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A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae in bud emergence and identification of a putative upstream regulator

1997; Springer Nature; Volume: 16; Issue: 16 Linguagem: Inglês

10.1093/emboj/16.16.4924

1460-2075

Joseph V. Gray,

Plant-Microbe Interactions and Immunity

Article15 August 1997free access A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae in bud emergence and identification of a putative upstream regulator Joseph V. Gray Corresponding Author Joseph V. Gray Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Robertson Institute, 54 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author Joseph P. Ogas Joseph P. Ogas Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Yoshiaki Kamada Yoshiaki Kamada Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, MD, 21205 USA Search for more papers by this author Marion Stone Marion Stone Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Robertson Institute, 54 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author David E. Levin David E. Levin Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, MD, 21205 USA Search for more papers by this author Ira Herskowitz Ira Herskowitz Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Joseph V. Gray Corresponding Author Joseph V. Gray Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Robertson Institute, 54 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author Joseph P. Ogas Joseph P. Ogas Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Yoshiaki Kamada Yoshiaki Kamada Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, MD, 21205 USA Search for more papers by this author Marion Stone Marion Stone Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Robertson Institute, 54 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author David E. Levin David E. Levin Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, MD, 21205 USA Search for more papers by this author Ira Herskowitz Ira Herskowitz Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Author Information Joseph V. Gray 1,2, Joseph P. Ogas1, Yoshiaki Kamada3, Marion Stone2, David E. Levin3 and Ira Herskowitz1 1Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA 2Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Robertson Institute, 54 Dumbarton Road, Glasgow, G11 6NU UK 3Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, MD, 21205 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:4924-4937https://doi.org/10.1093/emboj/16.16.4924 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The protein kinase C of Saccharomyces cerevisiae, Pkc1, regulates a MAP kinase, Mpk1, whose activity is stimulated at the G1–S transition of the cell cycle and by perturbations to the cell surface, e.g. induced by heat shock. The activity of the Pkc1 pathway is partially dependent on Cdc28 activity. Swi4 activates transcription of many genes at the G1–S transition, including CLN1 and CLN2. We find that swi4 mutants are defective specifically in bud emergence. The growth and budding defects of swi4 mutants are suppressed by overexpression of PKC1. This suppression requires CLN1 and CLN2. Inhibition of the Pkc1 pathway exacerbates the growth and bud emergence defects of swi4 mutants. We find that another dose-dependent suppressor of swi4 mutants, the novel gene HCS77, encodes a putative integral membrane protein. Hcs77 may regulate the Pkc1 pathway; hcs77 mutants exhibit phenotypes like those of mpk1 mutants, are partially suppressed by overexpression of PKC1 and are defective in heat shock induction of Mpk1 activity. We propose that the Pkc1 pathway promotes bud emergence and organized surface growth and is activated by Cdc28–Cln1/Cln2 at the G1–S transition and by Hcs77 upon heat shock. Hcs77 may monitor the state of the cell surface. Introduction The G1 phase of the cell cycle of the budding yeast, Saccharomyces cerevisiae, contains a commitment point, termed START, after which the cell is committed to a full round of cell division (reviewed in Reed, 1992). As the cell passes this point and