An Essential Domain within Cdc34p Is Required for Binding to a Complex Containing Cdc4p and Cdc53p in Saccharomyces cerevisiae*
- From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and the Walther Oncology Center, Indianapolis, Indiana, 46202-5122
Abstract
The CDC34 gene of the yeastSaccharomyces cerevisiae encodes a ubiquitin-conjugating protein that transfers ubiquitin onto substrates to signal rapid degradation via the proteasome. Cdc34p has been implicated in signaling the destruction of a variety of substrates including the cyclin-dependent kinase inhibitor, Sic1p, which must be degraded for cells to enter S-phase. Mutants lacking CDC34activity fail to degrade Sic1p and fail to enter S-phase, a phenotype that is also shared with cells lacking CDC4 andCDC53 activity. Here we demonstrate that Cdc4p, Cdc34p, and Cdc53p interact in vivo. We have mapped a Cdc4p/Cdc53p-binding region on Cdc34p; this region is essential for S-phase entry and thus the association of these three proteins is required for Sic1p degradation. All three proteins migrate in gel filtration to sizes that greatly exceed their actual size suggesting that they form stable associations with other proteins and we observe Cdc4p, Cdc34p, and Cdc53p fractionating into overlapping families of high molecular weight complexes. Finally, we demonstrate that Cdc4p, Cdc34p, and Cdc53p are stable throughout the cell cycle and that Cdc34p permanently resides as part of a complex throughout the cell cycle. This suggests that all Cdc34p substrates are ubiquitinated by a similar high molecular weight complex.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5122. Tel.: 317-274-3743; Fax: 317-274-4686; E-mail: neal{at}biochem4.iupui.edu.
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↵1 The abbreviations used are: Ub, ubiquitin; Ubc, ubiquitin conjugating family of proteins; E1, ubiquitin activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
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- Received September 24, 1997.
- Revision received December 10, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
