Abstract
The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system.
Highlights
The mitotic checkpoint system prevents premature separation of sister chromatids in mitosis and ensures the fidelity of chromosome segregation
To study the role of different subcomplexes of mitotic checkpoint proteins in Mitotic Checkpoint Complex (MCC) assembly and in Anaphase-Promoting Complex/Cyclosome (APC/C) inhibition, the subcomplexes were prepared from recombinant checkpoint proteins
In this study we investigated the roles of subcomplexes of mitotic checkpoint proteins in MCC assembly and in the regulation of APC/C activity by the use of purified subcomplexes
Summary
The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and ensures the fidelity of chromosome segregation. When the mitotic checkpoint is active, Cdc associates with a binding site at the N-terminal region of BubR1 in a process that requires Mad and that leads to MCC assembly [13, 15]. The binding of Cdc to the internal site has been reported to inhibit the APC/C in vitro by the sequestration of Cdc20 [16, 17], but the role of this process in the regulation of APC/C with bound Cdc (APC/CCdc20) is not clear It is not clear whether or not Mad2-containing MCC is the major checkpoint inhibitor of APC/C. The mitotic checkpoint system has important roles to ensure accurate segregation of chromosomes in mitosis This system regulates the activity of the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) by the formation of inhibitors including the Mitotic Checkpoint Complex (MCC). These results reveal important molecular mechanisms in the action of the mitotic checkpoint system
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