Genetic Analysis of the Shared Role of CLN3 and BCK2 at the G₁-S Transition in Saccharomyces cerevisiae

Corresponding author: Bruce Futcher, Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724., futcher{at}cshl.org (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The transcription complexes SBF and MBF mediate the G₁-S transition in the cell cycle of Saccharomyces cerevisiae. In late G₁, SBF and MBF induce a burst of transcription in a number of genes, including G₁- and S-phase cyclins. Activation of SBF and MBF depends on the G₁ cyclin Cln3 and a largely uncharacterized protein called Bck2. We show here that the induction of SBF/MBF target genes by Bck2 depends partly, but not wholly, on SBF and MBF. Unlike Cln3, Bck2 is capable of inducing its transcriptional targets in the absence of functional Cdc28. Our results revealed promoter-specific mechanisms of regulation by Cln3, Bck2, SBF, and MBF. We isolated high-copy suppressors of the cln3 bck2 growth defect; all of these had the ability to increase CLN2 expression. One of these suppressors was the negative regulator of meiosis RME1. Rme1 induces CLN2, and we show that it has a haploid-specific role in regulating cell size and pheromone sensitivity. Genetic analysis of the cln3 bck2 defect showed that CLN1, CLN2, and other SBF/MBF target genes have an essential role in addition to the degradation of Sic1.

THE molecular mechanisms that control the cell division cycle are remarkably conserved throughout eukaryotic organisms. In the cell cycle of both mammals and budding yeast, cells commit themselves to completion of a new round of division in late G₁. This event is termed the restriction point in mammalian cells and START in yeast (PARDEE et al. 1978 ; PRINGLE and HARTWELL 1981 ). At the molecular level, this commitment to a new round of the cell cycle is mediated via the carefully timed expression of a group of genes that mediate the G₁-S transition. In the budding yeast Saccharomyces cerevisiae, the transcription complexes SBF and MBF induce the late-G₁-specific expression of the G₁ cyclins CLN1, CLN2, and PCL1; the S-phase cyclins CLB5 and CLB6; a number of genes involved in DNA synthesis (including RNR1, POL1 etc.); and a number of genes involved in cell-wall biosynthesis (SPELLMAN et al. 1998 ; for review see KOCH and NASMYTH 1994 ). SBF consists of Swi6 and the DNA-binding protein Swi4, while MBF consists of Swi6 and the DNA-binding protein Mbp1 (which is related to Swi4). These complexes have distinct DNA-binding properties. The Swi4/6 Cell cycle Box (SCB) element PuNNPyCACGAAAA (NASMYTH 1985 ) is preferentially bound by SBF, whereas the MluI Cell cycle Box (MCB) element ACGCGTNA (JOHNSTON and LOWNDES 1992 ) is favored by MBF. SBF and MBF can, however, act on each other's recognition sequences to some extent (DIRICK et al. 1992 ). A striking example of this "cross-talk" was discovered in the CLN1 promoter, which has MCB-like sites, but is predominantly regulated by SBF (PARTRIDGE et al. 1997 ). Thus, "MCB-mediated regulation" is not synonymous with "MBF-mediated regulation," and "SCB-mediated regulation" is not synonymous with "SBF-mediated regulation." The important role of SBF and MBF in cell cycle progress is illustrated by the fact that a swi4 mbp1 double mutant (which lacks the DNA-binding components of SBF and MBF) is inviable with a predominantly G₁ cell-cycle arrest (KOCH et al. 1993 ). A swi4 swi6 mutant is likewise inviable with a G₁ arrest, probably because Mbp1 has no significant transcription-inducing activity in the absence of Swi6 (KOCH et al. 1993 ). However, swi6 and mbp1 swi6 mutants are viable because Swi4 can induce some transcription, even without Swi6 (NASMYTH and DIRICK 1991 ; KOCH et al. 1993 ).

SBF and MBF are bound to the promoters of their target genes in early G₁ phase, yet they do not induce expression of these genes (HARRINGTON and ANDREWS 1996 ; KOCH et al. 1996 ). Expression of SBF/MBF target genes at START involves at least two other genes, CLN3 and BCK2. CLN3 encodes a G₁ cyclin, which is associated with Cdc28. The Cln3-Cdc28 complex is an important activator of SBF-regulated genes (e.g., CLN1, CLN2, PCL1, PCL2, and HO) and MBF-regulated genes (e.g., CLB5, CLB6, and SWI4; TYERS et al. 1993 ; DIRICK et al. 1995 ; STUART and WITTENBERG 1995 ; SPELLMAN et al. 1998 ). The ability of Cln3 to activate SBF and MBF depends on Swi6 because swi6 is largely epistatic to cln3 (NASMYTH and DIRICK 1991 ; H. WIJNEN and B. FUTCHER, unpublished results). Bck2 is a poorly understood nonessential protein. It was originally found as a high-copy suppressor of an mpk1 (MAP kinase) deletion or a pkc1 (protein kinase C) deletion (LEE et al. 1993 ). The protein has no homologs in the database.

