Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex
INTRODUCTION Protein homeostasis in the endoplasmic reticulum (ER) is maintained by a quality control system. When a newly synthesized ER protein misfolds, it is ultimately retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a pathway referred to as ER-associated protein degradation (ERAD). ERAD alleviates cytotoxic stress imposed by protein misfolding and is implicated in numerous diseases. ERAD is found in all eukaryotic cells but is best studied for the ERAD-L pathway in Saccharomyces cerevisiae, which disposes of misfolded glycoproteins in the ER lumen. The glycan attached to these proteins is first trimmed by glycosidases to generate a terminal α1,6-mannose residue. This residue, together with an unfolded polypeptide segment, targets the substrate to the Hrd1 complex, which is composed of the multispanning ubiquitin ligase Hrd1 and four additional proteins (Hrd3, Der1, Usa1, and Yos9). The Hrd1 complex mediates the retrotranslocation of the polypeptide into the cytosol, where it is polyubiquitinated, extracted from the membrane by the Cdc48 adenosine triphosphatase complex, and, finally, degraded by the proteasome. RATIONALE The mechanism of ERAD-L remains poorly understood. Arguably the most mysterious aspect is how misfolded proteins cross the ER membrane, which normally presents a barrier to macromolecules. How ERAD-L substrates are recognized and distinguished from properly folding intermediates is also unclear. Answers to these questions require structural information on the Hrd1 complex. RESULTS Here, we report a structure of the active Hrd1 complex from S. cerevisiae, as determined by cryo–electron microscopy (cryo-EM) analysis of two subcomplexes. Our structures, biochemical data, and experiments in vivo indicate that the Hrd1 complex functions as a monomer in ERAD-L. Hrd3 and Yos9 jointly create a luminal binding site that recognizes misfolded glycoproteins. The α1,6-mannose residue binds to the mannose 6-phosphate receptor homology (MRH) domain of Yos9, and the polypeptide segment downstream of the glycan attachment site is likely accommodated in a groove of the luminal domain of Hrd3. Hrd1 and the rhomboid-like Der1 protein are linked by Usa1 on the cytosolic side of the membrane. Both Der1 and Hrd1 have lateral gates that face one another within the membrane and possess luminal and cytosolic cavities, respectively. Both proteins distort the membrane region between the lateral gates, making it much thinner than a normal phospholipid bilayer, an observation supported by molecular dynamics simulations. The structures and photocrosslinking experiments indicate that the retrotranslocation of an ERAD-L substrate is initiated by loop insertion of the polypeptide into the membrane, with one strand of the loop interacting with Der1 and the other with Hrd1. CONCLUSION Our results lead to a model for the mechanism of retrotranslocation through the Hrd1 complex. The pathway across the membrane is formed by two “half-channels” corresponding to the luminal and cytosolic cavities of Der1 and Hrd1, respectively. These half-channels are juxtaposed in a thinned membrane region. The substrate inserts into the retrotranslocon as a hairpin that is hydrophilic on both sides. These features contrast with the Sec61 channel, which accepts substrates with a hydrophobic signal or transmembrane segment forming one side of the loop. This segment exits the lateral gate into the lipid environment and is not translocated, while the other side of the loop moves through the membrane in an entirely hydrophilic environment. The structural features of the retrotranslocon can facilitate movement of a fully hydrophilic substrate through a thinned and thus distorted membrane, a paradigm that may be replicated in other protein translocation systems.