RESUMEN
Accurate mitosis depends on a surveillance system called the spindle assembly checkpoint. This checkpoint acts at kinetochores, which attach chromosomes to the dynamic tips of spindle microtubules. When a kinetochore is unattached or improperly attached, the protein kinase Mps1 phosphorylates kinetochore components, catalyzing the generation of a diffusible "wait" signal that delays anaphase and gives the cell time to correct the error. When a kinetochore becomes properly attached, its checkpoint signal is silenced to allow progression into anaphase. Recently, microtubules were found to compete directly against recombinant human Mps1 fragments for binding to the major microtubule-binding kinetochore element Ndc80c, suggesting a direct competition model for silencing the checkpoint signal at properly attached kinetochores. Here, by developing single-particle fluorescence-based assays, we tested whether such direct competition occurs in the context of native kinetochores isolated from yeast. Mps1 levels were not reduced on kinetochore particles bound laterally to the sides of microtubules or on particles tracking processively with disassembling tips. Instead, we found that Mps1 kinase activity was sufficient to promote its release from the isolated kinetochores. Mps1 autophosphorylation, rather than phosphorylation of other kinetochore components, was responsible for this dissociation. Our findings suggest that checkpoint silencing in yeast does not arise from a direct competition between Mps1 and microtubules, and that phosphoregulation of Mps1 may be a critical aspect of the silencing mechanism.
Asunto(s)
Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Humanos , Cinetocoros/química , Microtúbulos/metabolismo , Modelos Biológicos , Fosforilación , Unión Proteica , Saccharomycetales/metabolismoRESUMEN
Chromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.
Asunto(s)
Cromatina/genética , Cinetocoros , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Centrómero/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Microtúbulos/genética , Fosforilación , Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
The incorporation of histone variants into nucleosomes can alter chromatin-based processes. CENP-A is the histone H3 variant found exclusively at centromeres that serves as an epigenetic mark for centromere identity and is required for kinetochore assembly. CENP-A mislocalization to ectopic sites appears to contribute to genomic instability, transcriptional misregulation, and tumorigenesis, so mechanisms exist to ensure its exclusive localization to centromeres. One conserved process is proteolysis, which is mediated by the Psh1 E3 ubiquitin ligase in Saccharomyces cerevisiae (budding yeast). To determine whether there are features of the CENP-A nucleosome that facilitate proteolysis, we performed a genetic screen to identify histone H4 residues that regulate CENP-ACse4 degradation. We found that H4-R36 is a key residue that promotes the interaction between CENP-ACse4 and Psh1 Consistent with this, CENP-ACse4 protein levels are stabilized in H4-R36A mutant cells and CENP-ACse4 is enriched in the euchromatin. We propose that the defects in CENP-ACse4 proteolysis may be related to changes in Psh1 localization, as Psh1 becomes enriched at some 3' intergenic regions in H4-R36A mutant cells. Together, these data reveal a key residue in histone H4 that is important for efficient CENP-ACse4 degradation, likely by facilitating the interaction between Psh1 and CENP-ACse4.