In a cln3 null mutant, the expression of SBF- and MBF-regulated genes is delayed, but there is enough residual SBF/MBF activity to prevent a cell-cycle arrest. Similarly, in a bck2 single mutant, expression of SBF- and MBF-regulated genes is delayed, but not abrogated. Both cln3 and bck2 single mutants have a large-cell phenotype because of a delay at START. The cln3 bck2 double mutant is inviable (EPSTEIN and CROSS 1994 ) or nearly so (DI COMO et al. 1995 ), and it fails to express normal levels of CLN1, CLN2, and CLB5 (DI COMO et al. 1995 ). The phenotype is similar to that of a swi4 mbp1 mutant. Furthermore, the synthetic lethality of both swi4 mbp1 and cln3 bck2 strains can be overcome by overexpression of CLN2. It seems from this that BCK2 has a shared function with CLN3 in activating the expression of CLN1, CLN2, CLB5, and probably other SBF- and MBF-dependent genes (EPSTEIN and CROSS 1994 ; DI COMO et al. 1995 ). It is the timely activation of SBF- and MBF-dependent transcription that implements the G₁-S transition and determines the critical cell size for budding, DNA replication, and spindle pole body duplication. Thus, understanding how SBF and MBF become active is key to understanding the commitment to cell division. Here, we present a more detailed genetic analysis of the SBF/MBF activation function that is shared between CLN3 and BCK2.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, culture conditions, and plasmids:
The S. cerevisiae strains that were used in this study are listed in Table 1. We used standard methods for culture and manipulation of yeast (GUTHRIE and FINK 1991 ). YAP-based media were made by supplementing YEP-based media with filter-sterilized adenine to 0.004% w/v. We used carbon sources to a combined concentration of 2% w/v. Galactose treatment of raffinose-grown cultures was performed by adding galactose to a final concentration of 2% w/v. A list of the plasmids used in the course of this study is given in Table 2.

Northern analysis:
Northern analysis was performed essentially as indicated in TYERS et al. 1992 . Total RNA was isolated, quantitated, run on 1% agarose gels in the presence of 6.7% w/v formaldehyde, transferred to nylon membrane, cross-linked, and probed. The CLN1, CLN3, ACT1, and RNR1 fragments that were used as probes have been described before (TOKIWA et al. 1994 ). We used a 350-bp internal PstI fragment to probe for the PCL1 gene. Northern signals were quantitated using a PhosphorImager and normalized to the ACT1 signal after subtraction of background signals.

Cell size profiles, budding, and FACS analysis:
Analysis of the cell size distribution of yeast strains was done using cultures in mid-log phase. Samples of the cultures were resuspended in 10 ml isoton buffer, briefly sonicated, and immediately analyzed using a Coulter counter model ZM (70-µm aperture) and a Coulter channelyzer model 256. Yeast cultures that were to be compared for their cell size distribution were started at the same time in aliquots of the same batch of media. Cultures were grown to log phase, rediluted at equal densities, and allowed to grow for at least two additional doublings. When cultures reached mid-log phase, as judged by both spectrophotometric analysis and cell count, aliquots were taken for size analysis. The numerical values for cell sizes reported in Table 4 represent the estimated median values of profiles with an approximately symmetric distribution. For comparison of the cell-size profile of different genotypes, we used strains derived from the same genetic background. Budding analysis was performed by scoring a minimum of 200 cells from an aliquot of cells that had been sonicated previously. FACS analysis was performed on yeast cells stained with propidium iodide. After yeast cells had been harvested, washed, sonicated, and fixed overnight in 70% ethanol at 4°, they were resuspended in 50 mM sodium citrate, washed in the same buffer, sonicated, treated with RNAse A (final concentration 0.25 mg/ml) for 1 hr at 50°, and treated with proteinase K (final concentration 1 mg/ml) for an additional hour at 50°. Before analysis, the yeast cells were stained with propidium iodide at a final concentration of 16 µg/ml.

-Factor sensitivity assays:
-Factor sensitivity was assayed as described previously (DI COMO et al. 1995 ). Equal numbers of exponentially growing cells were spotted on plates (YAPD, SCM, or -Leu SCM) containing 0, 0.3, 1, 3, 10, or 30 µM -factor. Whenever -factor sensitivity of different genotypes was compared, strains derived from the same genetic background were used.

