title: The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint.
abstract: The cell wall integrity (CWI) checkpoint in the budding yeast Saccharomyces cerevisiae coordinates cell wall construction and cell cycle progression. In this study, we showed that the regulation of Hcm1, a late-S-phase transcription factor, arrests the cell cycle via the cell wall integrity checkpoint. Although the HCM1 mRNA level remained unaffected when the cell wall integrity checkpoint was induced, the protein level decreased. The overproduction of Hcm1 resulted in the failure of the cell wall integrity checkpoint. We identified 39 Hcm1 phosphorylation sites, including 26 novel sites, by tandem mass spectrometry analysis. A mutational analysis revealed that phosphorylation of Hcm1 at S61, S65, and S66 is required for the proper onset of the cell wall integrity checkpoint by regulating the timely decrease in its protein level. Hyperactivation of the CWI mitogen-activated protein kinase (MAPK) signaling pathway significantly reduced the Hcm1 protein level, and the deletion of CWI MAPK Slt2 resulted in a failure to decrease Hcm1 protein levels in response to stress, suggesting that phosphorylation is regulated by CWI MAPK. In conclusion, we suggest that Hcm1 is regulated negatively by the cell wall integrity checkpoint through timely phosphorylation and degradation under stress to properly control budding yeast proliferation.
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Negishi
authors.affiliation: Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
authors.forename: Jiri
authors.initials: J
authors.lastname: Veis
authors.affiliation: Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
authors.forename: David
authors.initials: D
authors.lastname: Hollenstein
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan.
authors.forename: Mizuho
authors.initials: M
authors.lastname: Sekiya
authors.affiliation: Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria Christian Doppler Laboratory for Proteome Analysis, University of Vienna, Vienna, Austria.
authors.forename: Gustav
authors.initials: G
authors.lastname: Ammerer
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan ohya@k.u-tokyo.ac.jp.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Ohya
mesh_terms: D000595:Amino Acid Sequence; D002473:Cell Wall / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015854:Up-Regulation
chemical_list: D051858:Forkhead Transcription Factors; C079874:HCM1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020928:Mitogen-Activated Protein Kinases