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

EMBO J. 2004 Aug 4;23(15):3133-43. doi: 10.1038/sj.emboj.7600322. Epub 2004 Jul 15.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Anaphase-Promoting Complex-Cyclosome
  • Animals
  • Binding Sites
  • Calcium-Binding Proteins / antagonists & inhibitors*
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism*
  • Carrier Proteins / metabolism*
  • Cdc20 Proteins
  • Cell Cycle Proteins / metabolism*
  • Cell Cycle*
  • HeLa Cells
  • Humans
  • Mad2 Proteins
  • Models, Molecular
  • Nuclear Magnetic Resonance, Biomolecular
  • Nuclear Proteins
  • Protein Binding
  • Protein Structure, Tertiary
  • Repressor Proteins
  • Spindle Apparatus / metabolism*
  • Substrate Specificity
  • Ubiquitin-Protein Ligase Complexes / metabolism
  • Xenopus laevis


  • Adaptor Proteins, Signal Transducing
  • Calcium-Binding Proteins
  • Carrier Proteins
  • Cdc20 Proteins
  • Cell Cycle Proteins
  • MAD2L1 protein, human
  • MAD2L1BP protein, human
  • Mad2 Proteins
  • Nuclear Proteins
  • Repressor Proteins
  • CDC20 protein, human
  • Ubiquitin-Protein Ligase Complexes
  • Anaphase-Promoting Complex-Cyclosome