STRUCUTRAL PREDICTION OF THE YEAST KINETOCHORE PROTEIN NDC10

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ndc10p and more than sixty other proteins function at the budding yeast kinetochore in chromosome segregation to ensure proper partitioning of genetic material. To avoid errors in segregation this process is highly regulated and intimately coordinated with other mitotic events through the action of cell cycle checkpoints. Post-translational modification of kinetochore proteins and other cell cycle machinery is one mechanism used by the cell to control these events. In a genome wide two-hybrid screen, multiple components of the sumoylation machinery (Smt3p, Nfi1p, and Ubc9p) were identified as two-hybrid interactors of Ndc10p. To identify lysine residues participating in sumoylation, we generated lysine to arginine point mutations in Ndc10p. Three key lysine residues were identified that affect Ndc10p sumoylation levels when mutated. When combined these mutations cause increased rates of chromosome segregation errors and perturbs the localization pattern of Ndc10p indicating a critical role for Ndc10p sumoylation in the fidelity of the mitotic process.To understand the effect these mutations have we propose to use structural predictions of protein structure on to which these mutations can be mapped.