Dimeric U be2g2 simultaneously engages donor and acceptor ubiquitins to form L ys48‐linked ubiquitin chains

Cellular adaptation to proteotoxic stress at the endoplasmic reticulum ( ER ) depends on L ys48‐linked polyubiquitination by ER ‐associated ubiquitin ligases ( E 3s) and subsequent elimination of ubiquitinated retrotranslocation products by the proteasome. The ER ‐associated E 3 gp78 ubiquitinates misfolded proteins by transferring preformed L ys48‐linked ubiquitin chains from the cognate E 2 U be2g2 to substrates. Here we demonstrate that U be2g2 synthesizes linkage specific ubiquitin chains by forming an unprecedented homodimer: The dimerization of U be2g2, mediated primarily by electrostatic interactions between two U be2g2s, is also facilitated by the charged ubiquitin molecules. Mutagenesis studies show that U be2g2 dimerization is required for ER ‐associated degradation ( ERAD ). In addition to E 2 dimerization, we show that a highly conserved arginine residue in the donor U be2g2 senses the presence of an aspartate in the acceptor ubiquitin to position only L ys48 of ubiquitin in proximity to the donor E 2 active site. These results reveal an unanticipated mode of E 2 self‐association that allows the E 2 to effectively engage two ubiquitins to specifically synthesize L ys48‐linked ubiquitin chains.