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Published November 3, 1995 | Published
Journal Article Open

Characterization of a Dominant Negative Mutant of the Cell Cycle Ubiquitin-conjugating Enzyme Cdc34


The yeast Saccharomyces cerevisiae CDC34 gene encodes a ubiquitin-conjugating enzyme that is required for the cell cycle G(1)/S transition. We show here that a dominant negative Cdc34 protein is generated by simultaneously replacing both Cys and Leu with Ser residues. Cys is an essential catalytic residue that forms a transient thiol ester with ubiquitin during catalysis, and Leu is highly conserved among all known ubiquitin-conjugating enzymes. Mutants that encode either an alanine or a serine at one or both of these two positions are inactive. Of these eight mutants, overexpression of CDC34-C95S,L99S in wild type strains was found to block cell growth. Although cells overexpressing Cdc34-C95S,L99S do not exhibit the characteristic multibudded phenotype of cdc34 temperature-sensitive or null mutants, this blockade is relieved by simultaneous overexpression of wild type Cdc34. Purified Cdc34-C95S,L99S protein can be shown to inhibit in vitro ubiquitination of the Cdc34-specific substrate, Cln2 protein. We suggest that Cdc34-C95S,L99S selectively sequesters a subset of Cdc34 substrates or regulators. These findings have implications for the structure/function relationships of ubiquitin-conjugating enzymes, and suggest a general method for identifying components and substrates of specific ubiquitination pathways of eukaryotes.

Additional Information

© 1995 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, June 14, 1995; and in revised form, August 22, 1995) We thank Drs. F. Boschelli, M. Goebl, and R. Needleman for gift of plasmids and strains, and Dr. N. Davis for critical reading of the manuscript. This research was supported by National Institutes of Health Grant GM 47604. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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