RESUMO
Cyclin-dependent kinase (CDK) activity initiates the eukaryotic cell division cycle by turning on a suite of gene expression in late G1 phase. In metazoans, CDK-dependent phosphorylation of the retinoblastoma tumor suppressor protein (Rb) alleviates repression of E2F and thereby activates G1/S transcription. However, in yeast, an analogous G1 phase target of CDK activity has remained elusive. Here we show that the cell size regulator Whi5 inhibits G1/S transcription and that this inhibition is relieved by CDK-mediated phosphorylation. Deletion of WHI5 bypasses the requirement for upstream activators of the G1/S transcription factors SBF/MBF and thereby accelerates the G1/S transition. Whi5 is recruited to G1/S promoter elements via its interaction with SBF/MBF in vivo and in vitro. In late G1 phase, CDK-dependent phosphorylation dissociates Whi5 from SBF and drives Whi5 out of the nucleus. Elimination of CDK activity at the end of mitosis allows Whi5 to reenter the nucleus to again repress G1/S transcription. These findings harmonize G1/S control in eukaryotes.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Anticorpos Monoclonais/metabolismo , Núcleo Celular/metabolismo , Tamanho Celular/genética , Cromatina/metabolismo , Cruzamentos Genéticos , Epistasia Genética , Fase G1 , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genes Reguladores , Modelos Biológicos , Fosforilação , Testes de Precipitina , Regiões Promotoras Genéticas , Proteínas/análise , RNA/análise , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Fase S , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Transcrição GênicaRESUMO
The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.