Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.
The ability of Candida albicans to switch cellular morphologies is crucial for its ability to cause infection. Because the cell cycle machinery participates in Saccharomyces cerevisiae filamentous growth, we characterized in detail the two C. albicans B-type cyclins, CLB2 and CLB4, to better understand the molecular mechanisms that underlie the C. albicans morphogenic switch. Both Clb2p and Clb4p levels are cell cycle regulated, peaking at G2/M and declining before mitotic exit. On hyphal induction, the accumulation of the G1 cyclin Cln1p was prolonged, whereas the accumulation of both Clb proteins was delayed when compared with yeast form cells, indicating that CLB2 and CLB4 are differentially regulated in the two morphologies and that the dynamics of cyclin appearance differs between yeast and hyphal forms of growth. Clb2p-depleted cells were inviable and arrested with hyper-elongated projections containing two nuclei, suggesting that Clb2p is not required for entry into mitosis. Unlike Clb2p-depleted cells, Clb4p-depleted cells were viable and formed constitutive pseudohyphae. Clb proteins lacking destruction box domains blocked cell cycle progression resulting in the formation of long projections, indicating that both Clb2p and Clb4p must be degraded before mitotic exit. In addition, overexpression of either B-type cyclin reduced the extent of filamentous growth. Taken together, these data indicate that Clb2p and Clb4p regulate C. albicans morphogenesis by negatively regulating polarized growth.
Cyclin-dependent kinases (CDKs) are key regulators of eukaryotic cell cycle progression. The cyclin subunit activates the CDK and also imparts to the complex, at least in some cases, substrate specificity. Saccharomyces cerevisiae, an organism in which the roles of individual cyclins are best studied, contains nine cyclins (three G1 cyclins and six B-type cyclins) capable of activating the main cell cycle CDK, Cdc28. Analysis of the genome of the pathogenic yeast Candida albicans revealed only two sequences corresponding to B-type cyclins, C. albicans Clb2 (CaClb2) and CaClb4. Notably, no homolog of the S. cerevisiae S-phase-specific cyclins, Clb5/Clb6, could be detected. Here, we performed an in vitro analysis of the activity of CaClb2 and CaClb4 and of three G1 cyclins, as well as an analysis of the phenotype of S. cerevisiae cells expressing CaClb2 or CaClb4 instead of Clb5. Remarkably, replacement of CLB5 by CaCLB4 caused rapid diploidization of S. cerevisiae. In addition, both in vivo and in vitro analyses indicate that, in spite of the higher sequence similarity of CaClb2 to Clb5/Clb6, CaClb4 is the functional homolog of Clb5/Clb6. The activity of a CaClb2/CaClb4 cyclin hybrid suggests that the cyclin box domain of CaClb4 carries the functional specificity of the protein. These results have implications for our understanding of the evolution of specificity of the cell cycle cyclins.
DNA replication in budding yeast cells depends on the activation of the Cdc28 kinase (Cdk1 of Saccharomyces cerevisiae) associated with B-type cyclins Clb1 to Clb6. Activation of the kinase depends on proteolysis of the Cdk inhibitor p40SIC1 in late G1, which is mediated by the ubiquitin-conjugating enzyme Cdc34 and two other proteins, Cdc4 and Cdc53. Inactivation of any one of these three proteins prevents p40SIC1 degradation and causes cells to arrest in G1 with active Cln kinases but no Clb-associated Cdc28 kinase activity. Deletion of SIC1 allows these mutants to replicate. p40SIC1 disappears at the G1/S transition and reappears only after nuclear division. Cell cycle-regulated proteolysis seems largely responsible for this pattern, but transcriptional control could also contribute; SIC1 RNA accumulates to high levels as cells exit M phase. To identify additional factors necessary for the inhibition of the Cdk1/Cdc28 kinase in G1, we isolated mutants that can replicate DNA in the absence of Cdc4 function. Mutations in three loci (SIC1, SWI5, and RIC3) were identified. We have shown that high SIC1 transcript levels at late M phase depend on Swi5. Swi5 accumulates in the cytoplasm during S, G2, and M phases of the cell cycle but enters the nuclei at late anaphase. Our data suggest that cell cycle-regulated nuclear accumulation of Swi5 is responsible for the burst of SIC1 transcription at the end of anaphase. This transcriptional control may be important for inactivation of the Clb/Cdk1 kinase in G2/M transition and during the subsequent G1 period.
