RESUMEN
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.
Asunto(s)
Pared Celular/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Pared Celular/química , Pared Celular/genética , Factores de Transcripción Forkhead/análisis , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Mutación , Fosforilación , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Regulación hacia ArribaRESUMEN
The cell wall integrity checkpoint coordinates cell wall synthesis and mitosis in the budding yeast, Saccharomyces cerevisiae. It has been reported that this checkpoint arrests the cell cycle at G2/M phase with repression of the M phase cyclin Clb2p at the transcriptional level, under perturbation of cell wall synthesis. We demonstrate that an override of this checkpoint with accumulation of CLB2 mRNA is induced when negative CLB2 transcription factors are deleted or when positive CLB2 transcription factors are overproduced in cell wall-defective cells. Our data imply that transcription factors for CLB2 are involved in the cell wall integrity checkpoint system and suggest that there are multiple regulation pathways of the checkpoint.
Asunto(s)
División Celular/fisiología , Pared Celular/metabolismo , Ciclina B/biosíntesis , Fase G2/fisiología , Proteínas de Saccharomyces cerevisiae/biosíntesis , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Pared Celular/genética , Ciclina B/genética , ARN de Hongos/biosíntesis , ARN de Hongos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Checkpoint controls ensure the completion of cell cycle events with high fidelity in the correct order. Here we show the existence of a novel checkpoint that ensures coupling of cell wall synthesis and mitosis. In response to a defect in cell wall synthesis, S. cerevisiae cells arrest the cell-cycle before spindle pole body separation. This arrest results from the regulation of the M-phase cyclin Clb2p at the transcriptional level through the transcription factor Fkh2p. Components of the dynactin complex are required to achieve the G2 arrest whilst keeping cells highly viable. Thus, the dynactin complex has a function in a checkpoint that monitors cell wall synthesis.