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

Guohong Xia1, Xuelian Luo, Toshiyuki Habu

  • 1Department of Pharmacology, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.

The EMBO Journal
|July 17, 2004
PubMed

Insights

The protein p31(comet) counteracts Mad2

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • The spindle checkpoint is crucial for accurate chromosome segregation during mitosis.
  • Mad2 binding to Cdc20 inhibits the anaphase-promoting complex/cyclosome (APC/C), delaying anaphase onset.
  • The precise mechanisms of spindle checkpoint silencing remain under investigation.

Purpose of the Study:

  • To investigate the role of p31(comet) in mitotic progression and spindle checkpoint regulation.
  • To elucidate the mechanism by which p31(comet) antagonizes Mad2's inhibitory function on APC/C.

Main Methods:

  • RNA interference (RNAi) in HeLa cells to deplete p31(comet).
  • In vitro assays using purified recombinant p31(comet) and Xenopus egg extracts.
  • Analysis of protein complex formation and APC/C activity.

Main Results:

  • Depletion of p31(comet) delayed mitotic exit in HeLa cells.
  • Recombinant p31(comet) antagonized Mad2 inhibition of APC/C(Cdc20) in vitro and in Xenopus extracts.
  • p31(comet) selectively binds to the Cdc20-bound conformation of Mad2 without disrupting Mad2-Cdc20 interaction.
  • p31(comet) enhances APC/C activity during checkpoint inactivation.

Conclusions:

  • p31(comet) acts as a negative regulator of the spindle checkpoint.
  • p31(comet) is essential for timely mitotic exit by counteracting Mad2's inhibitory role.
  • p31(comet) facilitates spindle checkpoint silencing.

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