We derived novel, testable predictions from a
mathematical model of the budding yeast cell cycle. A
key qualitative prediction of bistability was confirmed in a
strain simultaneously lacking cdc14 and G1
cyclins. The model correctly predicted quantitative
dependence of cell size on gene dosage of the G1 cyclin
CLN3, but it incorrectly predicted strong
genetic interactions between G1 cyclins and the anaphase-
promoting complex specificity factor Cdh1. To provide
constraints on model generation, we determined accurate
concentrations for the abundance of all nine
cyclins as well as the inhibitor Sic1 and the catalytic
subunit Cdc28. For many of these we determined abundance
throughout the cell cycle by centrifugal elutriation, in the
presence or absence of Cdh1. In addition, perturbations to
the Clb-kinase oscillator were introduced, and the effects
on cyclin and Sic1 levels were compared between model and
experiment. Reasonable agreement was obtained in many of
these experiments, but significant experimental
discrepancies from the model predictions were also observed.
Thus, the model is a strong but incomplete attempt at a
realistic representation of cell cycle control. Constraints
of the sort developed here will be important in development
of a truly predictive model.
The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.
Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase–cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15, cdc5, cdc14, dbf2, and tem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, and SIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1.
Microarray analysis of gene expression during the yeast division cycle has led to the proposal that a significant number of genes in Saccharomyces cerevisiae are expressed in a cell-cycle-specific manner. Four different methods of synchronization were used for cell-cycle analysis. Randomized data exhibit periodic patterns of lesser strength than the experimental data. Thus the cyclicities in the expression measurements in the four experiments presented do not arise from chance fluctuations or noise in the data. However, when the degree of cyclicity for genes in different experiments are compared, a large degree of non-reproducibility is found. Re-examining the phase timing of peak expression, we find that three of the experiments (those using α-factor, CDC28 and CDC15 synchronization) show consistent patterns of phasing, but the elutriation synchrony results demonstrate a different pattern from the other arrest-release synchronization methods. Specific genes can show a wide range of cyclical behavior between different experiments; a gene with high cyclicity in one experiment can show essentially no cyclicity in another experiment. The elutriation experiment, possibly being the least perturbing of the four synchronization methods, may give the most accurate characterization of the state of gene expression during the normal, unperturbed cell cycle. Under this alternative explanation, the observed cyclicities in the other three experiments are a stress response to synchronization, and may not reproduce in unperturbed cells.
The budding yeast Saccharomyces cerevisiae CLN1, CLN2, and CLN3 genes encode functionally redundant G1 cyclins required for cell cycle initiation. CLN1 and CLN2 mRNAs accumulate periodically throughout the cell cycle, peaking in late G1. We show that cell cycle-dependent fluctuation in CLN2 mRNA is regulated at the level of transcriptional initiation. Mutational analysis of the CLN2 promoter revealed that the major cell cycle-dependent upstream activating sequence (UAS) resides within a 100-bp fragment. This UAS contains three putative SWI4-dependent cell cycle boxes (SCBs) and two putative MluI cell cycle boxes (MCBs). Mutational inactivation of these elements substantially decreased CLN2 promoter activity but failed to eliminate periodic transcription. Similarly, inactivation of SWI4 decreased CLN2 transcription without affecting its periodicity. We have identified a second UAS in the CLN2 upstream region that can promote cell cycle-dependent transcription with kinetics similar to that of the intact CLN2 promoter. Unlike the major CLN2 UAS, this newly identified UAS promotes transcription in cells arrested in G1 by inactivation of cdc28. This novel UAS is both necessary and sufficient for regulated transcription driven by a CLN2 promoter lacking functional SCBs and MCBs. Although this UAS itself contains no SCBs or MCBs, its activity is dependent upon SWI4 function. The characteristics of this novel UAS suggest that it might have a role in initiating CLN2 expression early in G1 to activate the positive feedback loop that drives maximal Cln accumulation.