begins the G1–S transition, at least three processes are coordinately initiated; bud emergence/polarized surface growth, DNA synthesis (S phase) and duplication of the spindle pole body (Pringle and Hartwell, 1981). A key regulator of the G1–S transition is the cyclin-dependent kinase, Cdc28, which associates with its regulatory subunits, including the G1 cyclins Cln1, Cln2 and Cln3, to form active kinase complexes. Mutants defective in any one or pair of these G1 cyclins are viable; mutants lacking all three cannot perform the G1–S transition and arrest in G1 (reviewed in Reed, 1992). It is now clear that the G1 cyclins are not fully redundant but play specialized roles in the wild-type cell, but each can partially compensate for the others when they are absent due to mutation (Dirick et al., 1995; Stuart and Wittenberg, 1995). CLN3 is expressed throughout the cell cycle and Cdc28–Cln3 kinase is required for proper timing of transcription of many genes at the G1–S transition, including CLN1 and CLN2 (Dirick et al., 1995; Stuart and Wittenberg, 1995). CLN1 and CLN2 are expressed only as the cell enters the cell cycle and are implicated in promoting bud emergence and polarized cell growth (Lew and Reed, 1993; Benton et al., 1994). Cell cycle-specific transcription at the G1–S border is mediated by a pair of related transcription factors, one dependent on SWI4, the other on MBP1 (reviewed in Koch and Nasmyth, 1994). Swi4 functions at promoter elements found upstream of many genes, including CLN1, CLN2 and HO (Nasmyth and Dirick, 1991; Ogas et al., 1991; Andrews and Moore, 1992; Nasmyth, 1993), and is the main transcription factor driving expression of CLN1 and CLN2. Swi4 is required for the growth of haploids (S288C strain background) at high temperature and of a/α diploids at all temperatures (Ogas et al., 1991). Mutants lacking SWI4 exhibit reduced expression of CLN1 and CLN2 and appear to arrest in G1 under restrictive conditions. This growth defect is suppressed efficiently by overexpressing CLN1 or CLN2 (Ogas et al., 1991), indicating that the activity of the Cdc28–Cln1 and Cdc28–Cln2 kinase complexes is limiting for the growth of swi4 mutant cells. Mbp1 is a non-essential Swi4-like protein that functions at a related promoter element (McIntosh, 1993) and is responsible for most of the residual expression of CLN1 and CLN2 observed in swi4 mutants (Koch et al., 1993). Swi4 and Mbp1 are activated at the G1–S transition either directly or indirectly by the Cdc28–Cln3 kinase. We know little about the other in vivo substrates of the various Cdc28 kinase complexes, although the activity of the yeast protein kinase C (PKC) isozyme, Pkc1, is partially dependent on Cdc28 activity (Marini et al., 1996; Zarzov et al., 1996). Protein kinase C isozymes (reviewed in Newton, 1995) comprise a family of structurally related serine/threonine protein kinases whose activity is stimulated by diacylglycerol (DAG)/phospholipid and in some cases also by calcium. PKC isozymes are involved in intracellular signaling and have long been implicated in mammalian cell proliferation (Rosengurt et al., 1984; Kaibuchi et al., 1985; Persons et al., 1988), although the mechanism by which they do so is obscure. The PKC1 gene encodes the budding yeast homolog of mammalian PKC (Levin et al., 1990). Purified Pkc1 displays a serine/threonine protein kinase activity in vitro (Antonsson et al., 1994; Watanabe et al., 1994) that is stimulated by phospholipid when the kinase is complexed with the GTP-bound form of the GTPase Rho1 (Kamada et al., 1996). Genetic evidence also supports the model that Pkc1 is regulated by Rho1 in vivo (Nonaka et al., 1995; Kamada et al., 1996). Pkc1p regulates a kinase cascade that includes the MAP kinase Mpk1. Mpk1 activity is stimulated by at least two signals, the G1–S transition of the cell cycle and perturbations to cell surface induced by heat and hypo-osmotic shocks (Davenport et al., 1995; Kamada et al., 1995; Zarzov et al., 1996). Mutants defective in the Pkc1 pathway lyse preferentially as small-budded cells either at high temperature, in