High-copy suppression screen:
To find genes acting as quantitative expression determinants of the G₁- and S-phase cyclins (QED genes), we transformed strain YHW204 (cln3 bck2 {pRS313/MET3-CLN2}) with a genomic library made in vector YEp213 (CAMERON et al. 1988 ). We directly plated the transformation mixture on -Leu SCM glucose plates that contained 2 mM methionine. We screened an estimated number of 44,000 transformants that gave rise to 84 potential suppressors. After continued growth in liquid -Leu 2 mM Met SCM glucose media, the putative suppressors were screened for the ability to lose the pRS313/MET3-CLN2 plasmid. In fact, all putative suppressors were shown to be independent from the MET3-CLN2 plasmid. We succesfully isolated plasmids from 76 potential suppressors. The 76 plasmids were divided into 14 groups according to their restriction digest pattern with HindIII or XhoI plus HindIII. Representative plasmids from each of these groups were sequenced to identify the genomic inserts. At the same time, these representatives were retransformed into strain YHW204 to test if their rescuing effect was reproducible. Of the 12 digest groups that represented reproducible high-copy suppressors, 4 had inserts that included the CLN3 gene, another 4 had inserts carrying RME1, 3 more had inserts containing truncations of the CLN2 gene, and the last digest group had inserts that contained BCK2.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A cln3 bck2 double mutant arrests in G₁:
BCK2 has been isolated as a gene required for normal growth in the absence of CLN3 (EPSTEIN and CROSS 1994 ; DI COMO et al. 1995 ). EPSTEIN and CROSS 1994 found that cln3 bck2 spores failed to germinate, whereas DI COMO et al. 1995 found that they could recover some cln3 bck2 progeny that was viable, but displayed a severe growth defect. This discrepancy is perhaps caused by differences between the strain backgrounds used. We made our own cln3 bck2 mutant cells in the context of several synthetic genes that could rescue the severe growth defect (GAL-CLN3, GAL-CLN1, or MET-CLN2). We found that if expression of the rescuing gene was turned off, virtually all cells arrested in G₁. This terminal phenotype is consistent with a shared role for CLN3 and BCK2 in the expression of G₁- and S-phase cyclins, as well as a number of genes required for DNA synthesis.

Dependence of Bck2 function on SBF and MBF:
DI COMO et al. 1995 showed that overexpression of BCK2 could induce a twofold increase in expression from a lacZ reporter driven by four tandem, artificial SCB or MCB elements. This induction was completely dependent upon SWI4 and SWI6 for SCB elements, and upon SWI6 for MCB elements. This suggested that Bck2 could act via SBF and MBF. On the other hand, the same study showed that the presence or absence of SWI4 or SWI6 had little effect on the ability of overexpressed BCK2 to induce expression of several natural SBF/MBF target genes (DI COMO et al. 1995 ), which suggested that neither SBF nor MBF were necessary for BCK2 function. That is, while Bck2 might be able to act via SBF and MBF, it might also be able to act in other ways, possibly through other promoter-bound proteins.

To look more deeply into this situation, we first asked about the functional relevance of overexpressed BCK2 in strains lacking either SWI4 or SWI6. As shown in Figure 1, we found that high-copy BCK2 was capable of reducing cell size to a similar degree in wild-type, swi4, and swi6 mutant cells. In addition, high-copy BCK2 was able to increase -factor resistance to a similar degree in wild-type, swi4, and swi6 mutant cells. (Decreased cell size and increased -factor resistance are both suggestive of a functionally relevant increase in the expression of CLN1 or CLN2.) Thus, by these phenotypic assays, neither SBF nor MBF is required for Bck2 to act.

View larger version (110K): In this window In a new window Download PPT slide   Figure 1. Control of cell size and -factor sensitivity by BCK2 in the absence of SWI4 or SWI6. Isogenic strains W303Va (wt), YHW95 (swi4), and YHW626 (swi6) were transformed with either a high-copy BCK2 plasmid (QED64) or a vector control (YEp213). Transformants were grown to mid-log phase in -Leu SCM glucose media and analyzed for their cell size (A–C). At the same time, an aliquot of 1000 cells was taken from each culture and spotted on YAPD plates containing 0, 3, or 30 µM -factor. The picture displayed in D was taken after 2.5 days of incubation at 30°.

The swi4 and swi6 strains above do contain Mbp1 and Swi6, or Swi4 and Mbp1, respectively, and we wished to see whether these proteins might be important for BCK2 function. Because Bck2 can induce expression of CLN1 and CLN2 in the absence of either SWI4 or SWI6, and because overexpression of CLN1 or CLN2 can rescue the viability of swi4 swi6 and swi4 mbp1 strains (Table 3), we asked whether these strains could also be rescued by overexpression of BCK2. We found that they could not (Table 3), suggesting that the ability of Bck2 to rescue Cln-deficient strains requires at least Swi4, or Mbp1 plus Swi6 (MBF).

We decided to quantitate the effect that overexpression of Bck2 has on the expression of SBF/MBF target genes in a swi4 mbp1 strain. Because of the synthetic lethality of swi4 and mbp1, we used CLN2 expressed from the MET3 promoter to keep the cells alive. Strains were transformed with YCp50, YCp50/GAL-BCK2, or YEp24/GAL-CLN3, and the transformants were grown to log phase in -Ura -Met raffinose medium. Galactose was added to a final concentration of 2%, and samples were taken at various times for Northern analysis. To overcome possible confounding effects of MET3-CLN2 expression, this analysis was repeated with an alternative protocol in which methionine (2 mM final concentration) was added to the cultures 3 hr before galactose treatment to shut off MET3-CLN2; the results obtained were similar in both protocols. We found that in a swi4 strain, overexpression of BCK2 can induce expression of the CLN1, PCL1, and RNR1 genes, in agreement with previously published data (Figure 2; H. WIJNEN and B. FUTCHER, data not shown; DI COMO et al. 1995 ). In the swi4 mbp1 double mutant, Bck2 was a much less potent activator of transcription: compared to results in the swi4 strain, the galactose-induced overexpression of BCK2 in the swi4 mbp1 double mutant gave delayed and reduced induction of PCL1 expression, delayed and reduced induction of CLN1 expression, and failed to give any induction of RNR1 expression (Figure 2; H. WIJNEN and B. FUTCHER, data not shown). It appears, therefore, that in the complete absence of SBF, MBF, and Swi4, Bck2's activity is limited to a partial induction of some of its target genes.

