Intragenic suppression among CDC34 (UBC3) mutations defines a class of ubiquitin-conjugating catalytic domains.

Ubiquitin-conjugating (E2) enzymes contain several regions within their catalytic domains that are highly conserved. However, within some of these conserved regions are several residues that may be used to define different classes of catalytic domains for the E2 enzymes. One class can be defined by the Ubc1 protein, which contains K-65, D-90, and D-120, while the corresponding positions within the Cdc34 (Ubc3) protein, which defines a second class of enzymes, contain S-73, S-97, and S-139, respectively. The presence of these differences within otherwise highly conserved regions of this family suggests that these residues may be critical for the specificity of Cdc34 function or regulation. Therefore, we have constructed a series of cdc34 alleles encoding mutant proteins in which these serine residues have been changed to other amino acid residues, including alanine and aspartic acid. In vivo complementation studies showed that S-97, which lies near the active site C-95, is essential for Cdc34 function. The addition of a second mutation in CDC34, which now encoded both the S97D and S73K changes, restored partial function to the Cdc34 enzyme. Moreover, the deletion of residues 103 to 114 within Cdc34, which are not present in the Ubc1-like E2s, allowed the S73K/S97D mutant to function as efficiently as wild-type Cdc34 protein. Finally, the cloning and sequencing of the temperature-sensitive alleles of CDC34 indicated that A-62 is also unique to the Cdc34 class of E2 enzymes and that mutations at this position can be detrimental to Cdc34 function. Our results suggest that several key residues within conserved regions of the E2 enzyme family genetically interact with each other and define a class of E2 catalytic domains.