Conformation-specific binding of p31^comet antagonizes the function of Mad2 in the spindle checkpoint

The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase in response to misaligned sister chromatids during mitosis. Upon checkpoint activation, Mad2 binds directly to Cdc20 and inhibits the anaphase-promoting complex or cyclosome (APC/C). Cdc20 binding triggers a dramatic conformational change of Mad2. Consistent with an earlier report, we show herein that depletion of p31^comet (formerly known as Cmt2) by RNA interference in HeLa cells causes a delay in mitotic exit following the removal of nocodazole. Purified recombinant p31^comet protein antagonizes the ability of Mad2 to inhibit APC/C^Cdc20 in vitro and in Xenopus egg extracts. Interestingly, p31^comet binds selectively to the Cdc20-bound conformation of Mad2. Binding of p31^comet to Mad2 does not prevent the interaction between Mad2 and Cdc20 in vitro. During checkpoint inactivation in HeLa cells, p31^comet forms a transient complex with APC/C^Cdc20-bound Mad2. Purified p31^comet enhances the activity of APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2-Cdc20 interaction. Therefore, our results suggest that p31^comet counteracts the function of Mad2 and is required for the silencing of the spindle checkpoint.