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Vol. 13, Issue 5, 1501-1511, May 2002
Department of Molecular Biology and Genetics, Cornell University,
Ithaca, New York 14853
The spindle checkpoint prevents anaphase from occurring until all
chromosomes have attached properly to the mitotic spindle. The
checkpoint components Mad1 and Mad2 associate with unattached kinetochores and are probably involved in triggering the
checkpoint. We now demonstrate that in Xenopus egg
extracts Mad1 and Mad2 form a stable complex, whereas a fraction of
Mad2 molecules is not bound to Mad1. The checkpoint establishment and
maintenance are lost upon titrating out free Mad2 with an excess of
Mad1 or a truncated Mad1 (amino acids 326-718, Mad1C) that contains
the Mad2-binding region. Mad1N (amino acids 1-445) that binds
kinetochores, but not Mad2, reduces Mad1 and Mad2 at
kinetochores and abolishes checkpoint maintenance.
Furthermore, the association between Mad2 and Cdc20, the activator for
the anaphase-promoting complex, is enhanced under
checkpoint-active condition compared with that at metaphase.
Immunodepletion analysis shows that the Mad1-free Mad2 protein is
unable to bind Cdc20, consistent with the model that
kinetochore localization of Mad2 facilitates the formation of Mad2-Cdc20 complex. This study demonstrates that the ratio between
Mad1 and Mad2 is critical for maintaining a pool of Mad1-free Mad2 that
is necessary for the spindle checkpoint. We propose that Mad2 may
become activated and dissociated from Mad1 at kinetochores and is replenished by the pool of Mad1-free Mad2.
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