Structural basis and pathological implications of the dimeric OS9-SEL1L-HRD1 ERAD Core Complex

Domain: neuroscience
Published: 2026-01-27
Editorial outcome: major_revision

Abstract
The SEL1L-HRD1 complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD), a critical quality-control pathway that clears misfolded ER proteins. However, the molecular organization and pathogenic mechanisms of mammalian ERAD have remained elusive. Here, we report the cryo-EM structure of the core mammalian ERAD complex, comprising the ER lectin OS9, SEL1L, and the E3 ubiquitin ligase HRD1. The structure, validated by mutagenesis and crosslinking assays, reveals a dimeric assembly of the core complex in which SEL1L and OS9 form a claw-like configuration in the ER lumen that mediates substrate engagement, while HRD1 dimerizes within the membrane that may facilitate substrate translocation. Furthermore, pathogenic SEL1L mutations at the SEL1L-OS9 (Gly585Asp) and SEL1L-HRD1 (Ser658Pro) interfaces disrupt complex formation and impair ERAD activity. A newly identified disease-associated HRD1 variant (Ala91Asp), located in transmembrane helix 3, impairs HRD1 dimerization and substrate processing. These findings provide structural and functional insights for mammalian SEL1L-HRD1 ERAD and elucidate how mutations destabilizing this machinery contribute to human disease.

Introduction
In eukaryotes, approximately 30% of newly synthesized proteins are directed to the endoplasmic reticulum (ER), where they fold and mature into their functional forms1,2. Proteins that fail to fold properly are eliminated via ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded proteins from the ER lumen to the cytosol for proteasomal degradation3–7. Suppressor/Enhancer of Lin-12-like protein 1-like (SEL1L) and the E3 ubiquitin ligase HMG-CoA reductase degradation protein 1 (HRD1) complex, homologous to the yeast Hrd3-Hrd1 pair, forms the core of the mammalian ERAD machinery and represents its most extensively characterized module8–12. The discovery of pathogenic SEL1L and HRD1 variants in patients with ERAD-associated neurodevelopmental disorders with onset at infancy (ENDI syndrome)13–15, along with the severe phenotypes observed in global and cell type-specific Sel1L- or Hrd1-knockout (KO) mice12,16–30, underscores the critical role of ERAD in health and disease. However, the molecular mechanism by which these variants impair ERAD remains poorly understood, primarily due to the lack of structural information on the human complex.

Hrd3/SEL1L and Hrd1/HRD1 form the ERAD core, with HRD1 acting as the E3 ubiquitin ligase31 and SEL1L stabilizing HRD119,32 and promoting complex assembly33,34. Additional cofactors include ER lectins Yos9/OS935–37 and XTP3B (also known as ERLEC1)38,39, Der1/Derlin40–42, and Usa1/HERP43, which assist in substrate recognition, retrotranslocation, and ubiquitination. Previous cryo-electron microscopy (cryo-EM) studies of yeast ERAD revealed both a homo-dimeric Hrd3-Hrd1 complex44 and monomeric Yos9-Hrd3-Hrd1 and Hrd3-Hrd1-Usa1-Derl subcomplexes45. While the dimeric structure suggested a protein-conducting channel formed by the Hrd1 dimer, the dimer interface was closed, and the aqueous channel interior was instead exposed to the surrounding lipid layer44. It was later proposed that this conformation is a detergent artifact with low ubiquitination activity45, favoring the monomeric model in yeast. In this monomeric model, Hrd1 and Der1 form complementary “half-channels” within a thinned ER membrane, while Hrd3 and Yos9 act as luminal substrate receptors45. Whether similar assemblies exist in mammals remains unknown.

Notably, both size-exclusion chromatography and chemical-mediated crosslinking assays of endogenous SEL1L-HRD1 complex from human cells support HRD1 oligomerization46 and SEL1L dimerization47,48. These findings raise the possibility that unlike the yeast complex, SEL1L-HRD1 dimerization is functionally important in mammals. In this work, we characterize the architecture of the mammalian ERAD core complex, which is essential for understanding the molecular basis of ERAD-related diseases.

Results
The OS9-SEL1L-HRD1 complex forms the core of mammalian ERAD machinery
To define the composition of the SEL1L-HRD1 ERAD core complex, we independently analyzed SEL1L and HRD1 interactomes in HEK293T cells using immunoprecipitation (IP) followed by LC-MS/MS (Supplementary Fig. 1a)49. Each dataset was generated in triplicate using stringent filtering criteria, with SEL1L or HRD1 KO cells as negative controls. We identified 46 high-confidence SEL1L interactors and 24 HRD1 interactors (Supplementary Fig. 1b, c and Supplementary Tables 1-2). Cross-comparison revealed 10 shared interactors (Supplementary Fig. 1d and Supplementary Table 3), with OS9, XTP3B, SEL1L, HRD1, and FAM8A1 emerging as the most prominent components (Fig. 1a). OS9 and XTP3B are ER-resident lectins with partially redundant roles in HRD1-mediated ERAD39,50,51, with OS9 serving as the major partner34. Consistent with a previous study52, FAM8A1 deletion did not affect SEL1L-HRD1 protein levels or their function towards endogenous substrates such as IRE1α, CD147 and OS9 (Supplementary Figs. 1e–h).

To further define the core components of the complex, we co-expressed HRD1, SEL1L, OS9, HERP1, UBE2J1, and DERL2 in HEK293F cells, followed by HRD1 pull-downs. The OS9-SEL1L-HRD1 subcomplex co-purified robustly under multiple detergent conditions and maintained a stable stoichiometric ratio, except in Triton X-100 and NP-40 where complex integrity was lost (Supplementary Figs. 2a, b). In contrast, Derlin-2, HERP1 and UBE2J1 were not detected under any conditions tested (Supplementary Figs. 2a, b).

We next expressed a known ERAD substrate pro-arginine vasopressin (proAVP) carrying the Gly57Ser disease mutation53,54, tagged with an ER-retention signal KDEL to prevent secretion (proAVP G57S*, Supplementary Figs. 2c, d). Co-expression of OS9, SEL1L, and HRD1 in HEK293F cells resulted in ubiquitination of proAVP G57S* in an E3-dependent manner, as shown by the loss of ubiquitination with a ligase-dead HRD1 mutant (C291A/C294A; C2A)55 (Fig. 1b and Supplementary Figs. 2e, f). Notably, both Derlin-1 and Derlin-2 were required for efficient ERAD of proAVP G57S* (Supplementary Figs. 2g–i). Taken together, these data establish OS9-SEL1L-HRD1 as the core mammalian ERAD complex.

OS9-SEL1L-HRD1 ERAD core complex forms a dimer
To determine the molecular organization of the core complex, we co-expressed full length OS9, SEL1L and FLAG-tagged HRD1 in HEK293F cells. After membrane solubilization in lauryl maltose neopentyl glycol (LMNG), the complex was purified using FLAG-based immunoprecipitation followed by size-exclusion chromatography. This procedure yielded a single, well-defined peak containing all three components – OS9, SEL1L and HRD1 in near-stoichiometric amounts (Fig. 1c and Supplementary Fig. 2j). Peak fractions (#10-11, Fig. 1c) were collected, concentrated and immediately prepared for sample freezing and subsequent single-particle cryo-EM analysis (Supplementary Figs. 3a, b).

Initial 2D image analysis revealed the presence of a homogenous set of particles, most of which display visible C2 symmetry (Supplementary Fig. 3a). Subsequent 3D reconstruction with the selected particles revealed that the OS9-SEL1L-HRD1 complex adopts a dimeric architecture, with an overall resolution of 3.6Å (Supplementary Figs. 3a, b and Supplementary Table 4). We then performed a focused 3D refinement using a mask around the visible regions of OS9 and in the structure (Supplementary Fig. 3b). This process led to improvements in local resolutions to 3.3Å for the OS9-SEL1L interface (Fig. 1d and Supplementary Fig. 3b). Model building into the EM density maps was guided using AlphaFold3-predicted structures56, followed by manual adjustments based on visible side chain densities for SEL1L-OS9 and the transmembrane helices of HRD1. This enabled accurate modeling of the three core components: OS9, SEL1L, and HRD1 (Fig. 1d, e and Supplementary Figs. 4a–e). The final 3D model captured all major domains of the core ERAD complex: the eight transmembrane helices of HRD1 (residues 1-265, blue), the luminal domain of SEL1L (residues 119-725, yellow), and two conserved domains of OS9 – the mannose-6-phosphate receptor homology (MRH) domain (residues 88-236) and a structured region comprising four β-sheets and an α-helix (termed “4β”, residues 28-87, 237-259, 633-652) (green) (Fig. 1d, e and Supplementary Figs. 4b–e). Regions not resolved in the map included the transmembrane domain of SEL1L, the cytosolic regions of both SEL1L and HRD1 (including the E3 ligase RING domain), and the remaining flexible portions of OS9’s luminal domain (dotted lines, Supplementary Fig. 4a).

SEL1L disease variants disrupt the OS9-SEL1L-HRD1 core complex
Our cryo-EM map of the OS9-SEL1L subcomplex revealed a crab claw-like configuration with a central pore (red asterisk, Fig. 2a), suggestive of a conduit for OS9-bound substrate delivery to HRD1. To probe this configuration, we mapped two disease-causing SEL1L variants, G585D and S658P13,49, to the structure. SEL1L-G585 resides at the SEL1L-OS9 interface (Fig. 2b, c and Supplementary Fig. 5a), while SEL1L-S658 lies near the SEL1L-HRD1 interface (Fig. 2d, e and Supplementary Fig. 5a). In SEL1L-/- HEK293T cells, re-expression of SEL1L-G585D selectively reduced OS9 binding by ~90% (lane 3, Fig. 2f), while S658P disrupted HRD1 interaction by ~70% (lane 4). The double mutant (G585D/S658P) nearly abolished both interactions (lane 5, Fig. 2f). Notably, neither mutation impaired binding to the E2 conjugating enzyme UBE2J1 (lane 2-5, Fig. 2f). These results demonstrate that disease-associated SEL1L variants selectively disrupt OS9 and HRD1 engagement, underscoring the OS9-SEL1L-HRD1 assembly as the structural and functional core of the mammalian ERAD machinery.

In vivo formation of OS9-SEL1L dimers
Given that the yeast Yos9-Hrd3 structure is monomeric45, we next tested whether the mammalian OS9-SEL1L protomer adopts a closed dimer interface (Fig. 3a). Within this interface, OS9 residue D135 lies in close proximity to SEL1L residues D439 and M440 from the opposing protomer (Fig. 3b, c). To test whether this dimer exists in vivo rather than an artifact of detergent-based purification, we performed native disulfide crosslinking assays using Cys substitution at these position, with the more distal N442C serving as a negative control57. In the oxidizing ER lumen58, such spatial proximity enables disulfide bond formation between engineered Cys57. To avoid artifactual crosslinking during lysis, cells were directly lysed in denaturing SDS buffer lacking reducing agents and containing a cysteine-alkylating reagent N-ethylmaleimide (NEM), instead of non-denaturing detergents such as Triton X-100 or NP-40. Under these conditions, disulfide-linked complexes formed between OS9-D135C and SEL1L-D439C or M440C, were markedly reduced with SEL1L-N442C, and were absent with WT controls (lanes 7-8 vs. 9-10, Fig. 3d, e). Coomassie staining after HRD1-FLAG IP further confirmed OS9-SEL1L complex formation under native conditions (lane 1 vs. 2, Fig. 3f). As expected, all disulfide-linked species were eliminated by reducing agents (lane 2-3 vs. 7-8, Fig. 3d; lane 1 vs. 3, Fig. 3f). Moreover, 3D variability analysis reveals coordinated motions within the OS9-SEL1L luminal “substrate claw,” which alternates between compact and open conformations that likely facilitate dynamic substrate capture and handover to HRD1 (Supplementary Figs. 5b, c, Supplementary Movie 1). Together, these data demonstrate in vivo disulfide-mediated dimerization of the OS9-SEL1L complex.

In vivo formation of HRD1 dimers
Our cryo-EM structure revealed that HRD1 dimerizes through interactions centered on transmembrane helix 3 (TM3) (Fig. 4a). In this interface, residues T93, F95 and R96 are positioned in close proximity (red spheres), while V94 and D97 are oriented away from the interface (gray spheres) (Fig. 4b and Supplementary Fig. 6a). Several of these residues are evolutionarily conserved (Fig. 4c). To test their functional contribution, we introduced Cys substitutions as performed above for SEL1L-OS9. T93C, F95C, and R96C, but not V94C and D97C, efficiently formed disulfide-linked HRD1 dimers, reaching ~50% dimerization (lanes 3, 5, 6, Fig. 4d). To further assess endogenous HRD1 dimerization, we treated HEK293T cells with the membrane-permeable crosslinker dithiobis-succinimidyl propionate (DSP), previously used to detect HRD1 dimers47,48. HRD1 dimers were readily observed in WT cells but markedly reduced in SEL1L-deficient cells (Supplementary Figs. 6b, c). In both crosslinking assays, dimeric species were eliminated by reducing agents, confirming disulfide/DSP-dependent linkage (Fig. 4d and Supplementary Figs. 6b, c). Together, these findings indicate that the OS9-SEL1L-HRD1 complex can assemble into a relatively stable dimeric form in vivo.

TM3-mediated HRD1 dimerization is required for function
Because yeast Hrd1 dimers are considered inactive45, we next investigated the functional relevance of HRD1 dimers in mammalian cells. Our cryo-EM structure indicates that HRD1 homodimerization is stabilized by a polar interaction at T93 and a π-stacking interaction involving Y83 between opposing TM3 helices (Fig. 5a and Supplementary Fig. 7a). Unlike Y83, T93 is highly conserved from yeast to humans and appears to be the dominant stabilizing residue (Fig. 4c and Supplementary Fig. 7a). To test this, we introduced T93A and T93F mutants to disrupt the polar interaction and assessed dimerization using crosslinking assays. Both mutations abolished R96C-mediated disulfide-linked dimers (lane 4-5 vs. 3, Fig. 5b) and reduced DSP-crosslinked dimers by ~10-fold (lanes 3-4 vs. 2, Supplementary Fig. 7b). Consistently, both T93A and T93F markedly reduced HRD1-HRD1 interaction in transfected HEK293T cells (Fig. 5c), demonstrating that T93 is essential for TM3-mediated HRD1 dimerization.

