STRUCUTRAL PREDICTION OF THE YEAST KINETOCHORE PROTEIN NDC10

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。Ndc10p和其他60多种蛋白质在出芽酵母的染色体分离中起着着丝点的作用，以确保遗传物质的适当分配。为了避免分离过程中的错误，这一过程受到高度调控，并通过细胞周期检查点的作用与其他有丝分裂事件密切协调。着丝点蛋白的翻译后修饰和其他细胞周期机制是细胞用来控制这些事件的一种机制。在全基因组双杂交筛选中，sumoylation机制的多个组分（Smt3p， Nfi1p和Ubc9p）被鉴定为Ndc10p的双杂交相互作用物。为了鉴定参与sumo化的赖氨酸残基，我们在Ndc10p中产生了赖氨酸到精氨酸的点突变。三个关键赖氨酸残基在突变时影响Ndc10p的sumo化水平。当这些突变结合在一起时，会导致染色体分离错误率增加，并扰乱Ndc10p的定位模式，这表明Ndc10p的聚合化在有丝分裂过程的保真度中起着关键作用。为了理解这些突变的影响，我们建议使用蛋白质结构的结构预测，这些突变可以映射到蛋白质结构上。

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.