Determinants of Swe1p degradation in Saccharomyces cerevisiae

Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCF(Met30) acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCF(Met30)) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist.     

Clb2 and the APC/CCdh1 regulate Swe1 stability

Swe1/Wee1 regulates mitotic entry by inhibiting Clb2-Cdk1 and its accumulation is involved in stress induced G2 arrest. The APC/C^Cdh1 substrates Cdc5, Clb2 and Hsl1 regulate Swe1 degradation. We observed that clb2Dcdh1D double mutant S. cerevisiae does not express any detectable levels of Swe1, presumably due to its constitutive degradation. This effect of Cdh1 inactivation is due to stabilization of Cdc5 and Hsl1, as expression of the non-degradable Cdc5^T29A in clb2D cells prevented Swe1 accumulation. Strikingly, expression of non-degradable Hsl1^mdb/mkb prevented Swe1 accumulation even in wild type Clb2 cells. Interestingly Swe1 accumulation could be reconstituted in all these mutants by eliciting a replication fork stress with hydroxyurea. Cells expressing the Clb2^ME mutant, that cannot bind Swe1, behaved like clb2D cells, and failed to accumulate Swe1 in the absence of Cdh1 or the presence of Cdc5^T29A. This suggests that for Swe1 to accumulate it must interact with Clb2. We further show that in the absence of Clb2, Hsl1 is no longer essential for Swe1 degradation. We hypothesize that Clb2-Cdk1 protects Swe1 from premature degradation until its Hsl1 mediated de-protection, which enables its Cdc5 mediated degradation. Swe1 levels are thus regulated by monitoring the levels of three major mitotic regulators.     

Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p regulation and Bfa1p/Bub2p-independent cytokinesis

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G(2)/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Delta defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.     

Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae

Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes. Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation. We have investigated the basis for the developmental stage-specific differences in the function of these two proteins. Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane. In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p. Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells. Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation. Additionally, the N terminus of Spo20p is inhibitory in vegetative cells. Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus. Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells.     

Identification of novel activation mechanisms for FLO11 regulation in Saccharomyces cerevisiae

Adhesins play a central role in the cellular response of eukaryotic microorganisms to their host environment. In pathogens such as Candida spp. and other fungi, adhesins are responsible for adherence to mammalian tissues, and in Saccharomyces spp. yeasts also confer adherence to solid surfaces and to other yeast cells. The analysis of FLO11, the main adhesin identified in Saccharomyces cerevisiae, has revealed complex mechanisms, involving both genetic and epigenetic regulation, governing the expression of this critical gene. We designed a genomewide screen to identify new regulators of this pivotal adhesin in budding yeasts. We took advantage of a specific FLO11 allele that confers very high levels of FLO11 expression to wild "flor" strains of S. cerevisiae. We screened for mutants that abrogated the increased FLO11 expression of this allele using the loss of the characteristic fluffy-colony phenotype and a reporter plasmid containing GFP controlled by the same FLO11 promoter. Using this approach, we isolated several genes whose function was essential to maintain the expression of FLO11. In addition to previously characterized activators, we identified a number of novel FLO11 activators, which reveal the pH response pathway and chromatin-remodeling complexes as central elements involved in FLO11 activation.     

APC/CCdh1 specific degradation of Hsl1 and Clb2 is required for proper stress responses of S. cerevisiae

