Recovery of the Yeast Cell Cycle from Heat Shock-induced G1 Arrest Involves a Positive Regulation of G1Cyclin Expression by the S Phase Cyclin Clb5
1999; Elsevier BV; Volume: 274; Issue: 34 Linguagem: Inglês
10.1074/jbc.274.34.24220
ISSN1083-351X
Autores Tópico(s)DNA Repair Mechanisms
ResumoIn the yeast Saccharomyces cerevisiae, heat shock stress induces a variety of cellular responses including a transient cell cycle arrest before G1/S transition. Previous studies have suggested that this G1 delay is probably attributable to a reduced level of the G1 cyclin gene (CLN1 and CLN2) transcripts. Here we report our finding that the G1 cyclin Cln3 and the S cyclin Clb5 are the key factors required for recovery from heat shock-induced G1 arrest. Heat shock treatment of G1 cells lacking either CLN3 or CLB5/CLB6 functions leads to prolonged cell cycle arrest before the initiation of DNA synthesis, concomitant with a severe deficiency in bud formation. The inability of the clb5 clb6 mutant to resume normal budding after heat shock treatment is unanticipated, since the S phase cyclins are generally thought to be required mainly for initiation of DNA synthesis and have no significant roles in bud formation in the presence of functional G1cyclins. Further studies reveal that the accumulation of G1cyclin transcripts is markedly delayed in the clb5 clb6 mutant following heat shock treatment, indicating that the CLN gene expression may require Clb5/Clb6 to attain a threshold level for driving the cell cycle through G1/S transition. Consistent with this assumption, overproduction of Clb5 greatly enhances the transcription of at least two G1cyclin genes (CLN1 and CLN2) in heat-shocked G1 cells. These results suggest that Clb5 may positively regulate the expression of G1 cyclins during cellular recovery from heat shock-induced G1 arrest. Additional evidence is presented to support a role for Clb5 in maintaining the synchrony between budding and DNA synthesis during normal cell division as well. In the yeast Saccharomyces cerevisiae, heat shock stress induces a variety of cellular responses including a transient cell cycle arrest before G1/S transition. Previous studies have suggested that this G1 delay is probably attributable to a reduced level of the G1 cyclin gene (CLN1 and CLN2) transcripts. Here we report our finding that the G1 cyclin Cln3 and the S cyclin Clb5 are the key factors required for recovery from heat shock-induced G1 arrest. Heat shock treatment of G1 cells lacking either CLN3 or CLB5/CLB6 functions leads to prolonged cell cycle arrest before the initiation of DNA synthesis, concomitant with a severe deficiency in bud formation. The inability of the clb5 clb6 mutant to resume normal budding after heat shock treatment is unanticipated, since the S phase cyclins are generally thought to be required mainly for initiation of DNA synthesis and have no significant roles in bud formation in the presence of functional G1cyclins. Further studies reveal that the accumulation of G1cyclin transcripts is markedly delayed in the clb5 clb6 mutant following heat shock treatment, indicating that the CLN gene expression may require Clb5/Clb6 to attain a threshold level for driving the cell cycle through G1/S transition. Consistent with this assumption, overproduction of Clb5 greatly enhances the transcription of at least two G1cyclin genes (CLN1 and CLN2) in heat-shocked G1 cells. These results suggest that Clb5 may positively regulate the expression of G1 cyclins during cellular recovery from heat shock-induced G1 arrest. Additional evidence is presented to support a role for Clb5 in maintaining the synchrony between budding and DNA synthesis during normal cell division as well. kilobase pair(s) Cells from various organisms are equipped through evolution with the ability to react to an abrupt elevation of temperature in their environment with what has been collectively termed heat shock response to increase their chance of survival in such an environment (1Craig E.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 501-537Google Scholar, 2Lindquist S. Annu. Rev. Biochem. 