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Published August 1997 | Published
Journal Article Open

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.

Additional Information

Copyright © 1997 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0). Submitted April 14, 1997; Accepted May 13, 1997 We thank Drs. Judy Callis, Vince Chau, Bill Dunphy, Bryan Jensen, Doug Kellogg, Randy King, Michael Mendenhall, Matthias Peter, and Michael Weinreich for their gifts of plasmids, strains, antibodies, recombinant baculoviruses, and purified proteins. We also thank Greg Reynard for constructing epitope-tagged SICI expression plasmids, members of the Deshaies lab for stimulating discussions, and Julie Archer, Craig Correll, and Svetlana Lyapina for critical comments on the manuscript. R.J.D. was supported by National Institutes of Health grant GM-52466-02, a Lucille P. Markey Charitable Trust Scholar Award, and the Searle Scholar's Award. R.M.R.F. is a Howard Hughes Medical Institute Predoctoral Fellow.

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