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Abstract

Misfolded endoplasmic reticulum (ER) proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the ER membrane, and degraded by the proteasome1-4, a pathway termed ER-associated protein degradation (ERAD). Proteins with misfolded domains in the ER lumen or membrane are discarded through the ERAD-L and -M pathways, respectively. In S. cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain5,6. Hrd1 is the crucial membrane component for retro-translocation7,8, but whether it forms a protein-conducting channel is unclear. Here, we report a cryo-electron microscopy (cryo-EM) structure of S. cerevisiae Hrd1 in complex with its ER luminal binding partner Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight trans-membrane segments, five of which form an aqueous cavity extending from the cytosol almost to the ER lumen, while a segment of the neighboring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features in protein-conducting conduits that facilitate polypeptide movement in the opposite direction, that is, from the cytosol into or across membranes9-11. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the ER membrane.