SigmaR1 is an auxiliary translocon factor with lipid-binding activity that regulates protein and lipid droplet homeostasis

Domain: biology
Published: 2025-12-31
Editorial outcome: major_revision

Abstract
Sigma Non-Opioid Intracellular Receptor 1 (SigmaR1) is a member of the sigma family of receptors that interacts with a variety of psychotomimetic drugs and is involved in a wide range of cellular and physiological functions. Despite its increasing importance in human physiology and disease, the subcellular localization of SigmaR1 and its molecular function remain poorly defined. Using endogenous tagging and cell fractionation, we show that SigmaR1 is a type II integral ER membrane protein that is specifically enriched at ER sheets. A short region at the N-terminus of SigmaR1 promotes its ER-sheet localization. Importantly, our biochemical studies demonstrate that SigmaR1 directly interacts with components of the translocon complex including TRAPα and Nicalin. In addition, we found that a β-barrel at the C-terminal of SigmaR1 binds phosphatidylcholine (PC), and the binding of PC strengthens the association of SigmaR1 with the translocon complex. SigmaR1 knockout systematically impaired cellular protein and lipid homeostasis, resulting in accumulation of lipid droplets in hepatocytes. Collectively, we propose that SigmaR1 is an auxiliary translocon factor that binds lipids to regulate protein and lipid droplet homeostasis, which may underlie the broad and vital roles of SigmaR1 in physiology and disease.

Introduction
Despitethecontinuityoftheendoplasmicreticulum(ER)membranesystem, the ER has a distinct morphological domain consisting of sheets and tubules that differ in their characteristic membrane curvature1–3, which can be clearly observed in electron microscopy (EM) studies2,4–6. Critical proteins drive the formation of these structural morphologies, which in turn generate rough and smooth functional domains of the ER7–10. ER sheets, which have a higher density of ribosomes and associated protein translocation machinery than ER tubules, are also referred to as rough ER1. Accordingly, the flat and stacked membrane of the rough ER can also better accommodate the bulky membrane-bound polyribosomes that support protein synthesis1,8,11–13.

In mammalian cells, the import of proteins into the ER begins before the polypeptide chain is completely synthesized, i.e., the import is a co-translational process. During co-translational translocation, the signal recognition particles interact with their cognate receptor to deliver the ribosome nascent chain complex into the rough ER and bind to the translocon complex14,15. The translocon complex is a large multi-subunit channel that transports the nascent peptides across ER membrane and undergo post-translational modifications and folding16–21. The eukaryotic translocon is composed of Sec61 core channels associated with multiple co-factors, including the translocon-associated protein (TRAP) complex22, translocation-associated membrane protein (TRAM)23,24, Sec62/6325, oligosaccharyl transferase (OST)21, and signal peptidase complex26. A ribosome-associated complex consisting of the core Sec61 channel and five accessory factors—TMCO1, CCDC47, Nicalin, TMEM147, and NOMO-has recently been characterized and plays a specific role in the biogenesis of multipass membrane proteins27–30. Nevertheless, the molecular composition of the translocon complex remains incompletely clear.

Sigma Non-Opioid Intracellular Receptor 1 (SigmaR1) is a member of the Sigma family of receptors that interact with a wide range of psychotomimetic drugs. SigmaR1 has been shown to play an important role in the cellular functions of various tissues associated with the endocrine, immune, and nervous system. Mutations of SigmaR1 have been associated with distal hereditary motor neuropathy, type II and juvenile amyotrophic lateral sclerosis. In addition, SigmaR1 regulates protein stability by modulating the unfolded protein response31,32. SigmaR1 is also reported to play a role in calcium signaling by modulating of the IP3R3-dependent calcium efflux at the ER33. Although the cellular and physiological importance of SigmaR1 has been increasingly appreciated, the subcellular localization and molecular function of SigmaR1 have not yet been fully elucidated.

In agreement with a recent study34, we demonstrate that SigmaR1 is specifically enriched in ER sheets. This localization is facilitated by a short N-terminal region, which we found mediates direct interactions with the translocon components TRAPα and Nicalin. In addition, the C-terminal β-barrel structure of SigmaR1 binds phospholipids, including phosphatidylcholine (PC), and the binding strengthens the association of SigmaR1 with the translocon complex. SigmaR1 knockout (KO) systematically impaired cellular protein and lipid homeostasis, leading to dramatic accumulation of lipid droplets (LDs) in hepatocytes. Together, these results elucidate the molecular mechanisms underlying the diverse roles of SigmaR1 in physiology and disease.

Results
SigmaR1 is an ER sheet protein
Using high-resolution confocal microscopy, we investigated the subcellular localization of SigmaR1. SigmaR1-GFP was specifically enriched in ER sheets, as demonstrated by its strong co-localization with the ER-sheet marker Halo-Sec61β35 (Fig. 1a). This enrichment was significantly greater than its co-localization with the ER-tubule markers Reticulon-47, Reep57, and Tex236 (Fig. 1b–e). Furthermore, in accord with the recent study34, overexpression of SigmaR1 appears to increase the abundance of ER sheets, evident from their expansion into the cell periphery (Fig. 1a).

To avoid potential overexpression artifacts, we endogenously tagged the N-terminus of SigmaR1 with monomeric superfolder GFP (sfGFP A206K) in HeLa cells using CRISPR-Cas9, generating an sfGFP-SigmaR1 knock-in (KI) line (Supplementary Fig. 1a). The functionality of the construct was confirmed later in this manuscript (Fig. 6n). We chose HeLa cells due to the expression level of SigmaR1 was significantly higher compared to other cell lines commonly used in our laboratory (Supplementary Fig. 1b). The KI line (clone-2, used throughout this study) was validated by immunoblotting (Supplementary Fig. 1c). Live-cell and immunofluorescence imaging revealed that endogenous sfGFP-SigmaR1 was preferentially enriched in ER sheets, as evidenced by strong colocalization with the sheet marker Sec61β but minimal overlap with the tubule marker Reticulon-4 (Fig. 1f–i). Together, these results support that SigmaR1 is an ER sheet-resident protein.

To corroborate these findings, we investigated the distribution of endogenous, untagged SigmaR1 by cell fractionation. We selected rat liver tissue for this assay due to its high endogenous SigmaR1 expression (Supplementary Fig. 1d) and high abundance of rough ER37–40. Consistent with our imaging results, SigmaR1 was predominantly enriched in the rough ER fractions (3, 4 and 5), which were marked with the ER-sheet proteins Sec61β and Calnexin, but absent from fractions containing the tubular markers Reticulon-4 and Reep5 (Fig. 1j, k; Supplementary Fig. 1e). These results provide independent biochemical evidence that SigmaR1 is specifically localized to ER sheets.

The rough ER is characterized by the presence of membrane-bound ribosomes in the form of a thread-like structure, the polysome. Polysomes not only promote the formation of ER sheet but also facilitate the localization of certain membrane proteins to ER sheets8. We therefore investigated whether the polysome is required for the ER-sheet localization of SigmaR1. Live-cell imaging showed that localization of SigmaR1 was not significantly affected by treatment of puromycin (Puro) (Supplementary Fig. 1f, g), which releases nascent polypeptide chains from the ribosome and disassembles polysomes41. Similarly, cycloheximide (CHX), which halts polypeptide chain elongation but leaves the polysomes intact42,43, also had no substantial effect (Supplementary Fig. 1f, g). These results indicate that the localization of SigmaR1 to ER sheet is independent of polysome.

We next investigate whether the role of SigmaR1 in maintaining ER sheet-to-tubule ratio is responsive to cellular stress. Starvation triggers the expansion of ER sheet44—a phenomenon we could recapitulate in our system (Fig. 1l, m). Importantly, the starvation-induced expansion of ER sheet was significantly inhibited upon SigmaR1 depletion (Fig. 1l, m), indicating that starvation-induced ER sheet formation requires SigmaR1.

A short region at the N-terminus promotes the ER-sheet localization of SigmaR1
To determine the mechanism underlying the specific targeting of SigmaR1 to ER sheets, we investigated its membrane topology. SigmaR1 has been reported as a type II integral membrane protein45,46. We therefore sought to confirm this topology in our cellular system (Supplementary Fig. 2a). We utilized a live-cell assay based on a high-affinity interaction between a Flag tag and a genetically encoded cytosol-localized Halo-Flag frankenbody (a single-chain variable fragment)47. When co-expressed, Flag-SigmaR1, but not SigmaR1-Flag, recruited the frankenbody (Supplementary Fig. 2b, c), confirming that the N-terminus is cytosolic and the C-terminus is luminal. These results validate the canonical type II topology of SigmaR1 in the ER.

To identify the ER-sheet targeting determinant, we dissected the SigmaR1 protein. We found that a conserved N-terminal region containing the transmembrane domain (TM) (residues 1-30; SigmaR1-NT) was sufficient for ER-sheet localization, albeit to a slightly lesser extent than the full-length protein (Fig. 2a, b, e & f; Supplementary Fig. 2d). In contrast, a construct containing only the TM domain (residues 10-30; SigmaR1-TM) failed to target sheets and instead colocalized with the tubule marker Reticulon-4, regardless of the position of the GFP tag (Fig. 2c, e, f; Supplementary Fig. 2e). This implicated the upstream residues 2-8 as critical. Consistent with this, their deletion (SigmaR1Δ2-8) abolished specific ER-sheet targeting (Fig. 2d–f), demonstrating that this short N-terminal segment is important for ER sheet localization.

We next investigated whether the SigmaR1-NT is able to redirect other proteins to ER sheet. To test this, we generated a chimera in which SigmaR1-NT was fused to a truncated version of the tubular ER protein Tex2 [Tex2Δ(NT-TM); Supplementary Fig. 2f, g]. This chimeric protein [SigmaR1-NT-Tex2Δ(NT-TM)-GFP] was highly enriched on ER sheets (Fig. 2g). In contrast, the short N-terminal segment (residues 2-8) alone was insufficient to redirect Tex2 to sheets (Fig. 2h, i). These results demonstrate that both the short N-terminal sequence and the TM domain are required for efficient ER-sheet targeting.

SigmaR1 directly interacts with the translocon complex
To elucidate the functional significance of the ER sheet localization of SigmaR1, we sought to identify its interacting partners. We performed coimmunoprecipitation (CoIP) using SigmaR1-GFP as bait, followed by mass spectrometry (MS) analysis. Consistent with its ER localization, functional enrichment analysis revealed that the identified SigmaR1-interacting proteins were predominantly associated with ER-related cellular components in the GO database (Supplementary Fig. 3a) and metabolic pathways in the KEGG database (Supplementary Fig. 3b). Interestingly, our analysis identified numerous translocon components, including TRAPα, Nicalin, SEC11A, CCDC47, TMEM147, NOMO1, RRBP1, TRAM1, and TMCO1, as potential SigmaR1-interacting partners (Supplementary Fig. 3c). Consistent with this finding, all of these proteins exhibited strong colocalization with SigmaR1 on ER sheets, with the exception of TRAM1. TRAM1 was mainly found in distinct punctate structures in addition to its ER sheet localization (Fig. 3a, Supplementary Fig. 3d-h).

To validate these interactions, we performed GFP-Trap assays. Consistent with our imaging data, SigmaR1 interacted with Halo-tagged Nicalin, TRAPα, SEC11A, TMEM147, NOMO1, and TMCO1, as well as GFP-tagged RRBP1 and TRAM1 (Supplementary Fig. 4a-h). Importantly, immunoprecipitation using our sfGFP-SigmaR1 KI cell line revealed specific interactions with Nicalin, TRAPα, SEC11A, and CCDC47 at endogenous levels, but not with NOMO1, RRBP1, SRP54, or TRAM1; a weak interaction was detected with TMEM147 (Fig. 3b, Supplementary Fig. 4i). Consistent with the crucial role of residues 2-8 in targeting SigmaR1 to ER sheets, GFP-Trap assays demonstrated that deletion of this short sequence significantly impaired its interaction with endogenous Nicalin (Fig. 3c).

We next employed in vitro GST pull-down assays to determine if these interactions were direct. Recombinant GST tagged SigmaR1 (GST-SigmaR1) bound directly to His-Nicalin and His-TRAPα (Supplementary Fig. 4j, k), but not to His-SEC11A or His-CCDC47 (Supplementary Fig. 4l, m). To further validate these direct interactions, we purified His-Flag-SigmaR1 alongside GFP-TRAPα and GFP-Nicalin from HEK293F cells, and found that purified SigmaR1 bound directly to both GFP-TRAPα and GFP-Nicalin, but not to GFP alone (Fig. 3d). Importantly, the deletion of the residues 2-8 in SigmaR1 significantly reduced its binding to these translocon components (Fig. 3d). Collectively, these results demonstrate that SigmaR1 directly interacts with specific components of the translocon complex, and indicate an essential role of the NT sequence (residues 2-8) of SigmaR1 in its binding to the translocon complex.

To validate the specificity of the association of SigmaR1 with the translocon, we employed sucrose gradient centrifugation. Using solubilized ER microsomes isolated from the sfGFP-SigmaR1 KI cell line, we compared SimgaR1 distribution in ribosome-bound and ribosome-free fractions. Endogenous SigmaR1 was specifically co-enriched with the translocon components Nicalin and TRAPα in the ribosome-bound ER sheet fractions (Fig. 3e). GFP-Trap assays further confirmed that the interactions between SigmaR1 and these two proteins occurred specifically within these ER sheet fractions (Fig. 3f).

It should be noted that no interaction was detected between SigmaR1 and the core channel of the translocon complex, Sec61β, in the ribosome-bound ER fraction (Supplementary Fig. 4n). This finding was further corroborated by in vitro pull down assays, which showed no binding between His‑Flag‑SigmaR1 and GFP‑Sec61β (Supplementary Fig. 4o). Together, these results suggest that SigmaR1 is not a structural subunit of the core translocon channel.

Furthermore, we performed fluorescence Lifetime Imaging Microscopy (FLIM) to validate a specific interaction between SigmaR1 and Nicalin at ER sheets. The FLIM results demonstrated that mClover2 fluorescence lifetime was significantly reduced in a cell co-expressing SigmaR1-mClover2 and mRuby2-Nicalin (yellow asterisk) compared to that in cells expressing SigmaR1-mClover2 alone (white asterisk) in the same imaging field (Fig. 3g, top panel; 3h). In contrast, the mClover2 fluorescence lifetime was not significantly impacted in cells co-expressing SigmaR1-mClover2 and Reep5-mRuby2 (Fig. 3g, middle panel; 3h), indicating no direct interaction between these two proteins. Consistent with our co-IP and pull down data, a slight reduction in lifetime was observed with the translocon channel Sec61β-mRuby2, suggesting these proteins are co-enriched in the translocon complex but do not directly interact (Fig. 3g, bottom panel; 3h). Taken together, these findings support that SigmaR1 is an auxiliary component that physically engages with the translocon machinery.

SigmaR1 binds to PC via a C-terminal β-barrel structure
SigmaR1 harbored a V-shaped double helix bundle and a cupin-like β-barrel structure in the C-terminal region48–50. Notably, the β-barrel exhibits a wide, flat, and hydrophobic membrane-proximal surface that forms a large binding cavity, suggesting a potential site for hydrophobic lipid interactions (Fig. 4a). To test this hypothesis, we analyzed lipids bound to endogenous SigmaR1 in the SigmaR1 KI cell line using non-targeted lipidomics (LC-MS/MS). This revealed associations with various phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingolipids/ceramides, and cholesterol (Fig. 4b), with a marked preference for PC (Fig. 4b). Molecular docking simulations further supported these findings, demonstrating a favorable binding mode between the β-barrel of trimeric SigmaR1 and PC (Fig. 4c; Supplementary Fig. 5a, b).

To determine whether SigmaR1 binds glycerophospholipids directly, we performed in vitro protein-lipid co-migration assays using purified GST tagged SigmaR1 with a deletion of the TM (GST-SigmaR1ΔTM). Using native gel electrophoresis, we found that GST-SigmaR1-ΔTM co-migrated with nitrobenzoxazole (NBD)-labeled PC, phosphatidic acid (PA), and phosphatidylserine (PS), demonstrating direct binding to these lipids. In contrast, it did not bind to cholesterol (Fig. 4d). The lack of cholesterol binding is consistent with previous reports that cholesterol association is mediated by the TM domain of SigmaR151, which was absent in this construct. Notably, the purified SigmaR1 also failed to bind PE and ceramide in vitro (Fig. 4d), suggesting that its cellular associations with these lipids are likely indirect.

To determine whether the β-barrel domain of SigmaR1 mediates lipid binding, we first purified the GST-SigmaR1Δ2-30 mutant, which is deficient in ER-sheet targeting. This mutant retained the ability to bind lipids, though with reduced affinity compared to the wild-type protein (Fig. 4e). In addition, a truncation mutant containing only the β-barrel domain (GST-SigmaR1-80-168) could bind these lipids (Supplementary Fig. 5c), suggesting that this structural region may mediate lipid interactions. We further generated four space-filling mutants (Mut1, 2, 3 and 4) of SigmaR1 in which several conserved residues with small hydrophobic side chains of the β-barrel structure were substituted with bulky tryptophan residues (Fig. 4f), based on a previous study52. We purified these mutants and found that all the four mutants reduced the lipid binding affinity to varying degrees, with the Mut2 being most efficient (Supplementary Fig. 5d-g).

To further validate these results, we performed in vitro liposome pelleting assays (Fig. 4g). The GST-SigmaR1, but not GST-SigmaR1-Mut2 (104V, 105L, 106L, 107F-W), was efficiently pelleted by sucrose-loaded liposomes (Fig. 4h, i), confirming that lipid interaction is mediated specifically by the β-barrel domain of SigmaR1.

We next investigated the functional significance of lipid binding of SigmaR1. In vitro pull down assays showed that PC binding enhances the binding of SigmaR1 to the set two translocon components Nicalin and TRAPα (Fig. 4j). This regulatory relationship supports that SigmaR1 functions not as a static structural element, but as a dynamic auxiliary factor whose interaction with the translocon complex is modulated by lipid binding.

SigmaR1 KO impairs cellular protein homeostasis
Given the direct interaction of SigmaR1 with the translocon complex, we next investigated the function of SigmaR1 relative to protein homeostasis in SigmaR1 KO HeLa cells generated by CRISPR-Cas9 (Supplementary Fig. 6a, b). Of note, SigmaR1 KO did not affect the levels of other translocon components (Supplementary Fig. 6c), suggesting that SigmaR1 is not directly involved in the stability of the translocon complex.

Importantly, we observed a marked increase in phosphorylated eIF2α—a key integrator of the stress response—in SigmaR1 KO cells, despite unchanged total eIF2α levels (Fig. 5a). As eIF2α phosphorylation globally represses translation in response to cell stress, this indicates that SigmaR1 is critical for maintaining protein homeostasis. Notably, this effect is not indirectly caused by ER stress, as SigmaR1 deficiency neither induced nor potentiated thapsigargin-triggered ER stress. This was evidenced by unaltered levels of the chaperone BiP (Fig. 5a) and ER whorls (marked by Sec61β) (Supplementary Fig. 6d, e), both established markers of ER stress53.

