Abstract
The yeast ubiquitin (Ub) conjugating enzyme CDC34 plays a crucial role in the progression of the cell cycle from the G1 to S phase. In an effort to identify proteins that interact with CDC34 we undertook a genetic screen to isolate genes whose increased expression suppressed the cell cycle defect associated with the cdc34-2 temperature-sensitive allele. From this screen, the poly-Ub gene UBI4 was identified as a moderately strong suppressor. The fact that the overexpression of a gene encoding a single Ub protein could also suppress the cdc34-2 allele indicated that suppression was related to the increased abundance of Ub. Ub overexpression was found to suppress two other structurally unrelated cdc34 mutations, in addition to the cdc34-2 allele. In all three cases, suppression depended on the expression of Ub with an intact carboxyl terminus. Only the cdc34-2 allele, however, could be suppressed by Ub with an amino acid substitution at lysine 48 which is known to be involved in multi-Ub chain assembly. Genetic results showing allele specific suppression of cdc34 mutations by various Ub derivatives suggested a specific noncovalent interaction between Ub and CDC34. Consistent with this prediction, we have shown by chemical cross-linking the existence of a specific noncovalent Ub binding site on CDC34. Together, these genetic and biochemical experiments indicate that Ub suppression of these cdc34 mutations results from the combined contributions of Ub-CDC34 thiol ester formation and a noncovalent interaction between Ub and CDC34 and therefore suggest that the correct positioning of Ub on a surface of the ubiquitin conjugating enzyme is a requirement of enzyme function.
Highlights
The yeast ubiquitin (Ub) conjugating enzyme CDC34 plays a crucial role in the progression of the cell cycle from the G1 to S phase
Genetic results showing allele specific suppression of cdc34 mutations by various Vb derivatives suggested a specific noneovalent interaction between Vb and CDC34. Consistent with this prediction, we have shown by chemical crosslinking the existence of a specific noncovalent Vb binding site on CDC34. These genetic and biochemical experiments indicate that Vb suppression of these cdc34 mutations results from the combined contributions of Vb·CDC34 thiol ester formation and a noncovalent interaction between Vb and CDC34 and suggest that the correct positioning ofVb on a surface of the ubiquitin conjugating enzyme is a requirement of enzyme function
In recent work we have shown that the ability of CDC34 to self-associate in vitro is dependent on the same region of the tail that is required for its cell cycle function in vivo [12]
Summary
The yeast ubiquitin (Ub) conjugating enzyme CDC34 plays a crucial role in the progression of the cell cycle from the G1 to S phase. In an effort to identify proteins that interact with CDC34 we undertook a genetic screen to isolate genes whose increased expression suppressed the cell cycle defect associated with the cdc temperature-sensitive allele. In this study we show that increased Ub expression can suppress the cell cycle defects associated with three structurally unrelated mutant cdc alleles These genetic results in conjunction with Vb-CDC34 cross-linking studies, indicate that in addition to the previously reported CDC34-CDC34 interaction [10, 12], CDC34 function requires the correct orientation of Ub on the E2 surface
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