1986; 55: 1151-1191Crossref PubMed Google Scholar, 3Lindquist S. Curr. Opin. Genet. Dev. 1992; 2: 748-755Crossref PubMed Scopus (109) Google Scholar, 4Schoffl F. Prandl R. Reindl A. Plant Physiol. 1998; 117: 1135-1141Crossref PubMed Scopus (342) Google Scholar). Heat shock treatment of yeast cells, for instance, induces several observable responses including a reprogramming of gene expression, acquisition of thermotolerance, and a transient cell cycle arrest at G1 (1Craig E.A. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 501-537Google Scholar, 5Barnes C.A. Johnston G.C. Singer R.A. J. Bacteriol. 1990; 172: 4352-4358Crossref PubMed Google Scholar, 6Finkelstein D.B. Strausberg S. McAlister L. J. Biol. Chem. 1982; 257: 8405-8411Abstract Full Text PDF PubMed Google Scholar, 7Shin D.Y. Matsumoto K. Iida H. Uno I. Ishikawa T. Mol. Cell. Biol. 1987; 7: 244-250Crossref PubMed Scopus (99) Google Scholar). The reprogramming of gene expression allows biased synthesis of proteins and metabolites with protective functions against the heat insult. An important group of proteins that are induced to high levels immediately after heat exposure in yeast as well as other organisms is known to be the heat shock proteins, which act as molecular chaperones to help minimize the detrimental effects of protein denaturation caused by heat shock treatment (3Lindquist S. Curr. Opin. Genet. Dev. 1992; 2: 748-755Crossref PubMed Scopus (109) Google Scholar, 8Georgopoulos C. Welch W.J. Annu. Rev. Cell Biol. 1993; 9: 601-634Crossref PubMed Scopus (1002) Google Scholar, 9Hendricks J.P. Hartl F.U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1477) Google Scholar, 10Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1895) Google Scholar, 11Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 372: 475-478Crossref PubMed Scopus (742) Google Scholar). The heat shock-induced reprogramming of gene expression also results in a temporal reduction in the expression of proteins whose functions are not in immediate need for thermotolerance acquisition. This down-regulation of gene expression may be the reason behind the transient cell cycle arrest at G1 in yeast cells, since two G1 cyclin genes required for G1/S transition, CLN1 and CLN2, are found to be repressed after heat shock treatment (12Rowley A. Johnston G.C. Butler B. Werner-Washburne M. Singer R.A. Mol. Cell. Biol. 1993; 13: 1034-1041Crossref PubMed Scopus (94) Google Scholar). A stress-inducible protein, Xbp1, has been proposed to function in the repression of gene expression following heat shock treatment (13Mai B. Breeden L. Mol. Cell. Biol. 1997; 17: 6491-6501Crossref PubMed Scopus (89) Google Scholar). Apart from down-regulating the expression of many genes, heat shock stress is also believed to result in a down-regulation of the cyclic AMP (cAMP)-Ras pathway (7Shin D.Y. Matsumoto K. Iida H. Uno I. Ishikawa T. Mol. Cell. Biol. 1987; 7: 244-250Crossref PubMed Scopus (99) Google Scholar, 14Attfield P.V. Nat. Biotech. 1997; 15: 1351-1357Crossref PubMed Scopus (362) Google Scholar), which may affect cell cycle progression through the G1 cyclin Cln3 (15Hall D.D. Markwardt D.D. Parviz F. Heideman W. EMBO J. 1998; 17: 4370-4378Crossref PubMed Scopus (96) Google Scholar). The heat shock-induced G1 arrest in yeast lasts for a period of approximately 1 h (7Shin D.Y. Matsumoto K. Iida H. Uno I. Ishikawa T. Mol. Cell. Biol. 1987; 7: 244-250Crossref PubMed Scopus (99) Google Scholar). Once the heat shock proteins are induced and thermotolerance is acquired, the normal cell cycle resumes. Normal cell cycle progression in yeast relies on sequential activation of the cyclin-dependent kinase Cdc28 by the cell cycle stage-specific cyclins (16Futcher B. Yeast. 1996; 12: 1635-1646Crossref PubMed Scopus (95) Google Scholar, 17Lew D.J. Weinert T. Pringle J.R. Pringle J.R. Broach J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 607-695Google Scholar, 18Mendenhall M.D. Hodge A.E. Microbiol. Mol. Biol. Rev. 1998; 62: 1191-1243Crossref PubMed Google Scholar, 19Nasmyth K. Trends Genet. 1996; 12: 405-412Abstract Full Text PDF PubMed Scopus (295) Google Scholar). G1/S transition, often referred to as START, is dependent on three G1 cyclins: Cln1, Cln2, and Cln3 (16Futcher B. Yeast. 