The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase essential for the completion of mitosis in all eukaryotic cells. Substrates are recruited to the APC/C by activator proteins (Cdc20 or Cdh1), but it is not known where substrates are bound during catalysis. We explored this problem by analyzing mutations in the tetratricopeptide repeat (TPR)-containing APC/C subunits. We identified residues in Cdc23 and Cdc27 that are required for APC/C binding to Cdc20 and Cdh1 and for APC/C function in vivo. Mutation of these sites increased the rate of activator dissociation from the APC/C but did not affect reaction processivity, suggesting that the mutations have little effect on substrate dissociation from the active site. Further studies revealed that activator dissociation from the APC/C is inhibited by substrate, and that substrates are not bound solely to activator during catalysis but interact bivalently with an additional binding site on the APC/C core.
|Advisor:||Morgan, David O.|
|Commitee:||Narlikar, Geeta J., Weissman, Jonathan S.|
|School:||University of California, San Francisco|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- California|
|Source:||DAI-B 70/10, Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||Anaphase-promoting complex, Substrate binding, Tetratricopeptide repeats, Ubiquitination|
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