Separase (Esp1 in budding yeast) is a conserved protease that is necessary for chromosome segregation. Mice lacking separase function fail to survive, and cell lines in which separase is inactivated show gross chromosome mis-segregation. During mitosis and meiosis, separase cleaves one of the subunits of the cohesin complex that links the two sister chromatids together. This cleavage leads to the dissolution of cohesion, allowing the sister chromatids to be pulled apart by spindle microtubules. Separase is regulated at multiple levels, including phosphorylation, auto-cleavage, and sub-cellular localization. In addition, the activity of separase is regulated by an inhibitor, called securin (Pds1 in budding yeast), which binds to separase and blocks its active site. Evidence from both yeast and higher eukaryotes suggests that securin/Pds1 is not only an inhibitor but that its binding to separase is needed for separase activation. The mechanism of separase activation by Pds1, or by any other protein, is unknown. In this project, we aimed to uncover the mechanisms for separase activation, and in particular we are focusing on proteins and pathways that control separase's nuclear localization. Previous work from our lab and others showed that Pds1 is needed for Esp1's nuclear localization, but it is not known if Esp1 enters the nucleus unaccompanied by Pds1 and then gets sequestered in the nucleus through Pds1 binding, or whether Pds1 shuttles in and out of the nucleus, interacting with Esp1 in the cytoplasm and promoting its nuclear localization. There is also evidence to suggest that other proteins are involved in Esp1's nuclear localization: in mutants lacking Pds1, Esp1 enters the nucleus on schedule, albeit at reduced levels, and in wild type cells Esp1 lingers in the nucleus after Pds1 is removed by protein degradation. To gain a better understanding of how separase is activated and to uncover proteins involved in the nuclear localization of the budding yeast Esp1, we have taken a three-pronged approach. The first was a genetic based approach making use of several different esp1 mutant alleles. Some mutations are from existing esp1 allele and some were created in the lab. The existing mutations fell in different ESP1 regions. It should be noted that apart from the protease domain, the functions of most of the Esp1 protein domains are unknown. We also undertook a high copy suppressor screen, the idea being that by over expressing proteins that normally interact with Esp1 or lead to its activation, we will be able to overcome the defects of the various mutant alleles. Indeed, so far we have isolated multiple suppressors;some of which are common to all esp1 alleles and some are allele specific. Some suppressors act as protein chaperons, some increase the levels of Esp1 and some suppress by a yet unknown mechanism. This approach can lead to the identification of proteins that contribute to Esp1 activation and further our knowledge of separase function in higher eukaryotes. In the second approach to study the regulation of Esp1's nuclear localization we developed tools to detect Esp1 in live cells. We used these tools to determine the protein regions of Esp1 that are necessary for its nuclear and sub-nuclear localization, either in the presence or absence of Pds1. We then began to create esp1 mutants that are defective in sub-nuclear localization, with hope of using these mutants to determine the functional consequences at each localization site. The third approach involved the identification of proteins that interact with Esp1. We used a candidate approach for possible interacting proteins based on published and newly identified genetic interactions. In the future, mass spectrometry will be used to identify proteins that co-purify with Esp1, and those will be studied for their role in Esp1 localization and function. Progress was made on all three aims. However, due to a shortage in personnel, this project was put on hold indefinitely.