1996; 12: 1635-1646Crossref PubMed Scopus (95) Google Scholar, 17Lew D.J. Weinert T. Pringle J.R. Pringle J.R. Broach J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 607-695Google Scholar, 18Mendenhall M.D. Hodge A.E. Microbiol. Mol. Biol. Rev. 1998; 62: 1191-1243Crossref PubMed Google Scholar, 19Nasmyth K. Trends Genet. 1996; 12: 405-412Abstract Full Text PDF PubMed Scopus (295) Google Scholar). Cln3 is particularly important, since it controls the expression of other G1 cyclins, as well as Clb5 and Clb6, two S phase cyclins required for initiation of DNA synthesis (20Stuart D. Wittenberg C. Genes Dev. 1995; 9: 2780-2794Crossref PubMed Scopus (174) Google Scholar, 21Tyers M. Tokiwa G. Futcher B. EMBO J. 1993; 12: 1955-1968Crossref PubMed Scopus (393) Google Scholar). By a yet unknown mechanism, Cln3 activates the transcription factor SBF, which is composed of the Swi4 and Swi6 proteins (22Dirick L. Bohm T. Nasmyth K. EMBO J. 1995; 14: 4803-4813Crossref PubMed Scopus (279) Google Scholar, 23Koch C. Moll T. Neuberg M. Ahorn H. Nasmyth K. Science. 1993; 261: 1551-1557Crossref PubMed Scopus (314) Google Scholar, 24Nasmyth K. Dirick L. Cell. 1991; 66: 995-1013Abstract Full Text PDF PubMed Scopus (252) Google Scholar). SBF in turn drives the transcription of a set of genes including CLN1 and CLN2, leading to execution of START and budding (22Dirick L. Bohm T. Nasmyth K. EMBO J. 1995; 14: 4803-4813Crossref PubMed Scopus (279) Google Scholar, 23Koch C. Moll T. Neuberg M. Ahorn H. Nasmyth K. Science. 1993; 261: 1551-1557Crossref PubMed Scopus (314) Google Scholar, 24Nasmyth K. Dirick L. Cell. 1991; 66: 995-1013Abstract Full Text PDF PubMed Scopus (252) Google Scholar). In a parallel fashion, Cln3 is also thought to activate another transcription factor MBF, consisting of Mbp1 and Swi6, which then stimulates expression of the genes involved in DNA synthesis including CLB5 and CLB6 (22Dirick L. Bohm T. Nasmyth K. EMBO J. 1995; 14: 4803-4813Crossref PubMed Scopus (279) Google Scholar, 23Koch C. Moll T. Neuberg M. Ahorn H. Nasmyth K. Science. 1993; 261: 1551-1557Crossref PubMed Scopus (314) Google Scholar). Thus, the cyclin cascade initiated by Cln3 sets in motion two parallel cell cycle events downstream of START, i.e. budding and DNA replication, each controlled by a pair of functionally overlapping cyclins. Although G1 cyclin functions are sufficient for passage of START and bud formation, the initiation of DNA replication cannot properly take place without the functions of Clb5/Clb6. Loss of Clb5/Clb6 functions results in a delay in initiation of DNA synthesis but has no effects on the timing of bud emergence (25Schwob E. Nasmyth K. Genes Dev. 1993; 7: 1160-1175Crossref PubMed Scopus (406) Google Scholar), suggesting that Clb5/Clb6 are principally required for DNA synthesis control. Under special conditions, however, Clb5 has been shown to be more versatile, capable of performing some overlapping functions with the G1 cyclins. Overproduction of Clb5, for example, can rescue the lethality of the cln1 cln2 cln3 triple mutant (26Epstein C.B. Cross F.R. Genes Dev. 1992; 6: 1695-1706Crossref PubMed Scopus (299) Google Scholar). Similarly, elevation of the Clb5 activity through inactivation of Sic1, an inhibitor of the Clb5-Cdc28 kinase, also suppresses the START deficiency in the cln1 cln2 cln3 triple mutant (27Schneider B.L. Yang Q.H. Futcher A.B. Science. 1996; 272: 560-562Crossref PubMed Scopus (190) Google Scholar, 28Tyers M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7772-7776Crossref PubMed Scopus (127) Google Scholar). These observations suggest that, although Clb5 normally is not required for execution of START and budding, it is able to provide these functions if its activity is sufficiently increased. In the present study, the roles of various cyclins in the recovery of cell cycle from heat shock-induced G1 arrest have been examined. Our results suggest that Clb5 is one of the key factors required for this process. Clb5 facilitates the cell cycle recovery following heat shock by promoting initiation of DNA replication on one hand and positively regulating