To systematically identify pathways affected by SigmaR1 loss, we performed quantitative MS. SigmaR1 deficiency resulted in a substantial downregulation of ribosome-related proteins, as highlighted by enrichment analyses using the Gene Ontology (GO) and KEGG databases (Fig. 5b, Supplementary Fig. 6f). This is consistent with the role of SigmaR1 as an auxiliary factor of the translocon on ER sheets. KEGG analysis further implicated SigmaR1 in a broad range of physiological, pathological, and disease contexts, including infectious diseases (e.g., COVID-19), neurodegenerative disorders, cell cycle regulation, and endocytosis (Supplementary Fig. 6f). In addition, proteins involved in lipid metabolism were also affected (Supplementary Fig. 6f), suggesting a role for SigmaR1 in lipid regulation.

To investigate the mechanistic link between molecular function of SigmaR1 and protein homeostasis dysregulation, we performed rescue experiment in SigmaR1 KO HeLa cells by reconstituting expression of SigmaR1-GFP, an N-terminal deletion mutant (defective in ER sheet targeting), or the lipid-binding variant. Cells expressing these constructs were isolated by flow cytometry for subsequent proteomic analysis (Fig. 5c). Introduction of SigmaR1-GFP efficiently restored global protein homeostasis, particularly rescuing these ribosomal pathways (Fig. 5d; Supplementary Fig. 6g). In contrast, the NT-deletion mutant failed to fully restore the proteome (Fig. 5e; Supplementary Fig. 6h), underscoring the importance of ER sheet localization and translocon interaction for SigmaR1 function. Similarly, the lipid-binding mutant was also insufficient for complete rescue (Fig. 5f; Supplementary Fig. 6i), indicating that the ability of SigmaR1 to bind lipids is also involved in maintaining protein homeostasis.

SigmaR1 depletion resulted in an accumulation of lipid droplets in hepatocytes
Unexpectedly, small interfering RNA (siRNA)-mediated depletion of SigmaR1 triggered a dramatic accumulation of LDs in the hepatic cell line HepG2 cells following oleic acid (OA) stimulation (Fig. 6a; Supplementary Fig. 7a). To confirm the phenotype, we measured intracellular levels of triglycerides (TG) and cholesterol. In agreement with our imaging results, both TG and cholesterol were significantly increased in SigmaR1-depleted cells compared to controls (Fig. 6b, c).

We next confirmed that the ER sheet localization of SigmaR1 and the aberrant accumulation of LDs upon its depletion are conserved across other hepatic-related cells, including the Huh7 cell line (Supplementary Fig. 7b-d) and mouse primary liver cells (Fig. 6d–f; Supplementary Fig. 7e). In primary hepatocytes, siRNA-mediated depletion of SigmaR1 not only resulted in a striking increase in LD size but also significantly elevated levels of TG and cholesterol/esters levels following OA treatment (Fig. 6g, h).

Notably, this lipid accumulation phenotype exhibited cell-type specificity. Although proteomic data from SigmaR1-KO HeLa cells indicated a predisposition toward dysregulated lipid metabolism, these cells exhibited only moderate, statistically insignificant LD accumulation (Supplementary Fig. 7f–i). Similarly, SigmaR1 depletion in non-hepatic cell lines, including pancreatic PaCa2 cells (Supplementary Fig. 7j–l) and mouse neuroblastoma Neuro2a cells (Supplementary Fig. 7m–o), resulted in a moderate LD phenotype, despite robust conservation of its ER sheet localization. We attribute this cell-type dependent effect to the specialized role of hepatocytes in lipid synthesis, storage, metabolism and secretion, which appears to amplify the phenotypic severity of SigmaR1 loss.

Next, we performed transmission electron microscopy (TEM) to examine the ultrastructural details of LDs in SigmaR1-depleted HepG2 cells. TEM micrographs revealed a strong increase in LD size without a significant change in their number (Fig. 6i–l).

Importantly, the strong accumulation of LDs was specific to SigmaR1, as the introduction of SigmaR1-GFP substantially rescued the phenotype compared to an untransfected cell in the same imaging field (Fig. 6m). The sfGFP-tagged version of SigmaR1 (sfGFP-SigmaR1) also effectively reversed the LD accumulation in SigmaR1-depleted HepG2 cells (Fig. 6n), confirming its functionality.

Next, we investigated whether and to what extent LD accumulation was dependent on lipid-binding activity or ER sheet-localization of SigmaR1. Importantly, introduction of SigmaR1Δ81-160 (Fig. 6o) or lipid binding deficient mutant (GST-SigmaR1-Mut2; Fig. 6p, r) could not completely restore the LD defect. Similarly, the LD accumulation phenotype was not fully rescued by expression of SigmaR1Δ2-8, the mutant defective in ER sheet targeting and translocon association (Fig. 6q, r). These results suggest that both the ligand-binding and ER-sheet localization are required for the role of SigmaR1 in LD dynamics.

In addition, depletion of SigmaR1, but not other known ER sheet factors p180/RRBP1 and Climp63, significantly impacted LD dynamics. Notably, depletion of Kinectin, the kinesin adaptor associating with ER sheet, also caused accumulated LDs, but to a lesser extent compared to SigmaR1 depletion (Supplementary Fig. 8a-c). This suggests that the LD defect in SigmaR1-depleted cells is not merely a secondary consequence of an altered ER sheet-to-tubule ratio but rather points to a more direct, unique role for SigmaR1 in lipid storage.

Methods
Cell cultures, transfection and RNAi
African green monkey kidney fibroblast-like COS7 cell line (ATCC), human embryonic kidney 293T (ATCC), the derivative of the commonly used HEK293 cell line AD293 and HEK293F (ATCC), the mouse hippocampal neuroblast line Neuro-2a (ATCC), the human pancreatic cancer cell line PaCa2 (ATCC), the human cervical cancer HeLa (ATCC), and the human hepatocellular carcinoma cell lines [HepG2 (ATCC) and Huh-7 (ATCC)] were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (VivaCell) and 1% penicillin/streptomycin at 37°C with 5% CO2. Mouse hepatocytes were isolated from male C57 mouse by collagenase perfusion and were cultured in DMEM with 10% fetal bovine serum (PAN Seratech) at 37°C under 5% CO2 as described by previously76. All cells in this study were tested and confirmed to be mycoplasma-free. Transfection of plasmids and RNAi oligos was carried out with Lipofectamine 2000 and RNAi MAX, respectively. For transfection, cells were seeded at 4×105 cells per well in a six-well dishes approximately 16 h prior to transfection. Plasmid transfections were performed in OPTI-MEM (Gibco) with 2 μL Lipofectamine 2000 (Invitrogen) per well for 6 h, followed by trypsin (Gibco) digestion and re-plated onto glass-bottom confocal dishes at ~3.5×105 cells per well. Cells were imaged in live-cell medium (DMEM with 10% FBS and 20 mM Hepes without penicillin or streptomycin) approximately 16-24 h after transfection (individually or combined for co-transfection). For all transfection experiments in this study, 1000 ng DNA was used per 3.5 cm well. For siRNA transfections, cells were plated on 3.5 cm dishes at 30-40% density, and 2 μL Lipofectamine RNAimax (Invitrogen) and 50 ng siRNA were used per well. At 48 h after transfection, a second round of transfection was performed with 50 ng siRNAs. Cells were harvested and analyzed 24 h after the second transfection for suppression. All siRNAs used in all experiments were synthesized by RiboBio. siRNA targeting sequences for human SigmaR1#1: TGCAGTGGGTGTTCGTGAA, Sigmar1#2: TCACCCTCTTCTATACTCT, SigmaR1#3: GCGAAGAGATAGCGCAGTT. siRNA targeting sequences for mouse SigmaR1#1: CCCTGATTCTGACTATTAT, SigmaR1#2: GCTGGAACTCATATTCAGA, SigmaR1#3: CTCACCATATGCCTTATCT. siRNA targeting sequences for human Climp63: GGTGCAGTCTTTGCAAGCCACATTT. siRNA targeting sequences for human p180: GGCAGCAGTTGAGTGAAAT. siRNA targeting sequences for human Kinectin: GCAGTTCCATTCCCAGATA.

OA induced LD accumulation
To examine the colocalization of LD with different proteins or measure cellular triglyceride (TG) and cholesterol (CHO), cells were treated with 200 μM OA (O1008, Sigma-Aldrich) for 16 h.

Plasmid construction
SigmaR1 (NM_005866.4), TRAPα (NM_003144.5), SEC11A (NM_014300.4), Nicalin (NM_020170.4), CCDC47 (NM_020198.3), TMEM147 (NM_032635.4), TMCO1 (NM_019026.6), RRBP1 (NM_001042576.2), TRAM1 (NM_014294.6), NOMO1 (NM_014287.4) were cloned from HeLa or 293T cDNA. SigmaR1, CCDC47, TMEM147, TMCO1, RRBP1, TRAM1, NOMO1 were cloned into mEGFP-N1 or Halo-N1 between the HindIII and BamHI. GFP/Halo-SigmaR1, Halo-TRAPα, Halo-SEC11A, Halo-Nicalin were generated by cloning respective ORF into mEGFP-C1 or Halo-C1 between the SacI and BamHI/ The ORFs of SigmaR1, TRAPα, SEC11A, Nicalin were cloned into mEGFP-C1 or Halo-C1 between the SacI and BamHI. GST-SigmaR1ΔTM, GST-SigmaR1Δ2-30, GST-SigmaR1-β barrel and GST-SigmaR1-β barrel mut1-4 were cloned into PGEX-2T vector between the BamHI and EcoRI. 14xHis-NEDD8-TRAPα, SEC11A, Nicalin and CCDC47 were cloned into 14xHis-NEDD8 vector between the BamHI and HindIII. Reticulon 4-Halo, Reep5-Halo, Halo-Tex2, Halo-Sec61β and ER-eBFP2 were previously described77. All mutant constructs in this study were generated by PCR mediated site directed mutagenesis on the basis of their respective wild-type constructs. All constructs were confirmed by DNA sequencing. A list of plasmids and primers for DNA constructs were provided in Supplementary Table 1 and 2, respectively.

Generation of KO/KI cell line using CRISPR/Cas9
To make sfGFP-SigmaR1-KI HeLa cell line, two gRNAs (5’-CGCCGTGCCAGGCCGCCCGCCGG-3’ and 5’ -AGTGGGCCGTGGGCCGGCGGTGG-3’) were used to target the N-terminus of SigmaR1 gene. The sgRNA was annealed and ligated into the BsmBI-cleaved/cut pLenti-CRISPR-V2 vector (#52961, Addgene). Then the plasmid encoding sgRNA and the donor construct containing superfolded GFP (sfGFP) and two homology arms were transfected into HeLa cells with Lipofectamine 2000, and single clones were sorted 48 h after transfection. Four positive clones were verified by imaging and Western blots.

To generate the SigmaR1 KO HeLa cell line, 630 bp bases between exon 1 and exon 3 of the SigmaR1 gene were deleted using two gRNAs (5’-CGTGGGCCGGCGGTGGGCGTGGG-3’ and 5’-TCTCTGGCACCTTCCACCAGTGG-3’). Complementary gRNAs were annealed and subcloned into the pSpCas9 (BB) -2A-GFP (pX-458) vector (#48138, Addgene) between restriction sites of the BbsI endonuclease. After 48 h transfection, single-cell was sorted by fluorescence-based flow cytometry. Two independent clones were verified by PCR validation and Western blot. All single-guide RNAs (sgRNAs) were designed using CCTop (https://cctop.cos.uni-heidelberg.de:8043/index.html).

Antibodies and reagents
Anti-SigmaR1 (B-5) (sc-137075, Santa Cruz), anti-Sec61β (E-6) (sc-393633, Santa Cruz), anti-Tom20 (sc17764, Santa Cruz), anti-NOMO (B-10) (sc-390565, Santa Cruz) and anti-Nicalin (P09771, promab) were used at 1:500 for western blots. Anti-LAMP1 (H4A3) (sc-20011, Santa Cruz), anti-REEP5 (H-10) (sc-393508, Santa Cruz), anti-RRBP1 (A12239, ABclonal), anti-SEC11A (P02842, promab), anti-TRAPα (201011-T32, Sino Biological Inc), anti-CCDC47 (201332-T44, Sino Biological Inc), anti-TMEM147 (K113174P, Solarbio), anti-TRAM1 (K113836P, Solarbio) anti-Perilipin-2 (A6276, Abclonal), anti-BiP (A11366, Abclonal), eIF2α (9722, Cell Signaling Technology) and Phospho-eIF2α (Ser51) (9721, Cell Signaling Technology) were used at 1:1000 for western blots. Anti-Flag (F1804, Sigma-Aldrich) and anti-RPL10 (K009842P, Solarbio) were used at 1:1500 for western blots. Anti-Calnexin (ab22595, Abcam), anti-PMP70 (ab3421, Abcam) and anti-GM130 mAb (M179-3, MBL) were used at 1:2000 for western blots. Anti GFP-Tag pAb (AE011, ABclonal), anti-HaloTag pAB (G928A, Promega), anti-GAPDH (AB9485, Abcam), anti-tubulin (100109-MM05T, Sino Biological Inc), anti GST-Tag mAb (AE001, ABclonal) and anti-His (66005-1-Ig, Proteintech) were used at 1:5000 for western blots. Anti-Sec61β (15087-1-AP, Proteintech) were used at 1:20 for Immunofluorescence (IF). Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 546 (A10040, Invitrogen) was performed for IF. For immunoblotting, HRP-conjugated goat anti-mouse IgG (H+L) (AS003, ABclonal), HRP-conjugated goat anti-rabbit IgG (H+L) (AS014, ABclonal) were used as secondary antibodies. For live cell imaging, Janilia Fluo® 646 HaloTag® Ligand (GA1120, Promega) and BODIPY-558/568 C12 (D3835, Thermo Fisher Scientific) were added to added to OPTI-MEM (Invitrogen) medium in concentration of 20 nM, followed by incubation for 20 min. Cells were washed three times with PBS before imaging. For translation inhibition experiments, cells were treated with 200 μM cycloheximide (C112766, Aladdin) and 200 μM puromycin (P8230, Solarbio) in complete media for 20 min. For the ER stress test, cells were treated with EBSS buffer (G4213, Servicebio) for 4 h. Cells were treated with Thapsigargin (TG) (SC0389, Beyotime) for different durations before imaging. For in vitro lipid binding assay, the following lipids were purchased from Avanti Polar Lipids: NBD-PC (810133), NBD-PE (810144), Rh-PE (810150), NBD-PS (810198), NBD-PA (810138), NBD-ceramide (810211) and NBD-Cholesterol (810252).

Live imaging by confocal microscopy
Cells were grown on 35 mm glass-bottom confocal dishes (801001, NEST). Confocal dishes were loaded to a spinning disk confocal microscope (Dragonfly CR-DFLY-201-40, Andor) equipped with multiple excitation lasers (405-nm, 488-nm, 561-nm, 637-nm). Cells were imaged with the 60×/1.4 NA iPlan-Apochromat 60× oil objective using the 405-nm laser for BFP, 488-nm for GFP, 561-nm for mStrawberry, OFP, tagRFP or mCherry and 637-nm for Janilia Fluo® 646 HaloTag® Ligand. All analyses/quantifications were done on raw 16-bit images using ImageJ.

Immunofluorescence staining
Cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. After three PBS washes, cells were permeabilized with 0.1% Triton X-100 in PBS for 12 min on ice (4°C). Following another three PBS washes, cells were blocked with 1% BSA for 1 h at room temperature, then incubated with specific primary antibodies overnight at 4°C. After thorough PBS washing, cells were incubated with Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 546 (Invitrogen, A10040) for 1 h at room temperature protected from light. Finally, cells were washed three times with PBS, mounted with antifade reagent, and imaged.

Fluorescence lifetime imaging microscopy (FLIM)
Cos7 cells were transiently co-transfected with plasmids encoding SigmaR1-mClover2 and mRuby2-Nicalin (or Reep5-mRuby2/mRuby2-Sec61β) for 24 h. FLIM assays were performed using a Leica Stellaris STED laser confocal microscope. Data were analyzed using the TauSense FLIM module.

Quantitative RT-PCR
For quantification of SigmaR1 mRNA levels in different cells, total RNA from cells were extracted with Trizol reagent (15596026, Invitrogen). According to the reagent instructions, cDNA (1000 ng) was synthesized using gDNA Clear cDNA Synthesis Kit/HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R212, Vazyme), and qPCR experiments were performed using ChamQ universal SYBR Green SYBR qPCR Master Mix (Q711, Vazyme) via CFX connect™ fluorescent quantitative PCR detection system (BIO-RAD). Primers used for mRNA quantification were:
h-GAPDH: 5’-GACAAGCTTCCCGTTCTCAG-3’, 5’-GAGTCAACGGATTTGGTCGT-3’. h-SigmaR1: 5’-GGGCCAAACACATGGATG-3’, 5’-GCATAGGAGCGAAGAGTATAGAAG-3’.
h-Climp63: 5’- GCTCACCAAATCCATCAACG - 3’, 5’- TGCTTGTTCCCCTCAGATTC - 3’. h- p180: 5’- TTGAAAGTTCGGACCAGGTG-3’, 5’- CGAGCTGAGATTGAGATTCTAGG - 3’. h- Kinectin: 5’- TGGAATCAGAGCAGAAAAGGG-3’, 5’- GAACTGAAGCGGAGGTCTG-3’.

GST pull-down assay
GST or His-tagged proteins were constructed into pGEX-2T vector or 14×His-NEDD8 vector. E. coli Rosetta (DE3) or BL21 (DE3) transformed with GST-SigmaR1, 14×His- TRAPα, 14×His-SEC11A, 14×His-Nicalin, 14×His-CCDC47 plasmids were inoculated into 5 mL LB medium and grown to OD600 of 0.6-0.8 at 37°C, and then E. coli transformed with different plasmids was induced with the corresponding optimal induction concentration of IPTG for 16 h at 16°C. Bacteria were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris, pH 7.4, 300 mM NaCl, 0.1% NP-40, 1% Protease Inhibitor Cocktail (C0001, TargetMol), 1 mM DTT) and then lysed by sonication on ice. Lysates were centrifuged at 12,000×g for 30 min and supernatants were collected. After washing the GST agarose beads (C600031-0025, Sangon) three times with lysis buffer, the supernatant was incubated with the beads overnight at 4°C. Then GST agarose beads were washed three times with lysis buffer, collected, and resuspended in 50 μL of 2× SDS loading buffer. Samples were analyzed by Western blot.

