|Ph.D Student||Kaisari Sharon|
|Subject||Mechanisms of Formation and Disassembly of the Mitotic|
|Department||Department of Medicine||Supervisor||? 18? Avram Hershko|
|Full Thesis text|
The mitotic checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. When this checkpoint is active, a Mitotic Checkpoint Complex (MCC), composed of Mad2, BubR1, Bub3 and Cdc20, is assembled. MCC inhibits the ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C), whose action is necessary for anaphase initiation.
The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some sub-complexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. In the first part of this study, I used purified sub-complexes of mitotic checkpoint proteins to examine these problems. My results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. I also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in APC/C inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. Furthermore, 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.
When the checkpoint signal is turned off, MCC is disassembled, a process required for exit from checkpoint-arrested state. Different moieties of MCC are disassembled by different ATP-requiring processes. Previous work showed that Mad2 is released from MCC by the joint action of the TRIP13 AAA-ATPase and the Mad2-binding protein p31comet. In the second part of this work, I have isolated from extracts of HeLa cells an ATP-dependent factor that releases Cdc20 from MCC and identified it as CCT/TRiC chaperonin (Chaperonin Containing TCP1 or TCP1-Ring Complex), a complex known to function in protein folding. Bacterially expressed CCT5 chaperonin subunits, which form biologically active homo-oligomers, also promote the disassembly of MCC. CCT chaperonin further binds and disassembles sub-complexes of MCC that lack Mad2. Thus, the combined action of CCT chaperonin with that of TRIP13 ATPase promotes the complete disassembly of MCC, necessary for the inactivation of the mitotic checkpoint.