the case of the MAP kinase cascade components, or at all temperatures, in the case of pkc1Δ mutants (Lee and Levin, 1992; Levin and Bartlett-Heubusch, 1992; Lee et al., 1993b). These growth defects can be partially suppressed by osmotic stabilization. The primary defect in these mutants is in cell wall growth, organization of cortical actin, or both (Novick and Botstein, 1985; Costigan et al., 1992; Mulholland et al., 1994). A function for the Pkc1 pathway specifically at the G1–S transition has been suggested by a number of observations. First, Mpk1 activity is stimulated at the G1–S boundary, concomitant with bud emergence (Zarzov et al., 1996). Activation appears to be partially dependent on Cdc28 (Marini et al., 1996; Zarzov et al., 1996). Second, mpk1 mutants and certain pkc1 alleles exacerbate the growth defects of certain G1-defective cdc28 alleles (Mozzoni et al., 1993; Marini et al., 1996). Third, Pkc1 pathway mutants can be partially suppressed by overexpression of BCK2, a CLN3-independent activator of G1/S-specific transcription (Lee et al., 1993b; Di Como et al., 1995). Here we find that swi4 mutants are defective primarily in bud emergence and that overexpressing PKC1 suppresses this growth defect by a Cln1- and Cln2-dependent mechanism. Our observations suggest that the Pkc1 pathway functions downstream of Cln1 and Cln2 to control bud morphogenesis. We also describe a novel dose-dependent suppressor of swi4 mutations, HCS77, which encodes a putative transmembrane protein. Mutants lacking HCS77 display phenotypes similar to those of mutants defective in the known components of the Pkc1 pathway. Hcs77 appears to be a novel component of the Pkc1 pathway and is a candidate protein for sensing deformation of the plasma membrane induced by heat shock. Results swi4 mutants are defective in bud emergence Haploid swi4Δ mutants (S288C strain background) are temperature sensitive for growth, whereas a/α swi4Δ. swi4Δ diploids do not proliferate at any temperature. When an a/α swi4ts/swi4Δ strain is shifted to the restrictive temperature, the cells arrest as large, unbudded cells with a single nucleus (mixture of 1N and 2N DNA content) (Ogas et al., 1991). Growth can be restored to these strains by high-copy plasmids carrying one of the G1 cyclin genes, CLN1 or CLN2. It was concluded that swi4 mutants arrest in the G1 phase of the cell cycle under restrictive conditions (Ogas et al., 1991). In the course of analyzing other dose-dependent suppressors of the swi4 defect, we came to re-examine the terminal phenotype of SWI4-defective strains in the S288C background. Haploid swi4Δ cells growing at permissive temperature (25°C) in rich (YEPD) medium displayed a much higher fraction of unbudded cells (65%) than do wild-type cells (42%) (Figure 1A and B). The unbudded cells contained a single nucleus as judged by 4′,6′-diamidino-2-phenylindole (DAPI) staining (data not shown). Fluorescence activated cell sorting (FACS) analysis of these cells revealed, surprisingly, that the majority have 2N DNA content (Figure 1B). Hence a large proportion of swi4Δ cells under permissive conditions are unbudded but have undergone DNA synthesis. In contrast, for wild-type cells, the fraction of unbudded cells matches the fraction of cells with 1N DNA content (Figure 1A). These data demonstrate that, even under permissive conditions, swi4Δ cells are impaired primarily in bud emergence. When the exponentially growing swi4Δ haploid cells in YEPD medium were shifted to high (i.e. restrictive) temperature (37°C), they accumulated with a higher fraction of unbudded cells (79%), again with one nucleus and largely with 2N DNA content (data not shown). We conclude that swi4Δ mutants show a bud emergence defect at both permissive and non-permissive temperatures. Figure 1.Haploid swi4Δ cells are defective in bud emergence but not DNA synthesis at 25°C. Haploid strains JO31-3A (WT) and JO51-2C (swi4Δ) were grown to A600 = 0.4 in liquid YEPD medium at 25°C, fixed in ethanol (70%) and stained with propidium iodide. The FACS profiles of (A) JO31-3A and (B) JO51-2C