Abstract
Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol and degraded by the ubiquitin-proteasome system, a pathway termed luminal ER-associated protein degradation. Retrotranslocation is mediated by a conserved protein complex, consisting of the ubiquitin ligase Hrd1 and four associated proteins (Der1, Usa1, Hrd3, and Yos9). Photocrosslinking experiments provided preliminary evidence for the polypeptide path through the membrane but did not reveal specific interactions between amino acids in the substrate and Hrd1 complex. Here, we have used site-specific disulfide crosslinking to map the interactions of a glycosylated model substrate with the Hrd1 complex in live S. cerevisiae cells. Together with available electron cryo-microscopy structures, the results show that the substrate interacts on the luminal side with both a groove in Hrd3 and the lectin domain of Yos9 and inserts a loop into the membrane, with one side of the loop interacting with the lateral gate of Der1 and the other with the lateral gate of Hrd1. Our disulfide crosslinking experiments also show that two Hrd1 molecules can interact through their lateral gates and that Hrd1 autoubiquitination is required for the disassembly of these Hrd1 dimers. Taken together, these data define the path of a polypeptide through the ER membrane and suggest that autoubiquitination of inactive Hrd1 dimers is required to generate active Hrd1 monomers.