A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s.

Abstract

The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains coexist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature‐sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi‐ubiquitin chain synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2

“…In an elegant experiment the C-terminal (substrate binding) domain was fused to the catalytic domain of yeast UBC2/RAD6. Subsequently it was demonstrated that this hybrid molecule with ubiquitin conjugating activity resulting from the RAD6 portion and substrate recognition conferred by the CDC34 part is able to rescue cdc34 mutants [31,32]. No phenotype has yet been described for the deletion of the yeast UbcH2 homologue UBC8.…”

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

“…The molecular functions of only a handful of these extensions are known [38][39][40][41] , and to date, the structural basis for protein-protein interactions mediated by these extensions remain elusive. One of the best understood extensions is in the E2 Ubc2p, which interacts with the E3, Ubr1p.…”

The ubiquitin-proteasome pathway and synaptic plasticity

“…In addition to the structural elements mentioned above, unique parts of some E2s contribute to the specificity of E3 binding as well. For example, the C-terminal tail of a yeast E2 called Cdc34, which is dissimilar to that of a closely related E2 Ubc4, confers specificity of Cdc34 binding to an SCF ligase (Kolman et al 1992;Silver et al 1992). The E3s are the most specific to a given substrate, however.…”