cells are shown. The percentage of cells with 1N DNA content (± 4%) derived from these plots is shown as a bar (filled) and compared with the percentage of unbudded cells (± 2%) in the same samples determined by microscopy, also shown as a bar (hatched) on the same scale. Download figure Download PowerPoint High-copy plasmids carrying either CLN1 or CLN2 efficiently suppress the growth defects of swi4Δ and swi4ts mutants (Ogas et al., 1991). We found that these plasmids also corrected the bud emergence defect of haploid swi4Δ mutants grown exponentially at low temperature (25°C) on synthetic medium (54% unbudded) relative to the vector control (73% unbudded) (Table I; data not shown). We investigated if overproduction of other Cdc28-associated cyclins could rescue the swi4 growth defect efficiently. We found that CLN3-2 (DAF1-1), which encodes a hyperstable allele of Cln3 (Cross, 1988), or a high-copy plasmid bearing CLB5 are poor suppressors of the swi4 defect (Figure 2A; data not shown). These findings support the contention that the swi4 defect is due primarily to a Cln1- and Cln2-dependent process, most likely bud emergence. Figure 2.Overproduction of Pkc1 but not a Cln3 strain restores growth to SWI4-deficient cells. (A) Haploid strain JO51-2C (swi4Δ) was transformed with plasmids YCp50 (vector), pFC101 (pDAF1-1; DAF1-1 in YCp50), pB3.2A (pSWI4; SWI4 in YCp50) and pJO1 (pCLN1; CLN1 in YEp24). Transformants were selected on Ura drop-out plates at 25°C for 3 days and restruck on fresh drop-out plates which were then incubated at either 25 or 37°C for 3 days. Growth of this strain carrying YCp50 is indistinguishable from the same strain carrying the YEp24 vector (data not shown). (B) High-copy plasmids YEp352 (vector), pDL287 (pPKC1; PKC1 in YEp352), pJO16 (pSWI4; SWI4 in YEp24) or pJO21 (pCLN2; CLN2 in YEp24) were introduced into an a/α swi4ts. swi4Δ strain (JO184) by transformation. The transformants were streaked on Ura drop-out plates and incubated at 25 or at 37°C for 3 days. Note that JO184 carrying YEp24 is indistinguishable in phenotype from JO184 carrying YEp352 (data not shown). Download figure Download PowerPoint Table 1. Overexpression of PKC1 or CLN2 partially corrects the budding defect of haploid swi4Δ cells growing in liquid Ura drop-out medium at 25°C Strain Genotype Plasmid % budded cells JO31-3a a WT YEp352 (vector) 54 (± 3) JO51-2C a swi4Δ YEp352 (vector) 27 (± 4) JO51-2C a swi4Δ pDL289 (YEp352.PKC1) 44 (± 4) JO51-2C a swi4Δ YEp24 (vector) 27 (± 4) JO51-2C a swi4Δ pJO21 (YEp24.CLN2) 46 (± 4) Overproduction of Pkc1 suppresses the growth defect of swi4 mutants As noted above, haploid swi4 mutants are temperature sensitive for growth and exhibit a bud emergence defect; a/α swi4Δ. swi4Δ diploids display the same defect at all temperatures (Ogas et al., 1991; this work). We found that the presence of a high-copy plasmid carrying the PKC1 gene efficiently suppressed the growth defects of both temperature-sensitive and deletion mutants of SWI4 at 37°C on rich (YEPD) and synthetic media (Figure 2B; data not shown) and in both haploid and homozygous a/α diploid mutant strains (Figure 2B; data not shown). The suppression of the swi4 defect was due to overexpression of PKC1 and not an adjacent gene, since a plasmid bearing an epitope-tagged version of PKC1 under control of the GAL1,10 promoter (Watanabe et al., 1994) suppressed the mutant growth defect only when grown on galactose (data not shown). In contrast, expression of Pkc1-K853R, encoding a catalytically inactive version of the kinase (Watanabe et al., 1994), from the GAL1,10 promoter did not suppress the growth defect of haploid swi4Δ mutants (data not shown). We conclude that PKC1 is a dose-dependent suppressor of the swi4 growth defect. The suppression requires Pkc1 kinase activity and is neither allele specific nor cell type specific. Pkc1 is believed to control two downstream branches (Lee and Levin, 1992). One involves a kinase cascade culminating in a MAP kinase (Mpk1). The nature of the second branch is not known (Lee and Levin, 1992). Activation of