In all eukaryotes, entry into mitosis is triggered by mitotic cyclin-dependent kinases (mitotic CDKs). For cells to leave mitosis and enter the next G1 phase mitotic CDKs need to be inactivated. In budding yeast, the protein phosphatase Cdc14 plays a key role in promoting the inactivation of mitotic CDKs at the end of mitosis. The activity of this phosphatase is controlled by an inhibitory subunit Cfi1/Net1. Cdc14 is sequestered in the nucleolus and kept inactive by Cfi1/Net1 during G1, S phase and early mitosis but is released from its inhibitor during anaphase. The Cdc14 early anaphase release network (FEAR network) and the Mitotic Exit network (MEN) promote Cdc14 release from its inhibitor. The goal of this proposal is to understand how Cdc14 is released from its inhibitor by the FEAR network and the MEN and how Cdc14 returns into the nucleolus after mitotic exit is completed. First, we will, using a molecular biological and biochemical approach, determine whether and how phosphorylation controls the association of Cdc14 with its inhibitor and will identify the protein kinases responsible for phosphorylating these sites. Second, we will, using genetic and cell biological means, identify all FEAR network components and perform a detailed characterization of their function. Finally we will determine how an activator of APC/C-mediated protein degradation, CDH1, promotes the return of Cdc14 into the nucleolus by identifying the FEAR network and MEN components that are targeted for degradation by Cdhl and by determining whether their degradation is required for the re-sequestration of Cdc14 into the nucleolus after the completion' of mitotic exit. As the return of Cdc14 into the nucleolus is only delayed in cells lacking CDH1 we will also conduct a genetic screen to identify negative regulators of mitotic exit and identify the genes among them that promote the return of Cdc14 into the nucleolus in the absence of CDH1.
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