We next asked whether HRD1 dimerization is required for ERAD activity. Expression of either T93 mutant in HRD1-/- HEK293T cells significantly prolonged the half-life of proAVP G57S*, and impaired its ubiquitination, phenocopying the ligase-dead HRD1-C2A mutant (Fig. 5d–f and Supplementary. 7c). Similar defects were observed with another ER-retained model substrate, POMC C28F*59 (Supplementary Figs. 8a, b). These functional impairments were not due to disruption of the OS9-SEL1L-HRD1 complex, as both T93 mutants retained OS9 and SEL1L binding, as well as Derlin-2 (Fig. 5g, h). Thus, T93-dependent TM3-mediated HRD1 dimerization is essential for ERAD function.

A disease-associated HRD1 variant disrupts dimerization and impairs ERAD function
We recently learned of a novel HRD1 variant of uncertain significance, A91D, identified in a one-year-old boy with congenital heart defects and early-onset pulmonary dysfunction (Lifera Omics Database). A91 is conserved from humans to Arabidopsis but not in C. elegans and yeast (Fig. 4c). Our cryo-EM structure places this residue within TM3 of HRD1 at the dimer interface (Fig. 6a). Expression of HRD1-A91D in HRD1-/- HEK239T cells markedly prolonged the half-life and impaired ubiquitination of proAVP G57S* (Fig. 6b, c and Supplementary Fig. 9a). Similar results were obtained for POMC C28F* (Supplementary Figs. 8a and 9b).

Substituting a nonpolar residue (A) with a negatively charged residue (D) would be expected to introduce both steric hindrance and electrostatic repulsion, potentially destabilizing HRD1 dimers. To test this, we examined dimerization using the crosslinking assays described above. Both native disulfide crosslinking (R96C reporter) and DSP crosslinking showed ~50% reduction in dimer formation by A91D (Fig. 6d and Supplementary Fig. 9c). Importantly, A91D retained normal binding to OS9, SEL1L and Derlin-2 (Fig. 6e, f), indicating that the defect is specific to HRD1 dimerization rather than disruption of the core complex. These findings show that HRD1 A91D patient variant selectively impairs HRD1 dimerization and ERAD function, linking dimer-interface integrity to human disease pathogenesis.

Proposed substrate handling by the OS9-SEL1L-HRD1 core complex
To understand how the core complex engages misfolded substrates, we examined the spatial arrangement of OS9-SEL1L relative to the HRD1 channel. Our structure reveals that the distal end of the putative substrate-binding region of OS9-SEL1L lies ~58Å from the entrance of the HRD1 channel (Fig. 7a). The ~36Å OS9-SEL1L pocket aligns precisely with the 12Å pore entrance and the 18Å HRD1 dimer channel, positioning the luminal module for efficient substrate delivery. Electrostatic analysis shows that OS9-SEL1L contains a negatively charged substrate-binding pocket (Supplementary Fig. 10a). Similar to the inward-bent helices that generate the substrate cavity in the yeast Hrd1-Der1 complex45, the HRD1 dimer in our structure appears to induce local membrane thinning or bending at the dimer interface, producing a cavity evident in the micelle density (Fig. 7b and Supplementary Fig. 10b). Notably, this channel is enriched in methionine residues (Fig. 7c), reminiscent of the “methionine bristles” that line the pores of the EMC and YidC membrane insertases60,61 as well as the M-domain of the signal-recognition particle62,63 (Fig. 7d and Supplementary Fig. 10c). These observations reveal a previously unrecognized methionine-lined conduit within the HRD1 dimer that may facilitate substrate engagement or translocation during ERAD.

Comparison with existing ERAD core structures
Our cryo-EM structure establishes an HRD1 dimer architecture that differs substantially from previously reported yeast ERAD complexes in both yeast44,45 and mammals64 (Fig. 7e–i and Supplementary Figs. 10d–f). In our model, the putative translocation channel is formed between TM1-4 of one HRD1 protomer and TM8 of the other (asterisks, Fig. 7e and Supplementary Fig. 10d). This arrangement contrasts with the yeast Hrd1-Hrd3 dimer, which lacks such a channel (Fig. 7f and Supplementary Fig. 10e), and the Hrd1-Der1 complex, where half-channels are formed by TM2-4 of Hrd1 and TM1-2, TM5 of Der1 (asterisks, Fig. 7g and Supplementary Fig. 10f).

Our structure also differs from the recently reported mammalian XTP3B-SEL1L-HRD1 complex64, which captured an asymmetric dimer in which only one HRD1 protomer binds XTP3B-SEL1L (2:1:1 stoichiometry) (Fig. 7h). That study proposed that Derlin engagement may trigger HRD1 dimer dissociation, although Derlin itself was not visualized. Together, these observations suggest that the HRD1 dimer likely represents one of several coexisting assembly states, with transitions potentially regulated by substrate load, Derlin engagement, or broader ERAD pathway dynamics.

Methods
Cell lines
HEK293T cells (ATCC, CRL-3216) were cultured at 37°C with 5% CO2 in DMEM (Gibco) with 10% fetal bovine serum (Fisher Scientific). HEK293F cells (Gibco, A14527) were cultured at 37°C with 5% CO2 in Expi293™ Expression Medium (Gibco) with shaking.

Protein expression and purification
0.6 mg plasmid was transfected to 0.5L HEK293F cells by 1.6 mg polyethylenimine “Max” (PEIMax) (Polysciences) for protein expression. The medium was collected at 72 h post-transfection for protein purification. The pellet from 500 mL cells was resuspended in 30 mL buffer A (20 mM HEPES, 150 mM NaCl, pH 7.5) supplemented by protease inhibitor (Sigma) and phosphatase inhibitor cocktail (Sigma) and disrupted by sonication. After 3500 × g centrifugation for 5 min, the resulting supernatant was then incubated with 1% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace) at 4oC for 2 h. The insoluble fraction was removed by centrifugation (21,000 × g, 4oC, 30 min), and the supernatant was incubated with 1 mL anti-FLAG M2 resin (Sigma) at 4oC for 4 h. The beads were then washed with 20 column volume (CV) buffer B (20 mM HEPES, 150 mM NaCl, 0.01% LMNG, pH 7.5) by gravity flow. Proteins were eluted in 5 CV buffer B supplemented by 0.1 mg/mL 3xFLAG peptide (APExBIO). The eluate was concentrated and further purified by size-exclusion chromatography (SEC) (Superose 6 Increase, 10/300 GL column, Cytiva) in buffer C (20 mM HEPES, 150 mM NaCl, 0.02% glyco-diosgenin/GDN, pH 7.5) for cryo-EM.

Cryo-EM sample preparation and data acquisition
Protein samples were concentrated using 100 kDa filter (Amino) to 4 mg/mL for grid preparation. Aliquots of 3 μl protein samples in detergent were applied to Quantifoil R1.2/1.3 400 mesh Cu holy carbon grids (Quantifoil) glow discharged with amylamine, blotted for 8 s at 4°C and 100%, then plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI). Data was collected on the ThermoFisher Titan Krios equipped with a K3 direct electron detector. The microscope was operated at 300 kV at a nominal magnification of 130,000X with an energy filter slit width of 10 eV. A defocus range of −0.8 µm to −1.8 µm at 0.2 µm step sizes was used for data collection. Movies were collected at a dose of 60 e/Å2 across 40 frames per movie. A total of 6,350 movies were collected, and the calibrated pixel size was 0.652 Å.

Cryo-EM data processing
Cryo-EM data was processed using CryoSPARC v4.6.067. Movies were corrected for beam-induced motion and subjected to patch CTF estimation. Blob picker was initially used to select for particles with minimum and maximum diameters of 120 Å and 250 Å, respectively. 2D classification of the picked particles yielded classes with clear membrane-embedded protein domain elements at an approximate diameter of ~130 Å. These classes were used to generate templates and improve particle picking. Template picker was used to select for particles with a 200 Å diameter using the generated templates from selected 2D class averages. From the motion-corrected micrographs, 1,101,567 particles were extracted at a box size of 488 × 488 pixels. An initial 2D classification was conducted to select a subset of well-aligned particles from 2D class averages showing clear protein domain and secondary structure elements resolved within and outside the micelle. An initial model for the ERAD complex was then generated using ab initio reconstruction from 163,759 selected particles.

Subsequent 3D classifications were performed to recover additional ERAD particles for 3D homogenous refinements. Starting with the initial 1,101,567 extracted particles, iterative heterogenous refinements were conducted to sort for ERAD particles and remove junk or poorly aligning particles. A final set of 192,786 particles were reconstructed using non-uniform refinement applying C2 symmetry. The average resolution of the final map was 3.64 Å, as determined by the gold standard Fourier Shell Correlation (FSC) from two independently processed half-particle sets at an FSC threshold of 0.143. A mask was then created for the SEL1L and OS9 domains visible in the cryo-EM map. Symmetry expansion, followed by local 3D refinement focused on SEL1L-OS9 using the generated mask, was conducted to overcome symmetry-breaking features such as subunit flexibility between the individual components in the ERAD dimer and improve the map resolution in the OS9-SEL1L interaction interface to 3.30 Å. Local resolution of the focused and unfocused map was determined in cryoSPARC at an FSC threshold of 0.143.

Model building and refinement
The predicted Alphafold 356 model of OS9, SEL1L, and HRD1 was docked as a rigid body into the cryo-EM map using ChimeraX (v1.9). Regions that were not resolved in the EM map were removed from fitted model. We then broke the model into 5 rigid body elements: 2 OS9, 2 SEL1L, and the HRD1 dimer that were then adjusted into the cryo-EM map with COOT (v1.1.02). The transmembrane domains of HRD1 and secondary structural elements of SEL1L and OS9 were further rotated and positioned in the density map based on visible side chains in COOT. The final model was iteratively real space refined in PHENIX68 (v1.20.1) with 3 macrocycles applying Ramachandran and secondary structure restraints, and non-crystallographic symmetry constraints. Molprobity was used to validate the model and resolve clashes. All model and map figures were made with ChimeraX.

CRISPR/Cas9-based knockout (KO) HEK293T cells
To generate SEL1L-, HRD1-, FAM8A1-, Derlin-1-, and Derlin-2-deficient HEK293T cells, sgRNA oligonucleotides designed for human SEL1L (5’-GGCTGAACAGGGCTATGAAG-3’), human HRD1 (5’-GGACAAAGGCCTGGATGTAC-3’), FAM8A1 (gRNA1: 5’-GCGCGGCGGCTCCAATTTGT-3’, gRNA2: 5’-TTCGGCCTGGGGGTCGTCGC-3’), Derlin-1 (5’-GCTGCTGATCAGAATGGCGG-3’), and Derlin-2 (5’-GAAGGCCGAAGAAGTTCATG-3’) was inserted into lentiCRISPR v2 (plasmid 52961; Addgene). HEK293T cells grown in 10 cm petri dishes were transfected with indicated plasmids using 5 μl 1 mg/ml PEI (Sigma) per 1 μg of plasmids for HEK293T cells. Cells were cultured 24 h after transfection in medium containing 2 μg/ml puromycin for 24 h and then in normal growth media.

Plasmids
The following plasmids were used in the study (h denotes human genes; m denotes mouse genes): h-HRD1 cDNA with a C-terminal FLAG or Strep tag was cloned from HEK293T cDNA and inserted into the pcDNA3 to generate pcDNA3-h-HRD1(WT)-FLAG and pcDNA3-h-HRD1(WT)-Strep. The HRD1 mutant C2A (C291A/C294A), T93C, V94C, F95C, R96C, D97C, T93A, T93F and A91D were generated using the plasmid pcDNA-h-HRD1(WT)-FLAG or pcDNA3-h-HRD1(WT)-Strep as template. HRD1 T93A/R96C, T93F/R96C, and A91D/R96C mutants were generated using the plasmid pcDNA-h-HRD1(R96C)-FLAG as template. h-OS9 isoform 2 (OS9) cDNA was cloned from HEK293T cDNA and inserted into the pcDNA3 to generate pcDNA3-h-OS9(WT). The OS9 mutant D135C was generated using plasmid pcDNA3-h-OS9(WT) as the template. m-Sel1L cDNA, with/without a C-terminal FLAG tag was cloned from mouse liver cDNA and inserted into the pcDNA3 to generate pcDNA3-m-SEL1L(WT)-FLAG and pcDNA3-m-SEL1L(WT). The SEL1L-FLAG mutants S658P and G585D were generated using the plasmid pcDNA-m-SEL1L(WT)-FLAG as the template. The double mutations of SEL1L S658P/G585D was generated using the plasmid pcDNA-m-SEL1L(S658P)-FLAG as the template. The SEL1L mutants D439C, M440C and N442C were generated using the plasmid pcDNA-m-SEL1L(WT) as the template. The OS9 mutant D135C was generated using plasmid pcDNA3-h-OS9(WT) as the template. h-proAVP G57S with C-terminal 1xStrep or 10xHis tag and KDEL was cloned from the plasmid pcDNA3-h-proAVP G57S-HA53, to generate pcDNA3-h-proAVP G57S*. mPOMC C28F with 10xHis tag and KDEL in its C-terminal was cloned from the plasmid mPOMC C28F-HA59 to generate pcDNA3-m-POMC C28F*. All plasmids were validated by DNA sequencing. Residue numbering of SEL1L in figures corresponds to human SEL1L.

Western blot and antibodies (reducing SDS-PAGE)
HEK293T or HEK293F cells were harvested and snap-frozen in liquid nitrogen. The proteins were extracted by sonication in Western blot lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) with protease inhibitor (Sigma) and phosphatase inhibitor cocktail (Sigma). Lysates were incubated on ice for 15 min and centrifuged at 16,000 g for 10 min. Supernatants were collected and analyzed for protein concentration using the Bio-Rad Protein Assay Dye (Bio-Rad). 20–50 μg of protein were denatured at 95°C for 5 min or 37°C for 1 hr in 5x SDS sample buffer (250 mM Tris-HCl pH 6.8, 10% sodium dodecyl sulfate, 0.05% Bromophenol blue, 50% glycerol, and 1.44 M β-mercaptoethanol). Protein was separated on SDS-PAGE, followed by electrophoretic transfer to PVDF (Fisher Scientific) membrane. The blots were incubated in 2% BSA/Tri-buffered saline tween-20 (TBST) with primary antibodies overnight at 4°C: anti-HSP90 (Santa Cruz, #sc-13119, 1:5000), anti-GAPDH (Proteintech, #60004-1, 1:5000), anti-SEL1L (home-made, 1:10,000)69, anti-HRD1 (Proteintech, #13473-1, 1:2000), anti-OS9 (Abcam, #ab109510, 1:5000), anti-CD147 (Proteintech, #11989-1, 1:3,000), anti-IRE1α (Cell Signaling, #3294, 1:2,000), anti-UBE2J1 (Santa Cruz, #sc-377002, 1:3,000), anti-Derlin-2 (gift, 1:1000), anti-Derlin-1 (gift, 1:1000), anti-FLAG (Sigma, #F1804, 1:1000), anti-HERPUD1 (HERP1) (Abcam, #ab150424, 1:3000), anti-FAM8A1 (Proteintech, #24746-1-AP, 1:3000), anti-ubiquitin (Santa Cruz, #sc-8017, 1:1000), anti-His (Genscript, #A00174, 1:2000), anti-Strep (Sigma, #SAB2702216, 1:1000). Membranes were washed with TBST and incubated with secondary antibodies, HRP conjugated (Bio-Rad, 1:10,000) at room temperature for 1 h for ECL chemiluminescence detection system (Bio-Rad) development. Band intensity was determined using Imagelab (Bio-Rad, v6.1.0) software.