the expression of the G1cyclin genes to promote budding on the other. All yeast strains used in this study were derived from the wild type strain W303 and are listed in Table I. The rad24 and cln1 cln3 mutants were originally obtained from U. Surana and I. Herskowitz, respectively. YUS454 (cln1 cln2 cln3) contains the CLN3 gene under control of the methionine-repressible MET3 promoter for viability. Yeast extract-peptone, synthetic complete, and dropout media were prepared as described by Rose et al. (29Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Recombinant DNA methodology was performed as described by Sambrook et al. (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). PCR was performed with Vent polymerase (New England Biolabs) as recommended by the manufacturer. Genetic manipulations were performed according to standard methods (29Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Cell morphology and DNA content analysis by FACScan were performed as described previously (31Li X. Cai M. Mol. Cell. Biol. 1997; 17: 2723-2734Crossref PubMed Scopus (46) Google Scholar).Table IYeast strainsStrainRelevant genotypeW303 aMAT a ade2 ura3 leu2 trp1 his3YMC309 aMAT a rad9Δ∷TRP1 ade2 ura3 leu2 his3YMC431MAT a rad24 ade2 ura3 leu2 trp1 his3YMC406MAT a clb5Δ∷URA3 clb6Δ∷TRP1 ade2 leu2 his3YMC404 aMAT a clb3Δ∷HIS3 clb4Δ∷TRP1 ade2 ura3 leu2YMC432MAT a cln2Δ∷URA3 ade2 leu2 trp1 his3YMC433MAT a cln3Δ∷URA3 ade2 leu2 trp1 his3YMC434MAT a cln1Δ∷TRP1 cln2Δ∷URA3 ade2 leu2 his3YMC435MAT a swi4Δ∷TRP1 ade2 ura3 leu2 his3YMC436MAT a cln1Δ∷TRP1 cln3Δ∷URA3 ade2 leu2 his3YUS454MATa cln1Δ cln2Δ cln3Δ ade2 ura3 (pMET3-CLN3) Open table in a new tab Plasmids used in this study are listed in Table II. To generate the plasmids pMC229–233, polymerase chain reaction-amplified coding regions of CLN2, CLN3, CLB3, CLB5, and CLB6 were cloned individually into a centromere vector under control of the GAL1 promoter containing URA3 as a selectable marker. The plasmids pMC234 and pMC235 were generated by fusing the coding regions of CLN2 and CLN3 with GAL1 in a centromere vector containing the LEU2 gene.Table IIPlasmidsNameConstructpMC229GAL1-CLN2, URA3, yeast centromere vectorpMC230GAL1-CLN3, URA3, yeast centromere vectorpMC231GAL1-CLB5, URA3, yeast centromere vectorpMC232GAL1-CLB6, URA3, yeast centromere vectorpMC233GAL1-CLB3, URA3, yeast centromere vectorpMC234GAL1-CLN2, LEU2, yeast centromere vectorpMC235GAL1-CLN3, LEU2, yeast centromere vector Open table in a new tab Gene disruptions were performed using the one-step replacement method (32Rothstein R. Methods Enzymol. 1991; 194: 281-301Crossref PubMed Scopus (1105) Google Scholar). The CLN2 gene was disrupted by replacing its 1.3-kb1 Xho I–Nco I fragment by URA3 in wild type and a cln1 mutant to generate YMC432 and YMC434, respectively. The CLB3 gene was disrupted by replacing the 1-kb Hpa I–Bgl II fragment by HIS3, and the CLB4 gene was disrupted by replacing the 0.5-kb Stu I–Spe I fragment by TRP1. YMC404 (clb3Δ::HIS3 clb4Δ::TRP1) was generated by two consecutive disruptions. Similarly, YMC406 (clb5Δ::URA3 clb6Δ::TRP1) was made by consecutive disruptions of CLB5, whose 1-kb Pvu I–Fok I fragment was replaced by URA3, and CLB6, whose 0.9-kb Bst XI–Xba I fragment was replaced by TRP1. Overnight cultures were diluted to an A600 of 0.1 and were allowed to grow at 25 °C to an A600 of 0.3. At this point, α-factor was added to a final concentration of 5 μg/ml. When greater than 95% cells had been arrested in G1, the cultures were divided into two halves, with one shifted to 42 °C for 30 min and another remaining at 25 °C. The cells were then washed by filtration and resuspended in fresh medium at 25 °C. Samples taken at intervals were used for analysis of cell morphology, DNA content, and Northern blotting. In experiments where the expression of a gene was driven by the GAL1 promoter, the cells were first grown in medium containing raffinose as the sole carbon source. The cells were synchronized in G1 as described above. Galactose was then added to 2% to induce the gene expression for 15 min at 25 °C before being subjected to heat shock treatment. After α-factor was removed by filtration, the