Molecular insights into the regulation of kinetochore assembly by post-translational modifications

Eukaryotic cells require the correct segregation of chromosomes during mitosis in order to propagate. This involves biorientation of replicated sister chromatids by their attachment to the mitotic spindle. The molecular link between the centromeric regions of chromatids to the mitotic spindle is mediated by a multi-protein complex called the kinetochore. The yeast inner constitutive centromere associated network (CCAN) contains multiple subunits which must assemble on the correct section of DNA at the right time to allow progression through the cell cycle. Little is known about how centromeric replication is coupled with the assembly or propagation of kinetochores on both sister centromeres. There is accumulating evidence that post-translational modifications such as phosphorylation and ubiquitination regulate crucial aspects of kinetochore assembly. It is not well understood, however, which kinases contribute to kinetochore assembly and how phosphorylation is coordinated with additional posttranslational modifications. For example, previous studies have implicated the multisubunit Ubiquitin ligase SCF in the regulation of inner kinetochore complexes including the CCAN subcomplex COMA and the centromeric Cse4 nucleosome. How substrate recognition and ubiquitination is achieved in the context of multi-protein kinetochore complexes, and what specific aspect of kinetochore assembly is regulated by the UbiquitinProteasome system, however, is still unresolved. To gain a better understanding into how specific recognition of some kinetochore substrates is achieved, and how this relates to the process of kinetochore assembly, it is necessary to reconstitute these reactions in vitro with purified components. Here, the E3 ubiquitin ligase complex SCFCdc4 along with the E1 enzyme Uba1 and the E2 enzyme Cdc34 were reconstituted in vitro. Assays were established that faithfully recapitulate the phosphorylation-dependent ubiquitination of the model substrate Sic1 in vitro. Applying equivalent reaction conditions to the CCAN components Ame1-Okp1 or COMA failed to lead to efficient ubiquitination due to a lack of Cdc4 binding. This suggests that CDK phosphorylation of COMA alone is insufficient to convert it into an SCFCdc4 substrate and that other regulatory mechanisms may exist. To reveal such modes of regulation, genetic and cell biological approaches were used to investigate the regulation of the essential COMA subunits Ame1 and Okp1 in yeast cells. A systematic series of Okp1 truncation mutants showed that segments of the unstructured amino-terminus of Okp1 are required for cell cycle-dependent phosphorylation and normal growth. The polo-like kinase Cdc5 was identified as a candidate regulator of Ame1-Okp1. Temperature sensitive cdc5 mutants were found to influence Okp1 protein levels and stability, resembling the effects of Okp1 truncation mutants. In addition, Cdc5 phosphorylates Ame1-Okp1 in vitro and a variety of biochemical techniques were used to demonstrate that this activity is aided by Polo-box domain (PBD) iii dependent Cdc5 recruitment to Ame1-Okp1. Taken together, these results implicate Cdc5 as an important regulator of Ame1-Okp1 during kinetochore assembly.