GST-tagged protein purification
The constructed plasmids GST-SigmaR1, GST-SigmaR1Δ2-30, GST-β barrel and GST-β barrel Mut1 (83F-W, 95L, 96L-W), Mut2 (104V, 105L, 106L, 107F-W), Mut3 (128I, 129I-W) and Mut4 (152V, 153V-W) were transformed into E. coli Rossetta (DE3) cells and coated onto LB plates overnight. The following day, healthy monoclonal bacteria were picked and inoculated into a small amount of LB culture medium, and then fresh bacterial solution was inoculated into 3 L of fresh LB medium at a ratio of 1:100 and expanded the culture at 37°C. When the optical density at 600 nm (OD600) reached 0.6–0.8, protein expression was induced with 0.5 mM IPTG for 20 h at 16°C. The supernatant containing these proteins was incubated overnight with GST agarose beads after sonication and high-speed centrifugation. Finally, the proteins were eluted with washing buffer containing 20 mM GSH and stored at −80°C. Protein expression and size were confirmed by Coomassie staining and protein concentration was measured by Nanodrop.

In vitro protein-lipid co-migrating assays
The in vitro lipid binding assays were performed according to a previous study77 with modifications. Briefly, 19 μL purified GST-SigmaR1, purified GST-SigmaR1Δ2-30, purified GST-β and GST-β Mut1 (83F-W, 95L, 96L-W), Mut2 (104V, 105L, 106L, 107F-W), Mut3 (128I, 129I-W) and Mut4 (152V, 153V-W) (1 mg/mL) were mixed with 1 μL of methanol and NBD-labeled ceramide, PE, PS, PA, PC, cholesterol (1 mg/ml in methanol) in 20 μL total reaction volumes and incubated at 4°C for 3 h. Samples mixed with 5 μL of non-SDS 5x loading buffer were loaded onto 10% non-denaturing native PAGE gel for 4 h at a low current (80-100 V), during which low temperature and dark conditions were maintained. Finally, the NBD fluorescence was visualized using Bio-Rad ChemiDoc XRS+ 170-8265 and migration of total protein was observed by Coomassie staining.

Liposome pelleting assay
The purified protein was pre-cleared by ultracentrifugation at 20,000 g for 1 h. Various amounts of the pre-cleared protein were incubated with liposomes (250 μM, 70% DOPC, 30% PE, 0.75 M sucrose) in a shaking incubator at 30°C for 1 h. The mixtures were then centrifuged at 16,000 g for 15 min. Subsequently, both the supernatant and the pellet fractions were carefully collected and analyzed by Western blotting.

GFP-Trap assays
GFP-Trap was used to detect protein-protein interactions and GFP-Trap assays were performed according to the manufacturer’s protocol. For exogenous protein-protein interaction assays, 293T cells transfected with the indicated constructs were collected and resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) supplemented with 1% Protease Inhibitor Cocktail for 30 min on ice. 10% supernatant after high-speed centrifugation was saved for input sample of GFP-Trap, and the remaining supernatant samples were incubated with GFP-Nanoab-agarose beads (GNA-50-1000, LABLEAD) for 4 h at 4°C. The beads were washed three times with lysis buffer and collected. For endogenous protein-protein interaction assays, two equal amounts of cells were cultured and collected, one was HeLa cells transfected with mEGFP-C1 empty vector as the control group and the other was sfGFP-SigmaR1 KI #2 HeLa cells in the experimental group. Subsequently, the experimental samples were processed and collected according to the GFP-Trap experimental manufacturer‘s protocol. Western blot analysis of protein co-IP.

In vitro pull-down assay of his-flag-SigmaR1 and GFP-TRAPα/GFP-nicalin
His-Flag-SigmaR1 was purified from suspension HEK293F cells transiently transfected with a corresponding plasmid. Cells were harvested 72 h post-transfection and lysed via sonication in lysis buffer containing 150 mM NaCl, 25 mM HEPES, 10% glycerol, and 1 mM PMSF. The lysate was supplemented with detergents to final concentrations of 1% (w/v) n-Dodecyl-β-D-maltopyranoside (DDM) and 0.1% (w/v) cholesteryl hemisuccinate tris salt (CHS), followed by membrane solubilization for 2 h. The mixture was then centrifuged at 20,000×g for 1 h at 4°C. The supernatant was incubated with anti-Flag Magnetic Beads (P2115, Beyptime) for 3 h. Bound proteins were eluted using 3× FLAG peptide. All purification steps were carried out on ice or at 4°C to ensure protein stability.

HEK293F cells transiently transfected with GFP-TRAPα or GFP-Nicalin were lysed in a high-salt lysis buffer. GFP-Trap beads were used to capture the respective GFP-fusion proteins from the cell lysates. The beads were subsequently washed ten times with the high-salt lysis buffer to eliminate any potential interacting molecules.

For the pull-down assay, the purified His-Flag-SigmaR1 protein was incubated with immobilized GFP-TRAPα or GFP-Nicalin complexes for 4 h at 4°C. The beads were washed extensively with HNM buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.01% DDM). Finally, the beads were resuspended in 100 μL of 2× SDS sample buffer and boiled at 95°C for 10 min to elute bound proteins.

Transmission electron microscopy
HepG2 cells treated with either scrambled or SigmaR1 siRNA were fixed with 2.5% glutaraldehyde in 0.1 M Phosphate buffer, pH 7.4 for 2 h at room temperature. After washing three times with 0.1 M Phosphate buffer, cells were scraped and collected with 0.1 M phosphate buffer followed by centrifugation at 956×g. The pellet was resuspended in PBS (0.1 M), and centrifuged at 956 ×g for 10 min. This step was repeated three times. The samples were post-fixed with pre-cold 1% OsO4 in 0.1 M Phosphate buffer for 2–3 h at 4°C, followed by rinsing with PBS 3 times (3× 20 min). The samples were dehydrated in graded ethanol (50%, 70%, 85%, 90%, 95%, 2 × 100%) with 15 min for each condition. The penetrations were performed in an order of acetone-epoxy (2:1); acetone-epoxy (1:1); epoxy. Each round of penetration was performed at 37°C for 12 h. The samples were embedded in epoxy resin using standard protocols78. Sections parallel to the cellular monolayer were obtained using a Leica EM UC7 with the thickness of 60-100 nm and examined under Tecnai G2 20 TWIN (FEI) with accelerating voltage 200 kv. LDs and nucleus were identified based on their respective morphology. The number and size (diameter) of LDs were measured using the “measure” tool in ImageJ (NIH).

Mass spectrometry for identification of SigmaR1-GFP interacting proteins
Identification of Sigmar1-GFP interacting proteins by MS was described in our previous study77. For processing of samples before loading, bound proteins were first extracted from GFP-Trap agarose beads using SDT lysis buffer (4% SDS, 100 mM DTT, 100 mM Tris-HCl, pH 8.0), and then samples were boiled for 3 min and further sonicated. Undissolved beads were removed by centrifugation at 16,000×g for 15 min. Collect the supernatant containing protein. Subsequently, the FASP method was used for protein digestion: detergent, DTT, and IAA in UA buffer were added to blocked reduced cysteines. The proteins were digested with 2 µg trypsin (Promega) for overnight at 37°C. Peptides were collected by centrifugation at 16,000×g for 15 min. Finally, peptides were desalted with C18 StageTip for further LC-MS analysis. LC-MS/MS experiments were performed on a Q Exactive Plus mass spectrometer coupled to an Easy nLC (Thermo Fisher Scientific). Firstly, three independent sample were loaded onto the capture column (100 µm × 20 mm, 5 µm, C18, Dr. Maisch GmbH) in buffer A (0.1% formic acid in water). Reversed-phase high-performance liquid chromatography (RP-HPLC) separation was performed using a self-packed column (75 µm × 150 mm; 3 µm ReproSil Pur C18 beads, 120 Å; Dr. Maisch GmbH, Ammerbuch) at a flow rate of 300 nl/min. Specifically, the RP-HPLC mobile phase (A was 0.1% formic acid in water and B was 0.1% formic acid in 95% acetonitrile) eluted the sample from 2-4% buffer B gradient to 90% buffer B within 60 min. MS data were acquired using a data-dependent top 20 method dynamically choosing the most abundant precursor ions from the survey scan (350–1800 m/z) for HCD fragmentation. A locked mass of 445.120025 Da was used as the internal standard for mass calibration. Full MS scans were acquired at resolutions of 70,000 at m/z 200 and 17,500 at m/z 200. The maximum injection time was set to 50 ms for MS and MS/MS, respectively. Normalized collision energy was 27 and the isolation window was set to 1.6 Th. Dynamic exclusion duration was 60 s. MS data were analyzed using MaxQuant software version 1.6.1.0. MS data were searched against the UniProtKB human database (36,080 entries in total, downloaded on 25 June 2019). Trypsin was chosen as the digestion enzyme. A maximum of two missed cleavage sites and a mass tolerance of 4.5 ppm for precursor ions and 20 ppm for fragment ions were defined for database search. Carbamidomethylation of cysteine was defined as a fixed modification, whereas acetylation of the protein N-terminus and the oxidation of methionine were set as variable modifications for database search. Database search results were filtered and exported at <1% false discovery rate (FDR) at the peptide spectrum matching level and protein level, respectively.

Non-targeted lipidomics using LC-MS/MS
The identification of sfGFP-SigmaR1-associated lipids in the KI cells was described in our previous study77. In particular, to extract lipids, 1 mL methyl tert-butyl ether (MTBE) was added to GFP-Trap agarose beads (Chromoteck) and the samples were shaken for 1 h at room temperature. Next, phase separation was induced by adding 250 μl water, letting it sit for 10 min at room temperature, and centrifuging for 15 min at 14,000×g, 4°C. Due to the low density and high hydrophobicity of MTBE, lipids and lipophilic metabolites were mainly extracted to the upper MTBE-rich phase. The lipid was transferred to fresh tubes and dried with nitrogen.

In addition, to ensure data quality for metabolic profiling, quality control (QC) samples were prepared by pooling aliquots from representative samples for all of the analysis samples, and were used for data normalization. QC samples were prepared and analyzed with the same procedure as that for the experiment samples in each batch. Dried extracts were then dissolved in 50% acetonitrile. Each sample was filtered with a disposable 0.22 µm cellulose acetate and transferred into 2 mL HPLC vials and stored at −80°C until analysis. For UHPLC-MS/MS analysis, lipid analysis was performed on Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to a UHPLC system Ultimate 3000 (Thermo Fisher Scientific). Samples were separated using a Hypersil GOLD C18 column (100×2.1 mm, 1.9 μm) (Thermo Fisher Scientific). Mobile phase A was prepared by dissolving 0.77 g of ammonium acetate to 400 mL of HPLC-grade water, followed by adding 600 ml of HPLC-grade acetonitrile. Mobile phase B was prepared by mixing 100 ml of acetonitrile with 900 ml isopropanol. The flow rate was set as 0.3 ml/min. The gradient was 30% B for 0.5 min and was linearly increased to 100% in 10.5 min, and then maintained at 100% in 2 min, and then reduced to 30% in 0.1 min, with 4.5 min re-equilibration period employed. Both electrospray ionization (ESI) positive-mode and negative-mode were applied for MS data acquisition. The positive mode of spray voltage was 3.0 kV and the negative mode 2.5 kV. The ESI source conditions were set as follows: heater temperature of 300°C, Sheath Gas Flow rate, 45 arb, Aux Gas Flow Rate, 15 arb, Sweep Gas Flow Rate, 1 arb, Capillary Temp, 350°C, and S-Lens RF Level, 50%. The full MS scans were acquire data resolution of 70,000 at m/z 200, and 17,500 at m/z 200 for MS/MS scans. The maximum injection time was set to for 50 ms for MS and 50 ms for MS/MS. MS data was acquired using a data-dependent Top10 method dynamically choosing the most abundant precursor ions from the survey scan (200–1500 m/z) for HCD fragmentation. Stepped normalized collision energy was set as 15, 25, 35 and the isolation window was set to 1.6 Th. QC samples were prepared by pooling aliquots that were representative of all samples under analysis, and used for data normalization. Blank samples (75% acetonitrile in water) and QC samples were injected every six samples during acquisition.

For data preprocessing and filtering, lipids were identified and quantified using LipidSearch 4.1.30 (Thermo, CA). Mass tolerance of 5 ppm and 10 ppm were applied for precursor and product ions. Retention time shift of 0.25 min was performed in ‘alignment’. M-score and chromatographic areas were used to reduce false positives. The lipids with less than 30% relative standard deviation (RSD) of MS peak area in the QC samples were kept for further data analysis. SIMCA-P software (Version 14.0, Umetrics, Sweden) was used for all multivariate data analyses and modeling. Data were mean-centered using Pareto scaling. Models were built on principal component analysis (PCA), orthogonal partial least-square discriminant analysis (PLS-DA) and partial least-square discriminant analysis (OPLS-DA). All the models evaluated were tested for overfitting with methods of permutation tests. The descriptive performance of the models was determined by R2X(cumulative) [perfect model: R2X(cum)=1] and R2Y(cumulative) [perfect model: R2Y(cum)=1] values while their prediction performance was measured by Q2(cumulative) [perfect model: Q2(cum)=1] and a permutation test (n=200). The permuted model should not be able to predict classes-R2 and Q2 values at the Y-axis intercept must be lower than those of Q2 and the R2 of the non-permuted model. OPLS-DA allowed the determination of discriminating metabolites using the variable importance on projection (VIP). The VIP score value indicates the contribution of a variable to the discrimination between all the classes of samples. Mathematically, these scores are calculated for each variable as a weighted sum of squares of PLS weights. The mean VIP value is 1, and usually VIP values over 1 are considered as significant. A high score is in agreement with a strong discriminatory ability and thus constitutes a criterion for the selection of biomarkers. The discriminating metabolites were obtained using a statistically significant threshold of variable influence on projection (VIP) values obtained from the OPLS-DA model and two-tailed Student’s t-test (P-value) on the normalized raw data at a univariate analysis level. The P-value was calculated by one-way analysis of variance (ANOVA) for multiple groups analysis. Metabolites with VIP values greater than 1.0 and P-value less than 0.05 were considered to be statistically significant metabolites. Fold change was calculated as the logarithm of the average mass response (area) ratio between two arbitrary classes. On the other side, the identified differential metabolites were used to perform cluster analyses with R package.

Preparation of rough ER fraction from rat liver and sfGFP-Sigmar1-KI cell
Based on the protocol used in the preparation of crude microsomes from rat livers79, 5 female Balb/c rats (age, 6 weeks; weight, 80–100 g) were euthanized and the livers were collected quickly and washed with cold homogeneous medium (HM) (0.5 M sucrose, 1 mM dithiothreitol (DTT)). Firstly, Liver tissue was ground with a tissue grinder until a paste-like consistency form. The homogenate volume was diluted to 5 times the liver weight (i.e., 20% [w/v]) with HM and centrifuged at 1400×g for 10 min mainly to remove nuclei and cellular debris, and the supernatant was centrifuged at 13,800×g for 10 min at 4°C to remove mitochondria. After transferring the supernatant enriched in cytosol and ER into a 50 ml tube on ice, the pellet was resuspended with a 5 × volume of HM, ground 5 times, and then centrifuged at 13,800×g for 10 min at 4°C. The above step was repeated for once.

Subsequently, the supernatants (post-mitochondrial supernatant, PMS) were combined and kept on ice. Next, four sucrose gradients solutions were prepared using PMS and 2.5 M sucrose: PMS containing 1 M sucrose, PMS containing 1.35 M sucrose, PMS containing 1.55 M sucrose, and PMS containing 1.8 M sucrose. Finally, different sucrose gradients solutions (Layer the solutions from the bottom to the top starting with 0.67 mL of 1.8 M sucrose-PMS, 0.4 mL of 1.55 M sucrose-PMS, 1.5–2.5 mL of 1.35 M sucrose-PMS, and finally 0.4 mL of 1 M sucrose-PMS) were transferred in ultracentrifuge tubes (Ultra-Clear Beckman, USA) with a Ti60 rotor and centrifuged for at least 6 h at maximum 240,000×g at 4°C. A total of 11 samples within and between each layer were collected for Western blotting.

For the preparation of crude rough microsomes from culture cells80, we added cycloheximide to the culture medium to a final concentration of 10 µM, and incubated the culture at 37˚C for 10 min, and washed the cells three times with cold PBS containing a concentration of 10 µM cycloheximide. Finally, the collected cells were added 0.7 mL of HM (10 mM HEPES-KOH buffer (pH 7.5), 10 mM KCl, 1 mM MgCl2, 1 mM DTT, 0.5 mM PMSF) containing 3 units/mL RNase inhibitor and ground with the Dounce homogenizer until the cell homogenate was in a milky white state. After cell homogenization, the cell homogenate was centrifuged at 1400×g for 10 min mainly to remove nuclei and cellular debris, and the supernatant was centrifuged at 13,800×g for 10 min at 4°C to remove mitochondria. After transferring the supernatant enriched in cytosol and ER into a tube, 2.2 times the volume of 2.5 M sucrose was added to achieve a sucrose concentration of 1.8 M in the sucrose PMS mixture. Finally, different sucrose gradients solutions (layered from bottom to top, starting from 1.67 mL of 1.8 M sucrose-PMS mixture, 0.5 mL of HM containing 1.5 M sucrose, 0.5 mL of HM containing 1.3 M sucrose, 0.5 mL of HM containing 1 M sucrose, 0.5 mL of HM containing sucrose, and finally 0.33 mL of HM containing 0.25 M sucrose) were transferred to ultracentrifuge tubes (Ultra Clean Beckman, USA) with Ti60 rotors and centrifuged at 263,000×g for at least 6 h at 4°C. A total of 14 samples within and between each layer were collected for Western blotting.

Fluorescence-activated cell sorting (FACS) analysis
SigmaR1 KO #3 HeLa cells were transfected with either SigmaR1-GFP, SigmaR1-GFPΔ2-8, or SigmaR1-GFP lipid-binding mutant plasmids and cultured for 36 h. Cells (40 10 cm dishes with ~100% confluency for each condition) were collected, washed with PBS, and resuspended as single-cell suspensions. The suspensions were then filtered through a cell strainer to remove clumps and debris. GFP-positive cells were isolated using a BD FACSAria II cell sorter equipped with a FITC fluorescence channel, and collected for subsequent experiments.

Total triglyceride and cholesterol/cholesterol esters measurements
Cells were treated with 200 µM OA for 16 h, and cellular triglyceride and cholesterol levels were measured using total triglyceride (TG) assay kit (A110-1-1, NanJing JianCheng Bioengineering Institute) and total cholesterol assay kit (A111-1-1, NanJing JianCheng Bioengineering Institute), respectively. Briefly, cells were collected by centrifugation at 1000×g for 10 min at 4°C, then cells were resuspended in 500 μL of cold diluted PBS buffer and homogenized on ice by ultrasonic. For triglyceride and cholesterol assay, 10 μL standard or cell suspension was added to 96 plate and the reaction was initiated by adding 200 μL diluted Enzyme Mixture solution to each well. The plate was incubated for 30 min at 37°C, the absorbance at 500 nm was measured.

Image analysis
All image analysis and processing were performed using ImageJ (2.1.0/1.53c; National Institutes of Health). The colocalization analysis of Pearson’s correlation coefficient was performed using a plugin named JACoP in ImageJ.

