Functional redundancy between Cdc20 ubiquitination and p31comet

Abstract

The spindle checkpoint senses unattached kinetochores in a mitotic cell, and inhibits the function of Cdc20, the mitotic activator of the anaphase‐promoting complex or cyclosome (APC/C), thus delaying anaphase onset. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins and produce diffusible APC/C‐inhibitory signals. The mitotic checkpoint complex (MCC) consisting of BubR1 (Mad3 in yeast), Bub3, Mad2, and Cdc20 is a critical checkpoint inhibitor of APC/C. When all kinetochores achieve proper microtubule attachment and are under tension, the spindle checkpoint turns off and MCC disassembles to allow APC/C activation. Several mechanisms have been implicated in MCC disassembly, including p31comet‐and UbcH10‐dependent Cdc20 autoubiquitination and proteasomal degradation. However, the role of Cdc20 autoubiquitination in checkpoint inactivation has remained controversial, because preventing Cdc20 ubiquitination by mutating all lysines in Cdc20 to arginines does not prevent MCC disassembly.In this study, we have further investigated the roles and coordination of p31comet, proteasomal degradation, and Cdc20 ubiquitination in MCC disassembly. We show that both the proteasome and p31comet are required for timely MCC disassembly in HeLa cells by treatment of proteasome inhibitor MG132 or RNA interference (RNAi). Depletion of p31comet further delays MCC disassembly in cells treated with MG132, suggesting that p31comet has a proteasome‐independent role. Consistently, p31comet depletion delays Mad2–Cdc20 dissociation and mitotic exit in cells expressing Cdc20 mutants deficient for autoubiquitination. Taken together, our results reconcile earlier conflicting data, and suggest p31comet and Cdc20 ubiquitination act in somewhat redundant pathways to promote MCC disassembly.