View larger version (16K): In this window In a new window Download PPT slide   Figure 2. Transcriptional induction by Cln3 and Bck2 in the absence of SBF or both SBF and MBF. Strains YHW337 (swi4 CLN2::MET3-CLN2) and YHW603 (swi4 mbp1 CLN2::MET3-CLN2) were transformed with either plasmid pMT40 (GAL-CLN3-YEp24), CB2232 (GAL-BCK2-YCp50), or YCp50. Transformants were grown to log phase in -Ura -Met SCM media with 2% raffinose as carbon source. The cultures were then split in half. One half was treated by adding galactose to a final concentration of 2%, whereas the other half was given methionine to a final concentration of 2 mM, incubated for an additional 3 hr, and then treated with galactose. Samples were taken immediately before, and at 30, 65, and 360 min after addition of galactose. Aliquots were used for cell size, FACS, and Northern analyses. Northern blots for were probed for CLN1, PCL1, RNR1, ACT1, and CLN3. Quantitation is based on the PhosphorImager analysis of the Northern signals. The Northern signals were normalized to the signal for ACT1 after the background had been subtracted. The bars represent the ratio between the normalized signal after 65 min and the normalized signal after 0 min. Only data for the cultures treated with 2 mM methionine are shown. The parallel experiment in -Met media yielded comparable results. A complete data set is available upon request. The light gray bars correspond to the YCp50 transformants, the cross-hatched bars to the GAL-BCK2-YCp50 transformants, and the black bars to the GAL-CLN3-YEp24 transformants.

For comparison, we did similar experiments with the alternative activator, Cln3. In the swi4 strain, Cln3 can still induce the RNR1 and PCL1 promoters, but not the CLN1 promoter, whereas Bck2 can induce all three promoters (Figure 2; H. WIJNEN and B. FUTCHER, data not shown). In the swi4 mbp1 strain, Cln3 cannot induce transcription of any of the three promoters tested, while Bck2 still has some ability to induce CLN1 and PCL1 (Figure 2; H. WIJNEN and B. FUTCHER, data not shown). These data indicate that each of these promoters functions differently.

Bck2, but not Cln3, can function in a Cdc28-independent manner:
Transcriptional induction by SBF and MBF is strongly dependent upon Cdc28 (MARINI and REED 1992 ; KOCH et al. 1996 ). Given the fact that Bck2 function is at least partially dependent on SBF and MBF, we sought to determine whether the activity of Bck2 as a transcriptional activator also depends on CDC28. We monitored the effect of overexpressing BCK2 in the presence of the temperature-sensitive cdc28-4 allele. As illustrated in Figure 3, Cln3 is unable to induce CLN1, PCL1, and RNR1 at the restrictive temperature in this background. Bck2, on the other hand, can still induce expression of all three of these genes under these circumstances. Note that the induction of the CLN1 gene is smaller than that of the PCL1 or RNR1 genes. This may be caused by the relatively high basal level of expression of the CLN1 gene at the restrictive temperature. At least in the case of RNR1, Bck2 can apparently function in a manner that is Cdc28 independent, but still mediated by SBF or MBF (Figure 2; H. WIJNEN and B. FUTCHER, data not shown; Figure 3).

View larger version (97K): In this window In a new window Download PPT slide   Figure 3. Transcriptional induction by Cln3 and Bck2 in a cdc28-4 strain. Strain BF421-3c (cdc28-4) was transformed separately with plasmids YCp50, CB2232 (GAL-BCK2-YCp50), and pMT40 (GAL-CLN3-YEp24). Cultures of each of the resulting transformants were grown to log phase at 24° in -Ura SCM 2% raffinose media. Cultures were then split in half. One half was maintained at 24° and received galactose to a final concentration of 2%, whereas the other half was shifted to 37° and incubated for an additional 6 hr before galactose treatment. Samples for cell size, FACS, and Northern analyses were taken immediately before and at 30 and 65 min after galactose treatment. Northern analyses for CLN1, PCL1, RNR1, ACT1, and CLN3 are shown in A. In some cases, the signal for the endogenous CLN3 message is partially obscured by the GAL-CLN3 signal. Quantitation of the Northern signals is shown in B. The Northern signals were normalized to the signal for ACT1 after the background had been subtracted. The bars represent the ratio between the normalized signal after 65 min and the normalized signal after 0 min. The light gray bars correspond to the YCp50 transformants, the cross-hatched bars to the GAL-BCK2-YCp50 transformants, and the black bars to the GAL-CLN3-YEp24 transformants. Note that in vector control and GAL-CLN3 cells, galactose treatment at the restrictive temperature causes a relative reduction of RNR1 and PCL1 expression and, to a lesser degree, CLN1 expression. The level of induction of these genes by GAL-BCK2 at the restrictive temperature is, therefore, underestimated by this quantitation.