Proteomics analysis
For functional analysis of mass spectrometry data, the corresponding IDs of the screened proteins (any two groups of unique peptides enriched in GFP-C1 control group = 0, and any two groups of unique peptides enriched in Sigmar1-GFP experimental group ≠ 0) were converted to gene IDs by the DAVID knowledgebase tool81,82, and then enrichment analysis and mapping of GO function and KEGG pathway database of the converted genes were performed using the OmicShare tools (www.omicshare.com/tools).

Prediction of the conservation and the secondary structure of SigmaR1
In order to analyze the secondary structure and sequence conservation of SigmaR1, the sequence similarities and secondary structure of SigmaR1 were performed ESPript 3.x83.

Molecular docking
To analyze the binding affinities and modes of interaction between SigmaR1 (PDB: 6DJZ) and PC (CID: 10425706), CB-Dock2, the silico protein-ligand docking blind softwares were employed84,85.

Statistical analysis
All statistical analyses and p-value determinations were performed in GraphPad Prism 6. All the error bars represent Mean ± SD. To determine p-values, ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed among multiple groups and a two-tailed unpaired student t-test was performed between two groups.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript investigates the intriguing role of SigmaR1 in ER sheet localization and its interaction with the translocon complex, providing valuable insights into the regulation of protein and lipid homeostasis. The identification of SigmaR1 as a lipid-binding protein associated with ER sheet-specific functions represents a novel perspective on its physiological roles and broadens our understanding of the molecular machinery involved in ER morphology and lipid metabolism.\nHowever, despite the strengths of the study, several critical issues limit the overall impact and reliability of the findings. Additional experiments and improved discussion of mechanistic and physiological implications are needed to fully support the claims and enhance the impact of the study. In the current version, this manuscript is not suitable for publication in the Nature Communications.\n1. In Fig 1F, the colocalization of sfGFP-SigmaR1 KI#2 and Sec61β was not shown.\n2. In Fig S1F, it should be co-stained with sec61β to show whether the localization of SigmaR1 on the ER sheet is affected.\n3. Additional methods should be employed to confirm the location of SigmaR1 on the ER sheet, such as FRET, proximity ligation assay, or Co-IP.\n4. Fig 2, it is confused why Reticulon 4 is used to indicate the localization of SigmaR1. As the authors mentioned, Reticulon 4 is the marker of ER-tubular, while sec61β is the marker of ER sheet. In order to prove the localization of SigmaR1, Co-staining of sec61β and SigmaR1 is required.\n5. The short region (residues 2-8) and the TM domain mediated ER-sheet targeting and LD targeting were specific for SigmaR1 protein? or they can regulate a wide range of proteins localization?\n6. Refine the presentation of figures and ensure that all critical results are clearly illustrated and labeled. The description of Figure 2F-H is confused and does not correspond to the result. The serial numbers of the pictures in the last two Results sections are wrong. Fig4 should be Fig5 and Fig5 should be Fig6. The serial number of Fig I-M pictures in the last Result section is confused.\n7. While the manuscript presents compelling evidence of SigmaR1’s localization and function, the link between its lipid-binding activity and its role in ER morphology and lipid homeostasis is not fully explored. The mechanistic basis of how SigmaR1 coordinates these processes remains unclear.\n8. The use of HeLa and HepG2 cells limits the generalizability of the findings. Additional validation in other cell types, particularly those with distinct ER morphologies, could enhance the study's impact.\n9. The lipidomics results suggest a preference for PC binding but do not fully rule out interactions with other lipids or potential lipid-binding partners. Expanding these experiments would strengthen the conclusions. The figure legends were also missed in this part.\n10. The study does not include direct evidence of SigmaR1’s role in maintaining ER sheet-to-tubule ratios or its dynamics in response to cellular stress or lipid perturbations.", "reviewer_2": "The morphology of the ER is tightly linked to its function. In this manuscript, the authors investigated a recently identified ER-sheet forming protein SigmaR1, and found that it has some lipid binding activity and a broad correlation with protein and lipid homeostasis of the cell. Overall, the work is mostly descriptive at this stage and offers limited mechanistic insight into the function of SigmaR1.\n1. The membrane topology analysis appears to be complicated for SigmaR1. In reference #48, it has been shown that N-terminal large tag causes problem for the membrane orientation of SigmaR1. However, large tag GFP was positioned by the authors here to determine topology. Along this line, the usage of NT-GFP KI cells needs caution as the problem mentioned above and the potential impact on NT-mediated protein interactions described below. Finally, the authors need to clarify whether the SigmaR1-GFP used in the rescue assay in 6C is indeed CT-GFP but not NT-GFP. If so, the NT-GFP version has to be put into the same test before the KI cell data can be used.\n2. Similar tagging issue applies to Fig. 2. GFP or Halo appears at both ends. Functional tests are needed before these constructs can be used for analysis. In addition, the authors claim that a SigmaR1 truncation mutant lacking an NT region containing the short region (residues 2-8) and the TM was localized to LD. The image says otherwise. The overlap of signals is marginal. The link between SigmaR1 and the LD is tempting but the membrane topology favored here is not compatible with its binding or relocalizing to LDs. The spatial association between SigmaR1 and LDs thus lacks physical evidence.\n3. The interactions between SigmaR1 and translocon has been reported, but the mechanisms proposed here is problematic. In this case, the membrane topology of SigmaR1 is indeed critical. In the two AF predicted complexes, SigmaR1 is positioned in a non-type II orientation; its “cytosolic NT” is facing the luminal region of the binding partners. In addition, the NT soluble region and the following TM all have contacts with the binding partners. It is thus important to test whether these interactions are TM-dependent or soluble region-dependent.\n4. The part where the global protein expression is decreased is less informative. On one hand, if the translocon interactions are real, the changes are expected. On the other hand, limited mechanistic insight is offered here and the “phenotype” is not rescued in this case. For example, whether NT-deleted mutant or lipid-binding mutants would restore protein homeostasis.\n5. The part where lipid binding is detected and LDs are enlarged in KD cells is interesting, but not so surprising, and at the same time, also not so informative. As mentioned by the authors, the lipid binding has been reported for the receptor role of the protein. The author proposed that the LD phenotype is likely linked to ER morphology. In this case, one would wonder if the phenotype can be reversed by simply manipulating tubule-to-sheet ratio.\n6. A minor point, the topology has not been analyzed until the second part but appears in the first subtitle.", "reviewer_3": "The paper by Hu et al., uses live cell imaging, mass spectrometry, biochemical analysis, and AlphaFold modeling to characterize the ER membrane protein, SigmaR1. This study follows on the heels of another publication (Sawyer et al., Dev Cell 2024) showing that SigmaR1 promotes formation of rough ER (sheets) and opposes the formation of smooth ER (tubules). Hu et al. confirm SigmaR1 enrichment at ER sheets and attempt to go further with data in support of a model in which SigmaR1 binds lipids (particularly PC) and components of the ER translocon to regulate protein and lipid homeostasis. These data are flawed, and therefore I do not support its publication in Nature Communications.\nData claiming direct interaction between SigmaR1 and components of the ribosome-translocon complex are also unconvincing. The authors need to take a more rigorous approach, for example, by comparing SigmaR1 interaction partners in the ribosomal-bound and ribosome-free fractions derived from solubilized ER microsomes—by mass spec, co-IP and/or sucrose gradients.\nMechanistic insights derived from AlphaFold modeling are almost certainly incorrect. Although the authors fail to provide any details that might allow one to evaluate the quality of the predictions, the SigmaR1-Trapa and SigmaR1-Nicalin AlphaFold models are obviously incorrect based solely on topological considerations: the models show SigmaR1 with its N-terminus in the lumen—this is not a type II topology (Fig S4A,B and Fig S5G). It is also unclear why the authors focus on a SigmaR1 monomer, when previous structural studies indicate it functions as a trimer (e.g., Schmidt et al., 2016).\nThe proteomic analysis of SigmaR1 deletion is over-interpreted. Rather than provide support for a direct interaction between SigmaR1 and the translocon, these data likely reflect pleiotropy resulting from its effects on ER morphology.\nData showing retained lipid-binding activity of a SigmaR-80-168 mutant (Fig 4F) are not convincing. Although this construct retains the core beta-barrel, deletion of several helices that normally surround the barrel almost certainly render it insoluble and/or misfolded and unable to bind ligands. This raises significant concerns about the validity of the lipid binding assay used here. The data for a series of point mutants (Fig 4H-K) does little to alleviate this concern.", "reviewer_4": "In this study, using high-resolution microscopy in combination with endogenous labeling and membrane fractionation, authors further demonstrate that SigmaR1 is a type II integral protein specifically enriched at ER sheet which is consistent with others’ previous findings. Besides, the manuscript also presents interesting and valuable data on the functional significance of SigmaR1 in regulating protein and lipid homeostasis based on proteomic and biochemical studies which added more novelty to the work. Overall, the experimental design is strong, and the conclusions drawn are supported by the data. However, there are several areas and details where the paper could be improved:\n1. In Figure 1A-D, to show the colocalization of SigmaR1 and different ER maker proteins by living cell imaging, all proteins including the ER maker proteins were overexpressed which may affect their natural locations, so it may be more convincing if we could label the endogenous marker protein at lease for the ER-sheet marker Sec61β as in Figure 1F for the SigmaR1 and show the colocalization of endogenous proteins.\n2. Although Figure1G shows a statistically better colocalization of SigmaR1 and Sec61β, the related images are missing in Figure1F.\n3. The content of fraction 0-11 in Figure 1H should be clearly indicated somewhere in the paper. According to the WB data, the 7-10 lane seems very likely to be fraction of ER tubules because of the high level of Reep5, but the Reticulon 4 did not show any enrichment in those lanes which may make people doubt if the Reticulon 4 is a good marker for ER tubules when Reticulon 4 was used quite a lot in the rest of the studies.\n4. Figure1/S1 and related paragraphs are insufficient to support the subtitle that “SigmaR1 is a type II integral protein enriched at ER sheet.”, but together with data in Figure2/S2 may better support the conclusion that SigmaR1 is a type II integral protein.\n5. To support that “A short region at the N-terminus is sufficient and required for the ER-sheet localization of SigmaR1.”, ER tubular protein Reticulon 4 is used as a marker, I wondered why it’s preferred over ER sheet protein Sec61β which might be more straightforward. Figure 2E shows that WT SigmaR1 is worse colocalized with tubular protein compared to 1-30, which indicate WT may have a better colocalization with ER sheet, so it seems that the 30-223 is still helpful in SigmaR1’s ER sheet targeting and the N-terminus of SigmaR1 is partially contributing to its ER-sheet localization. In the previous paper “SigmaR1 shapes rough endoplasmic reticulum membrane sheets” which was published late last year on Developmental Cell, the Figure 3H-I also suggest that Lumenal SigmaR1 lacking its TM domain localized exclusively to ER sheets and increased ER sheet area and the mutations at C terminus which disrupted its oligomerization can dramatically decrease SigmaR1’s targeting to the ER-sheet.\n6. In Figure S2, authors tried to investigate the membrane topology by using the GFP and GFP nanobody system and suggest that the SigmaR1 is a type II integral protein at ER sheet. The data as in Figure S2 D-E support that the N terminus of SigmaR1 is exposed in the cytosol and the C-terminus is in ER lumen but based on the presented data we may not be able to rule out the possibility of the existence of the model in S2 F, because the mCh-GFPnb is cytosol resident. However, in the previous Nature paper “Crystal structure of the human σ1 receptor” (2017), researchers already used the X-ray crystallography solved the structure of SigmaR1 as a homotrimer and each subunit is a ∼25-kDa single-pass integral membrane protein that terminates with two α helices at its C terminus. Also, Multiple experimental approaches confirm that SigmaR1 is oriented as a type II topology membrane protein, for example the JBC paper “The sigma-1 receptor is an ER-localized type II membrane protein” (2021).\n7. Under the subtitle “A short region at the N-terminus is sufficient and required for the ER-sheet localization of SigmaR1.”, when Figure 2G was described, 2F was mis-referred to, when 2H was described, 2G was mis-cited, while 2F was not described in the paragraph. Besides, for 2F, SigmaR1-Halo is used but not SigmaR1-GFP as in other images, is it true or mis-labeled?\n8. For Figure S3L, both inputs are labeled TRAM1-GFP negative.\n9. For Figure 6B, the SigmaR1 MW is mis-labeled as ~60KD. Based on Figure 6C, it’s insufficient to conclude that “the strong accumulation of LDs was specific to SigmaR1”, because the WT SigmaR1 only partially rescued the phenotype, and the restored level of WT looks quite similar to that of SigmaR1-△2-8. It’s better to use both siRNA1 and 3 to rule out the possibility of off targets.\nIn addition, there are some inconsistencies existing. For example, a lot of different size of images and scale bar are used in the live imaging results; in Figure 4H’-K’, both “-” and “–” are used, also different font are used; in Figure 5, different font size are used, and some are barely to see; the triglycerides (TG) was mentioned in the paper but the TAG was frequently seen in the paper which I suppose to be the same; different cells lines including COS7, HeLa, HepG2, 293T etc. are used in the study, it would be a bit complex to integrate data, for example, the location of SigmaR1 to ER sheet was demonstrated in the COS7 and HeLa cells, while the SigmaR1 interacting with translocon complex and binding to PC was tested in HeLa and 293T cells, and it said because the lipids phenotype could not be seen in SigmaR1 KO HeLa cells so the KO was conducted in HepG2 although HepG2 has much lower SigmaR1 according to Figure S1B, but the quantitative MS study conducted with HeLa sample suggested SigmaR1 KO in HeLa cells also increased the lipid metabolism, based on which, it seems that SigmaR1’s regulation on lipids is not hepatocyte specific, after all, and it’s hard to conclude any relation between these findings like whether there’s any link between the translocon complex protein interaction and the lipid binding/regulation.\nAlso, there’re some repetitions in the manuscript. For example, for the paragraphs following “SigmaR1 KO impairs cellular protein homeostasis”, figures supporting this subtitle should be Figure5, but Figure4 are repeated cited there, and it’s the same problem for the paragraphs following “SigmaR1 KO resulted in an accumulation of lipid droplets in hepatocytes.”, the related figure should be Figure6 not Figure5. Also, sentence like “Recently, the Nicalin -TMEM147-NOMO1/2/3 complex, TMCO1 and CCDC47 have been recently identified as novel conserved components of the translocon complex during biogenesis of multipass membrane proteins.” should be double checked and improved. Moreover, there are two “Table 1. Plasmids used in this Study” as for both plasmids and primers.\nOverall, the structure of the paper is logical, the evidence from different methods can well support the conclusions, the discussion also emphasized the novelty and outlook of the work. Improvements on details may improve the quality of the paper.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We appreciate your thoughtful and constructive comments, which have helped us significantly improve the quality and impact of our work. We have carefully considered all points raised and have conducted additional experiments and analyses as suggested.\n1. In Fig 1F, the colocalization of sfGFP-SigmaR1 KI#2 and Sec61β was not shown.\nThank you for the comment. As suggested, we now add two additional imaging results showing the colocalization between sfGFP-SigmaR1 KI and Halo-Sec61β (Fig. 1F, I) in live cells or endogenous Sec61β in immunofluorescence (Fig. 1G, I).\n2. In Fig S1F, it should be co-stained with sec61β to show whether the localization of SigmaR1 on the ER sheet is affected.\nThank you for the comment. We now add a new dataset showing the colocalization of SigmaR1 and Sec61β in Fig. S1F, G of the revised manuscript.\n3. Additional methods should be employed to confirm the location of SigmaR1 on the ER sheet, such as FRET, proximity ligation assay, or Co-IP.\nWe now provide two lines of evidence to support the ER-sheet locaition of SigmaR1 and a direct and specific interaction between SigmaR1 and translocon components TRAPa and Niclain. First, we performed fluorescence Lifetime Imaging Microscopy (FLIM) to validate a specific interaction between SigmaR1 and Nicalin at ER sheets. The FLIM results demonstrated that mClover2 fluorescence lifetime was significantly reduced in a cell co-expressing SigmaR1-mClover2 and mRuby2-Nicalin (yellow asterisk) compared to that in another cell without mRuby2-Nicalin (white asterisk) in the same imaging field (Fig. 3F, top panel; 3G). In contrast, the mClover2 fluorescence lifetime was not significantly impacted in cells co-expressing SigmaR1-mClover2 and Reep5-mRuby2 (Fig. 3F, middle panel; 3G), indicating no direct interaction between these two proteins. Notably, a slight reduction in lifetime was observed with the ER sheet marker Sec61β-mRuby2, suggesting these proteins are co-enriched in the same domain but do not directly interact (Fig. 3F, bottom panel; 3G).\nSecond, the location of SigmaR1 on the ER sheet was further validated by sucrose gradient centrifuge. We compared SigmaR1 distribution in the ribosomal-bound and ribosome-free fractions derived from solubilized ER microsomes in the SigmaR1 KI line. We found that endogenous SigmaR1 was preferentially co-enriched with the two translocon proteins in ER sheet fractions specifically enriched with ribosomes (Fig. 3D). Furthermore, GFP trap assays demonstrated that the interaction between SigmaR1 and these two translocon proteins specifically occurred in the ER sheet fractions (Fig. 3E). As a control, no interaction between SigmaR1 and another ER sheet protein Sec61b was detected in the ribosome-bound ER fraction (Fig. S5N).\n4. Fig 2, it is confused why Reticulon 4 is used to indicate the localization of SigmaR1. As the authors mentioned, Reticulon 4 is the marker of ER-tubular, while sec61β is the marker of ER sheet. In order to prove the localization of SigmaR1, Co-staining of sec61β and SigmaR1 is required.\nThank you for this thoughtful suggestion. We now provide imaging results showing co-staining of sec61β and SigmaR1 in Fig.2 of revised manuscript.\n5. The short region (residues 2-8) and the TM domain mediated ER-sheet targeting and LD targeting were specific for SigmaR1 protein? or they can regulate a wide range of proteins localization?\nWe appreciate this insightful comment. In response, we generated a chimera by fusing the ER-sheet targeting module of SigmaR1—comprising a short region (residues 2-8) and its transmembrane domain—to the tubular ER protein Tex2. This chimera was highly enriched in ER sheets, whereas the short region alone was insufficient for targeting (Fig. 2G, H; S3A, B). Due to time limitations in the revision process, we were unable to extend this analysis to more proteins. Nonetheless, this successful proof of concept supports the efficiency of the SigmaR1 module in directing heterologous ER proteins to sheets.\nWe do not believe SigmaR1 directly targets lipid droplets (LDs) for three reasons. First, as a type II integral ER protein with a large luminal domain (residues 31 to the C-terminus), SigmaR1 is physically excluded from associating with cytosolic LDs. Second, endogenous SigmaR1 was absent from isolated LD fractions (Fig. S3F). Third, full-length SigmaR1 did not colocalize with LDs; LD association was only observed in truncation mutants like SigmaR1-Δ2-30 (Fig. S3C-E). We speculate that this N-terminal truncation, which is essential for ER sheet targeting, prevents proper membrane insertion, resulting in artificial LD localization. Although we have preliminary evidence that SigmaR1 may associate with ER lumen-resident lipoprotein particles in hepatocytes via its C-terminal α5 helix, these findings are beyond the scope of this manuscript and will be presented in a future study.\n(Unpublished result)\n6. Refine the presentation of figures and ensure that all critical results are clearly illustrated and labeled. The description of Figure 2F-H is confused and does not correspond to the result. The serial numbers of the pictures in the last two Results sections are wrong. Fig4 should be Fig5 and Fig5 should be Fig6. The serial number of Fig I-M pictures in the last Result section is confused.