Phosphoregulation of yeast kinetochore interactions

The kinetochore is a large macromolecular structure assembled at the centromere which facilitates interactions between chromosomes and spindle microtubules. This process is necessary for the correct segregation of sister chromatids and to avoid aneuploidy and decreased viability for daughter cells. Due to the fact that many cancers are either the cause or result of aneuploidy it is critical that the molecular functioning of the kinetochore be determined; however, the complexity of the kinetochore in higher eukaryotes poses a formidable challenge. To this end the budding yeast Saccharomyces cerevisiae has been used as a model organism. S. cerevisiae offers many unique features which allow the study of complex processes which are often difficult to elucidate in higher eukaryotes: in particular the ability of budding yeast to be subjected to high throughput analysis. The process of segregating chromosomes into daughter cells is highly regulated by mitotic kinases. Ndc80 is regulated by phosphorylation from the yeast Aurora B kinase, Ipl1. Using the yeast two hybrid assay in combination with phosphomimetic analysis, it was shown that Ndc80 has multiple interactions which are altered due to the presence or absence of phosphorylation, indicating the connectivity of kinetochore subunits. Next, a relatively uncharacterized protein, Cnn1, was investigated. Cnn1 is a newly described kinetochore protein whose function has not yet been determined. We showed that the N-terminus of Cnn1 physically interacts with both Spc24 and Spc25 of the Spc24/Spc25 dimer of the Ndc80 complex, and this interaction was abolished by the presence of Mps1 phosphorylation at four residues in the amino terminus of Cnn1. In addition, it was shown that Cnn1 interacts with the globular domain of Spc24, but not the globular domain of Spc25, indicating that the interaction seen between Cnn1 and Spc25 may be bridged by endogenous Spc24. In order to further understand the role of Cnn1 a synthetic dosage lethality screen was performed in which Cnn1 was overexpressed in a background of gene deletions. In this screen we found a significant enrichment for components of the spindle assembly checkpoint. These data, in combination with the two-hybrid data, suggest a role for Cnn1 at the kinetochore involved in the process of sister chromatid segregation and under the control of the mitotic kinase Mps1