Mass spectrometry
SEL1L- and HRD1- IP-MS in HEK293T cells have been described previously49 and were performed with three independent biological replicates. Briefly, IP of endogenous SEL1L or HRD1 in WT, SEL1L-/- or HRD1-/- HEK293T cells were performed using 10 mg proteins from each sample lysed with the IP buffer (150 mM NaCl, 0.2% NP-40, 0.1% Triton X-100, 25 mM Tris-HCl pH 7.5) supplemented with protease inhibitors, protein phosphatase inhibitors, and 10 mM N-ethylmaleimide. The cell lysates were first incubated anti-SEL1L (home-made) or anti-HRD1 (home-made or Cell Signaling #14773) at 4°C overnight, followed by incubation with Protein A agarose (Invitrogen, #20333) at 4°C for 2 h. An IP reaction with IgG using cell lysate of SEL1L-/- or HRD1-/- cells was included as a negative control for SEL1L- or HRD1-IP, respectively. The mass spectrometry was conducted by the Proteomics Resource Facility at the University of Michigan Medical School. The beads were resuspended in 50 µl of 0.1 M ammonium bicarbonate buffer (pH ~8). Cysteines were reduced by adding 50 µl of 10 mM DTT and incubating at 45°C for 30 min. Samples were cooled to room temperature and alkylation of cysteines was achieved by incubating with 65 mM 2-Chloroacetamide, under darkness, for 30 min at room temperature. An overnight digestion with 1 µg sequencing-grade modified trypsin was carried out at 37°C with constant shaking in a Thermomixer. Digestion was stopped by acidification and peptides were desalted using SepPak C18 cartridges using manufacturer’s protocol (Waters). Samples were completely dried using vacufuge. Resulting peptides were dissolved in 0.1% formic acid/2% acetonitrile solution and were resolved on a nano-capillary reverse phase column (Acclaim PepMap C18, 2 micron, 50 cm, Thermo Scientific) using a 0.1% formic acid/2% acetonitrile (Buffer A) and 0.1% formic acid/95% acetonitrile (Buffer B) gradient at 300 nl/min over a period of 180 min (2–25% buffer B in 110 min, 25–40% in 20 min, 40–90% in 5 min followed by holding at 90% buffer B for 10 min and re-equilibration with Buffer A for 30 min). Eluent was directly introduced into Q exactive HF mass spectrometer (Thermo Scientific, San Jose CA) using an Easy-Spray source. MS1 scans were acquired at 60 K resolution (AGC target = 3×106; max IT=50 ms). Data-dependent collision induced dissociation MS/MS spectra were acquired using Top speed method (3 s) following each MS1 scan (NCE ~28%; 15 K resolution; AGC target 1×105; max IT 45 ms). Proteins were identified by searching the MS/MS data against UniProt entries using Proteome Discoverer (v2.4, Thermo Scientific). Search parameters included MS1 mass tolerance of 10 ppm and fragment tolerance of 0.2 Da; two missed cleavages were allowed; carbamidomethylation of cysteine was considered fixed modification and oxidation of methionine, deamidation of asparagine and glutamine were considered as potential modifications. False discovery rate (FDR) was determined using Percolator and proteins/peptides with an FDR of ≤ 1% were retained for further analysis.

SEL1L- or HRD1-interacting proteins were selected based on the peptide spectrum matches (PSMs) from the label-free IP-MS results. For each protein hit, the PSM value from the IgG must be 0; the PSMs ratio of the bait KO sample (negative control) to WT sample must be smaller than the ratio of the corresponding bait. Proteomic data has been deposited into a public database PRIDE (PXD043674 and PXD041882).

Native disulfide crosslinking (non-reducing SDS-PAGE)
HEK293T or HEK293F cells transfected with the indicated OS9, SEL1L, or HRD1 variants were harvested and snap-frozen in liquid nitrogen. Cell pellets were resuspended in 1x non-reducing SDS loading buffer (50 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate, 0.01% Bromophenol blue, 10% glycerol), supplemented with 15 mM N-ethylmaleimide (NEM) to alkylate free cysteines and preserve native disulfide-linked species, and proteins were extracted by sonication. Lysates were incubated at room temperature for 15 min, followed by centrifugation at 16,000 × g for 10 min. The resulting supernatants were collected for SDS-PAGE and immunoblot analysis.

DSP crosslinking (non-reducing SDS-PAGE)
HEK293T cells transfected with the indicated HRD1 variants were treated with 2 mM DSP crosslinker (Thermo Science) or DMSO at room temperature for 2 h. The crosslinking reaction was quenched by adding 20 mM Tris-HCl (pH 7.5). Treated cells were then harvested and snap-frozen in liquid nitrogen. Cell pellets were resuspended in 1x non-reducing SDS loading buffer (50 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate, 0.01% Bromophenol blue, 10% glycerol), and proteins were extracted by sonication. Lysates were incubated at room temperature for 15 min, followed by centrifugation at 16,000 × g for 10 min. The resulting supernatants were collected for SDS-PAGE and immunoblot analysis.

Co-immunoprecipitation (Co-IP) in HEK293T cells
HEK293T were snap-frozen in liquid nitrogen and whole cell lysate was prepared in IP buffer (150 mM NaCl, 0.2% NP-40, 0.1% Triton X-100, 25 mM Tris-HCl pH 7.5) for anti-SEL1L-FLAG or anti-HRD1-FLAG IP supplemented with protease inhibitors, and protein phosphatase inhibitors. A total of ~5 mg protein lysates were incubated with 10 μl anti-FLAG agarose (Sigma, #A2220) antibody overnight at 4°C with gentle rocking. The incubated agaroses were washed three times with IP buffer and eluted in 0.1 mg/ml 3xFLAG peptides. The resulting elution was incubated with 5x SDS sample buffer (250 mM Tris-HCl pH 6.8, 10% sodium dodecyl sulfate, 0.05% Bromophenol blue, 50% glycerol, and 1.44 M β-mercaptoethanol) at room temperature for 15 min, followed by SDS-PAGE and immunoblot analysis.

Denaturing immunoprecipitation for ubiquitination assay
HEK293T cells were transfected with proAVP(G57S)-Strep and the indicated HRD1 variants. HEK293F cells were transfected with proAVP(G57S)-Strep, and OS9-SEL1L-HRD1 WT/C2A proteins. The transfected cells were treated with DMSO or 10 μM MG132 for 2 h. Treated cells were snap-frozen in liquid nitrogen and whole cell lysates were prepared in the Triton X-100 lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) with 1% SDS and 5 mM DTT, and denatured at 95°C for 10 min and centrifuged at 16,000 × g for 10 min. Subsequently, supernatants were diluted 1:10 with NP-40 lysis buffer and incubated with 10 μl Strep-Tatin agarose (IBA, #6-6350) overnight at 4°C with gentle rocking. The incubated agaroses were washed three times with the Triton X-100 lysis buffer and eluted in 5 mM Biotin. The resulting elution was incubated with 5x SDS sample buffer (250 mM Tris-HCl pH 6.8, 10% sodium dodecyl sulfate, 0.05% Bromophenol blue, 50% glycerol, and 1.44 M β-mercaptoethanol) at 95oC for 5 min followed by SDS-PAGE and Immunoblot. The samples were loaded into a 6-15% gradient SDS-PAGE gel for separation.

SEL1L and HRD1 disease variants
SEL1L G585D and S658P have been reported previously13,49,70. We recently identified through Lifera Omics Database a novel variant of HRD1 A91D, with unknown significance in a one-year-old boy with congenital heart defects and early-onset pulmonary dysfunction. Human studies were approved by Health Sciences Research University of Virginia (HSR230351).