\nWe apologze for these mistakes and any confusion. We now thoroughly correct these mistakes.\n7. While the manuscript presents compelling evidence of SigmaR1’s localization and function, the link between its lipid-binding activity and its role in ER morphology and lipid homeostasis is not fully explored. The mechanistic basis of how SigmaR1 coordinates these processes remains unclear.\nThank you for this insightful comment. We now add two lines of data illustrating how SigmaR1 coordinates ER morphology and LD dynamics. First, we performed loss of fucntion assays, and found that depletion of SigmaR1 but not other known ER sheet factors p180, Climp63 or kinectin, significantly impacted LD dynamics, indicating that ER sheet-to-tubule ratio does not solely account for LD defect in SigmaR1-depleted cells.\nSecond, we generated a point mutant in the background of the full length protein, in which several hydrophobic residues were mutated to W in the binding domain (residues 80-168) for steric hinderance (V104W, L105W, L106W, F107W). We performed in vitro lipid binding assays to confirm the reduced lipid binding ability of the point mutant. Rescue experiment demonstrated that this lipid binding mutant did not fully restore the LD accumulation phenotype.\nOverall, we propose that the role for SigmaR1 in LD dynamics depends on an orchastrated interplay between lipid binding ability and translocon interaction in ER sheet. One plausible possibility is that lipid binding triggers conformational change and regulates the interaction of SigmaR1 with the translocon complex, consequently modulating translocon activity. This hypothesis will be tested in a future study.\n8. The use of HeLa and HepG2 cells limits the generalizability of the findings. Additional validation in other cell types, particularly those with distinct ER morphologies, could enhance the study's impact.\nThanks for the thoughtful suggestion. As suggested, we confirmed the ER sheet localization of SigmaR1 and the LD accumulation phenotype in multiple cell lines, including hepatic cells (Huh7), pancreatic cancer line PaCa2, and Neuro2a cells. Notably, this lipid accumulation phenotype exhibited cell-type specificity. SigmaR1 depletion resulted in an aberrant LD accumulation in Huh7 cell line (Fig. S9B-D). In contrast, SigmaR1 depletion in non-hepatic cell lines—including pancreatic PaCa2 cells (Fig. S9J-L) and mouse neuroblastoma Neuro2a cells (Fig. S9M-O)—resulted in a moderate LD phenotype, despite robust conservation of its ER sheet localization. We attribute this cell-type-dependent effect to the specialized role of hepatocytes in enhanced lipid synthesis, storage, metabolism and secretion, which appears to amplify the phenotypic severity of SigmaR1 loss.\nIn addition, we performed transmission electron microscopy (TEM) to examine the ultrastructural details of LDs in SigmaR1-depleted HepG2 cells. TEM micrographs revealed a strong increase in LD diameter without a significant change in their number (Fig. 6I-L).\n9. The lipidomics results suggest a preference for PC binding but do not fully rule out interactions with other lipids or potential lipid-binding partners. Expanding these experiments would strengthen the conclusions. The figure legends were also missed in this part.\nWe thank the reviewer for this critical insight. We now provide four additional lines of evidence to strengthen this conclusion. First, to avoid artificial effect induced by overexpression, we performed lipidomics analyses to identify SigmaR1-associated lipids in the KI cell line. Lipidomics confirmed the preference for PC binding by endogenous SigmaR1. The original lipidomic data is now replaced by this result as Fig. 4B in the revised manuscript.\nSecond, the direct binding of SigmaR1 to PC was further validated by in vitro liposome pelleting assay (Fig. 4G, H).\nLast, molecular docking predicts the PC-binding in the hydrophobic pocket in the β-barrel of SigmaR1 (Fig. S7A, B; 4C).These new results confirm that SigmaR1 binds PC in the β-barrel structure. We now add these new results in the revised manuscript.\n10. The study does not include direct evidence of SigmaR1’s role in maintaining ER sheet-to-tubule ratios or its dynamics in response to cellular stress or lipid perturbations.\nThis is a great comment. As suggested, we explored a potential role for SigmaR1 under several cellular stresses related to ER dynamics, including ER stress (+Thapsigargin), lipotoxicity (+Palmitic acid), starvation (EBSS). Among these stresses, only starvation was reported to trigger the formation of ER sheet (36520888), which can be recapitulated in our hands. Importantly, the starvation-induced ER sheet formation was significantly inhibited upon SigmaR1 depletion (Fig. 1L, M), indicating that starvation-induced ER sheet formation depends on SigmaR1.\nReviewer #2 (Remarks to the Author):\nThe morphology of the ER is tightly linked to its function. In this manuscript, the authors investigated a recently identified ER-sheet forming protein SigmaR1, and found that it has some lipid binding activity and a broad correlation with protein and lipid homeostasis of the cell. Overall, the work is mostly descriptive at this stage and offers limited mechanistic insight into the function of SigmaR1.\nWe thank the reviewer for this insightful comment and agree entirely that understanding the mechanistic function of SigmaR1 is a crucial next step, and we believe that our work serves as an important foundation by establishing SigmaR1 as a novel translocon component at ER sheet and demonstrating its broad correlation with cellular homeostasis. In response to your comment, we have performed additional experiments to provide more mechanistic insight into SigmaR1 function.\n1. The membrane topology analysis appears to be complicated for SigmaR1. In reference #48, it has been shown that N-terminal large tag causes problem for the membrane orientation of SigmaR1. However, large tag GFP was positioned by the authors here to determine topology. Along this line, the usage of NT-GFP KI cells needs caution as the problem mentioned above and the potential impact on NT-mediated protein interactions described below. Finally, the authors need to clarify whether the SigmaR1-GFP used in the rescue assay in 6C is indeed CT-GFP but not NT-GFP. If so, the NT-GFP version has to be put into the same test before the KI cell data can be used.\nWe thank the reviewer for these critical comments, and these comments can be further divided into three sub-questions.\n1) The membrane topology analysis appears to be complicated for SigmaR1. In reference #48, it has been shown that N-terminal large tag causes problem for the membrane orientation of SigmaR1. However, large tag GFP was positioned by the authors here to determine topology.\nWe appreciate these insightful suggestions. We agree that the placement of large tags on the NT of SigmaR1 requires careful validation, and we provide additional data to strengthen our conclusions. First, taking advantage of a newly developed genetically expressed Flag frankenbody, we used the small Flag tag to confirm the topology of SigmaR1. We found that, consistent with GFP tagged versions, Flag-SigmaR1 but not SigmaR1-Flag, was recognized by cytosolic Halo-frankenbody-Flag, confirming the topology described in the manuscript.\n2) Along this line, the usage of NT-GFP KI cells needs caution as the problem mentioned above and the potential impact on NT-mediated protein interactions described below.\nThanks for these comments. We provided two lines of evidence to prove the functionality of sfGFP-SigmaR1. First, the sfGFP-SigmaR1 displayed an ER sheet localization similar with SigmaR1-GFP. Second, introduction of the sfGFP-SigmaR1 efficiently restored the LD accumulation in SigmaR1-depleted cells (Fig. 6N).\n(Unpublished result)\nImportantly, we purified His-Flag tagged SigmaR1 proteins from HEK293F cells and performed in vitro pulldown assays with two translocon proteins (Nicalin and TRAPα). The binding between His-Flag-SigmaR1 and these two translocon proteins were confirmed (Fig. 3C). Therefore, these results confirmed that a generic binding between SigmaR1 and Nicalin and TRAPα.\n3) Finally, the authors need to clarify whether the SigmaR1-GFP used in the rescue assay in 6C is indeed CT-GFP but not NT-GFP. If so, the NT-GFP version has to be put into the same test before the KI cell data can be used.\nThank you for these comments. We confirm that CT-GFP (SigmaR1-GFP) was used in Fig. 6C. As noted previously, the sfGFP-SigmaR1 construct was also functional in our rescue experiments. Although the NT-GFP fusion was problematic in reference #48, the sfGFP-SigmaR1 variant functioned correctly in our hands. We speculate that the superior folding efficiency of monomeric sfGFP may explain this discrepancy.\n2. Similar tagging issue applies to Fig. 2. GFP or Halo appears at both ends. Functional tests are needed before these constructs can be used for analysis. In addition, the authors claim that a SigmaR1 truncation mutant lacking an NT region containing the short region (residues 2-8) and the TM was localized to LD. The image says otherwise. The overlap of signals is marginal. The link between SigmaR1 and the LD is tempting but the membrane topology favored here is not compatible with its binding or relocalizing to LDs. The spatial association between SigmaR1 and LDs thus lacks physical evidence.\nWe thank the reviewer for this critical comments, and these comments can be further divided into three sub-questions.\n1) Similar tagging issue applies to Fig. 2. GFP or Halo appears at both ends. Functional tests are needed before these constructs can be used for analysis.\nThanks for these comments. As mentioned above, we have confirmed the functionality of sfGFP-SigmaR1. We now add a new dataset showing the SigmaR1 truncation (residues 10-30) tagged with GFP at its C terminal (SigmaR1-10-30-GFP) as Fig. 2C.\nIn case of SigmaR1-Halo shown in Fig.2F, we now add a new imaging result showing the spatial relationship between SigmaR1-GFP and lipid droplets as Fig.S3E. The original image with SigmaR1-Halo was removed.\n2) In addition, the authors claim that a SigmaR1 truncation mutant lacking an NT region containing the short region (residues 2-8) and the TM was localized to LD. The image says otherwise. The overlap of signals is marginal.\nWe apologize for the confusion. We mistakenly cited Fig. 2F (it should be Fig. 2G, H in the original manuscript). We now correct the mis-citing problem, and move these results to the supplementary section as Fig. S3C-E in the revised manuscript.\n3) The link between SigmaR1 and the LD is tempting but the membrane topology favored here is not compatible with its binding or relocalizing to LDs. The spatial association between SigmaR1 and LDs thus lacks physical evidence.\nThis is an insightful comment! We agree with you that SigmaR1 does not physically target LD based on three lines of evidence. First, our results showed that SigmaR1 is a type-II ER integral protein with a large portion of this protein (from resides 31 to C terminus) resident in ER lumen, which physically exclude its association with cytosolic LDs. Second, we isolated LD fractions and found that endogenous SigmaR1 was absent in this fraction as shown in Fig.S3F of revised manuscript. Third, as seen in Fig. S3E of the revised manuscript, SigmaR1 was not cololcaized with LDs, and LD association can only be seen in SigmaR1 truncations for example SigmaR1-△2-30 in Fig. S3C, D. Given the essential role of this N-terminus region in ER sheet targeting, we speculate that this truncation mutant can not be properly insert into ER membranes, resulting in artificial targeting to LDs.\nThat said, we have some clue that SigmaR1 may associate with ER lumen-resident LDs (lipoprotein particles) in primary liver cells, and the α5 in the C-terminal region appears to be important for the assocaition. We decide to save these results for the next study and not to add them in this manucript.\n(Unpublished result)\n3. The interactions between SigmaR1 and translocon has been reported, but the mechanisms proposed here is problematic. In this case, the membrane topology of SigmaR1 is indeed critical. In the two AF predicted complexes, SigmaR1 is positioned in a non-type II orientation; its “cytosolic NT” is facing the luminal region of the binding partners. In addition, the NT soluble region and the following TM all have contacts with the binding partners. It is thus important to test whether these interactions are TM-dependent or soluble region-dependent.\nWe thank the reviewer for this critical comments, and these comments can be further divided into two sub-questions.\n1) The interactions between SigmaR1 and translocon has been reported, but the mechanisms proposed here is problematic. In this case, the membrane topology of SigmaR1 is indeed critical. In the two AF predicted complexes, SigmaR1 is positioned in a non-type II orientation; its “cytosolic NT” is facing the luminal region of the binding partners.\nWe appreciate this excellent suggestion. We apologize for the error in the orientation of the predicted model. The corrected model, now presented in Fig. S10F of the revised manuscript, properly shows the C-terminus of SigmaR1 residing in the ER lumen and the N-terminus facing the cytosol to contact its binding partners.\n2) In addition, the NT soluble region and the following TM all have contacts with the binding partners. It is thus important to test whether these interactions are TM-dependent or soluble region-dependent.\nThis is a great suggestion. In the original manuscript, we found that the region containing residues 2-8 was critical for SigmaR1 targeting to ER sheet, as the deletion of this region led to the re-distribution of SigmaR1-△2-8-GFP to the whole ER network including ER tubules (Fig. 2D). Consistently, our coIP assays showed deletion of the residues 2-8 reduced the interaction between SigmaR1 and the translocon proteins TRAPα and Nicalin (Fig. S6E, F). Importantly, deletion of the TM domain caused mis-location of SigmaR1 from the ER to LD surface (Fig. S3C, D), thereby completely losing the interaction with ER-resident tranlocon complex.\nImportantly, we generated a chimera by fusing the ER-sheet targeting module of SigmaR1—comprising a short region (residues 2-8) and its transmembrane domain—to the tubular ER protein Tex2. This chimera was highly enriched in ER sheets, whereas the short region alone was insufficient for targeting (Fig. 2G, H; S3A, B). Due to time limitations in the revision process, we were unable to extend this analysis to more proteins. Nonetheless, this successful proof of concept supports the efficiency of the SigmaR1 module in directing heterologous ER proteins to sheets. Therefore, we believe that the interactions between SigmaR1 and translocon depends on both the soluble N-terminus region and the TM domain.\n4. The part where the global protein expression is decreased is less informative. On one hand, if the translocon interactions are real, the changes are expected. On the other hand, limited mechanistic insight is offered here and the “phenotype” is not rescued in this case. For example, whether NT-deleted mutant or lipid-binding mutants would restore protein homeostasis.\nWe thank the reviewer for these critical comments, and these comments can be further divided into two sub-questions.\n1) The part where the global protein expression is decreased is less informative.\nWe agree that a deeper mechanistic investigation was warranted. In response, we measured markers of the integrated stress response and found a significant increase in phosphorylated eIF2α, while total eIF2α level remained unchanged in SigmaR1 KO cells (Fig. 5A). Since eIF2α phosphorylation is known to globally suppress protein synthesis in response to cellular stress, these findings suggest a role for SigmaR1 in the maintenance of protein homeostasis. Notably, SigmaR1 deficiency did not induce or exacerbate ER stress triggered by thapsigargin, as demonstrated by unaltered BiP expression in immunoblots (Fig. 5A) and ER whorls (marked by Sec61β) (Fig. S8B, C), which are established hallmarks of ER stress. Therefore, these results rule out the possibility that this effect is indirectly caused by ER stress. We have added this data in the result section of revised manuscript.\n2) On one hand, if the translocon interactions are real, the changes are expected. On the other hand, limited mechanistic insight is offered here and the “phenotype” is not rescued in this case. For example, whether NT-deleted mutant or lipid-binding mutants would restore protein homeostasis.\nThis is an excellent suggestion. To investigate the mechanistic link between molecular function of SigmaR1 and protein homeostasis dysregulation, we performed rescue experiment in SigmaR1 KO HeLa cells by reconstituting expression of SigmaR1-GFP, an N-terminal deletion mutant (defective in ER sheet targeting), or the lipid-binding Mut-2 variant. Cells expressing these constructs were isolated by flow cytometry for subsequent proteomic analysis (Fig. 5C). Introduction of SigmaR1-GFP efficiently restored global protein homeostasis, particularly rescuing ribosomal pathways (Fig. 5D; S8E). In contrast, either the NT-deletion mutant or the lipid-binding mutant (Mut-2) failed to fully restore the proteome (Fig. 5E, F; S8F, G), underscoring the importance of ER sheet localization/ translocon interaction and lipid binding for SigmaR1 function. These results support that the role for SigmaR1 depends on an orchastrated interplay between lipid binding ability and translocon interaction in ER sheet. Our hypothesis is that lipid binding triggers conformational change and regulates the interaction of SigmaR1 with the translocon complex, consequently modulating translocon activity. This hypothesis will be tested in a future study.\n5. The part where lipid binding is detected and LDs are enlarged in KD cells is interesting, but not so surprising, and at the same time, also not so informative. As mentioned by the authors, the lipid binding has been reported for the receptor role of the protein. The author proposed that the LD phenotype is likely linked to ER morphology. In this case, one would wonder if the phenotype can be reversed by simply manipulating tubule-to-sheet ratio.\nThank you for this critical comments, and these comments can be further divided into two sub-questions.\n1) The part where lipid binding is detected and LDs are enlarged in KD cells is interesting, but not so surprising, and at the same time, also not so informative. As mentioned by the authors, the lipid binding has been reported for the receptor role of the protein.\nWe appreciate the reviewer's perspective that the lipid-binding capability itself is known, and we have now toned down any language that might have over-interpreted the novelty of this specific finding. Our focus is now more clearly on the novel interaction with the translocon and the disrupted lipid homeostasis/ enlarged LDs and the functional link between them.\n2) The author proposed that the LD phenotype is likely linked to ER morphology. In this case, one would wonder if the phenotype can be reversed by simply manipulating tubule-to-sheet ratio.\nFollowing the reviewer's excellent suggestion, we now add two lines of data illustrating how SigmaR1 coordinates ER morphology and LD dynamics. First, we performed loss of fucntion assays, and found that depletion of SigmaR1, but not other known ER sheet factors p180 and Climp63, significantly impacted LD dynamics. In addition, depletion of another ER sheet factor Kinectin caused accumulated LDs, but to a lesser extent compared to SigmaR1 depletion. These results indicate that ER sheet-to-tubule ratio does not solely account for LD defect in SigmaR1-depleted cells.\nSecond, SigmaR1 point mutants with reduced PC binding but maintaining ER sheet localization failed to rescue the LD defect in SigmaR1-depleted cells (Fig. 6P, R), indicating that ligand-binding of SigmaR1 is also required for LD dynamics. As mentioned above, we believe that the role for SigmaR1 depends on an orchastrated interplay between ligand binding ability and translocon interaction on ER sheet, and lipid binding may trigger conformational change and regulates the interaction of SigmaR1 with the translocon complex, consequently modulating translocon activity. We will test this hypothesis in next study.\n6. A minor point, the topology has not been analyzed until the second part but appears in the first subtitle.\nThank you for this point. We now change the first subtitle.\nReviewer #3 (Remarks to the Author):\nThe paper by Hu et al., uses live cell imaging, mass spectrometry, biochemical analysis, and AlphaFold modeling to characterize the ER membrane protein, SigmaR1. This study follows on the heels of another publication (Sawyer et al., Dev Cell 2024) showing that SigmaR1 promotes formation of rough ER (sheets) and opposes the formation of smooth ER (tubules). Hu et al. confirm SigmaR1 enrichment at ER sheets and attempt to go further with data in support of a model in which SigmaR1 binds lipids (particularly PC) and components of the ER translocon to regulate protein and lipid homeostasis. These data are flawed, and therefore I do not support its publication in Nature Communications.\nWe thank the reviewer for critical assessment of our manuscript. We have taken these comments seriously and have performed significant new experiments and analyses to address the concerns raised. We believe the manuscript is now substantially improved and hope the reviewer finds our responses satisfactory.\nData claiming direct interaction between SigmaR1 and components of the ribosome-translocon complex are also unconvincing. The authors need to take a more rigorous approach, for example, by comparing SigmaR1 interaction partners in the ribosomal-bound and ribosome-free fractions derived from solubilized ER microsomes—by mass spec, co-IP and/or sucrose gradients.