Defining the Ubiquitin and E2-Enzyme Requirements for APC/C-Mediated Degradation of Cyclin B1
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CitationDimova, Nevena Varbinova. 2012. Defining the Ubiquitin and E2-Enzyme Requirements for APC/C-Mediated Degradation of Cyclin B1. Doctoral dissertation, Harvard University.
AbstractPost-translational modification of proteins with ubiquitin regulates many aspects of cell physiology, including protein degradation. A uniform polyubiquitin chain that is linked through Lys48 has been widely accepted as central for recognition and destruction by the 26S proteasome. Work in more recent years has demonstrated that the repertoire of proteolytic signals may encompass chains of other linkage types, including Lys11-linked ubiquitin chains and short assemblies of mixed linkage. In this dissertation I examine whether catalysis mediated by the Anaphase-Promoting Complex/Cyclosome (APC/C) is dependent on polyubiquitination and whether the proteolytic machinery exerts a requirement for specific ubiquitin linkages to efficiently degrade cyclin B1. In chapter II, I describe a novel method in which Xenopus cell-cycle extracts are made largely dependent on exogenous ubiquitin by inhibiting ubiquitin recycling, allowing us to evaluate the relative contribution of distinct ubiquitin linkages in APC/C-mediated ubiquitination and degradation. Utilizing this approach, in chapter III, I found that the conjugation of single ubiquitin moieties to multiple lysine residues in cyclin promotes efficient degradation of cyclin B1 in mitotic Xenopus extracts. Lysine11-ubiquitin chain-formation becomes essential to proteasomal targeting only when the number of available lysine residues in cyclin B1 is restricted. Analysis in a reconstituted system revealed that APC/C catalyzes multiple monoubiquitination with rapid kinetics and species bearing four or more monoubiquitins on distinct lysines are recognized by ubiquitin receptors. These multiply monoubiquitinated species are rapidly degraded by purified proteasomes. In chapter IV, I examine the role of distinct E2 enzymes in APC/C-dependent proteolysis. I demonstrate that the chain-extending E2 UBE2S and long Lys11-linked ubiquitin assemblies are dispensable for cyclin B1 degradation, but become increasingly important with restriction of the number of ubiquitination sites. Our findings support a model where through attachment of monoubiquitin to multiple lysine residues, and possibly elaboration of some short chains, UBCH10, or possibly members of the UBC4/5 family, cooperate with the APC/C to generate a robust proteolytic signal on cyclin B1.
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