Isolation of high-copy suppressors of the cln3 bck2 synthetic growth defect:
To learn more about the shared function of Cln3 and Bck2, we did a screen for high-copy suppressors of the synthetic lethality of cln3 and bck2. Strain YHW204 (cln3 bck2 {pRS313/MET3-CLN2}) was transformed with a library of yeast genomic fragments in vector YEp213 and plated directly onto -Leu 2 mM Met SCM plates to select for suppression of the cln3 bck2 growth defect. Transformants were retested for rescue after loss of the pRS313/MET3-CLN2 plasmid. Rescuing library plasmids were isolated and grouped according to restriction digest pattern. Representatives of each group were selected for sequence analysis and retransformation of YHW204. We isolated four genes: CLN3 (14 isolates), BCK2 (2 isolates), truncated CLN2 (6 isolates), and RME1 (52 isolates).

High-copy suppression of cln3 bck2 by CLN2-1:
When we sequenced the genomic inserts of the high-copy suppressors, we found six copies of the CLN2 gene, all of which were truncated before the normal stop codon. To determine whether this observation reflected the inability of full-length CLN2 to rescue, we constructed a high-copy, full-length CLN2 and tested it for rescue of strain YHW204. High-copy, full-length CLN2 is indeed not able to rescue strain YHW204 (Table 3). Five of the six rescuing copies of CLN2 that we isolated were truncated at the same HindIII site as the stabilizing CLN2-1 mutation described previously (HADWIGER et al. 1989 ). Considering the fact that expression of wild-type CLN2 from heterologous promoters (MET3, Schizosaccharomyces pombe ADH) also suppresses the cln3 bck2 growth defect (Table 3; DI COMO et al. 1995 ), we infer that it is the stabilizing effect of these CLN2-1 truncations, rather than a qualitative difference between wild-type Cln2 and Cln2-1, that allows rescue. Our sixth high-copy suppressor that contained CLN2 was truncated at amino acid 511 and ends with the heterologous sequence DKKH. This allele of CLN2, which we termed CLN2C, maintains virtually all of the sequences that have been previously implicated in the regulation of Cln2 stability (LANKER et al. 1996 ), yet its rescuing ability mimicks that of stabilizing versions of CLN2. Perhaps the Cln2C protein is stable; alternatively, the terminal sequence KKH may act like the terminal sequence of Cln3 (KKTR) and preferentially target Cln2C to the nucleus (N. EDGINGTON and B. FUTCHER, unpublished results). That is, CLN2C might be a mimic of CLN3. We attribute the inability of wild-type CLN2 to act as a high-copy suppressor to the strong dependence of CLN2 transcription on Cln3 and Bck2. Low-copy MET3-CLN2, but not high-copy CLN2-1, can rescue the synthetic lethality of cln3 bck2 swi6 and swi4 mbp1 strains (Table 3). This result could reflect a more severe defect in transcription from the CLN2 promoter in these strains.

RME1 acts as a high-copy suppressor of cln3 bck2:
The majority of the plasmids isolated as suppressors of cln3 bck2 contained the RME1 gene. RME1 encodes a zinc finger transcriptional regulator that is preferentially expressed in haploid cells (MITCHELL and HERSKOWITZ 1986 ). Apart from its initial identification as a negative regulator of IME1 expression (KASSIR et al. 1988 ; COVITZ and MITCHELL 1993 ), RME1 has, more recently, also been isolated as a high-copy suppressor of the temperature sensitivity of a swi6 swi4^ts strain (TOONE et al. 1995 ). The ability of RME1 to suppress the conditional lethality of swi6 swi4^ts was found to depend on induction of the CLN2 gene. In fact, Rme1 was shown to bind directly to an element in the CLN2 promoter (TOONE et al. 1995 ). To confirm that Rme1's ability to suppress the cln3 bck2 growth defect also depended on induction of the CLN2 gene, we tested the ability of high-copy RME1 to suppress the growth defect of a cln3 bck2 cln2 strain. High-copy RME1 was, indeed, not capable of rescuing cln3 bck2 in the absence of CLN2 (Table 3). We considered the possibility that, vice versa, CLN2-1's ability to act as a high-copy suppressor depended on the presence of a wild-type RME1 gene. High-copy CLN2-1 was, however, capable of rescuing the growth defect of a cln3 bck2 rme1 strain (Table 3), indicating that there was still some expression from the CLN2 promoter in the combined absence of Cln3, Bck2, and Rme1. In light of Rme1's ability to function in the absence of Swi6 (TOONE et al. 1995 ), we postulated that high-copy RME1 might also rescue a cln3 bck2 swi6 strain. This proved indeed to be the case (Table 3). Finally, we found that high-copy RME1 is capable of rescuing even a swi4 mbp1 strain (Table 3), showing that Rme1 and SBF/MBF are completely independent regulators of the CLN2 promoter (Table 3).

