Insights into ubiquitin‐conjugating enzyme/ co‐activator interactions from the structure of the Pex4p:Pex22p complex

Ubiquitin‐conjugating enzymes (E2s) coordinate distinct types of ubiquitination via specific E3 ligases, to a large number of protein substrates. While many E2 enzymes need only the presence of an E3 ligase for substrate ubiquitination, a number of E2s require additional, non‐canonical binding partners to specify their function. Here, we have determined the crystal structure and function of an E2/co‐activator assembly, the Pex4p:Pex22p complex. The peroxisome‐associated E2 enzyme Pex4p binds the peroxisomal membrane protein Pex22p through a binding site that does not overlap with any other known interaction interface in E2 enzymes. Pex22p association enhances Pex4p's ability to transfer ubiquitin to a substrate in vitro , and Pex22p binding‐deficient forms of Pex4p are unable to ubiquitinate the peroxisomal import receptor Pex5p in vivo . Our data demonstrate that the Pex4p:Pex22p assembly, and not Pex4p alone, functions as the E2 enzyme required for Pex5p ubiquitination, establishing a novel mechanism of E2 enzyme regulation.