E3 ubiquitin protein ligase complexes that regulate G1-phase in budding yeast

grantor: University of TorontoCommitment to the mitotic cell cycle occurs in late G1-phase, at a transition point called Start in budding yeast. The rate-limiting G1 cyclins (Cln1, 2, 3) trigger passage through Start by activating the cyclin-dependent kinase (CDK) Cdc28. A periodic wave of G1 cyclin-CDK activity at the Start is achieved through the induction of cyclin gene expression coupled with the rapid degradation of cyclin proteins. This thesis describes my research into the role of regulated ubiquitin mediated proteolysis of the G1 cyclins and other cell cycle regulators in progression through Start. Enzymes of the ubiquitin pathway transfer ubiquitin to the target protein in an E1 [right arrow] E2 [right arrow] E3 enzymatic cascade. Multiple rounds of ubiquitination tag the target with a poly-ubiquitin chain, which is recognized by the 26S proteasome, and the protein is rapidly degraded. E3 ubiquitin ligase complexes function to direct the transfer of ubiquitin from the ubiquitin E2 protein to the target substrate. In Chapter 2, I show that the protein Cdc53 is required for the degradation of the G1 cyclin Cln2. I also demonstrate that Cln2 instability is independent of B-type cyclin activity and of the CDK inhibitor Sic1, and describe how On instability is essential for proper G1-phase control. In Chapter 3, I describe the isolation of the Cdc53-interacting proteins, Skp1, and the F-box proteins Cdc4 and Met30. Cdc53 assembles with each F-box protein through Skp1, forming multiple E3 ubiquitin ligases called SCF (Skp1-Cdc53-F-box protein) complexes. I show that the F-box protein component of each SCF complex directs E3 complex specificity 'in vivo'. For example, SCFGrr1 specifically targets Cln2 for degradation, while SCFMet30 is specifically required for regulation of the 'MET' gene network. In Chapter 4, I continue the study of one SCF complex, SCFMet30, and demonstrate that it has an essential function in passage through Start. Conditional 'met30'[Delta] mutants arrest as large, unbudded cells with undetectable levels of 'CLN1' and 'CLN2' mRNA transcripts. I suggest that the G1 arrest phenotype of a 'met30 '[Delta] mutant arises from expression of a possible Start "inhibitor" gene under the regulation of the 'MET' gene transcription factor, Met4, which may interfere with accumulation of critical G1-phase mRNA transcripts.Ph.D