Phenotypes associated with rme1:
In spite of the characterization of RME1 function with respect to both repression of IME1 and induction of CLN2, no phenotype has been observed for either deletion or overexpression of RME1 in otherwise wild-type haploid cells. Assuming that haploid rme1 mutants might mimic some of the phenotypes associated with deleting CLN2, we asked whether cell size and -factor sensitivity were affected by changes in RME1 dosage. Haploid cells lacking RME1 have a small but significant increase in cell size (Figure 4A; Table 4), whereas haploid cells with increased RME1 dosage have a significantly smaller cell size (Table 4). In the absence of CLN2, overexpression of RME1 does not noticeably affect cell size regulation, whereas deletion of RME1 still results in a small increase in cell size (Table 4). Deletion of RME1 also led to a larger cell size in yeast strains lacking BCK2, SWI4, or both (Table 4). We could not find an effect of rme1 on cell size in diploid cells (Table 4), as is expected from the fact that RME1 is strongly repressed in MATa/MAT cells (MITCHELL and HERSKOWITZ 1986 ). Yeast strains that lack RME1 display a subtly increased sensitivity to mating pheromone. This effect can be observed by assaying growth on plates that contain various concentrations of -factor (Figure 4B and Figure C). We spotted aliquots of ~1000 cells from various RME1 and rme1 strains on plates with 0, 0.3, 1, 3, 10, or 30 µM -factor. Growth of rme1 and bck2 rme1 strains was generally slower than that of their RME1 counterparts at -factor concentrations of 1, 3, and 10 µM. Disruption of RME1 in a swi4 background resulted in slower growth at 0.3, 1, and 3 µM -factor. These differences in growth were subtle and somewhat variable. We could not easily determine whether disruption of RME1 affects the pheromone sensitivity of bck2 swi4 cells because the triple mutant displays synthetic slow growth on plates (H. WIJNEN and B. FUTCHER, unpublished results). Disruption of RME1 had no effect on the kinetics of pheromone arrest and short-term recovery in liquid culture (H. WIJNEN and B. FUTCHER, unpublished results).

View larger version (66K): In this window In a new window Download PPT slide   Figure 4. Rme1 regulates cell size and pheromone sensitivity in haploid yeast. (A) Mid-log phase cell size profiles of isogenic wild-type (W303Va) and rme1 (YHW329) strains in YAPD media are compared. (B) Strains YHW329 (rme1), YHW369 (bck2 rme1), and YHW375 (swi4 rme1) were transformed with either YCplac111 or pHW264 (YCplac111-RME1). Transformants were grown to log phase in -Leu SCM media. A total of 1000 cells were taken from each culture and spotted on -Leu SCM plates containing various amounts of -factor. The pictures in B were taken after the following incubation times at 30°. All strains on 0 µM -factor, 1 day; rme1 on 10 µM -factor and rme1 swi4 on 3 µM -factor, 3.5 days; bck2 rme1 on 10 µM -factor, 5.5 days. (C) Wild-type (W303Va) and rme1 (YHW329) strains were grown to mid-log phase in SC media and aliquots of 1000 cells were spotted on YAPD plates with the indicated concentration of -factor. The pictures were taken after the following incubation times at 30°. YAPD with 0 µM -factor, 1 day; 0.3 µM -factor, 1.5 days; 1 µM -factor, 2 days; 3 µM -factor, 3.5 days; 10 µM -factor, 5.5 days.

Overexpression of components of SBF/MBF in a cln3 bck2 context:
Because Cln3's function appears to be completely mediated by SBF and MBF, and because Bck2's function is at least partially mediated by Swi4 and Mbp1, we considered the possibility that overexpression of components of SBF and MBF could suppress the cln3 bck2 growth defect. As shown in Table 3, high-copy overexpression of SWI4, but not MBP1 or SWI6, could partially suppress cln3 bck2. The rescuing effect of SWI4 was increased if it was overexpressed from the heterologous S. pombe ADH promoter or if the region of the gene encoding the carboxyl terminus was deleted (SWI4C; Table 3). Versions of Swi4 lacking their carboxy-terminal Swi6-interaction domain have been shown to activate transcription in a Swi6-independent manner (SIDOROVA and BREEDEN 1993 ). Interestingly, increased expression of wild-type Swi4 can result in the accumulation of Swi4 degradation products with carboxy-terminal truncations (SIDOROVA and BREEDEN 1993 ). An additional indication that Swi6 is not required for the rescue of cln3 bck2 by increased expression of Swi4 is provided by the observation that overexpression of SWI4 from the GAL1-10 promoter can rescue a cln3 bck2 swi6 strain (Table 3).

Deletion of SIC1 does not suppress the cln3 bck2 phenotype:
The only nonredundant, essential function of Cln1, Cln2, and Cln3 is to target the cdk inhibitor Sic1 for degradation (SCHNEIDER et al. 1996 ; TYERS 1996 ). Because the lack of CLN1 and CLN2 expression is largely responsible for the cln3 bck2 growth defect, we hypothesized that deletion of SIC1 could potentially alter this phenotype. Therefore, we constructed a cln3::GAL-CLN3 bck2 sic1 strain and monitored its ability to grow in the absence of galactose. The results (Figure 5) show that deletion of SIC1 does not significantly alter the cln3 bck2 growth defect (Figure 5C) and only marginally alters the terminal arrest phenotype of this strain (Figure 5A and Figure B). cln3::GAL-CLN3 bck2 sic1 arrests in raffinose with mostly unbudded cells with a 1-N DNA content. Thus, apart from their role in the degradation of Sic1, Cln1 and Cln2 contribute to at least one other essential function that is required for cell cycle progress, such as budding. The fact that the importance of this function is uncovered in cln3 bck2 cells, but not cln1 cln2 cells, suggests that other genes that are normally regulated by Bck2 and Cln3 also contribute to this.