\nWe appreciate this great comment. We now provide two lines of evidence to support a direct and specific interaction between SigmaR1 and translocon components TRAPa and Niclain. First, we compared SigmaR1 interaction partners in the ribosomal-bound and ribosome-free fractions derived from solubilized ER microsomes by sucrose gradient centrifuge. We found that endogenous SigmaR1 was preferentially co-enriched with the two translocon proteins in ER sheet fractions enriched with ribosomes (Fig. 3D). Furthermore, GFP trap assays demonstrated that the interactions between SigmaR1 and these two translocon proteins specifically occurred in the ER sheet fractions (Fig. 3E). As a control, no interaction between SigmaR1 and another ER sheet protein Sec61β was detected in the ribosome-bound ER fraction.\nSecond, we performed fluorescence Lifetime Imaging Microscopy (FLIM) to validate a specific interaction between SigmaR1 and Nicalin at ER sheets. The FLIM results demonstrated that mClover2 fluorescence lifetime was significantly reduced in a cell co-expressing SigmaR1-mClover2 and mRuby2-Nicalin (yellow asterisk) compared to that in another cell without mRuby2-Nicalin (white asterisk) in the same imaging field (Fig. 3F, top panel; 3G). In contrast, the mClover2 fluorescence lifetime was not significantly impacted in cells co-expressing SigmaR1-mClover2 and Reep5-mRuby2 (Fig. 3F, middle panel; 3G), indicating no direct interaction between these two proteins. Notably, a slight reduction in lifetime was observed with the ER sheet marker Sec61β-mRuby2, suggesting these proteins are co-enriched in the same domain but do not directly interact (Fig. 3F, bottom panel; 3G).\nMechanistic insights derived from AlphaFold modeling are almost certainly incorrect. Although the authors fail to provide any details that might allow one to evaluate the quality of the predictions, the SigmaR1-Trapa and SigmaR1-Nicalin AlphaFold models are obviously incorrect based solely on topological considerations: the models show SigmaR1 with its N-terminus in the lumen—this is not a type II topology (Fig S4A,B and Fig S5G). It is also unclear why the authors focus on a SigmaR1 monomer, when previous structural studies indicate it functions as a trimer (e.g., Schmidt et al., 2016).\nThank you for this critical comments, and these comments can be further divided into two sub-questions.\n1) Mechanistic insights derived from AlphaFold modeling are almost certainly incorrect. Although the authors fail to provide any details that might allow one to evaluate the quality of the predictions, the SigmaR1-Trapa and SigmaR1-Nicalin AlphaFold models are obviously incorrect based solely on topological considerations: the models show SigmaR1 with its N-terminus in the lumen—this is not a type II topology (Fig S4A,B and Fig S5G).\nWe sincerely thank the reviewer for this critical comment. We deeply apologize for the oversight in our original AlphaFold modeling. You are correct—the initial models incorrectly placed the N-terminus of SigmaR1 in the ER lumen, which is inconsistent with its established type II transmembrane topology (cytosolic N-terminus, luminal C-terminus). We have now regenerated the protein complex predictions using AlphaFold2 Multimer (v2.2.0) with explicit structural constraints to enforce the correct topology. In the revised models, the N-terminus of SigmaR1 is oriented toward the cytosol and mediates contact with its binding partners, while the C-terminus remains luminal, consistent with biological evidence. The corrected model has been included as Fig. S10F. In addition, we have expanded the Methods section to provide a full description of the modeling parameters.\n2) It is also unclear why the authors focus on a SigmaR1 monomer, when previous structural studies indicate it functions as a trimer (e.g., Schmidt et al., 2016).\nThank you for this comment. We now provide a new topologically-correct model of SigmaR1 in its trimeric form. We show that the binding sites we propose for both lipids and translocon components remain solvent-accessible and are not occluded by the trimeric interface. This suggests our proposed mechanistic insights are compatible with the functional trimeric state of SigmaR1.\nThe proteomic analysis of SigmaR1 deletion is over-interpreted. Rather than provide support for a direct interaction between SigmaR1 and the translocon, these data likely reflect pleiotropy resulting from its effects on ER morphology.\nWe totally agree with you that a direction and specific interaction between SigmaR1 and the translocon is premise for the following functional study. As mentioned above, we now provide several new datasets to support a direct and specific interaction between SigmaR1 and translocon compoents.\nWe also performed a deeper mechanistic investigation. Importantly, we measured markers of the integrated stress response and found a significant increase in phosphorylated eIF2α, while total eIF2α level remained unchanged in SigmaR1 KO cells (Fig. 5A). Since eIF2α phosphorylation is known to globally suppress protein synthesis in response to cellular stress, these findings suggest a role for SigmaR1 in the maintenance of protein homeostasis. Notably, SigmaR1 deficiency did not induce or exacerbate ER stress triggered by thapsigargin, as demonstrated by unaltered BiP expression in immunoblots (Fig. 5A) and ER whorls (marked by Sec61β) (Fig. S8B, C), which are established hallmarks of ER stress. Therefore, these results rule out the possibility that this effect is indirectly caused by ER stress. We have added this data in the result section of revised manuscript.\nIn addition, to investigate the mechanistic link between molecular function of SigmaR1 and protein homeostasis dysregulation, we performed rescue experiment in SigmaR1 KO HeLa cells by reconstituting expression of SigmaR1-GFP, an N-terminal deletion mutant (defective in ER sheet targeting), or the lipid-binding Mut-2 variant. Cells expressing these constructs were isolated by flow cytometry for subsequent proteomic analysis (Fig. 5C). Introduction of SigmaR1-GFP efficiently restored global protein homeostasis, particularly rescuing ribosomal pathways (Fig. 5D; S8E). In contrast, the NT-deletion mutant failed to fully restore the proteome (Fig. 5E; S8F), underscoring the importance of ER sheet localization and translocon interaction for SigmaR1 function. Similarly, the lipid-binding mutant (Mut-2) was also insufficient for complete rescue (Fig. 5F; S8G), indicating that SigmaR1's ability to bind lipids is also involved in maintaining protein homeostasis. These results support that the role for SigmaR1 depends on an orchastrated interplay between lipid binding ability and translocon interaction in ER sheet. Our hypothesis is that lipid binding triggers conformational change and regulates the interaction of SigmaR1 with the translocon complex, consequently modulating translocon activity. This hypothesis will be tested in a future study.\nData showing retained lipid-binding activity of a SigmaR-80-168 mutant (Fig 4F) are not convincing. Although this construct retains the core beta-barrel, deletion of several helices that normally surround the barrel almost certainly render it insoluble and/or misfolded and unable to bind ligands. This raises significant concerns about the validity of the lipid binding assay used here. The data for a series of point mutants (Fig 4H-K) does little to alleviate this concern.\nWe thank the reviewer for raising this important point regarding the SigmaR1-80-168 mutant. We appreciate the concern that large deletions can cause misfolding. We now present a new line of evidence to strenghen the conclusion. We generated a point mutant (Mut-2) in the background of the full length protein, in which several hydrophobic residues were mutated to W in the binding domain (residues 80-168) for steric hinderance (V104W, L105W, L106W, F107W). We performed in vitro lipid binding assays to confirm the reduced lipid binding ability of the point mutant (Fig. 4G, H). Rescue experiment demonstrated that this lipid binding mutant did not fully restore the LD accumulation phenotype (Fig. 6P, R).\nReviewer #4 (Remarks to the Author):\nIn this study, using high-resolution microscopy in combination with endogenous labeling and membrane fractionation, authors further demonstrate that SigmaR1 is a type II integral protein specifically enriched at ER sheet which is consistent with others’ previous findings. Besides, the manuscript also presents interesting and valuable data on the functional significance of SigmaR1 in regulating protein and lipid homeostasis based on proteomic and biochemical studies which added more novelty to the work. Overall, the experimental design is strong, and the conclusions drawn are supported by the data. However, there are several areas and details where the paper could be improved:\nWe are grateful to you for all these insightful comments, which have undoubtedly improved the clarity and rigor of our manuscript. We hope that our revisions have fully addressed all of these concerns.\n1. In Figure 1A-D, to show the colocalization of SigmaR1 and different ER maker proteins by living cell imaging, all proteins including the ER maker proteins were overexpressed which may affect their natural locations, so it may be more convincing if we could label the endogenous marker protein at lease for the ER-sheet marker Sec61β as in Figure 1F for the SigmaR1 and show the colocalization of endogenous proteins.\nThank you for this nice comment. As suggested, we now add two additional imaging results showing the colocalization between sfGFP-SigmaR1 KI and Halo-Sec61β (Fig. 1F, I) in live cells or endogenous Sec61β in immunofluorescence (Fig. 1G, I).\n2. Although Figure1G shows a statistically better colocalization of SigmaR1 and Sec61β, the related images are missing in Figure1F.\nThank you for this suggestion. As mentioned above (point#1), we now add the image showing the spatial relationship between sfGFP-SigmaR1-KI and Sec61β.\n3. The content of fraction 0-11 in Figure 1H should be clearly indicated somewhere in the paper. According to the WB data, the 7-10 lane seems very likely to be fraction of ER tubules because of the high level of Reep5, but the Reticulon 4 did not show any enrichment in those lanes which may make people doubt if the Reticulon 4 is a good marker for ER tubules when Reticulon 4 was used quite a lot in the rest of the studies.\nThis is a nice suggestion. We now add a description of fraction 0-11 in figure legend (now as Fig.1J in revised manuscript). Following your suggestion, we now provide a new dataset to compared SigmaR1 distribution in the ribosomal-bound and ribosome-free fractions derived from solubilized ER microsomes by sucrose gradient centrifuge. In this assay, both RTN4 and Reep5 were used to label smooth ER fractions (tubular ER). Consistent with the results shown in Fig. 1H of the origenial manuscript, we found that endogenous SigmaR1 was preferentially co-enriched with two translocon proteins Nicalin and TRAPα in ER sheet fractions specifically enriched with ribosomes (Fig. 3D).\n4. Figure1/S1 and related paragraphs are insufficient to support the subtitle that “SigmaR1 is a type II integral protein enriched at ER sheet.”, but together with data in Figure2/S2 may better support the conclusion that SigmaR1 is a type II integral protein.\nWe apologize for this confusion about the subtitle. We now modify the subtitle of Fig.1 by removing type II integral protein.\n5. To support that “A short region at the N-terminus is sufficient and required for the ER-sheet localization of SigmaR1.”, ER tubular protein Reticulon 4 is used as a marker, I wondered why it’s preferred over ER sheet protein Sec61β which might be more straightforward. Figure 2E shows that WT SigmaR1 is worse colocalized with tubular protein compared to 1-30, which indicate WT may have a better colocalization with ER sheet, so it seems that the 30-223 is still helpful in SigmaR1’s ER sheet targeting and the N-terminus of SigmaR1 is partially contributing to its ER-sheet localization. In the previous paper “SigmaR1 shapes rough endoplasmic reticulum membrane sheets” which was published late last year on Developmental Cell, the Figure 3H-I also suggest that Lumenal SigmaR1 lacking its TM domain localized exclusively to ER sheets and increased ER sheet area and the mutations at C terminus which disrupted its oligomerization can dramatically decrease SigmaR1’s targeting to the ER-sheet.\nThank you for these insightful comments. These comments were divided into two sub-questions.\n1) To support that “A short region at the N-terminus is sufficient and required for the ER-sheet localization of SigmaR1.”, ER tubular protein Reticulon 4 is used as a marker, I wondered why it’s preferred over ER sheet protein Sec61β which might be more straightforward.\nThank you for this comment. We used Reticulon 4 as a tubular marker specifically to demonstrate that SigmaR1 is excluded from ER tubules, which is a strong indicator of its enrichment in sheets. We agree that co-staining with a canonical sheet marker like Sec61β would provide complementary evidence. We have now performed these experiments and included new images showing the strong co-localization of the SigmaR1 N-terminal fragment (1-30) with Sec61β in Fig. 2B-F. This further strengthens our conclusion.\n2) Figure 2E shows that WT SigmaR1 is worse colocalized with tubular protein compared to 1-30, which indicate WT may have a better colocalization with ER sheet, so it seems that the 30-223 is still helpful in SigmaR1’s ER sheet targeting and the N-terminus of SigmaR1 is partially contributing to its ER-sheet localization. In the previous paper “SigmaR1 shapes rough endoplasmic reticulum membrane sheets” which was published late last year on Developmental Cell, the Figure 3H-I also suggest that Lumenal SigmaR1 lacking its TM domain localized exclusively to ER sheets and increased ER sheet area and the mutations at C terminus which disrupted its oligomerization can dramatically decrease SigmaR1’s targeting to the ER-sheet.\nThis is an insightful comment. We agree that the luminal region of SigmaR1 may also retain some additional affinity for ER sheets. Our point is that the N-terminus is the primary and strongest targeting determinant, as it is both necessary and sufficient for SigmaR1 targeting to ER sheets. To further strenghen this point, we generated a chimera by fusing the ER-sheet targeting module of SigmaR1—comprising a short region (residues 2-8) and its transmembrane domain—to the tubular ER protein Tex2. This chimera was highly enriched in ER sheets, whereas the short region alone was insufficient for targeting (Fig. 2G, H; S3A, B). Due to time limitations in the revision process, we were unable to extend this analysis to more proteins. Nonetheless, this successful proof of concept supports the efficiency of the SigmaR1 module in directing heterologous ER proteins to sheets. Therefore, we believe that the N-terminus of SigmaR1 is the primary and strongest targeting determinant. That said, we have toned down the language throughout the manuscript from 'is sufficient and required to 'promote' to more precisely reflect our findings and acknowledge your point.\n6. In Figure S2, authors tried to investigate the membrane topology by using the GFP and GFP nanobody system and suggest that the SigmaR1 is a type II integral protein at ER sheet. The data as in Figure S2 D-E support that the N terminus of SigmaR1 is exposed in the cytosol and the C-terminus is in ER lumen but based on the presented data we may not be able to rule out the possibility of the existence of the model in S2 F, because the mCh-GFPnb is cytosol resident. However, in the previous Nature paper “Crystal structure of the human σ1 receptor” (2017), researchers already used the X-ray crystallography solved the structure of SigmaR1 as a homotrimer and each subunit is a ∼25-kDa single-pass integral membrane protein that terminates with two α helices at its C terminus. Also, Multiple experimental approaches confirm that SigmaR1 is oriented as a type II topology membrane protein, for example the JBC paper “The sigma-1 receptor is an ER-localized type II membrane protein” (2021).\nWe thank the reviewer for these critical and insightful comments. We agree that because the mCh-GFPnb is cytosolic, our assay can only confirm the cytosolic exposure of the N-terminus. We apologize for any over-interpretation in our original wording. Given that the type II topology of SigmaR1 has been conclusively established by previous structural and biochemical studies (including the excellent papers by Schmidt et al., Nature 2017 and Sharma et al., JBC 2021 cited by the reviewer), the goal of our experiment was not to define a new topology. Instead, we sought to provide independent, live-cell imaging validation of this known type II topology under the conditions of our study. This confirmation is a crucial foundational step for our subsequent functional analyses.\nWe will revise the manuscript to fully address this comment:\n1) We will modify the text in the Results section to state: \"To confirm the established type II topology of SigmaR1 (Schmidt et al., 2017; Sharma et al., 2021) in our cellular system, we...\"\n2) We will alter the conclusion to state that our data \"is consistent with the canonical type II topology\" rather than stating it as a novel finding.\n3) We now remove the speculative Model S2F, as it is effectively ruled out by the existing structural data, not by our own experiment.\n7. Under the subtitle “A short region at the N-terminus is sufficient and required for the ER-sheet localization of SigmaR1.”, when Figure 2G was described, 2F was mis-referred to, when 2H was described, 2G was mis-cited, while 2F was not described in the paragraph. Besides, for 2F, SigmaR1-Halo is used but not SigmaR1-GFP as in other images, is it true or mis-labeled?\nThank you for these insightful comments. These comments were divided into two sub-questions.\n1) Under the subtitle “A short region at the N-terminus is sufficient and required for the ER-sheet localization of SigmaR1.”, when Figure 2G was described, 2F was mis-referred to, when 2H was described, 2G was mis-cited, while 2F was not described in the paragraph.\nWe apologize for these citing errors. We now fix them. In addition, Fig. 2F (now as Fig. S3E in revised manuscript) have been described in the last paragraph of this section (lines 186-190 of the revised manuscript).\n2) Besides, for 2F, SigmaR1-Halo is used but not SigmaR1-GFP as in other images, is it true or mis-labeled?\nThank you for the comment. We indeed used SigmaR1-Halo in this experiment. Since the other reviewer raises a concern about the functionality of this construct, we now add a new imaging result using SigmaR1-GFP as Fig. S3E of the revised manuscript.\n8. For Figure S3L, both inputs are labeled TRAM1-GFP negative.\nWe apologize for this typo. We now correct it.\n9. For Figure 6B, the SigmaR1 MW is mis-labeled as ~60KD. Based on Figure 6C, it’s insufficient to conclude that “the strong accumulation of LDs was specific to SigmaR1”, because the WT SigmaR1 only partially rescued the phenotype, and the restored level of WT looks quite similar to that of SigmaR1-△2-8. It’s better to use both siRNA1 and 3 to rule out the possibility of off targets.\nIn addition, there are some inconsistencies existing. For example, a lot of different size of images and scale bar are used in the live imaging results; in Figure 4H’-K’, both “-” and “–” are used, also different font are used; in Figure 5, different font size are used, and some are barely to see; the triglycerides (TG) was mentioned in the paper but the TAG was frequently seen in the paper which I suppose to be the same; different cells lines including COS7, HeLa, HepG2, 293T etc. are used in the study, it would be a bit complex to integrate data, for example, the location of SigmaR1 to ER sheet was demonstrated in the COS7 and HeLa cells, while the SigmaR1 interacting with translocon complex and binding to PC was tested in HeLa and 293T cells, and it said because the lipids phenotype could not be seen in SigmaR1 KO HeLa cells so the KO was conducted in HepG2 although HepG2 has much lower SigmaR1 according to Figure S1B, but the quantitative MS study conducted with HeLa sample suggested SigmaR1 KO in HeLa cells also increased the lipid metabolism, based on which, it seems that SigmaR1’s regulation on lipids is not hepatocyte specific, after all, and it’s hard to conclude any relation between these findings like whether there’s any link between the translocon complex protein interaction and the lipid binding/regulation.\nAlso, there’re some repetitions in the manuscript. For example, for the paragraphs following “SigmaR1 KO impairs cellular protein homeostasis”, figures supporting this subtitle should be Figure5, but Figure4 are repeated cited there, and it’s the same problem for the paragraphs following “SigmaR1 KO resulted in an accumulation of lipid droplets in hepatocytes.”, the related figure should be Figure6 not Figure5. Also, sentence like “Recently, the Nicalin -TMEM147-NOMO1/2/3 complex, TMCO1 and CCDC47 have been recently identified as novel conserved components of the translocon complex during biogenesis of multipass membrane proteins.” should be double checked and improved. Moreover, there are two “Table 1. Plasmids used in this Study” as for both plasmids and primers.\nWe appreciate all these great comments. These comments were divided into five sub-questions.\n1) For Figure 6B, the SigmaR1 MW is mis-labeled as ~60KD.\nWe apologize for this confusion. In Fig. 6B (now Fig. S9A of the revised manuscript), efficiency of siRNA-mediated SigmaR1 depletion was tested by examining the level of ectopically expressed SigmaR1-GFP using GFP antibody because we did not have SigmaR1 antibody at that time. The MW of SigmaR1-GFP is ~60 kDa. Later on, we purchased a commercial SigmaR1 antibody which works well in our hands. We used this antibody in the following experiments. Therefore, we now change the label to “anti-GFP” in Fig. S9A of revised manuscript.