Covalent modification of a protein with the 76-residue protein ubiquitin (Ub), thereby changing the stability, localization, or activity of the target protein, regulates almost all aspects of eukaryotic cellular function. This reaction requires the sequential actions of three types of enzymes;an activating enzyme (E1) forms a thioester linkage between its catalytic cysteine and the carboxyl group of Gly76 in ubiquitin to activate it;a conjugating enzyme (E2) that receives ubiquitin from the E1;a ubiquitin ligase (E3) that transfers the ubiquitin molecule from the E2 to a substrate. For every eukaryotic species, there are two E1 enzymes, whereas approximately dozens of E2 enzymes (Table 1) and thousands of E3 ligases are found. The E3 ligases can be classified into two major catagories, the HECT domain (homologous to E6-associated protein C-Terminus)-containing ligases and those carrying a catalytic RING (really interesting new gene) domain. We have established an in vitro ubiquitination system using purified gp78c, a cytosolic domain of an ER-associated RING ligase and ube2g2. The assay allows us to assemble Lys48 linked polyubiquitin chains. Using this system, we have demonstrated that polyubiquitin chains can be preassembled on the catalytic cysteine of Ube2g2 before being transferred to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled on an E2 active site is unclear. We recently discovered that gp78 forms an oligomer via two oligomerization sites, one of which being a hydrophobic segment in the gp78 cytosolic domain (gp78C). We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules using a novel interaction that is primarily mediated by an Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that the formation of such a gp78-Ube2g2 hetero-oligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2 to form active site-linked polyubiquitin chains. More recently, in collaboration with Dr. Wei Li, we demonstrate that Ube2g2 synthesizes linkage specific ubiquitin chains by forming an unprecedented homodimer: The dimerization of Ube2g2, mediated primarily by electrostatic interactions between two Ube2g2s, is also facilitated by the charged ubiquitin molecules. Mutagenesis studies show that Ube2g2 dimerization is required for ER-associated degradation (ERAD). In addition to E2 dimerization, we show that a highly conserved arginine residue in the donor Ube2g2 senses the presence of an aspartate in the acceptor ubiquitin to position only Lys48 of ubiquitin in proximity to the donor E2 active site. These results reveal an unanticipated mode of E2 self-association that allows the E2 to effectively engage two ubiquitins to specifically synthesize Lys48-linked ubiquitin chains.
