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Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint.

ABSTRACT: 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.

SUBMITTER: Xia G

PROVIDER: S-EPMC514937 | biostudies-other | 2004 Aug

REPOSITORIES: biostudies-other

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p31(comet) inactivates the chemically induced Mad2-dependent spindle assembly checkpoint and leads to resistance to anti-mitotic drugs.

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