IDENTIFICATION OF A POSITIVE REGULATOR OF THE CELL-CYCLE UBIQUITIN-CONJUGATING ENZYME CDC34 (UBC3)

The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerev isiae plays an essential role in the progression of cells from the G(1 ) to S phase of the cell division cycle. Using a high-copy suppression strategy, we have identified a yeast gene (UBS1) whose elevated expre ssion suppresses the conditional cell cycle defects associated with cd c34 mutations. The UBS1 gene encodes a 32.2-kDa protein of previously unknown function and is identical in sequence to a genomic open readin g frame on chromosome II (GenBank accession number Z36034). Several li nes of evidence described here indicate that Ubs1 functions as a gener al positive regulator of Cdc34 activity. First, overexpression of UBS1 suppresses not only the cell proliferation and morphological defects associated with cdc34 mutants but also the inability of cdc34 mutant c ells to degrade the general amino acid biosynthesis transcriptional re gulator, Gcn4. Second, deletion of the UBS1 gene profoundly accentuate s the cell cycle defect when placed in combination with a cdc34 temper ature-sensitive allele. Finally, a comparison of the Ubs1 and Cdc34 po lypeptide sequences reveals two noncontiguous regions of similarity, w hich, when projected onto the three-dimensional structure of a ubiquit in-conjugating enzyme, define a single region situated on its surface. While cdc34 mutations corresponding to substitutions outside this reg ion are suppressed by UBS1 overexpression, Ubs1 fails to suppress amin o acid substitutions made within this region. Taken together with othe r findings, the allele specificity exhibited by UBS1 expression sugges ts that Ubs1 regulates Cdc34 by interaction or modification.