\n2) Based on Figure 6C, it’s insufficient to conclude that “the strong accumulation of LDs was specific to SigmaR1”, because the WT SigmaR1 only partially rescued the phenotype, and the restored level of WT looks quite similar to that of SigmaR1-△2-8. It’s better to use both siRNA1 and 3 to rule out the possibility of off targets.\nWe thank the reviewer for this critical insight and excellent suggestion. To rule out off-target effects, we have now repeated the key experiment using a second independent siRNA (siRNA3) targeting SigmaR1. As shown in the new figure below (Fig. 6A), the phenotype of LD accumulation was consistently observed with both siRNA1 and siRNA3. These new data confirm that the LD accumulation phenotype is due to the specific knockdown of SigmaR1 and not an off-target effect.\nIn addition, we performed transmission electron microscopy (TEM) to examine the ultrastructural details of LDs in SigmaR1-depleted HepG2 cells. TEM micrographs revealed a strong increase in LD diameter without a significant change in their number (Fig. 6I-L).\n3) In addition, there are some inconsistencies existing. For example, a lot of different size of images and scale bar are used in the live imaging results; in Figure 4H’-K’, both “-” and “–” are used, also different font are used; in Figure 5, different font size are used, and some are barely to see; the triglycerides (TG) was mentioned in the paper but the TAG was frequently seen in the paper which I suppose to be the same;\nWe apologize for these inconsistencies. We now correct them.\n4) different cells lines including COS7, HeLa, HepG2, 293T etc. are used in the study, it would be a bit complex to integrate data, for example, the location of SigmaR1 to ER sheet was demonstrated in the COS7 and HeLa cells, while the SigmaR1 interacting with translocon complex and binding to PC was tested in HeLa and 293T cells,\nThank you for these great comments! We would like to clarify our rationale, which we believe turns the use of different cell lines from a complexity into a strength. Our strategy was to use the most appropriate and well-established cellular system for each specific experimental technique to ensure the highest data quality and reliability. Importantly, SigmaR1 is a ubiquitously expressed protein, and its core functions in ER morphology and proteostasis are expected to be conserved across mammalian cell types. COS7 and HeLa for Imaging (Localization): These cells are large, flat, and optically clear, making them ideal for high-resolution microscopy to clearly resolve ER structures (sheets vs. tubules). This is a standard choice in the field for such experiments. HEK293T for Biochemistry (Interactome, Binding): HEK 293T/293F cells are renowned for their high transferability and protein expression yields, which is essential for successful affinity purification-mass spectrometry and subsequent biochemical assays like lipid binding. This ensures we could reliably detect interactions even for lower-abundance complexes. HepG2 for Lipid-Related Studies: As a hepatocyte-derived cell line, HepG2 has a highly active lipid metabolism and is a standard model for studying lipid droplet biology and related processes, making it highly relevant for our investigations into SigmaR1's role in lipid homeostasis.\nTo further strengthen our conclusion, we confirmed the ER sheet localization of SigmaR1 and the LD accumulation phenotype in multiple cell lines, including hepatic cells (Huh7), pancreatic cancer line PaCa2, and mouse neuroblastoma Neuro2a cells. Notably, this lipid accumulation phenotype exhibited cell-type specificity. SigmaR1 depletion resulted in an aberrant LD accumulation in Huh7 cell line similar as HepG2 (Fig. S9B-D). In contrast, SigmaR1 depletion in non-hepatic cell lines—including pancreatic PaCa2 cells (Fig. S9J-L) and Neuro2a cells (Fig. S9M-O)—resulted in a moderate LD phenotype, despite robust conservation of its ER sheet localization. We attribute this cell-type-dependent effect to the specialized role of hepatocytes in enhanced lipid synthesis, storage, metabolism and secretion, which appears to amplify the phenotypic severity of SigmaR1 loss.\nIn addition, we have now provided a new dataset showing that a direct interaction between Flag-His-SigmaR1 and two translocon components (Nicalin and TRAPa) using in vitro pulldown assay, confirming that this interaction is not unique to HEK293T cells.\n5) and it said because the lipids phenotype could not be seen in SigmaR1 KO HeLa cells so the KO was conducted in HepG2 although HepG2 has much lower SigmaR1 according to Figure S1B, but the quantitative MS study conducted with HeLa sample suggested SigmaR1 KO in HeLa cells also increased the lipid metabolism,\nWe thank the reviewer for this critical comment. We sincerely apologize for the lack of clarity in our original text. Our statement that \"the lipid phenotype could not be seen\" referred specifically to the absence of enlarged LDs as pronounced as those observed in SigmaR1-depleted HepG2 cells. Although the proteomic data indicated a molecular predisposition toward dysregulated lipid metabolism in HeLa KO, HeLa KO cells exhibited only moderate LD accumulation, which was not considered statistically significant (Fig. S5B). We attribute this to the specialized role of HepG2 cells in lipid storage and metabolism, which appears to amplify the phenotypic consequences of SigmaR1 loss. In response to your comment, we have revised our language throughout the manuscript, replacing “hepatocyte-specific” with more accurate phrasing such as “more pronounced in hepatocytes.” This adjustment better reflects our findings and acknowledges the point you raised.\n6) after all, and it’s hard to conclude any relation between these findings like whether there’s any link between the translocon complex protein interaction and the lipid binding/regulation.\nWe agree that this link needs to be clearly articulated. We believe that that these are not separate functions, but that they are integrated, and cellular function of SigmaR1 depends on an orchestrated interplay between ligand binding ability and translocon interaction in ER sheet. Our model is that lipid binding may trigger conformational change and regulates the interaction of SigmaR1 with the translocon complex, consequently modulating translocon activity. We will test this hypothesis in next study.\n7) Also, there’re some repetitions in the manuscript. For example, for the paragraphs following “SigmaR1 KO impairs cellular protein homeostasis”, figures supporting this subtitle should be Figure5, but Figure4 are repeated cited there, and it’s the same problem for the paragraphs following “SigmaR1 KO resulted in an accumulation of lipid droplets in hepatocytes.”, the related figure should be Figure6 not Figure5.\nWe apologize for these inconsistencies. We now correct them.\n8) Also, sentence like “Recently, the Nicalin -TMEM147-NOMO1/2/3 complex, TMCO1 and CCDC47 have been recently identified as novel conserved components of the translocon complex during biogenesis of multipass membrane proteins.” should be double checked and improved.\nThank you for careful reading and for catching this oversight. The sentence has been corrected to read: \"A ribosome-associated complex consisting of the core Sec61 channel and five accessory factors—TMCO1, CCDC47, Nicalin, TMEM147, and NOMO—has recently been characterized and is proposed to play a role in the biogenesis of multipass membrane proteins.\" In addition, we have taken the opportunity to perform a thorough review of the entire manuscript to improve sentence structure and eliminate any similar instances of redundant or awkward phrasing.\n9) Moreover, there are two “Table 1. Plasmids used in this Study” as for both plasmids and primers.\nWe apologize for the typo. We now correct it.\nOverall, the structure of the paper is logical, the evidence from different methods can well support the conclusions, the discussion also emphasized the novelty and outlook of the work. Improvements on details may improve the quality of the paper.\nWe appreciate your insightful and constructive comments. We have taken your advice and have conducted a thorough, detailed revision to improve the quality and impact of our work.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn the revised manuscript, the authors have provided detailed answers to my previous questions, the data and writing were also well improved. There were still some issues that need to be explained and noted.\n1. In this study, SigmaR1 was described as a component of the translocon complex using multiple approaches such as co-IP, FLIM and sucrose gradient centrifugation. However, precise role of SimgaR1 in the translocon complex is still not clear. Whether SigmaR1 plays a role as a component or an associated factor of translocon complex needs to be clarified.\nWe sincerely thank the reviewer for their positive assessment of our revised manuscript and for raising this important question regarding the precise role of SigmaR1 within the translocon complex. In response to this comment, we performed additional in vitro pulldown assays, which showed that His‐Flag‐SigmaR1 does not bind directly to GFP‐Sec61β (Fig. S5O in the revised manuscript). This result aligns with our earlier co‐IP and FLIM data and supports the conclusion that SigmaR1 is not a core structural component of the translocon channel.\nHowever, through multiple independent approaches—including co‐IP, sucrose gradient centrifugation, and FLIM—we consistently observed that SigmaR1 stably and directly interacts with key translocon‐associated factors, such as Nicalin and TRAPα, under physiological conditions. These findings support that SigmaR1 is an auxiliary component that physically engages with the translocon machinery. Notably, we now provide additional mechanistic insight through in vitro pulldown assays showing that phosphatidylcholine (PC) binding enhances SigmaR1's binding to these translocon components (Fig. 4J). This regulatory relationship aligns with our conclusion of SigmaR1 not as a static structural element, but as a dynamic auxiliary factor whose interaction with the translocon complex is modulated by lipid binding.\nCritically, although SigmaR1 does not form part of the central pore, its association is functionally essential. Loss of SigmaR1 leads to pronounced defects in both protein and lipid homeostasis, underscoring its importance in supporting translocon function. Thus, while SigmaR1 acts as a modulator rather than a structural subunit, its physical integration within the translocon environment is required for proper cellular proteostasis.\nWe have therefore modified our terminology throughout the manuscript to describe SigmaR1 as an \"auxiliary translocon factor\" rather than a core translocon component, and have also incorporated the new PC binding data to strengthen this model. Thank you once again for this insightful comment, which has helped us refine our model and improve the clarity of our conclusion.\n2. Line 181, 284 and 290, figures were mis-matched. Fig.3G should be Fig.2G, for example.\nWe apologize for this oversight. We have now carefully double-checked all figure references throughout the manuscript and have corrected the mismatched citations.\nReviewer #2 (Remarks to the Author):\nIn this revision, the authors have addressed most of my concerns. They corrected the topological mistake by confirming SigmaR1 is a type II membrane protein. Not sure what happened in the first place, but the current prediction and topological tests are in line with the subsequent mechanistic probing of the protein. The following points should be considered before the paper can be accepted.\n1. The interactions with translocon components are critical here. The authors tried to confirm a direct binding by purifying SigmaR1. Notably, the pull-down experiments using purified protein would eliminate topological restrictions. The co-IP in Fig. S6E,F are important and therefore can be improved or at least made certain for the reproducibility. Along the line, it would be helpful to purify the His-SigmaR1 with the deletion of 2-8 and repeat the pull-down assay.\nThank you for these insightful comments, which can be divided into two sub-questions.\n“The interactions with translocon components are critical here. The authors tried to confirm a direct binding by purifying SigmaR1. Notably, the pull-down experiments using purified protein would eliminate topological restrictions. The co-IP in Fig. S6E,F are important and therefore can be improved or at least made certain for the reproducibility.”\nAs suggested, we validated the result shown in the original Fig. S6 by performing coIP assays. Consistent with our previous data, deletion of residues 2-8 in SigmaR1 significantly reduce its interaction with endogenous Nicalin. This data is incoporated as Fig. 3C in the revised manuscript, and the data in the original Fig. S6E, F is now removed.\n“Along the line, it would be helpful to purify the His-SigmaR1 with the deletion of 2-8 and repeat the pull-down assay.”\nThis is a great comment. We performed in vitro pulldown assays, and found that the deletion of residues 2-8 in SigmaR1 significantly reduced its binding to these translocon components Nicalin or TRAPα (Fig. 3D). Together, these results confirmed an essential role of the NT sequence (residues 2-8) of SigmaR1 in its binding to the translocon complex.\n2. The rendering of the structural models using PyMOL can be improved. For example, in Fig. S6, the stick presentation of key residues usually only shows side chain atoms; nitrogen and oxygen atoms are colored in blue and red; and for structural models, “set valence, 0” is needed to avoid double-lined sticks.\nWe thank the reviewer for this thoughtful suggestion regarding the PyMOL rendering of our structural models. In light of these technical comments, and considering that another reviewer also found the topology presentation in Figs. S6A and S6B potentially confusing, we have elected to remove these predicted structural models from the revised manuscript. As these predicted results were not central to our main conclusions, their removal allows us to focus the narrative on more definitive experimental results while avoiding potential misinterpretation. We appreciate the reviewer's insightful input, which has helped improve the clarity and focus of the manuscript.\n3. The model to connect lipid binding with translocon regulation is intriguing. However, at this stage, evidence is far from enough to support such model. Statements for the limitation is needed. Similarly, several constructs made along the way are now proven irrelevant. For example, the SigmaR1 construct lacking TM only causes confusion here. If the authors believe that the LD phenotype is triggered indirectly, it is not necessary to pin the molecule close to LDs in some way. It is recommended that authors clean up these data.\nThank you for these insightful comments, which can be divided into two sub-questions.\n“The model to connect lipid binding with translocon regulation is intriguing. However, at this stage, evidence is far from enough to support such model. Statements for the limitation is needed.”\nWe agree with the reviewer that further experimental evidence is required to fully validate the model connecting SigmaR1's lipid binding to its regulation of the translocon. In response to this comment, we performed additional in vitro pulldown assays to examine the effect of phosphatidylcholine (PC) on SigmaR1’s interaction with the translocon. We found that pre-incubation of SigmaR1 with PC strengthened its interaction with Nicalin and TRAPα (Fig. 4J), suggesting that lipid binding may regulate SigmaR1’s association with the translocon complex. Given the established importance of PC binding for SigmaR1’s role in cellular protein and lipid homeostasis, these results provide preliminary support for a functional link between lipid sensing of SigmaR1 and translocon regulation.\nThat said, we fully acknowledge that these findings represent an initial step and that more comprehensive studies will be needed to substantiate this model. As suggested, we have explicitly noted in the revised discussion section that this model remains a theoretical framework at this stage and warrants further investigation.\n“Similarly, several constructs made along the way are now proven irrelevant. For example, the SigmaR1 construct lacking TM only causes confusion here. If the authors believe that the LD phenotype is triggered indirectly, it is not necessary to pin the molecule close to LDs in some way. It is recommended that authors clean up these data.”\nThanks for this thoughtful comment. We now remove all these data from the revised manuscript.\nReviewer #3 (Remarks to the Author):\nThe author's evidence for one of their key conclusions--that SigmaR1 is a component of the ER translocon--remains unconvincing. Beyond other issues noted previously by this reviewer and others, the AlphaFold modeling remains a serious concern. The authors show at least two different models for the purported (monomeric) SigmaR1 binding to Nicalin and TRAPalpha, with SigmaR1 in different topologies in each (type III Fig S6 vs type II in Fig S10). Moreover, they do not provide critical details of the AlphaFold modeling (e.g., models colored by pLDDT, PAE matrices), nor any analysis of sequence conservation within the predicted interfaces, for either of the two different models. This makes it impossible to assess the confidence of either prediction--and indeed, it seems likely that they are both incorrect. This is a major issue since the experimental evidence for the proposed translocon interaction are also not convincing. For example, the low-resolution sucrose gradient shows SigmaR1 co-migrating with RPL10 and some known components of the ER. If SigmaR1 is a bona fide component of the ribosome-translocon complex, the authors should find other translocon components--most notably, subunits of the core translocon component, Sec61--in their pulldowns. However, they specifically state that SigmaR1 does not interact with Sec61b in the ribosome-bound fraction. Thus, the revised manuscript still does not support the author's claim that SigmarR1 is a component of the ER translocon. Therefore I cannot recommend it for publication.\nThank you for all these constructive comments, which can be divided into four sub-questions.\n“The author's evidence for one of their key conclusions--that SigmaR1 is a component of the ER translocon--remains unconvincing. Beyond other issues noted previously by this reviewer and others, the AlphaFold modeling remains a serious concern. The authors show at least two different models for the purported (monomeric) SigmaR1 binding to Nicalin and TRAPalpha, with SigmaR1 in different topologies in each (type III Fig S6 vs type II in Fig S10). Moreover, they do not provide critical details of the AlphaFold modeling (e.g., models colored by pLDDT, PAE matrices),”\nWe thank the reviewer for this thoughtful suggestion regarding the predicted models. In light of these technical comments and topological inconsistencies, we have elected to remove all AlphaFold-predicted models from the revised manuscript. As these predicted results were not central to our main conclusions, their removal allows us to focus the narrative on more definitive experimental results while avoiding potential misinterpretation.\nTo further validate our conclusion, we now provide two new lines of evidence. First, co-IP assays confirm that deletion of residues 2-8 in SigmaR1 significantly reduces its interaction with endogenous Nicalin (Fig. 3C).\nSecond, in vitro pulldown assays demonstrate that the same deletion markedly impairs direct binding to both GFP-Nicalin and GFP-TRAPα (Fig. 3D). Together, these converging biochemical results provide robust validation that the N-terminal region (residues 2-8) is essential for SigmaR1's association with translocon components, strengthening our central conclusion without reliance on structural predictions.\n“nor any analysis of sequence conservation within the predicted interfaces, for either of the two different models. This makes it impossible to assess the confidence of either prediction--and indeed, it seems likely that they are both incorrect.”\nThank you for the comment. Based on the crucial role of the NT region of SigmaR1 in binding to translocon, we performed sequence analysis of this region among several species, and found that this sequence is highly conserved in mammals (Fig. S2D).\n“This is a major issue since the experimental evidence for the proposed translocon interaction are also not convincing. For example, the low-resolution sucrose gradient shows SigmaR1 co-migrating with RPL10 and some known components of the ER.”\nThank you for raising this important methodological consideration. To address the concern regarding resolution, we have now performed an modified sucrose gradient centrifugation assay that separates ER membranes into fourteen distinct fractions. This higher-resolution approach clearly demonstrates that endogenous SigmaR1 co-migrates with both the ribosomal marker RPL10 and established rough ER components, while being definitively separated from known ER tubular proteins (now presented as Fig. 3E in the revised manuscript). These results provide stronger evidence for SigmaR1's specific association with the rough ER translocon machinery.\n“If SigmaR1 is a bona fide component of the ribosome-translocon complex, the authors should find other translocon components--most notably, subunits of the core translocon component, Sec61--in their pulldowns. However, they specifically state that SigmaR1 does not interact with Sec61b in the ribosome-bound fraction. Thus, the revised manuscript still does not support the author's claim that SigmarR1 is a component of the ER translocon. Therefore I cannot recommend it for publication.”\nThank you for this critical and constructive comment, which allows us to clarify the nature of SigmaR1's relationship with the translocon complex. In response to this comment, we performed additional in vitro pulldown assays, which showed that His‐Flag‐SigmaR1 does not bind directly to GFP‐Sec61β (Fig. S5O). This result aligns with our earlier co‐IP and FLIM data and supports the conclusion that SigmaR1 is not a structural component that directly associating with the translocon core.