The anaphase promoting complex (APC) is a central regulator of proteolysis in the metaphase-anaphase transition, where it mediates the transfer of ubiquitin from either of two carriers, UbcH5 and UbcH10 to several substrates. Recent evidence suggests that it may be equally important in controlling the transition from G1 to S phase. We propose here to examine how APC controls the rate and sequence of the proteolysis of its substrates from the end of mitosis until the onset of S-phase. UBCH10 autoregulation appears to be central to this process, especially the control of the timing of UBCH10 autoubiquitination and degradation. A striking feature of this process is the noncompetitive inhibition of UbcH10 by other substrates, which we wish to understand. We will also address how the spindle assembly checkpoint controls APC activity, and how cyclin A degradation escapes control by this checkpoint early in mitosis. In particular we propose detailed kinetics and binding experiments aimed at determining the sequence of steps in ubiquitination and protein degradation mediated in G1 by APC. These studies may also inform us of the special properties of polyubiquitination, as a control process, and lead to an understanding of those features important in maintaining fidelity and establishing temporal order. Finally we propose to study two APC substrates with important biological effects: one involved in chromatid cohesion and the other affecting the growth of the brain in a human genetic disease.
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