either or both branches may be required for suppression of the swi4 defect. A high-copy plasmid bearing MPK1 or a plasmid carrying the BCK1-20 allele which encodes a partially activated version of the MEK kinase did not suppress the growth defects of haploid swi4Δ mutants or of diploid a/α swi4ts/swi4Δ mutants (data not shown) at high temperature. However, these constructs are only weakly activating for the pathway (Lee and Levin, 1992; Lee et al., 1993b; D.Levin, unpublished data), and the BCK1-20 allele alone confers a growth defect at high temperature. The results are thus inconclusive. PKC1 overexpression bypasses the essential function of Swi4 High-copy PKC1 could suppress the swi4 mutant growth defect by substituting for or by bypassing the need for Swi4 function. To discriminate between these two possibilities, we determined whether overexpression of PKC1 restored transcription to Swi4-dependent promoters. The HO gene contains multiple SCB elements in its promoter region, and its transcription is absolutely dependent on Swi4 (reviewed in Nasmyth, 1993). We found that overexpression of PKC1 did not restore expression to the HO–lacZ gene in a haploid swi4Δ mutant grown at 25°C (Table II). Furthermore, overexpression of PKC1 in swi4 mutants or in wild-type cells did not boost expression of a synthetic reporter gene driven by multimerized SCB (or MCB) elements (data not shown). Elevated Pkc1 pathway activity thus did not simply replace Swi4 function but rather relieved the growth defect. Table 2. PKC1 overexpression does not restore HO–lacZ expression to a haploid swi4Δ mutant (JO51-2C) growing in liquid Ura drop-out medium at 25°C Plasmid Relative HO–lacZ expression pB3.2A (YCp50.SWI4) 1.00 YEp352 (vector) <0.001 pDL289 (YEp352.PKC1) <0.002 The Pkc1 pathway affects bud emergence We next tested if overexpression of PKC1 mimicked overexpression of CLN1 or CLN2 (see above) in correcting the bud emergence defect of swi4 mutants. We found that swi4Δ strains harboring a high-copy number PKC1 plasmid budded more efficiently (44% budded cells) than the vector control (27% budded cells) during exponential growth in synthetic medium under permissive conditions (25°C) (Table I). This effect mirrors that seen when CLN2 is overexpressed (see above; Table I). The congenic wild-type strain harboring the vector alone budded most efficiently (54% budded cells) (Table I). We conclude that overexpression of PKC1 mimics overexpression of CLN2 in partially restoring efficient budding to haploid swi4Δ cells. We tested if inhibition of the Pkc1 pathway exacerbates the bud emergence defect of swi4 mutants. We first constructed mutant strains in the S288C strain background defective in SWI4 and MPK1. Sporulation of a SWI4/swi4Δ MPK1. mpk1Δ diploid strain yielded swi4Δ and mpk1Δ segregants at the expected frequency on YEPD medium at 25°C, but only one in 30 swi4Δ mpk1Δ spores formed colonies under these conditions. Microscopic examination showed that the swi4Δ mpk1Δ double mutant spores germinated and divided to form microcolonies composed of very large unbudded cells (data not shown). These observations suggested that loss of Mpk1 activity indeed exacerbates the budding defect of swi4 mutants. We next determined whether inhibition of the Pkc1 pathway by expression of a catalytically inactive Pkc1 protein inhibited growth of swi4 mutant cells. Although expression of Pkc1-K853R from the GAL1,10 promoter did not inhibit growth of wild-type cells (Watanabe et al., 1994; Figure 8A), it strongly inhibited the growth of haploid swi4Δ cells at all temperatures (Figure 3A; data not shown). To determine the terminal phenotype of the swi4Δ mutant expressing Pkc1-K853R, transformants were grown to mid-log phase at 30°C in synthetic medium containing raffinose (uninduced condition) and then diluted into galactose-containing medium at the same permissive temperature (induced condition). Cells harboring either the vector control or overexpressing wild-type Pkc1 from the GAL1,10 promoter continued to grow and displayed no obvious phenotype (Figure 3B and C; data not