@article {Wueaaz2449,
	title = {Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex},
	journal = {Science},
	volume = {368},
	number = {6489},
	year = {2020},
	publisher = {American Association for the Advancement of Science},
	abstract = {Misfolded endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome in a process known as ER-associated protein degradation (ERAD). ERAD of misfolded luminal ER proteins (ERAD-L) is mediated by the Hrd1 complex, composed of the ubiquitin ligase Hrd1 and four additional proteins (Hrd3, Der1, Usa1, and Yos9). Wu et al. report a cryo{\textendash}electron microscopy structure of the active Hrd1 complex from yeast and, based on this structure, developed a model for how substrates are recognized and retrotranslocated. They propose that Hrd3 and Yos9 jointly create a luminal binding site for misfolded glycoproteins. Hrd1 and Der1 form {\textquotedblleft}half-channels{\textquotedblright} juxtaposed in a thinned section of the ER membrane, which allows a polypeptide loop of an ERAD-L substrate to move through it.Science, this issue p. eaaz2449INTRODUCTIONProtein 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.RATIONALEThe 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.RESULTSHere, we report a structure of the active Hrd1 complex from S. cerevisiae, as determined by cryo{\textendash}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.CONCLUSIONOur results lead to a model for the mechanism of retrotranslocation through the Hrd1 complex. The pathway across the membrane is formed by two {\textquotedblleft}half-channels{\textquotedblright} 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.Initiation of ERAD-L revealed by cryo-EM and photocrosslinking.(A) Side view of a space-filling model of the Hrd1 complex, based on structures of the Hrd1~Usa1-Der1-Hrd3 and Hrd3-Yos9 subcomplexes. (B) Hypothetical position of a glycosylated ERAD-L substrate in the Hrd1 complex (dashed blue line). Substrate-interacting amino acid residues in Hrd1 and Der1 (red and orange, respectively) were determined by photocrosslinking. N, N terminus; C, C terminus; DHFR, dihydrofolate reductase; TM, transmembrane helix. (C) Model for the first three stages of retrotranslocation.Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo{\textendash}electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two {\textquotedblleft}half-channels{\textquotedblright} with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.},
	issn = {0036-8075},
	doi = {10.1126/science.aaz2449},
	url = {https://science.sciencemag.org/content/368/6489/eaaz2449},
	author = {Wu, Xudong and Siggel, Marc and Ovchinnikov, Sergey and Wei Mi and Svetlov, Vladimir and Nudler, Evgeny and Maofu Liao and Hummer, Gerhard and Rapoport, Tom A.}
}
