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 p31comet (formerly known as Cmt2) by RNA interference in HeLa cells causes a delay in mitotic exit following the removal of nocodazole. Purified recombinant p31comet protein antagonizes the ability of Mad2 to inhibit APC/CCdc20 in vitro and in Xenopus egg extracts. Interestingly, p31comet binds selectively to the Cdc20-bound conformation of Mad2. Binding of p31comet to Mad2 does not prevent the interaction between Mad2 and Cdc20 in vitro. During checkpoint inactivation in HeLa cells, p31comet forms a transient complex with APC/CCdc20-bound Mad2. Purified p31comet enhances the activity of APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2-Cdc20 interaction. Therefore, our results suggest that p31comet counteracts the function of Mad2 and is required for the silencing of the spindle checkpoint.
- Anaphase-promoting complex
- Spindle checkpoint
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)