A key event in the progression through and exit from mitosis is the ubiquitin-dependent degradation of cyclins and other key proteins by the Anaphase Promoting Complex (ARC; also called the cyclosome).ARC substrates contain degradation motifs such as the Destruction Box and the KEN Box that are required for their ubiquitination and for their binding to the ARC activators, Cdc20 and Cdh1. Both motifs are required for efficient degradation, but either one alone suffices for binding to Cdhlp. We have proposed a pathway for the assembly of the APC-Cdhip-substrate complex in which APC-free Cdhlp first binds a substrate. Engagement of the substrate Destruction Box by Cdh1p stimulates the binding of Cdh1p to the ARC, presumably via a conformation change in Cdhlp. The current studies are aimed at furthering our understanding of APC-mediated protedlysis in budding yeast. In particular, we wish to identify novel ARC substrates, to understand how APC-Cdh1p/Cdc20p-substrate complexes are assembled and disassembled, and to understand how the spindle assembly checkpoint makes use of a KEN box to turn off APC-mediated proteolysis when chromosomes are not properly attached to the mitotic spindle. To achieve these goals, I propose the following Specific Aims: 1) To Identify Novel ARC Substrates. 2) To Assess the Generality of the APC-Cdhip-Substrate Assembly Pathway. 3) To Determine How D-Box Engagement Stimulates Cdh1p Binding to the ARC: 4) To Determine the Roles of Conserved Cdhlp Motifs and of Phosphcrylation in APC-Cdh1p-Substrate Assembly. 5) To Determine Whether Disassembly of the APC-Cdh1p-Substrate Complex is an Active Process. 6) To Determine the Function of the MadSp KEN Box in the Spindle Assembly Checkpoint. Degradation of key proteins is essential for cell division. Understanding this degradation is thus important for understanding normal cell proliferation, and how this process goes awry in disease states, particularly cancers.
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