The yeast Saccharomyces cerevisiae CDC34 gene encodes a ubiquitin-conjugating enzyme that is required for the cell cycle G/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.

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