SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities.
Traversal from G1 to S-phase in cycling cells of budding yeast is dependent on the destruction of the S-phase cyclin/CDK inhibitor SIC1. Genetic 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/CDC28 protein kinase and the CDC34 ubiquitin-conjugating enzyme. Ubiquitin chain formation is abrogated in cdc4ts mutant extracts and assembly restored by the addition of exogenous CDC4, suggesting a direct role for this protein in SIC1 multiubiquitination. Deletion analysis of SIC1 indicates that the N-terminal 160 residues are both necessary and sufficient 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.
Verma
R
R
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Feldman
R M
RM
Deshaies
R J
RJ
D064173:Anaphase-Promoting Complex-Cyclosome; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D019926:Cyclin B; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism*; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D015003:Yeasts
C076558:CLB5 protein, S cerevisiae; D018797:Cell Cycle Proteins; D019926:Cyclin B; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D004791:Enzyme Inhibitors; D044783:F-Box Proteins; D005656:Fungal Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D017489:CDC28 Protein Kinase, S cerevisiae; D008025:Ligases