Identification and characterization of novel kinetochore components in the yeast Saccharomyces cerevisiae

To maintain a high fidelity of chromosome transmission during mitosis, the genetic material must be segregated accurately to daughter cells. Failures in this process lead to aneuploidy and may contribute to the development of cancer. The multiprotein kinetochore complex contributes to faithful chromosome segregation by mediating the attachment of a specialized chromosomal region, the centromere, to the mitotic spindle. The goal of this project was to identify and characterize novel components of the budding yeast kinetochore. A subset of kinetochore proteins, referred to as the central kinetochore, provides a link between centromere DNA-binding proteins of the inner kinetochore and microtubule-binding proteins of the outer kinetochore. A combination of chromatin immunoprecipitation, in vivo localization and protein co-immunoprecipitation was used here to establish that yeast Chl4p and Iml3p are central kinetochore proteins, by demonstrating that they localize to the kinetochore and interact with known central kinetochore proteins; kinetochore components needed for these proteins to interact with centromere DNA were also identified. Chl4p is proposed to be an important structural component of the central kinetochore, as it is required for protein-protein interactions of several members of the Ctfl9 central kinetochore complex. These physical interaction dependencies provide insights into the molecular architecture and centromere DNA loading requirements of the kinetochore complex. Proper chromosome segregation is monitored by the spindle assembly checkpoint that delays anaphase if defects in kinetochore-microtubule attachments occur. Amelp is an essential component of the Ctfl9 complex. Here conditional amel mutants were used to probe the molecular localization of Amelp and investigate its function, amel mutants are defective in chromosome segregation, and Amelp likely participates in kinetochoremicrotubule attachment through its interaction with Ctfl9p. amel mutants are also sensitive to overexpression of a microtubule-associated protein, Biklp. In addition, amel mutants break through an initial spindle assembly checkpoint-dependent delay in cell cycle progression; this defect is restored by overexpression of another essential component of the Ctf 19 complex, Okplp. Finally, amel mutants seem to exhibit premature spindle elongation, even in the presence of a microtubule-depolymerizing drug, which frequently occurs in the mother cell and appears to be uncoupled from sister chromatid separation. Together these results indicate that Amelp functions in kinetochore-microtubule attachment and may contribute to the maintenance of the spindle checkpoint signal.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat