The meiotic role of SIK19p in yeast

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DESCRIPTION (provided by applicant): A fundamental difference between meiotic and mitotic chromosome segregation is that in meiosis I, sister chromatids move as a unit to one pole of the spindle rather than separating as they do in mitosis. Sustained linkage of sister chromatids through meiosis I is accomplished by association of the chromatids at the centromere region and development of kinetochores that allow both chromatids to be moved as a unit to one pole of the spindle. The localization of the meiosis-specific cohesin, Rec8p, to the centromeres is essential for maintenance of sister chromatid cohesion through meiosis I, and Mam1p is necessary for kinetochore development, but the molecular basis for the regulation of cohesion and sister kinetochores in meiosis remains a mystery. Recently, it has been demonstrated that the S1k19 protein of Saccharomyces cerevisiae is essential for the maintenance of sister chromatid association through meiosis I. Two possible roles have been suggested for Slkl9p in meiosis. The first is to promote sister chromatid cohesion at the centromeres, perhaps by protecting Rec8p in this region from degradation at the metaphase I to anaphase I transition. The second is to control sister kinetochore development, such that sister chromatids share a single functional kinetochore throughout meiosis I. This proposal addresses these models through four sets of experiments. First, experiments are proposed that use the chromatin immuno-precipitation method to explore the centromeric localization of Slkl9p and Mamip, and their dependence upon each other for association with the centromere. The role of the CDEII centromere element in meiotic kinetochore function will be explored genetically and through studies of its association with Slkl9p. Second, we will test whether Slk 19p controls sister chromatid centromere cohesion by monitoring its relationship with Rec8p and by using cell biological assays to monitor the establishment and maintenance of sister chromatid centromere association in slkl9 mutants. The third set of experiments is designed to determine the identities of the proteins that interact with Slkl9p in meiosis. A two-hybrid screen will be performed. Affinity chromatography will be used to purify Slkl9p and associated proteins from meiotic cells. A genetic approach will used to identify high copy suppressors of slkl9 partial loss-of-function mutants. Finally, we will explore the regulation of Slkl9p by: 1) Spo 13p 2) possible conjugation to ubiquin or ubiquitin-like proteins, 3) targetting for degradation by Cdh 1p and Amaip and 4) degradation by Espip, the protease that triggers anaphase I by clipping cohesin proteins.