INCREASED UBIQUITIN EXPRESSION SUPPRESSES THE CELL-CYCLE DEFECT ASSOCIATED WITH THE YEAST UBIQUITIN-CONJUGATING ENZYME, CDC34 (UBC3) - EVIDENCE FOR A NONCOVALENT INTERACTION BETWEEN CDC34 AND UBIQUITIN

The yeast ubiquitin (Uh) conjugating enzyme CDC34 plays a crucial role in the progression of the cell cycle from the G(1) to S phase, In an effort to identify proteins that interact with CDC34 we undertook a ge netic screen to isolate genes whose increased expression suppressed th e cell cycle defect associated with the cdc34-2 temperature-sensitive allele, From this screen, the poly-Ub gene UBI4 was identified as a mo derately strong suppressor, The fact that the overexpression of a gene encoding a single Ub protein could also suppress the cdc34-2 allele i ndicated that suppression was related to the increased abundance of Ub , Ub overexpression was found to suppress two other structurally unrel ated cdc34 mutations, in addition to the cdc34-2 allele, In all three cases, suppression depended on the expression of Ub with an intact car boxyl terminus, Only the cdc34-2 allele, however, could be suppressed by Ub with an amino acid substitution at lysine 48 which is known to b e involved in multi-Ub chain assembly, Genetic results showing allele specific suppression of cdc34 mutations by various Ub derivatives sugg ested a specific noncovalent interaction between Ub and CDC34, Consist ent with this prediction, we have shown by chemical crosslinking the e xistence of a specific noncovalent Ub binding site on CDC34, Together, these genetic and biochemical experiments indicate that Ub suppressio n of these cdc34 mutations results from the combined contributions of Ub-CDC34 thiol ester formation and a noncovalent interaction between U b and CDC34 and therefore suggest that the correct positioning of Uh o n a surface of the ubiquitin conjugating enzyme is a requirement of en zyme function.