We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.
Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.
The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2Δ mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.
In Saccharomyces cerevisiae (budding yeast), commitment to cell division in late G1 is promoted by the G1 cyclin Cln3 and its associated cyclin-dependent kinase, Cdc28. We show here that all known aspects of the function of Cln3 in G1 phase, including control of cell size, pheromone sensitivity, cell cycle progress, and transcription, require the protein Swi6. Swi6 is a component of two related transcription factors, SBF and MBF, which are known to regulate many genes at the G1-S transition. The Cln3-Cdc28 complex somehow activates SBF and MBF, but there was no evidence for direct phosphorylation of SBF/MBF by Cln3-Cdc28 or for a stable complex between SBF/MBF and Cln3-Cdc28. The activation also does not depend on the ability of Cln3 to activate transcription when artificially recruited directly to a promoter. The amino terminus and the leucine zipper of Swi6 are important for the ability of Swi6 to respond to Cln3 but are not essential for the basal transcriptional activity of Swi6. Cln3-Cdc28 may activate SBF and MBF indirectly, perhaps by phosphorylating some intermediary protein.
The Saccharomyces cerevisiae cell cycle is arrested in G1 phase by the mating factor pathway. Genetic evidence has suggested that the G1 cyclins Cln1, Cln2, and Cln3 are targets of this pathway whose inhibition results in G1 arrest. Inhibition of Cln1- and Cln2-associated kinase activity by the mating factor pathway acting through Far1 has been described. Here we report that Cln3-associated kinase activity is inhibited by mating factor treatment, with dose response and timing consistent with involvement in cell cycle arrest. No regulation of Cln3-associated kinase was observed in a fus3 kss1 strain deficient in mating factor pathway mitogen-activated protein (MAP) kinases. Inhibition occurs mainly at the level of specific activity of Cln3-Cdc28 complexes. Inhibition of the C-terminally truncated Cln3-1-associated kinase is not observed; such truncations were previously identified genetically as causing resistance to mating factor-induced cell cycle arrest. Regulation of Cln3-associated kinase specific activity by mating factor treatment requires Far1. Overexpression of Far1 restores inhibition of C-terminally truncated Cln3-1-associated kinase activity. G2/M-arrested cells are unable to regulate Cln3-associated kinase, possibly because of cell cycle regulation of Far1 abundance. Inhibition of Cln3-associated kinase activity by the mating factor pathway may allow this pathway to block the earliest step in normal cell cycle initiation, since Cln3 functions as the most upstream G1-acting cyclin, activating transcription of the G1 cyclins CLN1 and CLN2 as well as of the S-phase cyclins CLB5 and CLB6.
The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation.
B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37°C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.
G1 cyclins, in association with a cyclin-dependent kinase (CDK), are universal activators of the transcriptional G1-S machinery during entry into the cell cycle. Regulation of cyclin degradation is crucial for coordinating progression through the cell cycle, but the mechanisms that modulate cyclin stability to control cell cycle entry are still unknown. Here, we show that a lack of phosphate downregulates Cln3 cyclin and leads to G1 arrest in Saccharomyces cerevisiae. The stability of Cln3 protein is diminished in strains with low activity of Pho85, a phosphate-sensing CDK. Cln3 is an in vitro substrate of Pho85, and both proteins interact in vivo. More interestingly, cells that carry a CLN3 allele encoding aspartic acid substitutions at the sites of Pho85 phosphorylation maintain high levels of Cln3 independently of Pho85 activity. Moreover, these cells do not properly arrest in G1 in the absence of phosphate and they die prematurely. Finally, the activity of Pho85 is essential for accumulating Cln3 and for reentering the cell cycle after phosphate refeeding. Taken together, our data indicate that Cln3 is a molecular target of the Pho85 kinase that is required to modulate cell cycle entry in response to environmental changes in nutrient availability.