shown). In contrast, cells overexpressing Pkc1-K853R ceased proliferating (Figure 3B), accumulating preferentially as large unbudded cells (78% unbudded compared with 61% unbudded for the vector control) with a single nucleus as judged by DAPI staining (Figure 3B, data not shown). FACS analysis of the arrested cells indicates that most had 2N DNA content (data not shown). We conclude that overexpression of catalytically inactive Pkc1 exacerbates the bud emergence defect of swi4 mutants. Figure 3.Overexpression of inactive Pkc1 inhibits the growth of SWI4-deficient cells. Haploid swi4Δ strain (JO395-1A) was transformed with pBM743 (vector), pDL468 (pGALPKC1) and pDL469 [pGALPKC1(K853R)], and transformants selected on Ura drop-out plates containing glucose at 25°C. (A) The transformants were streaked on Ura drop-out plates containing galactose and incubated at 30°C for 3 days. The same strain transformed with pB3.2A (pSWI4) served as a positive control. (B) Transformants were grown in liquid Ura drop-out medium containing raffinose at 30°C until exponential growth phase. Cells were collected by centrifugation, washed and resuspended in Ura drop-out medium containing galactose and incubated at 30°C. The budding indices of the cultures were determined as a function of time in galactose-containing medium. (C) The morphology of cells containing the vector or expressing inactive Pkc1 after 15 h in galactose-containing medium are shown under Nomarski optics at the same magnification. Download figure Download PowerPoint Pkc1 suppression of the swi4 growth defect requires Cln1 and Cln2 Overproduction of Pkc1, Cln1 or Cln2 suppresses the swi4 growth defect by promoting bud emergence (see above). To explore the possibility that the Pkc1 pathway and these cyclins act in the same pathway, we tested if a subset of the G1 cyclins is required for the ability of PKC1 overexpression to suppress the swi4 growth defect. We utilized a congenic series of haploid strains containing a swi4ts allele in combination with deletions of one or more of the CLN genes and observed that overproduction of Pkc1 could efficiently suppress the growth defects of swi4ts strains deleted for any one of the G1 cyclin genes CLN1, CLN2 or CLN3 (Table III). In contrast, high-copy PKC1 did not suppress the growth defect of the congenic swi4ts cln1Δ cln2Δ triple mutant even at semi-permissive temperature (Table III). We also found that high-copy PKC1 did not suppress the growth defect of a swi4ts mbp1Δ double mutant, although expression of CLN2 could (Figure 4, Table III). Given that the residual transcription of CLN1 and CLN2 in swi4 mutants is dependent on the Mbp1 transcription factor, we conclude that residual expression of the CLN1 and CLN2 genes is absolutely required for PKC1 overexpression to suppress the swi4 defect. We have also found that high-copy PKC1 is an efficient suppressor of the growth defect of swi4ts pcl1Δ pcl2Δ mutants (J.V.Gray, unpublished work), indicating that the major Pho85-associated cyclins are not required for suppression by Pkc1 (Ogas et al., 1991; Espinosa et al., 1994; Measday et al., 1994). The above data indicate that the Pkc1 pathway requires the G1 cyclin proteins Cln1 and Cln2 (associated with Cdc28) to suppress the swi4 defect. Figure 4.Restoration of growth to SWI4-deficient strains at high temperature by PKC1 overexpression requires Mbp1. The haploid swi4ts mbp1Δ strain (JVG284) was transformed with the high-copy plasmids YEp352 (vector), pDL289 (pPKC1), pJO21 (pCLN2) and pB3.2A (pSWI4) as a positive control. The transformants were streaked on Ura drop-out plates and incubated for 3 days at the lowest restrictive temperature (30°C) for this strain. Download figure Download PowerPoint Table 3. Suppression of the growth defect of haploid swi4ts mutants at 37°C by PKC1 overexpression requires a subset of the G1 cyclins Strain Genotype Suppression of growth defect by 2μPKC1 (pDL289) JVG994 a swi4ts +++ JVG287 a swi4ts cln1Δ +++ JVG286 a swi4ts cln2Δ +++ JVG281 a swi4ts cln3Δ ++ JVG290 a swi4ts cln1Δ cln2Δ − JVG284 a swi4ts mbp1Δ − We have