View larger version (130K): In this window In a new window Download PPT slide   Figure 5. Arrest of cln3::GAL-CLN3 bck2 sic1 in raffinose. A log-phase culture of strain YHW958 in YAP 2% raffinose + 0.03% galactose was washed twice in YAP without a carbon source and then released in YAP 2% raffinose. Starting at the time of release, samples were taken every 2 hr for 12 hr. Samples for the different time points were analyzed for cell-cycle distribution, as measured by FACS analysis (A) or budding (B). In addition, the density (B) and the cell size profile (not shown) of the cultures were monitored to determine the time of arrest. (C) The ability to grow on YAP raffinose was tested for several derivatives of YHW38 (cln3::GAL-CLN3 bck2). (Clockwise from the top) YHW38{YCplac111}, YHW38, YHW958, and YHW38{pHW254}.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

It has become clear from previous studies (EPSTEIN and CROSS 1994 ; DI COMO et al. 1995 ) and our current results that Cln3 and Bck2 share a very important, if not essential, function in mediating the G₁-S transition of the cell cycle. This shared function is exerted by the induction of SBF/MBF target genes. Cln3 acts through SBF and MBF to induce SBF/MBF-regulated genes. Bck2's function is partly dependent upon SBF and MBF. Bck2 can work to some extent, at least on some promoters, even in a swi4 mbp1 double mutant. However, the amount of gene induction is reduced in this situation, and overexpressed BCK2 cannot suppress the lethality of either swi4 mbp1 or swi4 swi6.

What is the role of BCK2 in the cell? The phenotype of both a bck2 single mutant and a cln3 single mutant consists of a large cell size and an increased pheromone sensitivity (NASH et al. 1988 ; DI COMO et al. 1995 ). The large cell size almost certainly reflects an increased critical cell size for START. The cln3 bck2 double mutant is dead or nearly so, arrests in G₁ phase, largely fails to transcribe any tested SBF or MBF target gene, and the lethality is suppressed by expression of CLN2. That is, BCK2 and CLN3 provide two different but redundant methods of activating transcription of the SBF and MBF target genes. Judging by the large-cell phenotypes of the single mutants, the two genes are of similar importance. CLN3 acts as a link between cell growth, cell size, and various environmental conditions to help determine the correct time for START; we presume that BCK2 plays a similar role, but perhaps responds to different kinds of conditions.

BCK2 was originally isolated as a high-copy suppressor of cell lysis defects in mpk1 and pkc1 mutants (LEE et al. 1993 ). Although it is possible that the role of BCK2 in the protein kinase C (PKC) pathway is unrelated to its role as a transcriptional activator at START, we now know that several functions needed for cell-wall synthesis and maintenance are induced by SBF/MBF (SPELLMAN et al. 1998 ) and also induced by the PKC pathway (MAZUR et al. 1995 ; ZHAO et al. 1998 ). For example, the gene FKS1, encoding ß-1,3-glucan synthase, is under the control of SBF/MBF (MAZUR et al. 1995 ; SPELLMAN et al. 1998 ), while its homolog, FKS2, which encodes an alternative copy of ß-1,3-glucan synthase, is under control of the PKC pathway (MAZUR et al. 1995 ; ZHAO et al. 1998 ). Mutations in the PKC pathway cause cell lysis defects at least in part because of the insufficient expression of some cell-wall synthesis genes, and this can be suppressed by BCK2 because it activates expression of compensating activities via SBF and MBF. The effect of disruption or overexpression of BCK2 in strains with a compromised PKC pathway can be mimicked by disruption or overexpression of SWI4 (LEE et al. 1993 ; IGUAL et al. 1996 ). Thus, the isolation of BCK2 as a suppressor of mpk1 and pkc1 is consistent with the role of Bck2 in activating transcription of SBF/MBF targets.

Bck2 and Cln3 act via distinct mechanisms. This is apparent, not only because of their different degrees of dependence upon SWI4, SWI6, and MBP1, but also because of their different dependence on CDC28. Bck2 can function even in a cdc28-4 mutant at the restrictive temperature, whereas Cln3 cannot. Finally, promoter-specific mechanisms exist for both Cln3- and Bck2-mediated induction. Cln3 induces CLN1 in an SBF-dependent manner, PCL1 in a manner that requires either SBF or MBF, and RNR1 in manner that probably depends fully on MBF. On the other hand, Bck2's ability to induce CLN1 and PCL1 is only partly dependent upon SBF and MBF, whereas Bck2's ability to induce RNR1 probably depends fully upon MBF.