Statistics
The cryo-EM data collection, refinement and validation statistics are shown in Supplementary Table 4. All experiments have been repeated at least two to three times and/or performed with multiple independent biological samples from which representative data are shown.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this nice piece of work, the authors present the cryo-EM structure of the dimeric OS9-SEL1L-HRD1 complex, in which\nHRD1 dimerizes within the membrane to facilitate substrate translocation, while the SEL1L and OS9 form a claw-like ER\nluminal domain that mediates substrate recruitment. Importantly, a series of biochemical experiments using pathogenic\nSEL1L mutants and disease-associated HRD1 variants support the functional necessity of this dimer formation in ERAD.\nThe structure also reveals two methionine-rich crevices in the TM domain of the HRD1 dimer, which likely serves as\nconductive channels for membrane protein substrates, similar to those in the EMC.\nOverall, the authors appear to have conducted cryo-EM analysis of the dimeric ERAD core complex, along with biochemical\nexperiments based on the structural information, in a sound and rigorous manner. The present data provide new mechanistic\ninsights into the ERAD pathway mediated by the OS9-SEL1L-HRD1 complex, highlighting scientific significance of this\nwork. However, several important issues need to be addressed appropriately with some additional experiments to make this\npaper more convincing and compelling, as described below.\nMajor comments:\n1) The most important issue regarding the ERAD pathway mediated by the dimeric OS9-SEL1L-HRD1 complex is how\ndiverse ERAD substrates are dynamically and efficiently transferred to the retrotranslocation channel formed by the HRD1\ndimer through the OS9-SEL1L luminal domain. This reviewer is particularly interested in the dynamic and, if present,\ncooperative movements of the luminal and transmembrane domains within the ERAD core complex during the substrate\ntransfer. In this context, the authors should discuss in greater detail the relative arrangements and/or spatial proximity\nbetween the OS9-SEL1L luminal domain and the conductive channel of the HRD1 dimer. What is the distance between the\nend of the putative substrate binding region of the OS9-SEL1L luminal domain and the entrance of the conductive channel of\nthe Hrd1 dimer? How dynamic is the luminal domain relative to the transmembrane domain of HRD1? What are the size and\nchemical features of the putative substrate binding region of the OS9-SEL1L luminal domain? What is the diameter of the\nconductive channels formed by the HRD1 dimer? The model shown in Fig. 8 and the text should be revised by incorporating\nthese critical points.\n2) According to the data processing workflow shown in Extended Data Fig. 3, the authors merged Class 1 (far left) and Class\n2 (second from the left) during the 5th heterogenous refinement. However, these two classes appear to have distinct relative\narrangements or orientations of the SEL1L-OS9 luminal domains. Specifically, Class 2 appears to adopt a more open\nluminal domain arrangement than Class 1. Given that, the subsequent data processing of local refinement with a C2\nsymmetry should be performed separately for Class 1 and Class 2, rather than after merging them. This issue is closely\nrelated to the above comment 1.\n3) In the cryo-EM map of the dimeric OS9-SEL1L-HRD1 complex, the local resolution does not appear to be very high for the\nTM domain. The authors should examine whether the density map can further be improved by performing a focused\nrefinement using a mask around the TM domain of the complex as well.\n4) This reviewer has a serious concern regarding the experimental procedure used for the native disulfide crosslinking\nexperiments. The authors resuspended cell pellets in non-reducing SDS loading buffer that lacks thiol-blocking reagents.\nBecause the cytosol is a highly reducing environment due to the abundance of reductants such as glutathione (GSH), cell\nlysis often results in cleavage of disulfide bonds formed in the ER or other oxidative compartments. To prevent such artificial\nreduction, the lysis buffer should contain cysteine-blocking reagents such as NEM. The authors are therefore advised to\nrepeat this assay using a buffer supplemented with a cysteine-blocking reagent.\n5) In Fig 5, the effects of the T93A/F mutations on HRD1 dimerization are assessed solely based on disulfide bond formation\nof the R96C mutant. These data appear to be insufficient. Additional biochemical analysis, such as native-PAGE or size-\nexclusion chromatography, are required to strengthen the conclusion.\nMinor comments:\n6) Line 119; Insert a phrase “in a nearly equal amount” to the end of this sentence.\n7) The positions of the asterisks indicating the retrotranslocation channels formed by the HRD1 dimer do not appear to be\nidentical between Fig. 7a and Extended data Fig. 9a. This should be corrected.\n8) Lines 244 & 245; “Fig 7a” should read “Fig. 7b”. “Extended Data Fig. 9a” should read “Extended Data Fig. 9b”. In this\nconnection, one of the protomers in Fig. 7b should be labeled as Hrd3.", "reviewer_2": "This manuscript presents a cryo EM structure of a human OS9–SEL1L–HRD1 complex, proposes that the HRD1 dimer\nforms the retrotranslocation channel for ERAD L substrates, and supports this model with mutational and substrate\ndegradation data using proAVP(G57S).\nThis is a very valuable dataset and the structural work will be of high interest to the ERAD community. However, the current\nmechanistic conclusions are too far‑reaching, especially in light of the well‑established model for Hrd1 function in ERAD‑L\nin yeast, in which Hrd1 operates monomerically with the Derlin Der1 to form the retrotranslocation channel. I support\npublication in Nature Communications after revisions. The authors should either:\n(1) clearly qualify their model by stating that they have not excluded – and have not even functionally tested – Derlin\ninvolvement for their substrate; or\n(2) directly test Derlin dependence for proAVP(G57S) in their system, and ideally probe additional ERAD‑L substrates to\nassess whether their findings are general.\nAddressing this point, and engaging fully with the existing body of literature in this mature field, is essential before claiming a\nmechanistic divergence from the currently accepted paradigm.\nReasoning and major points:\n1. “Most conserved pathway” – but divergent proposed mechanism\nThe authors emphasize in the Abstract and Introduction that the SEL1L–HRD1 branch is “the most conserved” branch of\nERAD. This is correct: the Hrd1 pathway is the only known branch that handles ER‑luminal substrates (ERAD‑L) in yeast,\nanimals, and plants. Given such evolutionary conservation, conservation of mechanism would be expected for the core\nchannel function.\nIn yeast, ERAD‑L is most likely mediated by monomeric Hrd1 in complex with Der1, with which it forms complementary\n“half‑channels” in the membrane. This mechanism is supported by cryo‑EM structures, functional assays, and even\nin‑membrane substrate photocrosslinking (most importantly: Wu et al. 2020, Science, PMID: 32327568; Pisa and Rapoport\n2022, JBC, PMID: 35970394). The mechanism proposed here — a symmetric HRD1 dimer alone forming the pore with no\nDerlins — differs fundamentally from this well‑supported yeast model. The manuscript does not confront this conflict\nsufficiently or provide a clear rationale for such a dramatic mechanistic divergence in an otherwise highly conserved\npathway. If the differences between their structure and the yeast Hrd1–Der1 channel reflect a true evolutionary\ndivergence, they should explain why and discuss possible functional drivers. Alternatively, the dimer may represent one of multiple\nco‑existing states (as in Guo et al., Nat Commun 2025 PMID: 405938782025), rather than the active retrotranslocon.\n2. Derlin involvement not functionally addressed\nAlso in mammals, multiple studies (for example: Oda et al. 2006, JCB, PMID: 16449189; Huang et al. 2013 JBC, PMID:\n23867461) have shown that Derlin 2/3 can be required for luminal glycoprotein degradation. (Already Lilley and Ploegh\n(2005, PNAS, PMID: 16186509) showed that Derlin-2 forms a complex with HRD1/SEL1L complexes).\nThe authors purify a stable OS9–SEL1L–HRD1 \"core\" without Derlins and infer Derlins are dispensable. But: no Derlin\nknockdown/knockout tests are presented for proAVP(G57S). Furthermore, absence from a detergent purified prep does not\nimply in vivo absence. I find it likely that Derlin association is very sensitive to solubilization conditions and probably lost in\nmost purifications. Purification in Extended Data Fig. 2b in DMNG shows additional bands; judging from the molecular\nweight, these could be HERP1 and DERL2 - please comment.\nThe recent Guo et al. (Nat Commun, 2025, PMID: 40593878) structures seem to contradict the authors’ interpretation: they\nreconstituted Derlin containing HRD1–SEL1L–XTP3B complexes and showed that Derlin presence breaks the HRD1 dimer\nand induces major SEL1L rearrangements, even when Derlin helices are unresolved. This supports Derlins as functional\ncomponents in at least some mammalian ERAD L assemblies. These observations need to be discussed in detail in the\ncurrent manuscript, with explicit consideration of how the Guo et al. findings impact the interpretation of the OS9–SEL1L–\nHRD1 dimer as the active ERAD‑L translocon.\n3. Substrate choice and pathway assignment\nThe sole test substrate is proAVP(G57S). This is an established disease linked ERAD substrate, but the manuscript treats it\nimplicitly as a “pure” ER luminal (ERAD L) substrate without noting caveats. Crucially, it does not cite or discuss the detailed\nbiochemical study by Friberg, Spiess & Rutishauser (JBC 2004, PMID: 14996841). That work showed: (1) Most WT and\nmutant pre pro vasopressin enters the ER lumen, is cleaved by signal peptidase, and is N glycosylated confirming bona fide\nluminal targeting for the majority of molecules. (2) A significant fraction, however, never enters the ER due to inherent\ninefficiency of the native vasopressin signal peptide. These unglycosylated, uncleaved “pre provasopressin” molecules\n(Form 2) remain cytosolic and are also degraded by the proteasome. (3) Misfolded ER imported molecules are\nretrotranslocated and deglycosylated into the cytosol (Form 3), consistent with canonical ERAD L processing. These\nfeatures are present for both WT and mutant precursors, including in neuroendocrine like cells, so G57S has dual\nlocalisation: part ER luminal, part cytosolic. The current manuscript’s degradation and ubiquitination readouts therefore\nmonitor a mixed population, and the fraction of ER imported substrate is not established. This matters when making\nmechanistic claims about ERAD L. My recommendation is to cite Friberg et al., quantify or at least discuss the cytosolic\nmistargeted pool in the presented system, and clarify whether functional readouts here reflect specifically ER imported\nsubstrate.\n4. HRD1 dimerization: interpretation and scope\nThe TM3 mutagenesis data show T93A and T93F disrupt crosslinkable dimer formation and impair proAVP(G57S)\ndegradation without destabilising SEL1L/OS9 binding. However, (1) only one substrate is tested, and — see Friberg et al. —\nit is not exclusively ER luminal. (2) The claim that HRD1 forms a “stable, stoichiometric” dimer is too strong; crosslinking\ncaptures ~40% dimer. It is unclear if this limit is technical or if the rest of HRD1 is simply not engaged in homodimers.\n(3) TM3 mutations could disrupt not only HRD1-HRD1 pairing but also possible HRD1-Derlin contacts, if Derlins contribute to\nthe functional pore. Other subtle allosteric effects on translocation cannot be excluded. From the structural position of Ala91,\nit is not immediately clear by what direct mechanism this substitution would prevent dimerization. This raises the possibility\nof more subtle distortions that could destabilize dimers indirectly or affect other assemblies or interactions.\n5. Dimerization in comparison with the homologous yeast system\nThere is no debate that Hrd1 dimerizes in yeast. Multiple studies have demonstrated this biochemically and/or structurally\n(Horn et al., Mol Cell 2010; Carvalho et al., Cell 2010; Schoebel et al., Nature 2017; Wu et al., Science 2020). The point of\ncontention is not whether dimers exist, but whether they are the functional form for ERAD‑L. In active ERAD‑L in yeast, the\nprevailing model — backed by Wu et al., 2020 and earlier biochemical work — is that Hrd1 functions monomerically with\nDer1, with each protein contributing complementary membrane “half‑channels” to form the pathway for substrate\nretrotranslocation. Symmetric Hrd1–Hrd1 dimers are not thought to be required for luminal‑substrate channel activity.\nDimerization in vivo may nevertheless have other roles, for example in scaffolding, ERAD‑M substrates, or complex stability.\nPisa & Rapoport (2022, JBC, PMID: 35970394) is particularly relevant here and should be discussed explicitly. Using\ncross‑linking in various genetic backgrounds, they concluded that the symmetric Hrd1 dimer is likely off‑pathway or\nnon‑functional for ERAD‑L, and that the active retrotranslocon is the monomeric Hrd1–Der1 complex.\nThe current manuscript’s proposal of a symmetric HRD1 dimer as the mammalian ERAD‑L pore therefore directly\ncontradicts the functional interpretation from the yeast system. This is a mature field, and the authors – in my opinion –\ncannot simply propose a radical mechanistic departure from established, well‑supported models without thoroughly\nengaging with the existing scholarly debate. The authors should address this difference head-on, explaining why a\nsymmetric dimer would be active in mammals when an analogous arrangement in yeast is viewed as off‑pathway, and\nconsidering the plausible alternative that their observed dimer reflects one of several assembly states rather than the sole,\nphysiologically active channel.\n6. Statistical analysis of biochemical data\nSeveral key biochemical figures present quantitative data as averaged values from multiple independent experiments, but\nwithout any indication of variability between replicates or formal statistical analysis. For example, in the crucial Fig. 5d,\naverages from three independent experiments are indicated. Without measures of variance, it is not possible to assess the\nreproducibility and robustness of the reported differences.\nMinor:\n7. When referring to yeast complexes, use yeast gene/protein names (Yos9, Hrd1, Hrd3, Der1). Use mammalian names (OS‑9, HRD1, SEL1L, Derlin‑1/2/3) for human proteins. This avoids confusion when comparing yeast and mammalian results.", "reviewer_3": "The manuscript entitled Structural basis and pathological implications of the dimeric OS9-SEL1L2-HRD1 ERAD Core\nComplex presents the cryo-EM structure of a mammalian ERAD complex comprising OS9, SEL1L and HRD1. The authors\nconcentrate on an ERAD complex that excludes Derlins, EDEMs, and other ERAD components, based on their preliminary\nmass spectrometry and Western blot experiments, which identify the three-component complex as the most stable. They\npropose a model describing a structure formed by two identical monomers held together by the HRD1 dimer within the\nmembrane. The authors speculate that this structure may provide a binding site/conduit for the ERAD substrate. This work is\nsignificant for the field of ER protein degradation, despite the existence of previous studies on similar complexes in yeast or\neven mammals, because the structural features reported here are highly distinct. Although the data provide a valuable\ncontribution to our understanding of ERAD complex structures, the following points should be addressed prior to final\ndecision:\n1. To properly assess the ERAD complex analysis by Western blot, the experiments should be performed in triplicate and\nthe data shared with reviewers. All quantifications should come from independent experiments, not merely technical repeats,\nand should include appropriate statistical analysis.