\nHowever, through multiple independent approaches—including co‐IP, sucrose gradient centrifugation, and FLIM—we consistently observed that SigmaR1 stably and directly interacts with key translocon‐associated factors, such as Nicalin and TRAPα, under physiological conditions. These findings support that SigmaR1 is an auxiliary component that physically engages with the translocon machinery. Notably, we now provide additional mechanistic insight through in vitro pulldown assays showing that phosphatidylcholine (PC) binding enhances SigmaR1's binding to these translocon components (Fig. 4J). This regulatory relationship aligns with our model of SigmaR1 not as a static structural element, but as a dynamic auxiliary factor whose interaction with the translocon complex is modulated by lipid binding.\nCritically, although SigmaR1 does not form part of the central pore, its association is functionally essential. Loss of SigmaR1 leads to pronounced defects in both protein and lipid homeostasis, underscoring its importance in supporting translocon function. Thus, while SigmaR1 acts as a modulator rather than a structural subunit, its physical integration within the translocon environment is required for proper cellular proteostasis.\nWe have therefore modified our terminology throughout the manuscript to describe SigmaR1 as an \"auxiliary translocon factor\" rather than a core translocon component, and have incorporated the new PC binding data to strengthen this model. We believe these clarifications and additional data address your concern while maintaining the significance of our findings regarding SigmaR1's essential role in translocon function. Thank you once again for this insightful comment, which has helped us refine our model and improve the clarity of our conclusion.\nReviewer #4 (Remarks to the Author):\nThe revision has resolved all my prior critiques. I have no further questions.\nThank you for all the insightful suggestions, which substantially improve the quality and impact of our work!\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the revised manuscript, the authors have provided detailed answers to my previous questions, the data and writing were also well improved. There were still some issues that need to be explained and noted.\n1. In this study, SigmaR1 was described as a component of the translocon complex using multiple approaches such as co-IP, FLIM and sucrose gradient centrifugation. However, precise role of SimgaR1 in the translocon complex is still not clear. Whether SigmaR1 plays a role as a component or an associated factor of translocon complex needs to be clarified.\n2. Line 181, 284 and 290, figures were mis-matched. Fig.3G should be Fig.2G, for example.", "reviewer_2": "In this revision, the authors have addressed most of my concerns. They corrected the topological mistake by confirming SigmaR1 is a type II membrane protein. Not sure what happened in the first place, but the current prediction and topological tests are in line with the subsequent mechanistic probing of the protein. The following points should be considered before the paper can be accepted.\n1. The interactions with translocon components are critical here. The authors tried to confirm a direct binding by purifying SigmaR1. Notably, the pull-down experiments using purified protein would eliminate topological restrictions. The co-IP in Fig. S6E,F are important and therefore can be improved or at least made certain for the reproducibility. Along the line, it would be helpful to purify the His-SigmaR1 with the deletion of 2-8 and repeat the pull-down assay.\n2. The rendering of the structural models using PyMOL can be improved. For example, in Fig. S6, the stick presentation of key residues usually only shows side chain atoms; nitrogen and oxygen atoms are colored in blue and red; and for structural models, “set valence, 0” is needed to avoid double-lined sticks.\n3. The model to connect lipid binding with translocon regulation is intriguing. However, at this stage, evidence is far from enough to support such model. Statements for the limitation is needed. Similarly, several constructs made along the way are now proven irrelevant. For example, the SigmaR1 construct lacking TM only causes confusion here. If the authors believe that the LD phenotype is triggered indirectly, it is not necessary to pin the molecule close to LDs in some way. It is recommended that authors clean up these data.", "reviewer_3": "The author's evidence for one of their key conclusions--that SigmaR1 is a component of the ER translocon--remains unconvincing. Beyond other issues noted previously by this reviewer and others, the AlphaFold modeling remains a serious concern. The authors show at least two different models for the purported (monomeric) SigmaR1 binding to Nicalin and TRAPalpha, with SigmaR1 in different topologies in each (type III Fig S6 vs type II in Fig S10). Moreover, they do not provide critical details of the AlphaFold modeling (e.g., models colored by pLDDT, PAE matrices), nor any analysis of sequence conservation within the predicted interfaces, for either of the two different models. This makes it impossible to assess the confidence of either prediction--and indeed, it seems likely that they are both incorrect. This is a major issue since the experimental evidence for the proposed translocon interaction are also not convincing. For example, the low-resolution sucrose gradient shows SigmaR1 co-migrating with RPL10 and some known components of the ER. If SigmaR1 is a bona fide component of the ribosome-translocon complex, the authors should find other translocon components--most notably, subunits of the core translocon component, Sec61--in their pulldowns. However, they specifically state that SigmaR1 does not interact with Sec61b in the ribosome-bound fraction. Thus, the revised manuscript still does not support the author's claim that SigmarR1 is a component of the ER translocon. Therefore I cannot recommend it for publication.", "reviewer_4": "The revision has resolved all my prior critiques. I have no further questions.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely thank the reviewer for their positive assessment of our revised manuscript and for raising this important question regarding the precise role of SigmaR1 within the translocon complex. In response to this comment, we performed additional in vitro pulldown assays, which showed that His‐Flag‐SigmaR1 does not bind directly to GFP‐Sec61β (Fig. S5O in the revised manuscript). This result aligns with our earlier co‐IP and FLIM data and supports the conclusion that SigmaR1 is not a core structural component of the translocon channel.\nHowever, through multiple independent approaches—including co‐IP, sucrose gradient centrifugation, and FLIM—we consistently observed that SigmaR1 stably and directly interacts with key translocon‐associated factors, such as Nicalin and TRAPα, under physiological conditions. These findings support that SigmaR1 is an auxiliary component that physically engages with the translocon machinery. Notably, we now provide additional mechanistic insight through in vitro pulldown assays showing that phosphatidylcholine (PC) binding enhances SigmaR1's binding to these translocon components (Fig. 4J). This regulatory relationship aligns with our conclusion of SigmaR1 not as a static structural element, but as a dynamic auxiliary factor whose interaction with the translocon complex is modulated by lipid binding.\nCritically, although SigmaR1 does not form part of the central pore, its association is functionally essential. Loss of SigmaR1 leads to pronounced defects in both protein and lipid homeostasis, underscoring its importance in supporting translocon function. Thus, while SigmaR1 acts as a modulator rather than a structural subunit, its physical integration within the translocon environment is required for proper cellular proteostasis.\nWe have therefore modified our terminology throughout the manuscript to describe SigmaR1 as an \"auxiliary translocon factor\" rather than a core translocon component, and have also incorporated the new PC binding data to strengthen this model. Thank you once again for this insightful comment, which has helped us refine our model and improve the clarity of our conclusion.\n2. Line 181, 284 and 290, figures were mis-matched. Fig.3G should be Fig.2G, for example.\nWe apologize for this oversight. We have now carefully double-checked all figure references throughout the manuscript and have corrected the mismatched citations.\nReviewer #2 (Remarks to the Author):\nIn this revision, the authors have addressed most of my concerns. They corrected the topological mistake by confirming SigmaR1 is a type II membrane protein. Not sure what happened in the first place, but the current prediction and topological tests are in line with the subsequent mechanistic probing of the protein. The following points should be considered before the paper can be accepted.\n1. The interactions with translocon components are critical here. The authors tried to confirm a direct binding by purifying SigmaR1. Notably, the pull-down experiments using purified protein would eliminate topological restrictions. The co-IP in Fig. S6E,F are important and therefore can be improved or at least made certain for the reproducibility. Along the line, it would be helpful to purify the His-SigmaR1 with the deletion of 2-8 and repeat the pull-down assay.\nThank you for these insightful comments, which can be divided into two sub-questions.\n“The interactions with translocon components are critical here. The authors tried to confirm a direct binding by purifying SigmaR1. Notably, the pull-down experiments using purified protein would eliminate topological restrictions. The co-IP in Fig. S6E,F are important and therefore can be improved or at least made certain for the reproducibility.”\nAs suggested, we validated the result shown in the original Fig. S6 by performing coIP assays. Consistent with our previous data, deletion of residues 2-8 in SigmaR1 significantly reduce its interaction with endogenous Nicalin. This data is incoporated as Fig. 3C in the revised manuscript, and the data in the original Fig. S6E, F is now removed.\n“Along the line, it would be helpful to purify the His-SigmaR1 with the deletion of 2-8 and repeat the pull-down assay.”\nThis is a great comment. We performed in vitro pulldown assays, and found that the deletion of residues 2-8 in SigmaR1 significantly reduced its binding to these translocon components Nicalin or TRAPα (Fig. 3D). Together, these results confirmed an essential role of the NT sequence (residues 2-8) of SigmaR1 in its binding to the translocon complex.\n2. The rendering of the structural models using PyMOL can be improved. For example, in Fig. S6, the stick presentation of key residues usually only shows side chain atoms; nitrogen and oxygen atoms are colored in blue and red; and for structural models, “set valence, 0” is needed to avoid double-lined sticks.\nWe thank the reviewer for this thoughtful suggestion regarding the PyMOL rendering of our structural models. In light of these technical comments, and considering that another reviewer also found the topology presentation in Figs. S6A and S6B potentially confusing, we have elected to remove these predicted structural models from the revised manuscript. As these predicted results were not central to our main conclusions, their removal allows us to focus the narrative on more definitive experimental results while avoiding potential misinterpretation. We appreciate the reviewer's insightful input, which has helped improve the clarity and focus of the manuscript.\n3. The model to connect lipid binding with translocon regulation is intriguing. However, at this stage, evidence is far from enough to support such model. Statements for the limitation is needed. Similarly, several constructs made along the way are now proven irrelevant. For example, the SigmaR1 construct lacking TM only causes confusion here. If the authors believe that the LD phenotype is triggered indirectly, it is not necessary to pin the molecule close to LDs in some way. It is recommended that authors clean up these data.\nThank you for these insightful comments, which can be divided into two sub-questions.\n“The model to connect lipid binding with translocon regulation is intriguing. However, at this stage, evidence is far from enough to support such model. Statements for the limitation is needed.”\nWe agree with the reviewer that further experimental evidence is required to fully validate the model connecting SigmaR1's lipid binding to its regulation of the translocon. In response to this comment, we performed additional in vitro pulldown assays to examine the effect of phosphatidylcholine (PC) on SigmaR1’s interaction with the translocon. We found that pre-incubation of SigmaR1 with PC strengthened its interaction with Nicalin and TRAPα (Fig. 4J), suggesting that lipid binding may regulate SigmaR1’s association with the translocon complex. Given the established importance of PC binding for SigmaR1’s role in cellular protein and lipid homeostasis, these results provide preliminary support for a functional link between lipid sensing of SigmaR1 and translocon regulation.\nThat said, we fully acknowledge that these findings represent an initial step and that more comprehensive studies will be needed to substantiate this model. As suggested, we have explicitly noted in the revised discussion section that this model remains a theoretical framework at this stage and warrants further investigation.\n“Similarly, several constructs made along the way are now proven irrelevant. For example, the SigmaR1 construct lacking TM only causes confusion here. If the authors believe that the LD phenotype is triggered indirectly, it is not necessary to pin the molecule close to LDs in some way. It is recommended that authors clean up these data.”\nThanks for this thoughtful comment. We now remove all these data from the revised manuscript.\nReviewer #3 (Remarks to the Author):\nThe author's evidence for one of their key conclusions--that SigmaR1 is a component of the ER translocon--remains unconvincing. Beyond other issues noted previously by this reviewer and others, the AlphaFold modeling remains a serious concern. The authors show at least two different models for the purported (monomeric) SigmaR1 binding to Nicalin and TRAPalpha, with SigmaR1 in different topologies in each (type III Fig S6 vs type II in Fig S10). Moreover, they do not provide critical details of the AlphaFold modeling (e.g., models colored by pLDDT, PAE matrices), nor any analysis of sequence conservation within the predicted interfaces, for either of the two different models. This makes it impossible to assess the confidence of either prediction--and indeed, it seems likely that they are both incorrect. This is a major issue since the experimental evidence for the proposed translocon interaction are also not convincing. For example, the low-resolution sucrose gradient shows SigmaR1 co-migrating with RPL10 and some known components of the ER. If SigmaR1 is a bona fide component of the ribosome-translocon complex, the authors should find other translocon components--most notably, subunits of the core translocon component, Sec61--in their pulldowns. However, they specifically state that SigmaR1 does not interact with Sec61b in the ribosome-bound fraction. Thus, the revised manuscript still does not support the author's claim that SigmarR1 is a component of the ER translocon. Therefore I cannot recommend it for publication.\nThank you for all these constructive comments, which can be divided into four sub-questions.\n“The author's evidence for one of their key conclusions--that SigmaR1 is a component of the ER translocon--remains unconvincing. Beyond other issues noted previously by this reviewer and others, the AlphaFold modeling remains a serious concern. The authors show at least two different models for the purported (monomeric) SigmaR1 binding to Nicalin and TRAPalpha, with SigmaR1 in different topologies in each (type III Fig S6 vs type II in Fig S10). Moreover, they do not provide critical details of the AlphaFold modeling (e.g., models colored by pLDDT, PAE matrices),”\nWe thank the reviewer for this thoughtful suggestion regarding the predicted models. In light of these technical comments and topological inconsistencies, we have elected to remove all AlphaFold-predicted models from the revised manuscript. As these predicted results were not central to our main conclusions, their removal allows us to focus the narrative on more definitive experimental results while avoiding potential misinterpretation.\nTo further validate our conclusion, we now provide two new lines of evidence. First, co-IP assays confirm that deletion of residues 2-8 in SigmaR1 significantly reduces its interaction with endogenous Nicalin (Fig. 3C).\nSecond, in vitro pulldown assays demonstrate that the same deletion markedly impairs direct binding to both GFP-Nicalin and GFP-TRAPα (Fig. 3D). Together, these converging biochemical results provide robust validation that the N-terminal region (residues 2-8) is essential for SigmaR1's association with translocon components, strengthening our central conclusion without reliance on structural predictions.\n“nor any analysis of sequence conservation within the predicted interfaces, for either of the two different models. This makes it impossible to assess the confidence of either prediction--and indeed, it seems likely that they are both incorrect.”\nThank you for the comment. Based on the crucial role of the NT region of SigmaR1 in binding to translocon, we performed sequence analysis of this region among several species, and found that this sequence is highly conserved in mammals (Fig. S2D).\n“This is a major issue since the experimental evidence for the proposed translocon interaction are also not convincing. For example, the low-resolution sucrose gradient shows SigmaR1 co-migrating with RPL10 and some known components of the ER.”\nThank you for raising this important methodological consideration. To address the concern regarding resolution, we have now performed an modified sucrose gradient centrifugation assay that separates ER membranes into fourteen distinct fractions. This higher-resolution approach clearly demonstrates that endogenous SigmaR1 co-migrates with both the ribosomal marker RPL10 and established rough ER components, while being definitively separated from known ER tubular proteins (now presented as Fig. 3E in the revised manuscript). These results provide stronger evidence for SigmaR1's specific association with the rough ER translocon machinery.\n“If SigmaR1 is a bona fide component of the ribosome-translocon complex, the authors should find other translocon components--most notably, subunits of the core translocon component, Sec61--in their pulldowns. However, they specifically state that SigmaR1 does not interact with Sec61b in the ribosome-bound fraction. Thus, the revised manuscript still does not support the author's claim that SigmarR1 is a component of the ER translocon. Therefore I cannot recommend it for publication.”\nThank you for this critical and constructive comment, which allows us to clarify the nature of SigmaR1's relationship with the translocon complex. In response to this comment, we performed additional in vitro pulldown assays, which showed that His‐Flag‐SigmaR1 does not bind directly to GFP‐Sec61β (Fig. S5O). This result aligns with our earlier co‐IP and FLIM data and supports the conclusion that SigmaR1 is not a structural component that directly associating with the translocon core.\nHowever, through multiple independent approaches—including co‐IP, sucrose gradient centrifugation, and FLIM—we consistently observed that SigmaR1 stably and directly interacts with key translocon‐associated factors, such as Nicalin and TRAPα, under physiological conditions. These findings support that SigmaR1 is an auxiliary component that physically engages with the translocon machinery. Notably, we now provide additional mechanistic insight through in vitro pulldown assays showing that phosphatidylcholine (PC) binding enhances SigmaR1's binding to these translocon components (Fig. 4J). This regulatory relationship aligns with our model of SigmaR1 not as a static structural element, but as a dynamic auxiliary factor whose interaction with the translocon complex is modulated by lipid binding.\nCritically, although SigmaR1 does not form part of the central pore, its association is functionally essential. Loss of SigmaR1 leads to pronounced defects in both protein and lipid homeostasis, underscoring its importance in supporting translocon function. Thus, while SigmaR1 acts as a modulator rather than a structural subunit, its physical integration within the translocon environment is required for proper cellular proteostasis.\nWe have therefore modified our terminology throughout the manuscript to describe SigmaR1 as an \"auxiliary translocon factor\" rather than a core translocon component, and have incorporated the new PC binding data to strengthen this model. We believe these clarifications and additional data address your concern while maintaining the significance of our findings regarding SigmaR1's essential role in translocon function. Thank you once again for this insightful comment, which has helped us refine our model and improve the clarity of our conclusion.\nReviewer #4 (Remarks to the Author):\nThe revision has resolved all my prior critiques. I have no further questions.\nThank you for all the insightful suggestions, which substantially improve the quality and impact of our work!\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The revised manuscript has answered my previous critiques, I would recommend it for publication in the Nature Communications.", "reviewer_2": "My concerns have been addressed in this revision."}, "author_response": ""}]

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