SIC1 IS UBIQUITINATED IN-VITRO BY A PATHWAY THAT REQUIRES CDC4, CDC34, AND CYCLIN CDK ACTIVITIES/

Traversal from G(1) to S-phase in cycling cells of budding yeast is de pendent on the destruction of the S-phase cyclin/CDK inhibitor SIC1. G enetic data suggest that SIC1 proteolysis is mediated by the ubiquitin pathway and requires the action of CDC34, CDC4, CDC53, SKP1, and CLN/ CDC28. As a first step in defining the functions of the corresponding gene products, we have reconstituted SIC1 multiubiquitination in DEAE- fractionated yeast extract. Multiubiquitination depends on cyclin/CDC2 8 protein kinase and the CDC34 ubiquitin-conjugating enzyme. Ubiquitin chain formation is abrogated in cdc4(ts) mutant extracts and assembly restored by the addition of exogenous CDC4, suggesting a direct role for this protein in SIC1 multiubiquitination. Deletion analysis of SIC 1 indicates that the N-terminal 160 residues are both necessary and su fficient to serve as substrate for CDC34-dependent ubiquitination. The complementary C-terminal segment of SIC1 binds to the S-phase cyclin CLB5, indicating a modular structure for SIC1.