Cdh1 activates the Anaphase Promoting Complex/Cyclosome (APC/CCdh1) throughout G1 to degrade key cell cycle proteins. Cdh1 is not essential for cell proliferation, in spite of the fact that overexpression of some its degradation substrates is highly toxic. We report here that cdh1Δ cells are sensitive to stresses that activate the CWI (Cell Wall Integrity) and Hog1 MAP kinase pathways. Stresses did not activate APC/CCdh1 and cellular sensitivity was thus clearly due to constitutively elevated substrate levels. To explore the contribution of stabilization of individual APC/CCdh1 substrates to stress sensitivity, we generated cell lines expressing stabilized substrate mutants under their endogenous promoters. Cells expressing stabilized Hsl1 were sensitive to caffeine and failed to activate the Slt2 pathway. Cells expressing partially stable Clb2 were particularly sensitive to different stresses, possibly due to reduced Sic1 levels. Cells expressing stabilized Cdc5 were much less stress sensitive. Interestingly sensitivity of cdh1Δ cells does not seem to be restricted to G1 but is manifested also during S and G2 when the APC/CCdh1 is inactive anyway. We thus hypothesize that a role of G1 specific APC/CCdh1 activity is to reset substrate levels to enables appropriate regulation of substrate accumulation in the subsequent phases of the cell cycle.     

ESF1 is required for 18S rRNA synthesis in Saccharomyces cerevisiae

We report that Esf1p (Ydr365cp), an essential, evolutionarily conserved nucleolar protein, is required for the biogenesis of 18S rRNA in Saccharomyces cerevisiae. Depletion of Esf1p resulted in delayed processing of 35S precursor and a striking loss of 18S rRNA. Esf1p physically associated with ribosomal proteins and proteins involved in 18S rRNA biogenesis. Consistent with its role in 18S rRNA biogenesis, Esf1p also physically associated with U3 and U14 snoRNAs, but did not appear to be a core component of the SSU processome. These data indicate that Esf1p plays a direct role in early pre-rRNA processing.     

RSC1 and RSC2 are required for expression of mid-late sporulation-specific genes in Saccharomyces cerevisiae

Rsc1 and Rsc2 are alternative bromodomain-containing subunits of the ATP-dependent RSC chromatin remodeling complex in Saccharomyces cerevisiae. Smk1 is a sporulation-specific mitogen-activated protein kinase homolog that is required for the postmeiotic events of spore formation. In this study we show that RSC1 and RSC2 are haploinsufficient for spore formation in a smk1 hypomorph. Moreover, diploids lacking Rsc1 or Rsc2 show a subset of smk1-like phenotypes. High-copy-number RSC1 plasmids do not suppress rsc2-Delta/rsc2-Delta sporulation defects, and high-copy-number RSC2 plasmids do not suppress rsc1-Delta/rsc1-Delta sporulation defects. Mid-late sporulation-specific genes, which are normally expressed while key steps in spore assembly occur and which include genes that are required for spore wall formation, are not expressed in cells lacking Rsc1 or Rsc2. We speculate that the combined action of Rsc1 and Rsc2 at mid-late promoters is specifically required for the proper expression of this uniquely timed set of genes. Our data suggest that Smk1 and Rsc1/2 define parallel pathways that converge to provide signaling information and the expression of gene products, respectively, that are required for spore morphogenesis.     

Identification of a calcineurin-independent pathway required for sodium ion stress response in Saccharomyces cerevisiae.

The calcium-dependent protein phosphatase calcineurin plays an essential role in ion homeostasis in yeast. In this study, we identify a parallel ion stress response pathway that is independent of the calcineurin signaling pathway. Cells with null alleles in both STD1 and its homologue, MTH1, manifest numerous phenotypes observed in calcineurin mutants, including sodium, lithium, manganese, and hydroxyl ion sensitivity, as well as alpha factor toxicity. Furthermore, increased gene dosage of STD1 suppresses the ion stress phenotypes in calcineurin mutants and confers halotolerance in wild-type cells. However, Std1p functions in a calcineurin-independent ion stress response pathway, since a std1 mth1 mutant is FK506 sensitive under conditions of ion stress. Mutations in other genes known to regulate gene expression in response to changes in glucose concentration, including SNF3, RGT2, and SNF5, also affect cell growth under ion stress conditions. Gene expression studies indicate that the regulation of HAL1 and PMR2 expression is affected by STD1 gene dosage. Taken together, our data demonstrate that response to ion stress requires the participation of both calcineurin-dependent and -independent pathways.