cells were resuspended in fresh medium containing raffinose and galactose and incubated at 25 °C. Samples were collected at intervals for further analysis. In experiments showing that CLB5 overexpression overrides α-factor-induced G1 arrest, the G1-arrested cells were split into two parts with galactose added to one of them, and both cultures were continuously incubated with α-factor. Total RNA was isolated as described by Cross and Tinkelenberg (33Cross F.R. Tinkelenberg A.H. Cell. 1991; 65: 875-883Abstract Full Text PDF PubMed Scopus (249) Google Scholar), and Northern blot analyses were performed as described by Price et al. (34Price C. Nasmyth K. Schuster T. J. Mol. Biol. 1991; 218: 543-556Crossref PubMed Scopus (61) Google Scholar). To show RNA signals of various genes on the same blot, the DNA probes used earlier on RNA blot were stripped by heating at 80 °C for 30 min in a solution containing 0.1% SDS, 1 μm EDTA, and 10 μmTris-HCl (pH 7.2). After washing briefly in H2O, the blot was hybridized with another 32P-labeled probe. The RNA levels were quantitated with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Previous studies on yeast cell cycle response to heat shock treatment were performed with asynchronous cell populations (12Rowley A. Johnston G.C. Butler B. Werner-Washburne M. Singer R.A. Mol. Cell. Biol. 1993; 13: 1034-1041Crossref PubMed Scopus (94) Google Scholar). To better assess the duration of the G1 arrest period and the kinetics of the recovery, we used mating pheromone (α-factor)-synchronized cells for examination of their response to heat shock treatment. Wild type cells were synchronized by α-factor and divided into two halves with one shifted to 42 °C for 30 min, while another remained at 25 °C, followed by washing and resuspension in fresh medium devoid of the mating pheromone. As shown in Fig. 1, cells that had been exposed to high temperature initiated DNA replication and budding at a later time point than those that were not treated with heat shock. The delay was about 60 min (Fig. 1). This result confirms the previous finding that the cell cycle arrest induced by heat shock is prior to START, since budding and DNA replication were both delayed (12Rowley A. Johnston G.C. Butler B. Werner-Washburne M. Singer R.A. Mol. Cell. Biol. 1993; 13: 1034-1041Crossref PubMed Scopus (94) Google Scholar). It is well known that another type of stress, DNA damage, also induces cell cycle arrest in yeast, which is mediated through a control mechanism termed "checkpoint" (35Hartwell L.H. Weinert T.A. Science. 1989; 246: 629-634Crossref PubMed Scopus (2431) Google Scholar, 36Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2316) Google Scholar). The checkpoint genes such as RAD9 and RAD24 have been shown to be required for the transient cell cycle arrest at G1 caused by DNA damage (37Siede W. Friedberg A.S. Dianova I. Friedberg E.C. Genetics. 1994; 138: 271-281Crossref PubMed Google Scholar). To test whether the DNA damage checkpoint also functions in the G1 arrest caused by heat shock treatment, we examined the cell cycle response to heat shock stress in the rad9 and rad24 mutants. As shown in Fig. 1, both mutants exhibited a G1 arrest that lasted about 60 min, similar to the wild type cells, suggesting that the cell cycle arrest after heat shock is not mediated through DNA damage checkpoint genes. Decrease in the CLN1 and CLN2 transcript levels has been suggested as a possible cause of the cell cycle arrest in heat shock-treated cells (12Rowley A. Johnston G.C. Butler B. Werner-Washburne M. Singer R.A. Mol. Cell. Biol. 1993; 13: 1034-1041Crossref PubMed Scopus (94) Google Scholar). We therefore examined various cyclin mutants for their response to heat shock treatment. Cells lacking CLN2 responded to heat shock treatment with a G1arrest and recovered 60 min later, as did wild type cells (Fig.2). The cln1 mutant behaved similarly (data not shown). Deletion of both CLN1 and CLN2 genes caused a further delay of 30 min (Fig. 2). Nevertheless, the double mutant resumed cell cycle and divided normally after the 90-min delay (Fig. 2 and data not shown). Remarkably, the cln3 mutant failed