Negishi Takahiro, Veis Jiri, Hollenstein David, Sekiya Mizuho, Ammerer Gustav, Ohya Yoshikazu
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan.
Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.
Mol Cell Biol. 2016 Jan 4;36(6):941-53. doi: 10.1128/MCB.00952-15.
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.
在芽殖酵母酿酒酵母中，细胞壁完整性（CWI）检查点协调细胞壁构建和细胞周期进程。在本研究中，我们表明，S期晚期转录因子Hcm1的调控通过细胞壁完整性检查点使细胞周期停滞。尽管诱导细胞壁完整性检查点时HCM1 mRNA水平未受影响，但蛋白质水平下降。Hcm1的过量表达导致细胞壁完整性检查点失效。我们通过串联质谱分析鉴定了39个Hcm1磷酸化位点，包括26个新位点。突变分析表明，Hcm1在S61、S65和S66位点的磷酸化通过调节其蛋白质水平的适时下降，是细胞壁完整性检查点正常启动所必需的。CWI丝裂原活化蛋白激酶（MAPK）信号通路的过度激活显著降低了Hcm1蛋白质水平，而CWI MAPK Slt2的缺失导致在应激反应中无法降低Hcm1蛋白质水平，这表明磷酸化受CWI MAPK调控。总之，我们认为在应激条件下，细胞壁完整性检查点通过适时磷酸化和降解对Hcm1进行负调控，以适当控制芽殖酵母的增殖。
