Molecular dissection of the Erv41-Erv46 retrograde receptor reveals a conserved cysteine-rich region in Erv46 required for retrieval activity

The Erv41-Erv46 complex is a conserved retrograde cargo receptor that retrieves ER resident proteins from Golgi compartments in a pH-dependent manner. Here we functionally dissect the Erv46 subunit and define an approximately 60 residue cysteine-rich region that is unique to the Erv46 family of proteins. This cysteine-rich region contains two vicinal cysteine pairs in CXXC and CCXXC configurations that are each required for retrieval activity in cells. Mutation of the individual cysteine residues produced stable Erv46 proteins that were partially reduced and form mixed-disulfide species on nonreducing gels. Conserved hydrophobic amino acids within the cysteine-rich region of Erv46 were also required for retrieval function in cells. In vitro binding experiments showed that this hydrophobic patch is required for direct cargo binding. Surprisingly, the Erv46 cysteine mutants continued to bind cargo in cell-free assays and produced an increased level of Erv46-cargo complexes in cell extracts suggesting that disulfide linkages in the cysteine-rich region perform a role in releasing bound cargo. On the basis of these findings, we propose that both pH and redox environments regulate cargo binding to a hydrophobic site within the cysteine-rich region of Erv46.