The gene coding for human cyclin K was isolated as a CPR (cell-cycle progression restoration) gene by virtue of its ability to impart a Far− phenotype to the budding yeast Saccharomyces cerevisiae and to rescue the lethality of a deletion of the G1 cyclin genes CLN1, CLN2, and CLN3. The cyclin K gene encodes a 357-amino-acid protein most closely related to human cyclins C and H, which have been proposed to play a role in regulating basal transcription through their association with and activation of cyclin-dependent kinases (Cdks) that phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II). Murine and Drosophila melanogaster homologs of cyclin K have also been identified. Cyclin K mRNA is ubiquitously expressed in adult mouse and human tissues, but is most abundant in the developing germ cells of the adult testis and ovaries. Cyclin K is associated with potent CTD kinase and Cdk kinase (CAK) activity in vitro and coimmunoprecipitates with the large subunit of RNAP II. Thus, cyclin K represents a new member of the “transcription” cyclin family which may play a dual role in regulating Cdk and RNAP II activity.
Null mutations in three genes encoding cyclin-like proteins (CLN1, CLN2, and CLN3) in Saccharomyces cerevisiae cause cell cycle arrest in G1 (cln arrest). In cln1 cln2 cln3 strains bearing plasmids containing the CLN3 (also called WHI1 or DAF1) coding sequence under the transcriptional control of a galactose-regulated promoter, shift from galactose to glucose medium (shutting off synthesis of CLN3 mRNA) allowed completion of cell cycles in progress but caused arrest in the ensuing unbudded G1 phase. Cell growth was not inhibited in arrested cells. Cell division occurred in glucose medium even if cells were arrested in S phase during the initial 2 h of glucose treatment, suggesting that CLN function may not be required in the cell cycle after S phase. However, when the coding sequence of the hyperactive C-terminal truncation allele CLN3-2 (formerly DAF1-1) was placed under GAL control, cells went through multiple cycles before arresting after a shift from galactose to glucose. These results suggest that the C terminus of the wild-type protein confers functional instability. cln-arrested cells are mating competent. However, cln arrest is distinct from constitutive activation of the mating-factor signalling pathway because cln-arrested cells were dependent on the addition of pheromone both for mating and for induction of an alpha-factor-induced transcript, FUS1, and because MATa/MAT alpha (pheromone-nonresponsive) strains were capable of cln arrest in G1 (although a residual capacity for cell division before arrest was observed in MATa/MAT alpha strains). These results are consistent with a specific CLN requirement for START transit.
In Saccharomyces cerevisiae, the transition from the G1 phase of the mitotic cycle into S phase is controlled by a set of G1 cyclins that regulate the activity of the protein kinase encoded by CDC28. Yeast cells regulate progress through the G1/S boundary in response to nutrients, moving quickly through G1 in glucose medium and more slowly in poorer medium. We have examined connections between glucose and the level of the message encoding Cln3, a G1 cyclin. We found that glucose positively regulates CLN3 mRNA levels through a set of repeated AAGAAAAA (A2GA5) elements within the CLN3 promoter. Mutations in these sequences reduce both transcriptional activation and specific interaction between CLN3 promoter elements and proteins in yeast extracts. Creation of five point mutations, replacing the G’s within these repeats with T’s, in the CLN3 promoter substantially reduces CLN3 expression in glucose medium and inhibits the ability of the cells to maintain a constant size when shifted into glucose.
