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 G1/S transition. We show here that a dominant negative Cdc34 protein is generated by simultaneously replacing both Cys95 and Leu99 with Ser residues. Cys95 is an essential catalytic residue that forms a transient thiol ester with ubiquitin during catalysis, and Leu99 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 overxpression 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.
Banerjee
A
A
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Deshaies
R J
RJ
Chau
V
V
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D001665:Binding Sites; D002453:Cell Cycle; D003001:Cloning, Molecular; D003545:Cysteine; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression; D005799:Genes, Dominant; D005800:Genes, Fungal; D007930:Leucine; D008025:Ligases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D007930:Leucine; D003545:Cysteine