to recover from the heat shock-induced G1 arrest for at least 5 h (Fig. 2). The mutant cells showed no sign of DNA replication and virtually no bud formation during the entire period of the experiment (Fig. 2). The failure of the cln3 mutant to initiate DNA replication and budding was not because the mutant was particularly sensitive to the heat treatment, since the cln3 cell viability was not significantly affected by the heat shock treatment (data not shown). This result indicates that CLN3 is essential for cell recovery from heat shock-induced G1 arrest. A similar defect in recovery from the arrest was also observed in the clb5 clb6 double mutant except, in this case, the budding defect was less pronounced than that of the cln3 cells (Fig. 2). Nevertheless, the clb5 clb6 double mutant displayed a conspicuous budding defect after heat shock. 2 h after release from synchronization, for example, only 26% of the double mutant cells were budded, compared with over 50% in the populations of either the wild type or any of the cln1, cln2, clb5, and clb6 single mutants (Fig. 2, and data not shown). Furthermore, the budded cell population of the double mutant was limited to about 50% for at least 5 h (Fig. 2). It is evident, therefore, that the clb5 clb6 mutant was unable to recover from the heat shock-induced G1 arrest for a rather prolonged period. Both cln3 and clb5 clb6 mutants eventually resumed normal division without showing significant reductions in their viability (data not shown). In comparison, the clb3 clb4 double mutant, defective in another pair of S phase cyclins, Clb3/Clb4, (25Schwob E. Nasmyth K. Genes Dev. 1993; 7: 1160-1175Crossref PubMed Scopus (406) Google Scholar, 38Fitch I. Dahmann C. Surana U. Amon A. Goetsch L. Byers B. Futcher B. Mol. Cell. Biol. 1992; 3: 805-818Crossref Scopus (235) Google Scholar, 39Richardson H.E. Lew D.J. Henze M. Sugimoto K. Reed S.I. Genes Dev. 1992; 6: 2021-2034Crossref PubMed Scopus (209) Google Scholar), exhibited no defect in recovery from the G1 arrest (Fig. 2). The severe defect in recovery from heat shock-induced G1 arrest exhibited by the cln3 mutant is, by and large, within expectations, since this cyclin is the crucial factor in promoting the initiation of bud formation and DNA replication. However, the inability of the clb5 clb6 double mutant to resume normal budding was unexpected, because the Clb5/Clb6 cyclins are thought to be required mainly for DNA synthesis and not for bud formation when functional G1 cyclins are present (25Schwob E. Nasmyth K. Genes Dev. 1993; 7: 1160-1175Crossref PubMed Scopus (406) Google Scholar). To seek an explanation for this finding, we tested whether the CLN transcript levels were affected in the double mutant. As shown in Fig. 3 a, the CLN1 and CLN2 transcripts in the untreated wild type cells accumulated to a peak level 30 min after release from synchronization, followed by periodic oscillations. In the heat shock-treated wild type cells, the CLN1 and CLN2 transcripts lagged 30–60 min before reaching the peak levels (Fig. 3 a). Similarly, the accumulation of the CLB5 transcript was also delayed, albeit to a lesser extent compared with that of CLN1 and CLN2 (Fig.3 a). The CLN3 transcript was present in α-factor-treated cells and appeared to be the least affected by heat shock treatment (Fig. 3 a). In contrast, the clb5 clb6 mutant displayed a marked reduction in all G1cyclin transcripts after heat shock treatment (Fig. 3 b). The CLN1 and CLN2 transcripts in clb5 clb6 cells did not reach levels comparable with those in the untreated cells until 180 min after release from G1 synchronization (Fig.3 b). The level of the CLN3 transcript was also decreased significantly in the mutant after heat exposure (Fig.3 b). The clb5 clb6 mutant, nevertheless, produced the ACT1 and SSA4 transcripts in a manner similar to wild type cells after heat exposure, indicating that the mutant was not defective in the general and heat shock-induced transcriptions (Fig. 3). These results together suggest that the Clb5/Clb6 cyclins are involved in the recovery of CLN transcript abundance in cells that have undergone heat shock treatment. The observation