\n2. The authors claim that FAM8A1 deletion has no effect on ERAD function. However, the assessment of ERAD function\nbased on the Western blot shown in (Extended Data Fig. 1e) is inconclusive. The quantification of Ire1, Cd147, and OS9\nlevels in KO cell lines should be based on multiple replicates with statistical significance. Moreover, comparing protein\nlevels across different cell lines is not ideal, as it lacks direct kinetic information. Pulse-chase or cycloheximide chase assay\nwould be more informative in assessing ERAD function.\n3. The data on the disruption of the ERAD complex in the presence of SEL1L mutants are not entirely clear. For example,\ncontrary to the authors' conclusion, the double mutant 155 G585D/S658P does not completely abolish interactions with OS9\n(as seen in lane 5, Fig. 2f), as some interaction with OS9 is still detectable.\n4. To convincingly demonstrate ERAD function in Fig. 5d, the degradation of the ERAD substrate should be followed by\nchase experiments in cells expressing wild-type or mutant forms. Direct comparison of steady-state protein levels of ERAD\nsubstrates across different cell lines is prone to substantial errors.\n5. The authors should discuss how their structural findings compare to previously published data on ERAD complexes,\nparticularly those from mammalian and yeast systems. Several ERAD complex structures have been identified, and these\nstructures differ significantly from one another. A comparison with these existing models would help contextualize the current\nfindings and highlight both commonalities and differences."}, "author_response": "We thank all three reviewers for their insightful and constructive comments. We now have carefully addressed all\nthe comments from the reviewers, which have been instrumental to further improve our manuscript.\nReviewer #1 (Remarks to the Author):\nIn this nice piece of work, the authors present the cryo-EM structure of the dimeric OS9-SEL1L-HRD1\ncomplex, in which HRD1 dimerizes within the membrane to facilitate substrate translocation, while the\nSEL1L and OS9 form a claw-like ER luminal domain that mediates substrate recruitment. Importantly, a\nseries of biochemical experiments using pathogenic SEL1L mutants and disease-associated HRD1\nvariants support the functional necessity of this dimer formation in ERAD. The structure also reveals\ntwo methionine-rich crevices in the TM domain of the HRD1 dimer, which likely serves as\nconductive channels for membrane protein substrates, similar to those in the EMC.\nOverall, the authors appear to have conducted cryo-EM analysis of the dimeric ERAD core complex,\nalong with biochemical experiments based on the structural information, in a sound and rigorous\nmanner. The present data provide new mechanistic insights into the ERAD pathway mediated by the\nOS9-SEL1L-HRD1 complex, highlighting scientific significance of this work. However, several\nimportant issues need to be addressed appropriately with some additional experiments to make this\npaper more convincing and compelling, as described below.\nWe sincerely thank the reviewer for the positive and encouraging comments. We appreciate the\nrecognition of the rigor in our cryo-EM and biochemical analyses using disease variants, as well as the\nsignificance of our findings in elucidating the mechanism of the OS9-SEL1L-HRD1 complex in ERAD.\nMajor comments:\n1) The most important issue regarding the ERAD pathway mediated by the dimeric OS9-SEL1L-HRD1\ncomplex is how diverse ERAD substrates are dynamically and efficiently transferred to the\nretrotranslocation channel formed by the HRD1 dimer through the OS9-SEL1L luminal domain. This\nreviewer is particularly interested in the dynamic and, if present, cooperative movements of the luminal\nand transmembrane domains within the ERAD core complex during the substrate transfer. In this context,\nthe authors should discuss in greater detail the relative arrangements and/or spatial proximity between\nthe OS9-SEL1L luminal domain and the conductive channel of the HRD1 dimer. What is the distance\nbetween the end of the putative substrate binding region of the OS9-SEL1L luminal domain and the\nentrance of the conductive channel of the Hrd1 dimer? How dynamic is the luminal domain relative to\nthe transmembrane domain of HRD1? What are the size and chemical features of the putative substrate\nbinding region of the OS9-SEL1L luminal domain? What is the diameter of the conductive channels\nformed by the HRD1 dimer? The model shown in Fig. 8 and the text should be revised by incorporating\nthese critical points.\nAs suggested, we have expanded our discussion to address the relative arrangement, spatial proximity,\nand dynamic behavior of the OS9-SEL1L luminal domain in relation to the potential conductive channel\nof the HRD1 dimer. These key points have also been incorporated into the revised figures and text.\n(i) Spatial arrangement: Based on structural measurements, the distance between the end of the\nputative substrate-binding region of the OS9-SEL1L luminal domain and the entrance to the HRD1 dimer\nchannel is approximately 58 Å (Response Fig. 1a). The substrate-binding pocket within OS9-SEL1L\nmeasures 36 Å in diameter, whereas the entrance and conductive channel formed by the HRD1 dimer\nmeasure 12 Å and 18 Å, respectively (Response Fig. 1a).\n(ii) Chemical features: Electrostatic surface analysis revealed that the putative substrate-binding\npocket of the OS9-SEL1L complex is enriched in negative electrostatic potential (red), suggesting a\npreference for binding positively charged substrate regions (Response Fig. 1b).\n(iii) Conformational dynamics: To probe the dynamics within the ERAD core complex, we performed 3D\nvariability analysis in cryoSPARC, focusing on the luminal regions of SEL1L and OS9 to visualize\ncontinuous conformational heterogeneity (Response Fig. 1c). Using the 3D Variability Display in Simple\nmode, we generated 20 volumes along each variability component and examined low-pass–filtered\nmaps at 10 Å resolution. Representative first and last frames showing the most prominent\nconformational changes are displayed in Response Fig. 1c.\nWe observed clear intersubunit motions within the luminal domains of SEL1L and OS9 (Response Fig.\n1d), relative to the HRD1 dimer. Specifically, the SEL1L fibronectin type II (FNII) and Sel1-like repeat (SLR)\ndomains undergo rotations along multiple\naxes, while the OS9 MRH and 4β domains\nexhibit comparable flexibility. These\ncoordinated rotations generate alternating\ncompact and open conformations of the OS9-\nSEL1L “substrate claw,” which may facilitate\nsubstrate engagement and transfer to the\nHRD1 channel.\nWe have now updated our Results and added\nthe corresponding discussion. Response Fig.\n1a is now incorporated into Fig. 7a, Response\nFig. 1b is incorporated into Extended Data\nFig. 10a, and Response Fig. 1c-d is\nincorporated into Extended Data Fig. 5b-c in\nthe revised manuscript.\nResponse Figure 1. The distance and the dynamics of OS9-\nSEL1L-HRD1 ERAD core complex. (a) The measurements of\nthe distance between the end of the putative substrate-\nbinding region of the OS9-SEL1L luminal domain and the\nentrance of the HRD1 dimer channel, and the diameter of the\nconductive channels formed by the HRD1 dimer. (b)\nElectrostatic surface analysis of putative OS9-SEL1L\nsubstrate (green dotted line)-binding region. Red color\nindicates a region of negative potential charge, while the blue\ncolor indicates a region of positive potential charge. (c) 3D\nvariability analysis workflow used to determine OS9-SEL1L\ndynamics. (d) Conformational changes observed for the\nindicated OS9-SEL1L luminal domains using 10Å low-pass\nfiltered map for visualization.\nResults:\nLine 242: To understand how the core complex engages misfolded substrates, we examined the spatial\narrangement of OS9-SEL1L relative to the HRD1 channel. Our structure reveals that the distal end of the\nputative substrate-binding region of OS9-SEL1L lies ~58 Å from the entrance of the HRD1 channel (Fig.\n7a). The ~36 Å OS9-SEL1L pocket aligns precisely with the 12 Å pore entrance and the 18 Å HRD1 dimer\nchannel, positioning the luminal module for efficient substrate delivery. Electrostatic analysis shows\nthat OS9-SEL1L contains a negatively charged substrate-binding pocket (Extended Data Fig. 10a).\nLine 177: Moreover, 3D variability analysis reveals coordinated motions within the OS9-SEL1L luminal\n“substrate claw,” which alternates between compact and open conformations that likely facilitate\ndynamic substrate capture and handover to HRD1 (Extended Data Fig. 5b-c).\n2) According to the data processing workflow shown in Extended Data Fig. 3, the authors merged Class 1\n(far left) and Class 2 (second from the left) during the 5th heterogenous refinement. However, these two\nclasses appear to have distinct relative arrangements or orientations of the SEL1L-OS9 luminal domains.\nSpecifically, Class 2 appears to adopt a more open luminal domain arrangement than Class 1. Given\nthat, the subsequent data processing of local refinement with a C2 symmetry should be performed\nseparately for Class 1 and Class 2, rather than after merging them. This issue is closely related to the\nabove comment 1.\nWe thank the reviewer for their helpful suggestions on particle selection prior to reconstruction of the\ncomplex. We now have reanalyzed our dataset to assess whether processing particles with more\nconstrained intersubunit motion would improve local map quality. We first conducted a 3D variability\nanalysis using a focused mask on the full OS9-SEL1L-HRD1 dimer (Response Fig. 2a). Consistent with\nour earlier analysis, we observed flexibility within both SEL1L and OS9 protomers of the ERAD complex in\na subset of particles (grey classes, flexible areas highlighted in red-shaded regions, Response Fig. 2a).\nParticles exhibiting limited flexibility in the luminal domains of SEL1L and OS9 were combined and\nreconstructed using non-uniform refinement as implemented in CryoSPARC (Map 2) (Response Fig. 2a).\nWe then performed symmetry expansion to allow increase dataset based on group symmetry and then\nfollowed by local refinement on these particles to improve the local resolution. Comparison of the\nresulting maps to our previous map which used all ERAD particles, revealed no major differences in the\nrelative arrangement of the luminal domains (shown in overlay, Response Fig. 2b). However, we noted a\ndecrease in the overall map\nquality after classification,\nparticularly the loss of high-\nresolution features that were\npreviously visible (Response Fig.\n2c), which supports independent\ndynamics between SEL1L and\nHRD1 homodimers.\nResponse Figure 2. Particle classification\nscheme using 3D variability analysis in\nCryoSPARC. (a) Mask and workflow used\nfor the particle classification. Particles from\nclasses 1-3 (purple) were combined for the\nfinal reconstruction (Map 2). (b) Overlay of\nthe map reconstructed from all ERAD\nparticles and the resulting map following\nclassification. Maps were low pass filtered\nto 10Å for visualization. (c) Closeup of the\nSEL1L-OS9 interface comparing the local\nmap quality of the original map and Map 2\nfollowing classification.\n3) In the cryo-EM map of the dimeric OS9-SEL1L-HRD1 complex, the local resolution does not appear to\nbe very high for the TM domain. The authors should\nexamine whether the density map can further be\nimproved by performing a focused refinement using a\nmask around the TM domain of the complex as well.\nWe thank the reviewer for this insightful comment. We\nperformed a focused refinement as suggested by the\nreviewer using a mask around the HRD1 dimer after\nsignal subtraction of the SEL1L and OS9 luminal\ndomains (Response Fig. 3). This strategy did not yield\nimproved density and led to either an over-refinement of\nthe transmembrane domain of HRD1 (Response Fig. 3).\nResponse Figure 3. Focused refinement of HRD1 transmembrane\nhelices. (a) Masks generated for the signal subtraction of OS9-SEL1L\nluminal domains and the focused refinement of the HRD1 dimer. (b) Processing workflow used for the focused refinement of HRD1. (c)\nCloseup of the HRD1 dimer map following focused refinement.\n4) This reviewer has a serious concern regarding the experimental procedure used for the native disulfide\ncrosslinking experiments. The authors resuspended cell pellets in non-reducing SDS loading buffer that\nlacks thiol-blocking reagents. Because the cytosol is a highly reducing environment due to the\nabundance of reductants such as glutathione (GSH), cell lysis often results in cleavage of disulfide bonds\nformed in the ER or other oxidative compartments. To prevent such artificial reduction, the lysis buffer\nshould contain cysteine-blocking reagents such as NEM. The authors are therefore advised to\nrepeat this assay using a buffer supplemented with a cysteine-blocking reagent.\nAs suggested, we have now repeated the native disulfide crosslinking assays in the presence of the\ncysteine-blocking reagent NEM (at 15 mM) to ensure that the observed bands reflect bona fide disulfide-\nlinked species. Below we present a representative experiment examining HRD1 dimerization (Response\nFig. 4). Response Fig. 4a-b are now shown in Fig. 4d. We have updated all relevant figures associated\nwith the native disulfide crosslinking assays (Fig. 3d, 4d, 5b, 6d) in the revised manuscript.\nResponse Figure 4. Native disulfide crosslinking analysis of HRD1\ndimerization. (a-b) (Non-)Reducing and Western blot analysis of the native\ndisulfide crosslinking assays in HRD1-/- HEK293T cells transfected with the\nindicated HRD1 mutants, with quantitation of the percent of dimeric HRD1\nin total HRD1 shown in (b) (three independent repeats). Cell lysates were\ntreated with treated with 15 mM N-ethylmaleimide (NEM) to preserve native\ndisulfide-linked species during sample preparation. Values, mean ± SEM.\nn.s., not significant, ****p<0.0001 by One-way ANOVA analysis with\ncomparison to WT.\nResults:\nLine 169: To avoid artifactual crosslinking during lysis,\ncells were directly lysed in denaturing SDS buffer lacking\nreducing agents and containing a cysteine-alkylating\nreagent N-ethylmaleimide (NEM), instead of non-\ndenaturing detergents such as Triton X-100 or NP-40.\n5) In Fig 5, the effects of the T93A/F mutations on HRD1 dimerization are assessed solely based on\ndisulfide bond formation of the R96C mutant. These data appear to be insufficient. Additional\nbiochemical analysis, such as native-PAGE or size-exclusion chromatography, are required to strengthen\nthe conclusion.\nWe thank the reviewer for this constructive comment. In addition to the disulfide crosslinking assays,\nwe have now performed co-IP experiments by co-expressing the HRD1 and its T93A or T93F mutant with two\ndistinct epitope tags (FLAG and Strep) (Response Fig. 5a). These results demonstrate that both the T93A\nand T93F mutations significantly impair HRD1-HRD1 interactions (Response Fig. 5a-b). Response Fig. 5\nis now added into Fig. 5c.\nResponse Figure 5. HRD1 T93 is important for HRD1-HRD1\ninteraction in cells. (a-b) Immunoprecipitation of FLAG-agarose in\nHRD1-/- HEK293T cells transfected with Flag-tagged HRD1-WT, Strep-\ntagged HRD1 WT, T93A or T93F, to examine HRD1-HRD1 interaction,\nwith quantitation shown in (b) (three independent repeats). Values,\nmean ± SEM. ***p<0.001 and ****p<0.0001 by one-way ANOVA test\nwith comparison to WT (b).\nResults:\nLine 208: Consistently, both T93A and T93F\nmarkedly reduced HRD1-HRD1 interaction in\ntransfected HEK293T cells (Fig. 5c).\nMinor comments:\n6) Line 119; Insert a phrase “in a nearly equal amount” to the end of this sentence.\n7) The positions of the asterisks indicating the retrotranslocation channels formed by the HRD1 dimer do\nnot appear to be identical between Fig. 7a and Extended data Fig. 9a. This should be corrected.\n8) Lines 244 & 245; “Fig 7a” should read “Fig. 7b”. “Extended Data Fig. 9a” should read “Extended Data\nFig. 9b”. In this connection, one of the protomers in Fig. 7b should be labeled as Hrd3.\nWe thank the reviewer for catching these errors. We have corrected all the points above.