determined whether the Pkc1 pathway affects expression of CLN1 and CLN2 (and other Swi4-dependent genes including PCL1, PCL2 and FKS1) and of synthetic reporter constructs bearing lacZ under the control of multimerized SCB and MCB elements. In agreement with Igual et al. (1996) and Madden et al. (1997), we did not find convincing evidence that the Pkc1 pathway directly affected transcription at the G1–S boundary. The expression of some genes was somewhat reduced in mpk1Δ mutants, but only under restrictive conditions, probably as a secondary result of cell lysis (J.V.Gray, unpublished work). Furthermore, we found that the transcription factor Rlm1, which may be a target of Mpk1 kinase (Dodou and Treisman, 1997), was not required for overexpression of PKC1 to suppress the swi4ts growth defect efficiently (J.V.Gray, unpublished work). In addition, we found that overexpression of CLN1 or CLN2 from high-copy plasmids or from heterologous promoters did not suppress the growth defects of mutants defective in the Pkc1 pathway, including pkc1Δ, pkc1ts, bck1Δ and mpk1Δ in either the S288C or EG123 strain backgrounds (data not shown). These negative results are consistent with the view that the Pkc1 pathway does not act upstream of the G1 cyclins. The Pkc1 pathway is activated at the G1–S transition and functions downstream of Cdc28 kinase activity (Marini et al., 1996; Zarzov et al., 1996). Our findings are consistent with these observations and indicate that the Pkc1 pathway promotes bud emergence in a Cdc28–Cln1- and Cdc28–Cln2-dependent process, at least in a swi4 mutant background. To test if the Pkc1 pathway and the CLNs play redundant roles in SWI4+ cells, we examined the consequence of combining mutations in MPK1, CLN1, CLN2 and CLN3. Sporulation of a CLN3. cln3Δ MKP1. mpk1Δ diploid yielded healthy single and double mutant spores on YEPD medium at 25°C. Sporulation of a CLN1. cln1Δ CLN2. cln2Δ MPK1. mpk1Δ diploid strain yielded viable single and double mutant spores on YEPD at 25°C (Figure 5B) except for mpk1Δ cln1Δ cln2Δ segregants, which grew very slowly at 25°C and not at all at higher temperatures (Figure 5A), even when supplemented with sorbitol (10%). We conclude that mpk1Δ mutations are synthetically lethal with cln1Δ cln2Δ mutations, consistent with the view that the Pkc1 pathway and Cln1 and Cln2 function in the same process. These data also indicate that Cln1 and Cln2 also have Mpk1-independent functions and that Mpk1 has Cln1- and Cln2-independent functions (Figure 10). Figure 5.mpk1Δ is synthetic with cln1Δ.cln2Δ. The diploid strains JVG310 a/α MPK1. mpk1Δ CLN1. cln1Δ CLN2. cln2Δ and JVG341 a/α MPK1. mpk1Δ CLN3. cln3Δ were sporulated and the spores germinated on YEPD plates supplemented with sorbitol (10%). The resulting single, double and triple mutant haploids were identified by virtue of genetic markers, and the cells were streaked on YEPD plates and incubated at (A) 30°C and (B) 25°C for 3 days. Download figure Download PowerPoint HCS77 is a novel dose-dependent suppressor of swi4 and encodes a putative cell surface type I transmembrane protein HCS77 was found as a dose-dependent suppressor of the growth defect of a/α swi4ts/swi4Δ strains at high temperature (Ogas et al., 1991). Overexpression of HCS77 suppresses the growth defect of both swi4ts and swi4Δ alleles in both haploids and a/α diploids (Figure 6A; data not shown). In addition, the plasmid suppresses the growth defect of a/α swi4Δ. swi4Δ strains at ambient temperature. HCS77 is thus a dose-dependent, bypass suppressor of the swi4 defect. The suppression is not allele specific, cell type specific or temperature dependent. Figure 6.HCS77 is a dose-dependent suppressor of swi4 mutants which encodes a putative transmembrane protein and is required for growth at high temperature at low but not high osmolarity. (A) a/α swi4ts/swi4Δ cells (JO184) were transformed with the high-copy plasmids YEp24 (vector) or pJO36 (pHCS77; HCS77 in YEp24) and transformants selected on Ura drop-out medium at 25°C. Transformants were streaked on fresh plates and incubated at 25 or 37°C.