that the CLN transcript accumulation was delayed by heat shock treatment supports the notion that the cell cycle arrest following heat shock is attributable to the suboptimal level of G1 cyclin expression. Indeed, overexpression of CLN2 is able to abolish the G1 arrest induced by heat shock treatment in asynchronous cells (12Rowley A. Johnston G.C. Butler B. Werner-Washburne M. Singer R.A. Mol. Cell. Biol. 1993; 13: 1034-1041Crossref PubMed Scopus (94) Google Scholar). To better understand the mechanisms of the heat shock-induced G1 arrest, we examined the effects of overexpression of other cyclin genes on cell cycle response to heat shock. The CLN2, CLN3, CLB5, and CLB3 genes were each placed under control of p GAL1, a strong inducible promoter, and introduced into wild type cells. After synchronization in G1 by α-factor in a raffinose-containing medium, galactose was added for 15 min to induce the cyclin gene expression, followed by heat shock and release to fresh medium containing galactose. As shown in Fig.4, overexpression of each CLN2, CLN3, and CLB5 completely eliminated the G1 arrest. The cyclin-overproducing cells displayed cell cycle kinetics after heat shock treatment comparable with those of untreated wild type cells, as judged by the FACScan pattern and budding profile (Fig. 4). In contrast, overexpression of two other S phase cyclin genes, CLB3 and CLB4, generated no effect on the G1 arrest after heat shock (Fig.4, and data not shown), an observation in agreement with the results presented in Fig. 2. It has been suggested that the G1 cyclins are the rate-limiting factors in G1 cells for passage of START (16Futcher B. Yeast. 1996; 12: 1635-1646Crossref PubMed Scopus (95) Google Scholar). The finding that heat shock stress failed to result in cell cycle arrest in CLN overexpression cells suggests the same for the heat shock-induced G1 arrest. Since the Clb5/Clb6 cyclins are not rate-limiting factors for START (25Schwob E. Nasmyth K. Genes Dev. 1993; 7: 1160-1175Crossref PubMed Scopus (406) Google Scholar), the ability of overexpressed CLB5 to negate the heat shock-induced G1 arrest is indicative of an indirect role for Clb5/Clb6 in START promotion, possibly as stimulators of the CLN gene transcription. This possibility was investigated by analyzing the CLN transcript abundance in cells overexpressing CLB5. As shown in Fig.5 a, CLB5 overexpression reversed the delay of CLN1 and CLN2 transcripts caused by heat shock, making them appear at the same time as in the wild type cells that experienced no heat shock stress (Fig. 5 a). CLN3 transcript levels were not significantly affected by CLB5 overexpression (Fig.5 a). Overexpression of another B-type cyclin gene (CLB3) had no effect on the CLN1 transcription at all (Fig. 5 b), consistent with the results described above, namely that neither the mutations nor the overexpression of the CLB3/CLB4 genes conferred any impact on heat shock-induced G1 arrest. It is noteworthy that both CLN1 and CLN2 transcripts retained a periodic expression pattern throughout the cell cycle in the CLB5 overexpression cells. The finding that CLB5 overexpression stimulated CLN1/CLN2 gene transcription led us to test whether an increase in Clb5 activity will bring about G1/S transition in cln3 cells that had endured heat shock stress. In the absence of heat shock treatment, CLB5 overexpression resulted in slight advancement in cell cycle progression in cln3 cells (Fig.6 a). Heat shock treatment of cln3 cells led to persistent cell cycle arrest at G1, which could be effectively negated by CLB5 overexpression (Fig. 6 a), suggesting that the defect of heat-treated cln3 mutant in carrying out G1/S transition is likely to be derived from insufficient CLN and CLB5 transcriptions. This was ascertained by examining the levels of CLN1, CLN2, and CLB5 transcripts in heat-treated cln3 cells. As shown in Fig.6 b, heat shock treatment strongly prevented the accumulation of CLN1/CLN2/CLB5 transcripts, and no transcripts were detected for these three cyclins until 180 min after release from synchronization. Induction of CLB5 overexpres
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