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  • The EMBO Journal (2004) 23, 3133 - 3143
  • doi:10.1038/sj.emboj.7600322

Published online: 15 July 2004

Conformation-specific binding of p31comet antagonizes the function of Mad2 in the spindle checkpoint

Guohong Xia1, Xuelian Luo1,2, Toshiyuki Habu3, Josep Rizo1,2, Tomohiro Matsumoto3 and Hongtao Yu1

  1. Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA
  2. Department of Biochemistry, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA
  3. Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, Japan

Correspondence to:

Hongtao Yu, Department of Pharmacology, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA. Tel.: +1 214 648 9697; Fax: +1 214 648 2971; E-mail: hongtao.yu@utsouthwestern.edu

Received 6 February 2004; Accepted 21 June 2004

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/CCdc20in 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.

  • Keywords:

    • anaphase-promoting complex,
    • Mad2,
    • mitosis,
    • p31comet,
    • spindle checkpoint