\nReviewer #2 (Remarks to the Author)\nThis manuscript presents a cryo EM structure of a human OS9–SEL1L–HRD1 complex, proposes that the\nHRD1 dimer forms the retrotranslocation channel for ERAD L substrates, and supports this model with\nmutational and substrate degradation data using proAVP(G57S).This is a very valuable dataset and the\nstructural work will be of high interest to the ERAD community. However, the current\nmechanistic conclusions are too far-reaching, especially in light of the well-established model for Hrd1 function in\nERAD-L in yeast, in which Hrd1 operates monomerically with the Derlin Der1 to form the\nretrotranslocation channel. I support publication in Nature Communications after revisions.\nThe authors should either:\n(1) clearly qualify their model by stating that they have not excluded – and have not even functionally\ntested – Derlin involvement for their substrate; or\n(2) directly test Derlin dependence for proAVP(G57S) in their system, and ideally probe additional ERAD-L\nsubstrates to assess whether their findings are general.\nAddressing this point, and engaging fully with the existing body of literature in this mature field, is\nessential before claiming a mechanistic divergence from the currently accepted paradigm.\nWe thank the reviewer for his enthusiasm and constructive comments. In our revised manuscript, we\nhave now carefully toned down our mechanistic interpretation and discussed that our current data do not\nexclude the possible involvement of Derlin proteins in substrate retrotranslocation. In addition, we have\nprobed more substrates and incorporated more references as suggested by this reviewer.\nReasoning and major points:\n1. “Most conserved pathway” – but divergent proposed mechanism\nThe authors emphasize in the Abstract and Introduction that the SEL1L–HRD1 branch is “the most\nconserved” branch of ERAD. This is correct: the Hrd1 pathway is the only known branch that handles\nER-luminal substrates (ERAD-L) in yeast, animals, and plants. Given such evolutionary conservation,\nconservation of mechanism would be expected for the core channel function.\nIn yeast, ERAD-L is most likely mediated by monomeric Hrd1 in complex with Der1, with which it forms\ncomplementary “half-channels” in the membrane. This mechanism is supported by cryo-EM structures,\nfunctional assays, and even in-membrane substrate photocrosslinking (most importantly: Wu et al. 2020,\nScience, PMID: 32327568; Pisa and Rapoport 2022, JBC, PMID: 35970394). The mechanism proposed\nhere — a symmetric HRD1 dimer alone forming the pore with no Derlins — differs fundamentally from\nthis well-supported yeast model. The manuscript does not confront this conflict sufficiently or provide a\nclear rationale for such a dramatic mechanistic divergence in an otherwise highly conserved pathway. If\nthe differences between their structure and the yeast Hrd1–Der1 channel reflect a true evolutionary\ndivergence, they should explain why and discuss possible functional drivers. Alternatively, the dimer may\nrepresent one of multiple co-existing states (as in Guo et al., Nat Commun 2025 PMID: 405938782025),\nrather than the active retrotranslocon.\nWe fully agree that mechanistic conservation would indeed be expected for SEL1L-HRD1 ERAD. Using\ncryo-EM and biochemical analyses powered by the incorporation of patient disease variants, the current\nstudy showed a HRD1 dimer capable of forming a pore, independently of Derlin proteins. That said, we do\nnot exclude the possibility that alternative configurations, such as HRD1-Derlin complexes analogous to\nthose seen in yeast, may also exist under certain physiological contexts.\nGiven the differences in substrate diversity between yeast and mammalian cells, it is conceivable that\nHRD1 may adopt multiple assembly states tuned to distinct ERAD clients or cellular conditions. Indeed,\nin a recent study by Guo et al., Nat Commun, 2025, Derlin proteins were not resolved within the SEL1L-\nHRD1 assembly, suggesting that they may not form a stable component of this core complex under the\nexamined conditions. We are actively pursuing the configuration of endogenous ERAD complexes in\nmammals, which should clarify how these states interconvert and their relevance to substrate selection\nand retrotranslocation activity.\nIn the revised manuscript, we have toned down our mechanistic interpretation throughout the\nmanuscript and expanded the Discussion to include the roles of Derlin proteins and the HRD1 dimer in\nmammalian ERAD. Below are two examples:\nAbstract:\nLine 41: Furthermore, pathogenic SEL1L mutations at the SEL1L-OS9 (Gly585Asp) and SEL1L-HRD1\n(Ser658Pro) interfaces disrupt complex formation and impair ERAD activity. A newly identified disease-\nassociated HRD1 variant (Ala91Asp), located in transmembrane helix 3, impairs HRD1 dimerization and\nsubstrate processing, highlighting the functional relevance of this interface. These findings establish a\nstructural framework for mammalian SEL1L-HRD1 ERAD and elucidate how mutations destabilizing this\nmachinery contribute to human disease.\nResults:\nLine 267: Our structure also differs from the recently reported mammalian XTP3B-SEL1L-HRD1 complex 1,\nwhich captured an asymmetric dimer in which only one HRD1 protomer binds XTP3B-SEL1L (2:1:1\nstoichiometry) (Fig. 7h). That study proposed that Derlin engagement may trigger HRD1 dimer dissociation,\nalthough Derlin itself was not visualized. Together, these observations suggest that the HRD1 dimer likely\nrepresents one of several coexisting assembly states, with transitions potentially regulated by substrate\nload, Derlin engagement, or broader ERAD pathway dynamics.\n2. Derlin involvement not functionally addressed\nAlso in mammals, multiple studies (for example: Oda et al. 2006, JCB, PMID: 16449189; Huang et al.\ndegradation. (Already Lilley and Ploegh (2005, PNAS, PMID: 16186509) showed that Derlin-2 forms a\ncomplex with HRD1/SEL1L complexes). The authors purify a stable OS9–SEL1L–HRD1 \"core\" without\nDerlins and infer Derlins are dispensable. But: no Derlin knockdown/knockout tests are presented for\nproAVP(G57S).\nWe appreciate the reviewer’s insightful comment and agree that several prior studies have implicated\nDerlin-2 and -3 in mammalian ERAD of luminal glycoproteins – we have incorporated these references in\nthe revised manuscript. Indeed, the loss of both Derlin-1 and Derlin-2, but not each alone, led to marked\naccumulation of proAVP G57S* and inhibition of its degradation in HEK293T cells (Response Fig. 6),\nindicating that Derlin-1 and -2 proteins are indeed required for ERAD of proAVP G57S*. Functional and\nstructural interrogation of Derlin dependence, including targeted knockdown and knockout analyses, is\nan important next step. These studies are underway in our laboratory as part of a broader effort to define\nthe composition and dynamics of endogenous ERAD complexes in mammalian cells.\nResponse Figure 6. DERL1 and DERL2 are\nrequired for ERAD of proAVP G57S*. (a-b)\nCycloheximide (CHX) chase analysis of the\nsubstrates proAVP(G57S) in WT, HRD1-/- and\nDerlin-1-/-Derlin-2-/- HEK293T cells, with\nquantitation shown in (b). Values, mean ±\nSEM. ***p<0.001 and ****p<0.0001 by two-\nway ANOVA test with comparison to WT (b).\nFurthermore, absence from a\ndetergent purified prep does not\nimply in vivo absence. I find it likely\nthat Derlin association is very sensitive to solubilization conditions and probably lost in most\npurifications. Purification in Extended Data Fig. 2b in DMNG shows additional bands; judging from the\nmolecular weight, these could be HERP1 and DERL2 - please comment.\nWe appreciate the reviewer’s comments and agree that Derlin association is likely sensitive to detergent\nsolubilization and may be disrupted under certain purification conditions. Indeed, the additional faint\nbands observed in the DMNG preparation (Extended Data Fig. 2b) could correspond to accessory\nproteins such as HERP1 or Derlin-2, although their identities could not be confirmed by mass\nspectrometry due to low abundance.\nThe recent Guo et al. (Nat Commun, 2025, PMID: 40593878) structures seem to contradict the authors’\ninterpretation: they reconstituted Derlin containing HRD1–SEL1L–XTP3B complexes and showed that\nDerlin presence breaks the HRD1 dimer and induces major SEL1L rearrangements, even when Derlin\nhelices are unresolved. This supports Derlins as functional components in at least some mammalian\nERAD L assemblies. These observations need to be discussed in detail in the current manuscript, with\nexplicit consideration of how the Guo et al. findings impact the interpretation of the OS9–SEL1L–\nHRD1 dimer as the active ERAD-L translocon.\nWe appreciate the reviewer’s thoughtful comment. The reasons that Guo et al. (Nat. Commun., 2025;\nPMID: 40593878) were unable to resolve Derlin densities in their maps were likely due to Derlin proteins\nbeing substoichiometric, transiently associated, or conformationally heterogeneous under their\nreconstitution conditions. In addition, in the HRD1 structure used for Derlin study by Guo et al., HRD1\nwas truncated (residues 1-341), lacking the C-terminal cytosolic region beyond the RING domain.\nTherefore, the truncated HRD1 used in the Guo et al. reconstitution may not recapitulate the full\narchitecture or activity of the native HRD1-Derlin complex. Thus, the precise structural organization and\nstoichiometry of Derlin within the mammalian ERAD machinery remain undetermined.\nIn the revised Discussion, we have now compared our structure with the Derlin-associated complex\nreported by Guo et al. and highlighted the possibility that the HRD1 dimer we captured may represent a\ncoexisting or transitional state of the ERAD machinery. We have also refined our mechanistic\ninterpretation and expanded the Discussion to incorporate the potential roles of Derlin proteins in the\nrevised manuscript.\nResults:\nLine 267: Our structure also differs from the recently reported mammalian XTP3B-SEL1L-HRD1 complex 1,\nwhich captured an asymmetric dimer in which only one HRD1 protomer binds XTP3B-SEL1L (2:1:1\nstoichiometry) (Fig. 7h). That study proposed that Derlin engagement may trigger HRD1 dimer dissociation,\nalthough Derlin itself was not visualized. Together, these observations suggest that the HRD1 dimer likely\nrepresents one of several coexisting assembly states, with transitions potentially regulated by substrate\nload, Derlin engagement, or broader ERAD pathway dynamics.\nDiscussion:\nLine 303: Recent work by Guo et al. 1 reported a Derlin-engaged XTP3B-SEL1L-HRD1 structure, but the\nHRD1 construct used lacked a substantial portion of the cytosolic C-terminal region required for ERAD\nactivity and Derlin recruitment 2,3. Consistent with this, Derlin is absent from our purified OS9-SEL1L-HRD1\ncore complex even when Derlin and HERP1 are overexpressed, supporting a model in which Derlin\nfunctions downstream of HRD1 in mammals to recruit the cytosolic p97 ATPase, as previously suggested\n4-7. Together, these findings suggest that the HRD1 dimer captured here likely represents one of several\ncoexisting assembly states in the mammalian ERAD pathway, potentially corresponding to a\nretrotranslocation-competent intermediate.\n3. Substrate choice and pathway assignment\nThe sole test substrate is proAVP(G57S). This is an established disease linked ERAD substrate, but the\nmanuscript treats it implicitly as a “pure” ER luminal (ERAD L) substrate without noting caveats. Crucially,\nit does not cite or discuss the detailed biochemical study by Friberg, Spiess & Rutishauser (JBC 2004,\nPMID: 14996841). That work showed: (1) Most WT and mutant pre pro vasopressin enters the ER lumen,\nis cleaved by signal peptidase, and is N glycosylated confirming bona fide luminal targeting for the\nmajority of molecules. (2) A significant fraction, however, never enters the ER due to inherent\ninefficiency of the native vasopressin signal peptide. These unglycosylated, uncleaved “pre provasopressin”\nmolecules (Form 2) remain cytosolic and are also degraded by the proteasome. (3) Misfolded ER\nimported molecules are retrotranslocated and deglycosylated into the cytosol (Form 3), consistent with\ncanonical ERAD L processing. These features are present for both WT and mutant precursors, including\nin neuroendocrine like cells, so G57S has dual localisation: part ER luminal, part cytosolic. The current\nmanuscript’s degradation and ubiquitination readouts therefore monitor a mixed population, and the\nfraction of ER imported substrate is not established. This matters when making mechanistic claims\nabout ERAD L. My recommendation is to cite Friberg et al., quantify or at least discuss the cytosolic\nmistargeted pool in the presented system, and clarify whether functional readouts here reflect\nspecifically ER imported substrate.\nWe thank the reviewer for bring this to our attention. We have now cited the Friberg paper (PMID:\n14996841) in the revised manuscript. To prevent dual localization of proAVP(G57S), we have tagged a C-\nterminal ER-retention signal (KDEL) to proAVP(G57S) protien (proAVP G57S*) (Response Fig. 7a). We now\nshow that the majority of proAVP protein is N-glycosylated and retained\nwithin the ER as evidenced by its Endo H sensitivity (Response Fig. 7b).\nResponse Fig. 7 has been added into Extended Data Fig. 2c-d.\nResponse Figure 7. proAVP G57S* is an ER-retained ERAD substrate. (a) The diagram showing the\nmotifs of proAVP G57S* we used for this manuscript.SP, Signal Peptide; Y, N-glycosylation; KDEL,\nER-retention signal. Of note, we incorporated a C-terminal KDEL tag to efficiently retain the protein in\nthe ER. (b) Westen blot to analysis the N-glycosylation (gly) of proAVP(G57S) following EndoH and\nPNGase F digestions. SEL1L is used as a control.\nResults:\nLine 110: We next expressed a known ERAD substrate pro-arginine\nvasopressin (proAVP) carrying the Gly57Ser disease mutation 8,9, tagged with\nan ER-retention signal KDEL to present secretion (proAVP G57S*, Extended\nData Fig. 2c-d).\n4. HRD1 dimerization: interpretation and scope\nThe TM3 mutagenesis data show T93A and T93F disrupt crosslinkable dimer formation and impair\nproAVP(G57S) degradation without destabilising SEL1L/OS9 binding. However, (1) only one substrate is\ntested, and — see Friberg et al. — it is not exclusively ER luminal. (2) The claim that HRD1 forms a “stable,\nstoichiometric” dimer is too strong; crosslinking captures ~40% dimer. It is unclear if this limit is\ntechnical or if the rest of HRD1 is simply not engaged in homodimers. (3) TM3 mutations could disrupt\nnot only HRD1-HRD1 pairing but also possible HRD1-Derlin contacts, if Derlins contribute to the\nfunctional pore. Other subtle allosteric effects on translocation cannot be excluded. From the structural\nposition of Ala91, it is not immediately clear by what direct mechanism this substitution would prevent\ndimerization. This raises the possibility of more subtle distortions that could destabilize dimers indirectly\nor affect other assemblies or interactions.\nWe thank the reviewer for these valuable comments. (1) To strengthen our conclusions, we have\nexamined an additional ERAD-L substrate, POMC C28F, which we previously reported as a SEL1L-HRD1\nERAD-L substrate 10 and engineered to contain an ER-retention signal KDEL (POMC C28F*). Consistent\nwith the results observed for proAVP G57S*, expression of HRD1 T93A or T93F mutants in HRD1-/-cells led\nto a marked accumulation of POMC C28F* (Response Fig. 8a) and inhibited its turnover (Response Fig.\n8b). Response Fig. 8 are now updated in\nExtended Data Fig. 8 of the revised text.\nResponse Figure 8. HRD1 T93A/F mutants are\ndefective in ERAD. (a) Western blot analysis of ERAD\nsubstrate POMC C28F* in HRD1-/- HEK293T cells\ntransfected with POMC C28F*-HA and indicated HRD1\nmutants, with quantitation of POMC C28F* protein levels\nshown below (three independent repeats). (b)\nCycloheximide (CHX) chase analysis of POMC C28F* in\nHRD1-/-HEK293T cells transfected with POMC C28F*-HA\nand indicated HRD1 mutants, with quantitation shown\nbelow (three independent repeats). Values, mean ± SEM.