What might be the mechanism of Bck2 action? Bck2 is a large, nonabundant, serine-rich protein with no homologs in the database, so we have little clue as to its mechanism of action. Here, we have shown that Bck2 function depends partly, but not completely, on SBF and MBF. We favor a model in which Bck2 requires SBF and MBF, not for sending an activating signal to the promoters of SBF/MBF target genes, but rather for amplifying this signal at the promoters. Bck2 has no obvious DNA-binding domain, but it has been reported to have a potential transcriptional activation domain (LEVINE et al. 1996 ). It will be of interest to test whether Bck2 can function directly at the DNA.

The screen for suppressors of cln3 bck2 lethality was done partly in the hope that it might reveal the mechanism of Cln3 or Bck2 action. For instance, Cln3 or Bck2 might activate some intermediate protein, which in turn might activate SBF/MBF. The gene encoding this hypothetical intermediate protein might then have been found as a high-copy suppressor of cln3 bck2. However, the four suppressors we identified (CLN3, BCK2, CLN2-1, and RME1) do not seem to include any potential new activator of SBF/MBF.

Our characterization of RME1 has shown that it affects both cell size and -factor sensitivity in haploid yeast cells. All known phenotypes of overexpressing RME1 in haploid cells, including its effect on cell size, are dependent on CLN2. The fact that disruption of RME1 still has a small effect on cell size in the absence of CLN2 may be accounted for by its regulation of the CLN1 promoter (TOONE et al. 1995 ). All in all, both the cell-size and pheromone-sensitivity phenotypes associated with RME1 correspond well to Rme1's ability to regulate the expression of CLN2 and, to a lesser extent, CLN1 (TOONE et al. 1995 ). It has been shown recently that the G₁ cyclins are important inhibitors of meiosis (COLOMINA et al. 1999 ). The fact that RME1 acts as an inhibitor of meiosis in cells that are not MATa/MAT (MITCHELL and HERSKOWITZ 1986 ) may be partly because it induces CLN2 expression. That is, when cells are not heterozygous at the mating-type locus, they express RME1, which in turn causes a certain basal level of CLN2 expression regardless of CLN3 status, and this basal level of CLN2 may be important for preventing sporulation in some circumstances.

It has been demonstrated previously that the only nonredundant essential function of the Cln proteins in budding yeast is to target the B-type cyclin kinase inhibitor Sic1 for Cdc34-mediated proteolysis (SCHNEIDER et al. 1996 ; TYERS 1996 ). Deletion of sic1 or overexpression of CLB5 allows growth of a cln1 cln2 cln3 strain (EPSTEIN and CROSS 1994 ; SCHNEIDER et al. 1996 ; TYERS 1996 ), but not a cln3 bck2 strain or a swi4 swi6 strain (H. WIJNEN and B. FUTCHER, unpublished data). In fact, cln3 bck2 sic1 cells arrest in G₁ with a terminal phenotype that strongly resembles that of a cln3 bck2 strain. We conclude from these results that besides their role in Sic1 degradation, Cln1 and Cln2 contribute to at least one additional essential function. We postulate that this additional essential function is provided by CLN1 and CLN2 in conjunction with other SBF/MBF-regulated genes.

We have incorporated most of the conclusions of this study in the model shown in Figure 6. In this study, we have considered three pathways of regulating transcription at START: (1) Cln3 activates SBF and MBF and thereby activates transcription of CLN1, CLN2, PCL1, CLB5, CLB6, and a large number of other genes; (2) Bck2 functions in synergy with SBF and MBF at the promoters of SBF/MBF target genes; and (3) Rme1 functions independently from the other two pathways in activating transcription of CLN2 and possibly CLN1. The combined activity of these three pathways drives cell-cycle progress by inducing budding, DNA replication, and spindle pole body duplication. Apart from their regulation of Sic1 proteolysis, CLN1 and CLN2 share at least one additional essential function with other SBF/MBF target genes.

View larger version (43K): In this window In a new window Download PPT slide   Figure 6. Model for the transcriptional regulation of SBF/MBF target genes. We have described three pathways of regulating transcription at START: (1) Cln3 induces transcription of SBF/MBF target genes by activating SBF and MBF; (2) Bck2 functions in synergy with SBF and MBF at the promoters of SBF/MBF target genes; and (3) Rme1 acts independently from the other two pathways in activating transcription of CLN2 and, to a lesser extent, CLN1. The combined activity of these three pathways drives cell-cycle progress by inducing budding, DNA replication, and spindle pole body duplication. Cln1 and Cln2 drive cell-cycle progress by inducing the degradation of the S-phase inhibitor Sic1. We infer from our genetic analysis that besides their role in Sic1 degradation, CLN1 and CLN2 share at least one additional essential function with other SBF/MBF-regulated genes.

	ACKNOWLEDGMENTS

Martine Lessard provided excellent technical assistance. We thank Fred Cross and Kim Arndt for providing us with yeast strains, and Kim Arndt and Kim Nasmyth for sending us plasmids. This work was supported by grant GM-39978 from the National Institutes of Health.

Manuscript received March 17, 1999; Accepted for publication July 13, 1999.

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