The yeast Saccharomyces cerevisiae has three G1 cyclin (CLN) genes with overlapping functions. To analyze the functions of the various CLN genes, we examined mutations that result in lethality in conjunction with loss of cln1 and cln2. We have isolated alleles of RAD27/ERC11/YKL510, the yeast homolog of the gene encoding flap endonuclease 1, FEN-1.cln1 cln2 rad27/erc11 cells arrest in S phase; this cell cycle arrest is suppressed by the expression of CLN1 or CLN2 but not by that of CLN3 or the hyperactive CLN3-2. rad27/erc11 mutants are also defective in DNA damage repair, as determined by their increased sensitivity to a DNA-damaging agent, increased mitotic recombination rates, and increased spontaneous mutation rates. Unlike the block in cell cycle progression, these phenotypes are not suppressed by CLN1 or CLN2. CLN1 and CLN2 may activate an RAD27/ERC11-independent pathway specific for DNA synthesis that CLN3 is incapable of activating. Alternatively, CLN1 and CLN2 may be capable of overriding a checkpoint response which otherwise causes cln1 cln2 rad27/erc11 cells to arrest. These results imply that CLN1 and CLN2 have a role in the regulation of DNA replication. Consistent with this, GAL-CLN1 expression in checkpoint-deficient, mec1-1 mutant cells results in both cell death and increased chromosome loss among survivors, suggesting that CLN1 overexpression either activates defective DNA replication or leads to insensitivity to DNA damage.
Fission yeast replication checkpoint kinases Rad3p and Cds1p are essential for maintaining cell viability after transient treatment with hydroxyurea (HU), an agent that blocks DNA replication. Although current studies have focused on the cyclin-dependent protein kinase Cdc2p that is regulated by these checkpoint kinases, other aspects of their functions at the onset of S phase arrest have not been fully understood. In this study, we use genome-wide DNA microarray analyses to show that HU-induced change of expression profiles in synchronized G2 cells occurs specifically at the onset of S phase arrest. Induction of many core environmental stress response genes and repression of ribosomal genes happen during S phase arrest. Significantly, peak expression level of the MluI-like cell cycle box (MCB)-cluster (G1) genes is maintained at the onset of S phase arrest in a Rad3p- and Cds1p-dependent manner. Expression level maintenance of the MCB-cluster is mediated through the accumulation of Rep2p, a putative transcriptional activator of the MBF complex. Conversely, the FKH-cluster (M) genes are repressed during the onset of S phase arrest in a Rad3p-dependent manner. Repression of the FKH-cluster genes is mediated through the decreased levels of one of the putative forkhead transcription factors, Sep1p, but not Fkh2p. Together, our results demonstrate that Rad3p and Cds1p modulate transcriptional response during the onset of S phase arrest.
The Target of Rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is inhibited by the drug rapamycin. In the budding yeast Saccharomyces cerevisiae, translational defects associated with TORC1 inactivation inhibit cell cycle progression at an early stage in G1, but little is known about the possible roles for TORC1 later in the cell cycle. We investigated the rapamycin-hypersensitivity phenotype of cells lacking the S phase cyclin Clb5 (clb5Δ) as a basis for uncovering novel connections between TORC1 and the cell cycle regulatory machinery. Dosage suppression experiments suggested that the clb5Δ rapamycin hypersensitivity reflects a unique Clb5-associated cyclin-dependent kinase (CDK) function that cannot be performed by mitotic cyclins and that also involves motor proteins, particularly the kinesin-like protein Kip3. Synchronized cell experiments revealed rapamycin-induced defects in pre-anaphase spindle assembly and S phase progression that were more severe in clb5Δ than in wild type cells but no apparent activation of Rad53-dependent checkpoint pathways. Some rapamycin-treated cells had aberrant spindle morphologies, but rapamycin did not cause gross defects in the microtubule cytoskeleton. We propose a model in which TORC1 and Clb5/CDK act coordinately to promote both spindle assembly via a pathway involving Kip3 and S phase progression.
rapamycin; Kip3; Clb5; cell cycle; S phase; microtubules
Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.
Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.
► Cyclins gradually raise the active-site specificity of Cdk1 during the cell cycle ► A novel substrate docking motif is specific for G1-cyclin-Cdk1 complexes ► G1 cyclin-Cdk1 shows distinct features of substrate site consensus specificity ► Several G1, G2, and M phase-specific Cdk1 targets exist in the cell