\n****p<0.0001 by one-way ANOVA test with comparison to\nWT (a), and two-way ANOVA test with comparison to WT\n(b).\nResults:\nLine 216: Similar defects were observed with another ER-retained model substrate, POMC C28F* 10\n(Extended Data Fig. 8a-b).\nLine 228: Similar results were obtained for POMC C28F* (Extended Data Fig. 8a and 9b).\n(2) Regarding the statement that HRD1 forms a “stable, stoichiometric” dimer, we have removed it in the\nrevised text. Moreover, although our crosslinking assays capture approximately 40% of the HRD1\npopulation as dimers, we recognize that both native disulfide and DSP crosslinking methods have\ninherent technical limitations that may lead to incomplete efficiency.\n(3) To assess whether the TM3 mutants (T93A, T93F, A91D)\ndisrupt potential HRD1-Derlin interactions, we performed\nIP assays with Derlin-2. The results indicate that these\nTM3 mutations do not affect the HRD1-Derlin-2\ninteraction (Response Fig. 9a-b). We are currently\ninvestigating the detail molecular mechanisms underlying\nHRD1’s association with Derlin proteins. Hence, the data\non SEL1L and OS9 are shown in Fig. 5g and 6e in the\nrevised manuscript, while the data on Derlin-2 is not.\nFor the A91D mutant, replacing a nonpolar residue (A)\nwith a negatively charged residue (D) would introduce\nboth steric hindrance and electrostatic repulsion in HRD1\ndimer interface. Such changes could also locally perturb\nthe orientation of TM3, thereby weakening HRD1-HRD1\ninteraction. We have updated this in the revised text.\nResponse Figure 9. HRD1 TM3 mutants (T93A, T93F, A91D) do not affect\nthe interaction with SEL1L, OS9, and Derlin-2. (a-b) Immunoprecipitation of\nFLAG-agarose in HRD1-/- HEK293T cells transfected indicated HRD1 mutants\nto examine their interaction with SEL1L, OS9, and Derlin-2, with quantitation\nshown on the bottom (three independent repeats). Values, mean ± SEM. n.s.,\nnot significant, by one-way ANOVA test with comparison to each other (a), and Student t-test with comparison to WT (b).\nResults:\nLine 231: Substituting a nonpolar residue (A) with a negatively charged residue (D) would be expected to\nintroduce both steric hindrance and electrostatic repulsion, potentially destabilizing HRD1 dimers.\n5. Dimerization in comparison with the homologous yeast system\nThere is no debate that Hrd1 dimerizes in yeast. Multiple studies have demonstrated this biochemically\nand/or structurally (Horn et al., Mol Cell 2010; Carvalho et al., Cell 2010; Schoebel et al., Nature 2017;\nWu et al., Science 2020). The point of contention is not whether dimers exist, but whether they are the\nfunctional form for ERAD-L. In active ERAD-L in yeast, the prevailing model — backed by Wu et al., 2020\nand earlier biochemical work — is that Hrd1 functions monomerically with Der1, with each protein\ncontributing complementary membrane “half-channels” to form the pathway for substrate\nretrotranslocation. Symmetric Hrd1–Hrd1 dimers are not thought to be required for luminal-substrate\nchannel activity. Dimerization in vivo may nevertheless have other roles, for example in scaffolding,\nERAD-M substrates, or complex stability.\nPisa & Rapoport (2022, JBC, PMID: 35970394) is particularly relevant here and should be discussed\nexplicitly. Using cross-linking in various genetic backgrounds, they concluded that the symmetric Hrd1\ndimer is likely off-pathway or non-functional for ERAD-L, and that the active retrotranslocon is the\nmonomeric Hrd1–Der1 complex.\nThe current manuscript’s proposal of a symmetric HRD1 dimer as the mammalian ERAD-L pore therefore directly\ncontradicts the functional interpretation from the yeast system. This is a mature field, and the authors – in my opinion –\ncannot simply propose a radical mechanistic departure from established, well-supported models without thoroughly\nengaging with the existing scholarly debate. The authors should address this difference head-on, explaining why a\nsymmetric dimer would be active in mammals when an analogous arrangement in yeast is viewed as\noff-pathway, and considering the plausible alternative that their observed dimer reflects one of several\nassembly states rather than the sole, physiologically active channel.\nWe fully acknowledge and now have discussed extensively in the revised manuscript that our proposal of\na symmetric HRD1 dimer differs from the prevailing active yeast model, in which monomeric Hrd1\nfunctions with Der1 to form complementary “half-channels” for luminal substrate retrotranslocation.\nGiven that Pisa and Rapoport demonstrated that in yeast, TM3 of Hrd1 mediates both Hrd1-Der1 and\nHrd1-Hrd1 interactions, we now have examined whether disruption of the dimer interface in human\nHRD1 affects its association with Derlin proteins. As shown in Response Fig. 9a, T93 mutations has no\nimpact on the HRD1-Derlin-2 interaction, indicating that Derlin proteins likely do not engage HRD1 via\nTM3 and pointing to distinct molecular interfaces between the yeast and mammalian systems.\nMoreover, unlike the yeast Hrd1 dimer, which forms a laterally sealed, inactive configuration, our cryo-EM\nstructure reveals that the mammalian HRD1 dimer adopts an open architecture, with continuous\nmethionine-lined cavities spanning the dimer interface. This arrangement suggests the potential for a\nsubstrate-conducting channel rather than a mutually occluded assembly. Importantly, Derlin was not\ndetected in our purified complex, even when co-expressed with its co-factor HERP1, consistent with\nprior models that Derlin acts downstream of HRD1 to recruit the cytosolic ATPase p97 4-7.\nTaken together, while we agree that the HRD1 dimer may not represent the sole active form of the\nmammalian ERAD machinery, our data support the view that the dimeric OS9-SEL1L-HRD1 complex\nconstitutes a core or transitional assembly state, potentially competent for the retrotranslocation of\nERAD substrates. We have incorporated this discussion and clarified that our findings do not contradict\nthe established yeast model but rather reflect possible mechanistic divergence and specialization of the\nmammalian ERAD system.\nDiscussion:\nLine 298: The importance of HRD1 dimerization in ERAD has long been debated. In yeast, Hrd1 dimers are\nconsidered inactive because their lateral gates block Der1 engagement, which is required for\nretrotranslocating luminal ERAD-L substrates 11-13. In contrast, the mammalian HRD1 dimer visualized here\nadopts a fundamentally different arrangement: instead of two sealed protomers, it forms an open,\nmethionine-lined groove that extends across the interface, creating a plausible conduit for substrate\nmovement (Fig. 8a-b). Recent work by Guo et al. 1 reported a Derlin-engaged XTP3B-SEL1L-HRD1 structure,\nbut the HRD1 construct used lacked a substantial portion of the cytosolic C-terminal region required for ERAD\nactivity and Derlin recruitment 2,3. Consistent with this, Derlin is absent from our purified OS9-SEL1L-\nHRD1 core complex even when Derlin and HERP1 are overexpressed, supporting a model in which Derlin\nfunctions downstream of HRD1 in mammals to recruit the cytosolic p97 ATPase, as previously suggested\n4-7. Together, these findings suggest that the HRD1 dimer captured here likely represents one of several\ncoexisting assembly states in the mammalian ERAD pathway, potentially corresponding to a\nretrotranslocation-competent intermediate.\n6. Statistical analysis of biochemical data\nSeveral key biochemical figures present quantitative data as averaged values from multiple independent\nexperiments, but without any indication of variability between replicates or formal statistical analysis. For\nexample, in the crucial Fig. 5d, averages from three independent experiments are indicated. Without\nmeasures of variance, it is not possible to assess the reproducibility and robustness of the reported\ndifferences.\nIn the revised version, we have included quantifications from at least three independent experiments\nwith detailed descriptions of the statistical analyses provided in the corresponding figure legends.\nMinor:\n7. When referring to yeast complexes, use yeast gene/protein names (Yos9, Hrd1, Hrd3, Der1). Use\nmammalian names (OS-9, HRD1, SEL1L, Derlin-1/2/3) for human proteins. This avoids confusion when\ncomparing yeast and mammalian results.\nWe thank the reviewer for this great suggestion and have revised the text accordingly.\nReviewer #3 (Remarks to the Author)\nThe manuscript entitled Structural basis and pathological implications of the dimeric OS9-SEL1L2-HRD1\nERAD Core Complex presents the cryo-EM structure of a mammalian ERAD complex comprising OS9,\nSEL1L and HRD1. The authors concentrate on an ERAD complex that excludes Derlins, EDEMs, and other\nERAD components, based on their preliminary mass spectrometry and Western blot experiments, which\nidentify the three-component complex as the most stable. They propose a model describing a structure\nformed by two identical monomers held together by the HRD1 dimer within the membrane. The authors\nspeculate that this structure may provide a binding site/conduit for the ERAD substrate. This work is\nsignificant for the field of ER protein degradation, despite the existence of previous studies on similar\ncomplexes in yeast or even mammals, because the structural features reported here are highly distinct.\nAlthough the data provide a valuable contribution to our understanding of ERAD complex structures, the\nfollowing points should be addressed prior to final decision:\nWe thank the reviewer for these constructive comments. We have carefully addressed each point as\ndetailed below.\n1. To properly assess the ERAD complex analysis by Western blot, the experiments should be performed\nin triplicate and the data shared with reviewers. All quantifications should come from independent\nexperiments, not merely technical repeats, and should include appropriate statistical analysis.\nWe have included quantifications from at least three independent experiments with detailed descriptions\nof the statistical analyses provided in the corresponding figure legends.\n2. The authors claim that FAM8A1 deletion has no effect on ERAD function. However, the assessment of\nERAD function based on the Western blot shown in (Extended Data Fig. 1e) is inconclusive. The\nquantification of Ire1, Cd147, and OS9 levels in KO cell lines should be based on multiple replicates with\nstatistical significance. Moreover, comparing protein levels across different cell lines is not ideal, as it\nlacks direct kinetic information. Pulse-chase or cycloheximide chase assay would be more informative in\nassessing ERAD function.\nWe have now repeated and quantitated the protein levels of endogenous ERAD substrates (IRE1α, OS9,\nand CD147) in WT, SEL1L-/-, HRD1-/-, and FAM8A1-/-cells (Response Fig. 10a-b). We also performed a\ncycloheximide (CHX) chase assay to evaluate ERAD function in FAM8A1-/-cells (Response Fig. 10b-c).\nConsistent with previous findings 2 (PMID: 28827405), both the basal levels and degradation rates of\nendogenous ERAD substrates (IRE1α, OS9, and CD147) were comparable between FAM8A1-/-and WT\ncells, indicating that FAM8A1 deletion does not impair ERAD function in HEK293T cells. Response Fig. 10\nis now incorporated into Extended Data Fig. 1e-h in the revised manuscript. We have revised the\nmanuscript accordingly to include these\nCHX chase assays in the Results part.\nResponse Figure 10. FAM8A1 has no effect on SEL1L-\nHRD1 ERAD function. (a-b) Western blot analysis of\nSEL1L, HRD1, FAM8A1 and known endogenous\nsubstrates (OS9, IRE1α, CD147) in WT, SEL1L-/-, HRD1-/-\nand FAM8A1-/- HEK293T cells, with quantitation of\nSEL1L, HRD1 and known endogenous substrates shown\nin (b) (n = 3-4 independent samples each genotype,\nthree independent repeats). (c-d) Cycloheximide (CHX)\nchase analysis of known endogenous substrates IRE1α,\nOS9 and CD147 in WT, SEL1L-/-, HRD1-/- and FAM8A1-/-\nHEK293T cells, with quantitation shown in (d) (three\nindependent repeats). “1” and “2” in OS9 blot denote\nthe two OS9 isoforms. Values, mean ± SEM. n.s., not\nsignificant; *p<0.05, **p< 0.01, ***p<0.001 and\n****p<0.0001 by one-way ANOVA test with comparison\nto WT (b), and two-way ANOVA test with comparison\nto WT (d).\nResults:\nLine 99: Consistent with a previous study\n2, FAM8A1 did not affect SEL1L-HRD1\nprotein levels or their function towards\nendogenous substrates such as IRE1α,\nCD147 and OS9 (Extended Data Fig. 1e-h).\n3. The data on the disruption of the ERAD complex in the presence of SEL1L mutants are not entirely\nclear. For example, contrary to the authors' conclusion, the double mutant G585D/S658P does not\ncompletely abolish interactions with OS9 (as seen in lane 5, Fig. 2f), as some interaction with OS9 is still\ndetectable.\nWe thank the reviewer for this valuable comment. We have now repeated the co-IP experiments with\nquantitation, as shown in the Response Fig. 12. The results demonstrate that the G585D, S658P, and\nG585D/S658P mutants reduce the SEL1L-OS9, SEL1L-HRD1 or both interactions by approximately 70%-\n90%. We have revised the text accordingly. Response Fig. 11 is updated into Fig. 2f of the revised\nmanuscript.\nResponse Figure 11. SEL1L disease variants disrupt SEL1L-OS9 and SEL1L-\nHRD1 interaction. (a-b) Immunoprecipitation of FLAG-agarose in SEL1L-/-\nHEK293T cells transfected with indicated SEL1L variants to examine their\ninteraction with HRD1, OS9 and E2 UBE2J1, with quantitation shown in (b) (three\nindependent repeats). Values, mean ± SEM. n.s., not significant; *p<0.05,\n***p<0.001 and ****p<0.0001 by one-way ANOVA test with comparison to WT (b).\nResults:\nLine 154: In SEL1L-/- HEK293T cells, re-expression of SEL1L-\nG585D selectively reduced OS9 binding by ~ 90% (lane 3, Fig.\n2f), while S658P disrupted HRD1 interaction by ~ 70% (lane\n4). The double mutant (G585D/S658P) nearly abolished both\ninteractions (lane 5, Fig. 2f).\n4. To convincingly demonstrate ERAD function in Fig. 5d, the\ndegradation of the ERAD substrate should be followed by\nchase experiments in cells expressing wild-type or mutant\nforms. Direct comparison of steady-state protein levels of ERAD substrates across different cell lines is\nprone to substantial errors.\nWe have now performed the CHX chase assay for Fig 5, as shown in Response Fig. 12. The results\nindicate that T93 mutants impair the degradation of the ERAD substrate proAVP G57S*. Response Fig. 12\nis incorporated into Fig. 5d.\nResponse Figure 12. HRD1 T93A/F mutants inhibit\nthe degradation of proAVP(G57S). (a-b)\nCycloheximide (CHX) chase analysis of\nproAVP(G57S)*-His in HRD1-/-HEK293T cells\ntransfected with proAVP(G57S)*-His and indicated\nHRD1 mutants, with quantitation shown in (b) (three\nindependent repeats). Values, mean ± SEM. n.s., not\nsignificant; **p< 0.01, ***p<0.001 and ****p<0.0001\nby two-way ANOVA test with comparison to WT (b).\nResults:\nLine 212: Expression of either T93 mutant in HRD1-/- HEK293T cells impaired model substrate proAVP\nG57S* degradation and ubiquitination (Fig. 5d-f), and caused substrate accumulation (lanes 4-5 vs. 2,\nExtended Data Fig. 7c), phenocopying the ligase-dead HRD1-C2A mutant (Fig. 5d-e and Extended Data\nFig. 7c).\n5. The authors should discuss how their structural findings compare to previously published data on\nERAD complexes, particularly those from mammalian and yeast systems. Several ERAD complex\nstructures have been identified, and these structures differ significantly from one another. A comparison\nwith these existing models would help contextualize the current findings and highlight both\ncommonalities and differences.\nWe thank the reviewer for this great suggestion. We have now expanded both the Results and Discussion\nsections to include a detailed comparisons between our structure and previously reported ERAD\ncomplex from both yeast and mammalian systems (Response Fig. 13). We now describe how our HRD1\ndimer architecture differs from the yeast Hrd1-Hrd3 and Hrd1-Der1 complexes (Response Fig. 13a-f). We\nhave also incorporated direct comparison with the recently reported XTP3B-SEL1L-HRD1 structure and\nDerlin-engaged HRD1 complex by Guo et al. in Results and Discussion sections (Response 13g-h).