It has been known for some time that a cell blocked in S phase or in G2 continues to increase in mass and becomes enlarged (21). Evidently this unbalanced growth upon blockage of the cell cycle does not perturb the physiological status of the arrested cell sufficiently to trigger the induction of thermotolerance. Heat shock and thermotolerance. Acquired thermotolerance has been correlated with the expression of heat shock proteins (27, 28). Most of the major heat shock proteins are present at only low levels in yeast cells proliferating at 23°C, but within 1 h after transfer to 37°C, as cells become thermotolerant, heat shock proteins are some of the most abundant proteins present (11, 30). Indeed, this correlation was the basis for early proposals that heat shock proteins are involved in thermotolerance. Despite this correlation between heat shock protein production and thermotolerance, appreciation of the functions of particular heat shock proteins in thermotolerance induction in yeast cells has been elusive (8, 12, 28, 32, 41). It is only recently that one heat shock protein, Hspl04, has been shown to play a role in acquisition of thermotolerance; disrption of the nonessential HSP104 gene impairs the acquisition of thermotolerance by heat-shocked mutant cells (34a). However, this impairment is obvious only after prolonged incubation at the lethal temperature of 509C; the absence of the Hspl04 protein has no effect on the survival of heat-shocked mutant cells after short-term exposure to 50°C. This short-term, Hsp104-independent thermotolerance thus reveals a second, as-yetunidentified component of thermotolerance (34a). For the purposes of the study described here, cells were assessed for thermotolerance after a short-term incubation at an elevated, lethal temperature. Therefore, our procedure evaluated the role of heat shock proteins only in this initial component of thermotolerance. Treatment with o-phenanthroline resulted in short-term thermotolerance in the absence of the usual spectrum of heat shock proteins. This inhibitor may thus prove useful in the study of this initial phase of thermotolerance.
Studies that have not specifically resolved the initial from the delayed, HspI04-related thermotolerance have shown that thermotolerance in general can increase without an increased synthesis of all heat shock proteins (6). Moreover,
VOL. 172, 1990
cells that acquire thermotolerance by treatments other than heat do not always synthesize the full spectrum of heat shock proteins; in some cases only certain heat shock proteins are synthesized (for reviews, see references 7 and 25). Many individual heat shock genes are not required for thermotolerance. Cells bearing mutations in the heat shock genes HSP90 and HSC90 (28), UBI4 (13), HSP48 (H. Iida, cited in reference 28), HSP26 (32, 37), and members of the HSP70 gene family (42) nevertheless become thermotolerant after a mild heat treatment. Perhaps only a minor subset of yeast heat shock proteins functions in the acquisition of thermotolerance. Although the HsplO4 protein plays a role in thermotolerance (34a), it has been shown that thermotolerance can be acquired by yeast cells even in the presence of presumably nonfunctional heat shock proteins (15), and the acquisition of thermotolerance can occur in the absence of cytoplasmic and mitochondrial protein synthesis (29, 41). Yeast cells can also lose thermotolerance in the presence of elevated levels of the major heat shock proteins (5). Investigations of mammalian systems or Escherichia coli also suggest that thermotolerance need not be correlated with the induction of the spectrum of heat shock proteins. For example, addition of the protein synthesis inhibitor cycloheximide to rat embryonic fibroblasts does not prevent thermotolerance but does prevent heat shock protein production (43). Conversely, for E. coli cells the overexpression of heat shock proteins at non-heat-shock temperatures does not protect these cells from the effects of lethal temperatures (40). Further work is needed to resolve this issue. o-Phenanthroline induces stationary-phase arrest. We show here that o-phenanthroline brings about a stationary-phase state. The ability of o-phenanthroline to induce stationary phase suggests that this inhibitor mimics nutrient deprivation. The signal for nutrient supply, and thus for nutrient deprivation, is mediated in part by a cyclic AMP (cAMP)dependent signal transduction pathway that culminates in the activity of cAMP-dependent protein kinases (for a review, see reference 14). Disruption of the BCYJ gene, which codes for the regulatory subunit of cAMP-dependent protein kinases (38), leads to unregulated activation of protein kinases and the inability of bcyl disruption strains to respond in a regulated manner to nutrient deprivation (38). Treatment of a bcyl disruption strain with o-phenanthroline caused arrest of proliferation with cells blocked in the unbudded interval of the cell cycle (our unpublished results), which is the response of wild-type cells to o-phenanthroline treatment (22). Thus, the effects of o-phenanthroline are epistatic to those of the bcyl disruption, suggesting that o-phenanthroline brings about a stationary-phase state by affecting an activity that is regulated by the cAMP signal transduction pathway. It is not yet known whether o-phenanthroline interferes with the function of cAMP-dependent protein kinases directly or with some downstream activity. It is known, however, that o-phenanthroline, at the concentrations used here to bring about concerted blockage of start, inhibits rRNA gene expression (22) at the level of transcription initiation (40a), and also induces the general control response in S. cerevisiae (19) at the transcriptional level (40a). Although neither of these responses has been shown to play a role in the acquisition of stationary phase, the general control response is activated under starvation conditions and in this way may be a response that brings about the stationary-phase state. In any case, o-phenanthroline may prove useful in studies of stationary phase and nutrient signaling.
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