Creation of a Pluripotent Ubiquitin-Conjugating Enzyme

Abstract

We describe the creation of a pluripotent ubiquitin-conjugating enzyme (E2) generated through a single amino acid substitution within the catalytic domain of RAD6 (UBC2). This RAD6 derivative carries out the stress-related function of UBC4 and the cell cycle function of CDC34 while maintaining its own DNA repair function. Furthermore, it carries out CDC34's function in the absence of the CDC34 carboxy-terminal extension. By using sequence and structural comparisons, the residues that define the unique functions of these three E2s were found on the E2 catalytic face partitioned to either side by a conserved divide. One of these patches corresponds to a binding site for both HECT and RING domain proteins, suggesting that a single substitution in the catalytic domain of RAD6 confers upon it the ability to interact with multiple ubiquitin protein ligases (E3s). Other amino acid substitutions made within the catalytic domain of RAD6 either caused loss of its DNA repair function or modified its ability to carry out multiple E2 functions. These observations suggest that while HECT and RING domain binding may generally be localized to a specific patch on the E2 surface, other regions of the functional E2 face also play a role in specificity. Finally, these data also indicate that RAD6 uses a different functional region than either UBC4 or CDC34, allowing it to acquire the functions of these E2s while maintaining its own. The pluripotent RAD6 derivative, coupled with sequence, structural, and phylogenetic data, suggests that E2s have diverged from a common multifunctional progenitor.Ubiquitin-conjugating enzymes (E2s) comprise a class of eukaryotic enzymes that function at an intermediate step in the reaction pathway leading to protein ubiquitination. Initially, E2s accept ubiquitin from the active-site cysteine of a ubiquitin-activating enzyme (E1) to their own active-site cysteine via a transthiolation reaction. Subsequently, thiol ester-linked ubiquitin is donated by E2s in one of two possible ways. E2s may transfer ubiquitin directly to a lysine side chain found either on a protein substrate or on a ubiquitin moiety in a growing multiubiquitin chain through the formation of an isopeptide bond. Alternatively, E2s transfer ubiquitin to a cysteine of a ubiquitin protein ligase (E3) through a second transthiolation reaction. In the former case, an E3 functions to assist the E2 in the recognition of a protein substrate, while in the latter case, it is believed that the E3 directly links ubiquitin to either the protein substrate or a growing multiubiquitin chain. Assembly of a multiubiquitin chain on a protein substrate by either an E2 or an E3 targets the protein for degradation by the 26S proteosome (35,39,43).E2s have been shown to consist of a conserved catalytic domain that contains the active-site cysteine. This conservation is observed in both sequence and structural comparisons of E2s. For example, the 11 E2s and two ubiquitin-like conjugating enzymes (UBC9 and UBC12) from the yeast Saccharomyces cerevisiae exhibit...

Polyubiquitination by HECT E3s and the Determinants of Chain Type Specificity

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Tumor suppressor candidate TSSC5 is regulated by UbcH6 and a novel ubiquitin ligase RING105

“…E2 specificity can be altered by mutation. For example, mutation of the Rad6/Ubc2 catalytic core creates a pluripotent ubiquitin-conjugating enzyme that can complement functions of UBC4 and CDC34 proteins (Ptak et al, 2001). Alternatively, hCdc34/ UbcH3 may interact with RING105 but may require additional cofactors for ubiquitylation activity.…”

Functional interaction of 13 yeast SCF complexes with a set of yeast E2 enzymes in vitro

“…In yeast there are 11 E2 enzymes which, presumably in collaboration with specific E3 enzymes, are responsible for the ubiquitination of different subsets of target proteins 4. Structural and mutational analysis has shown that only one or a few residues on the E2 surface confer specificity for the E3 enzyme 5, 6…”