\nResponse Fig. 13a-c, 7g-h are shown in Fig. 7, and Response Fig. 13d-f are shown in Extended Data\nFig. 10d-f of the revised manuscript.\nResponse Figure 13. Structural comparisons of\nother ERAD complexes. (a-f) Comparisons of the\nhuman HRD1 dimer (hHRD1) (a, d), yeast Hrd1\ndimer (yHrd1) (PDB code: 5V6P) (b, e), and yeast\nHrd1-Der1 structures (PDB code: 6vjz) (c, f).\nAsterisks indicate the putative substrate\nretrotranslocation channels. (g-h) Comparison of\nOS9-SEL1L-HRD1 complex with XTP3B-SEL1L-HRD1\ncomplex (PDB: 8KET) (g), and Derlin-engaged XTP3B-\nSEL1L-HRD1 complex (PDB: 9UAV) (h).\nResults:\nLine 259: Our cryo-EM structure\nestablishes an HRD1 dimer\narchitecture that differs substantially\nfrom previously reported yeast ERAD\ncomplexes in both yeast 11,12 and\nmammals 1 (Fig. 7e-i and Extended Data\nFig. 10d-f). In our model, the putative\ntranslocation channel is formed\nbetween TM1-4 of one HRD1 protomer\nand TM8 of the other (asterisks, Fig. 7e\nand Extended Data Fig. 10d). This arrangement contrasts with the yeast Hrd1-Hrd3 dimer, which lacks such\na channel (Fig. 7f and Extended Data Fig. 10e), and the Hrd1-Der1 complex, where half-channels are\nformed by TM2-4 of Hrd1 and TM1-2, TM5 of Der1 (asterisks, Fig. 7g and Extended Data Fig. 10f).\nOur structure also differs from the recently reported mammalian XTP3B-SEL1L-HRD1 complex 1, which\ncaptured an asymmetric dimer in which only one HRD1 protomer binds XTP3B-SEL1L (2:1:1 stoichiometry)\n(Fig. 7h). That study proposed that Derlin engagement may trigger HRD1 dimer dissociation,\nalthough Derlin itself was not visualized. Together, these observations suggest that the HRD1 dimer likely\nrepresents one of several coexisting assembly states, with transitions potentially regulated by substrate\nload, Derlin engagement, or broader ERAD pathway dynamics.\nDiscussion:\nLine 303: Recent work by Guo et al. 1 reported a Derlin-engaged XTP3B-SEL1L-HRD1 structure, but the\nHRD1 construct used lacked a substantial portion of the cytosolic C-terminal region required for ERAD\nactivity and Derlin recruitment 2,3.\nReferences\n16, 6007, doi:10.1038/s41467-025-61143-z (2025).\nformation for ER-associated degradation (ERAD). J Cell Sci 130, 3322-3335,\ndoi:10.1242/jcs.206847 (2017).\nHRD1-dependent protein degradation at the endoplasmic reticulum. J Biol Chem 289, 4444-\n4454, doi:10.1074/jbc.M113.519561 (2014).\nextraction of misfolded proteins from the endoplasmic reticulum membrane. Proc Natl Acad Sci\nU S A 102, 14296-14301, doi:10.1073/pnas.0505014102 (2005).\nretrotranslocation at the endoplasmic reticulum membrane. Proc Natl Acad Sci USA 102,\n14132-14138, doi:10.1073/pnas.0505006102 (2005).\nretro-translocation from the ER lumen into the cytosol. Nature 429, 841-847,\ndoi:10.1038/nature02656 (2004).\n9, eadi5656, doi:10.1126/sciadv.adi5656 (2023).\nand systemic water homeostasis. J Clin Invest 127, 3897-3912 (2017).\nand pathogenic mutants by the proteasome. J Biol Chem 279, 19441-19447,\ndoi:10.1074/jbc.M310249200 (2004).\nfeeding, and age-associated obesity. J Clin Invest 128, 1125-1140, doi:10.1172/JCI96420\n(2018).\ncomplex with Hrd3. Nature 548, 352-355, doi:10.1038/nature23314 (2017).\nubiquitin ligase complex. Science 368, doi:10.1126/science.aaz2449 (2020).\nduring endoplasmic reticulum-associated protein degradation. J Biol Chem 298, 102373,\ndoi:10.1016/j.jbc.2022.102373 (2022)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "After careful reading of the revised manuscript, this reviewer found that the authors have addressed all of this reviewer’s comments appropriately and can now recommend this revised version for publication in this journal. One minor suggestion is that presenting the conformational dynamics within the ERAD complex, as revealed by 3D variability analysis, in supplementary movies would make the paper clearer and more fascinating for readers.", "reviewer_2": "The authors have addressed my previous comments through textual revisions and additional experiments. My main criticism of the original submission was that, in interpreting the structure and functional experiments, the authors did not sufficiently consider a role for Derlins in retrotranslocation. In the revised manuscript, the authors have tempered the claim that the reported structure represents the active retrotranslocon in ERAD-L. Furthermore, they also present additional data in the rebuttal letter (as Response Figures) that address the question of Derlin involvement in retrotranslocation. Specifically, Response Fig. 6 shows that Derlins are essential for ERAD of this substrate. Response Fig. 9 shows that mutations that disrupt Hrd1 dimer formation and block ERAD of POMC C28F* and proAVS G57S*, do not affect the association of Derlin-2 with Hrd1. Thus, although the current manuscript does not resolve whether retrotranslocation is mediated by a homodimeric Hrd1 assembly, a heterodimeric Hrd1–Derlin assembly, or both, the data represent an important contribution to this question. This aspect is now sufficiently discussed in lines 298-311. I recommend including Response Figs. 6 and 9 as supplemental figures because they illustrate that Derlins contribute to ERAD of this substrate.\nMinor comment:\nIn Line 308, the authors state that the cited publications suggest Derlins function downstream of Hrd1. I do not agree with that interpretation. For example, Ye et al. (PNAS, 2005; PMID: 16186510) indicates that Derlins engage substrates in early retrotranslocation intermediates. Rao et al. (Ref. 66) describes a putative retrotranslocon formed by Derlin-1 tetramers and therefore also does not support the claim that Derlins act downstream of Hrd1. I suggest revising the sentence to acknowledge that multiple studies place Derlins at or near the retrotranslocation step—potentially upstream of or in parallel with Hrd1—rather than downstream.", "reviewer_3": "The authors have included in the revised version the original blots upon which their conclusions are based. However, a major concern arises from examining the blots intended to demonstrate reproducibility. Specifically, in Figure 3d, the non-reducing gel (anti-OS9) shown for experimental replicates 1 and 3 appears to be the exact same gel presented twice(and HRD1 and HSP90 are shown only for 1 experiment) . The authors must explain how this duplication occurred. Beyond the fact that the experiments were clearly not performed in triplicate—resulting in invalid quantification and statistical analysis—this raises serious concerns about which data in the manuscript are reliable and which may be fabricated.\nAdditional points regarding the revised manuscript:\nThe findings do not support the claim that this study reveals the “structural framework for mammalian SEL1L-HRD1 ERAD.”\nAt minimum, Derlin1—absent from this study—could dramatically alter the structural configuration. The results shown reflect a state of the ERAD machinery lacking key components, not necessarily the native physiological assembly. It remains unresolved whether the functional entity is a dimeric conduit or a monomer cooperating with a Derlin-based conduit, as proposed by Wang et al. (Communications Biology, 8:1481, 2025). Therefore, the manuscript should be revised to clarify that the described structure represents one possible configuration in the absence of certain ERAD elements.\nExperimental interpretations:\nAlthough the revised data support the role of dimerization in efficient ERAD of model substrates, the cycloheximide (CHX) experiments should be properly referred to as turnover assays. The correct interpretation is that the ERAD substrates exhibit a shorter half-life in the presence of a dimeric complex, rather than simply “accumulating” in its absence. The current explanation, which focuses on protein accumulation, is imprecise. For example, lines 212–217 describing proAVP G57S* and POMC C28F* degradation need to be rewritten to reflect this mechanistic clarity.\nTerminology and reagent consistency:\nThere is inconsistency in naming and describing materials such as Strep-Tactin (IBA, #6-6350). The manuscript and Methods annex should explicitly acknowledge that these refer to the same reagent."}, "author_response": "We thank the reviewer for their positive assessment and helpful suggestion. We now have generated a Supplementary Movie 1 based on the 3D variability analysis as suggested.\nWe thank the reviewer for this great comment. As recommended, we have now incorporated the above mentioned data (Response Fig. 1) in the revised manuscripts.\nResponse Fig. 1a-b are included in Fig. 5g-h, 6e-f, and Response Fig. 1c-e are shown in Extended Data Fig. 2g-i\nResults:\nLine 115: Notably, both Derlin-1 and Derlin-2, but neither alone, were required for efficient ERAD of proAVP G57S* (Extended Data Fig. 2g-i).\nLine 218: These functional impairments were not due to disruption of the OS9-SEL1L-HRD1 complex, as both T93 mutants retained OS9 and SEL1L binding, as well as Derlin-2 (Fig. 5g-h).\nLine 236: Importantly, A91D retained normal binding to OS9, SEL1L and Derlin-2 (Fig. 6e-f), indicating that the defect is specific to HRD1 dimerization rather than disruption of the core complex.\nResponse Fig. 1. Interaction between Derlin-2 and HRD1 TM3 mutants (T93A, T93F, A91D). (a-b) Immunoprecipitation of FLAG-agarose in HRD1-/- HEK293T cells transfected with indicated HRD1 mutants to examine their interaction with SEL1L, OS9 and Derlin-2, with quantitation shown on the right (three independent repeats). (c) Western blot analysis of Derlin-1 and Derlin-2 proteins to verify the Derlin-1 and Derlin-2 double knockout HEK293T cells. (d-e) Turnover assays of proAVP G57S* in WT, HRD1-/- and Derlin-1;Derlin-2-/- HEK293T cells transfected with proAVP G57S*-His, assessed by cycloheximide (CHX) chase, with quantitation shown in (e) (three independent repeats). Values, mean ± SEM. n.s., not significant, **p< 0.01, ***p<0.001 and ****p<0.0001 by one-way ANOVA test with comparison to each other (a), Student t-test with comparison to WT (b), and two-way ANOVA test with comparison to WT (e).\nMinor comment:\nIn Line 308, the authors state that the cited publications suggest Derlins function downstream of Hrd1. I do not agree with that interpretation. For example, Ye et al. (PNAS, 2005; PMID: 16186510) indicates that Derlins engage substrates in early retrotranslocation intermediates. Rao et al. (Ref. 66) describes a putative retrotranslocon formed by Derlin-1 tetramers and therefore also does not support the claim that Derlins act downstream of Hrd1. I suggest revising the sentence to acknowledge that multiple studies place Derlins at or near the retrotranslocation step—potentially upstream of or in parallel with Hrd1—rather than downstream.\nWe thank the reviewer for these comments. We have now revised the sentence in Line 308 to more accurately reflect prior studies, acknowledging that Derlin function at or near the retrotranslocation step and may act in parallel with HRD1 rather than downstream.\nWe sincerely apologize for the duplicated blots in the Source Data. This duplication arose from an error during figure assembly: because the membranes from Exp. 1 and the correct Exp. 3 (Response Fig. 2a) were present within the same original image file (Response Fig. 2b), an incorrect crop was inadvertently selected, resulting in the unintended duplication shown in Exp. 3. We have now corrected the image for Exp. 3 (Response Fig. 2a).\nImportantly, the underlying raw data and the quantified source data were derived from the correct Exp.3. In addition, the corresponding HRD1 and HSP90 blots for Exp.2 and Exp.3 have been included in the updated source data (Response Fig. 2c). We have carefully verified the revised Source Data to ensure that all panels now accurately reflect the original experimental results.\nResponse Figure 2. Uncropped gels for Figure 3d.\nAdditional points regarding the revised manuscript:\nThe findings do not support the claim that this study reveals the “structural framework for mammalian SEL1L-HRD1 ERAD.” At minimum, Derlin1—absent from this study—could dramatically alter the structural configuration. The results shown reflect a state of the ERAD machinery lacking key components, not necessarily the native physiological assembly. It remains unresolved whether the functional entity is a dimeric conduit or a monomer cooperating with a Derlin-based conduit, as proposed by Wang et al. (Communications Biology, 8:1481, 2025). Therefore, the manuscript should be revised to clarify that the described structure represents one possible configuration in the absence of certain ERAD elements.\nWe agree with the reviewer that incorporation of Derlin-1 could substantially alter the structural organization of the ERAD machinery. Our data support a requirement for HRD1 dimerization for efficient ERAD activity in the contexts examined here; however, we agree that it does not exclude the possibility that HRD1 may function as a monomer in association with Derlin in other physiological assemblies. Determining whether the active retrotranslocon functions as a dimeric conduit or as a monomer cooperating with Derlin is a very important question that requires future structural and functional studies.\nWe have now changed “Structural framework” to “Structural basis” in the revised manuscript and revised the Discussion accordingly to clarify this scope of our findings and acknowledge these alternative models.\nAbstract:\nLine 44: These findings establish a structural basis for mammalian SEL1L-HRD1 ERAD and elucidate how mutations destabilizing this machinery contribute to human disease.\nDiscussion:\nLine 308: Although our data indicate that dimerization of the OS9-SEL1L-HRD1 core complex is critical for ERAD, incorporation of Derlin is likely to remodel its structural and functional organization. We therefore propose that the structure presented here represents one conformational state of the mammalian HRD1 ERAD complex. Future studies will be required to determine whether the functional translocation unit operates as a dimeric conduit or as a monomer in association with Derlin, as proposed by Wang et al 1.\nLine 321: Together, our study elucidates the molecular organization of the mammalian ERAD core complex, identifies a functionally essential HRD1 dimeric state, and provides a structural basis for understanding substrate recognition and retrotranslocation – processes whose dysregulation underlies a broad spectrum of human diseases.\nExperimental interpretations:\nAlthough the revised data support the role of dimerization in efficient ERAD of model substrates, the cycloheximide (CHX) experiments should be properly referred to as turnover assays. The correct interpretation is that the ERAD substrates exhibit a shorter half-life in the presence of a dimeric complex, rather than simply “accumulating” in its absence. The current explanation, which focuses on protein accumulation, is imprecise. For example, lines 212–217 describing proAVP G57S* and POMC C28F* degradation need to be rewritten to reflect this mechanistic clarity.\nWe have revised the relevant text accordingly.\nResults:\nLine 213: We next asked whether HRD1 dimerization is required for ERAD activity. Expression of either T93 mutant in HRD1-/- HEK293T cells significantly prolonged the half-life of proAVP G57S*, and impaired its ubiquitination (Fig. 5d-f, and Extended Data Fig. 7c), phenocopying the ligase-dead HRD1-C2A mutant (Fig. 5d-e and Extended Data Fig. 7c). Similar defects were observed with another ER-retained model substrate, POMC C28F* (Extended Data Fig. 8a-b).\nLine 227: Expression of HRD1-A91D in HRD1-/- HEK239T cells markedly prolonged the half-life and impaired ubiquitination of proAVP G57S* (Fig. 6b-c and Extended Data Fig. 9a). Similar results were obtained for POMC C28F* (Extended Data Fig. 8a and 9b).\nTerminology and reagent consistency:\nThere is inconsistency in naming and describing materials such as Strep-Tactin (IBA, #6-6350). The manuscript and Methods annex should explicitly acknowledge that these refer to the same reagent.\nWe have revised the manuscript and Methods to explicitly clarify that Strep-Tactin refers to the same reagent throughout the study."}]

Decision letter