Membralin Selects Foreign Glycoproteins from the Endoplasmic Reticulum to Lysosomes for Degradation

Membralin Selects Foreign Glycoproteins from the Endoplasmic Reticulum to Lysosomes for Degradation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Membralin Selects Foreign Glycoproteins from the Endoplasmic Reticulum to Lysosomes for Degradation Yong-Hui Zheng, Jing Zhang, Xiaoran Lu, Sunan Li, Tao Wang, Iqbal Ahmad This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6498082/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The endoplasmic reticulum (ER) plays a central role in protein synthesis and folding. Membralin is a multi-pass membrane protein involved in ER-associated degradation (ERAD). Here, we demonstrate that Membralin assembles a protein degradation machinery across the ER membrane, specifically targeting class I fusion proteins expressed by major human viruses. Membralin interacts with MAN1B1 and p97/VCP through its luminal and cytoplasmic loops, respectively. Importantly, Membralin also contains an LC3-interacting region (LIR) in its cytoplasmic tail. The expression of these viral glycoproteins induces ER stress, prompting MAN1B1 to trim mannose residues extensively. Subsequently, Membralin recruits p97/VCP and initiate ER-phagy via its LIR, leading to degradation. This pathway specifically recognizes dense N -glycans and is selective, as it does not degrade misfolded domestic proteins. Collectively, our study reveals a cell-autonomous immunity inside the ER orchestrated by Membralin, underscoring its important role in the clearance of foreign glycoproteins to maintain cellular homeostasis. Biological sciences/Cell biology/Organelles/Endoplasmic reticulum Biological sciences/Microbiology/Virology/SARS-CoV-2 Membralin ERLAD reticulophagy class I fusion proteins MAN1B1 TMEM259 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The endoplasmic reticulum (ER) is essential for protein synthesis, producing both secreted and integral membrane proteins. It plays a critical role in ensuring that newly synthesized proteins are properly folded into their native conformations. When terminally misfolded proteins accumulate, the ER experiences stress, which can be mitigated by the ER quality control systems, including ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) 1 . Membralin/TMEM259, encoded by TMEM259 , is crucial for motor neuron survival as it maintains optimal expression levels of nicastrin, a core component of the γ-secretase complex 2 . It is a multi-pass membrane protein in the ER and implicated in alleviating ER stress by interacting with proteins in the ERAD network 3 . Mannosyl-oligosaccharide 1,2-α-mannosidase (MAN1B1) is a key enzyme in the ERAD process that specifically targets glycoproteins by co-opting with three ER degradation-enhancing α -mannosidase-like (EDEM) proteins. During glycoprotein folding in the ER, MAN1B1 primarily removes a single 1,2-α-linked mannose residue from Man9GlcNAc2 on N -glycan precursors, yielding Man8GlcNAc2. For terminally misfolded glycoproteins, MAN1B1 and EDEM proteins further trim two or three additional 1,2-α-linked mannose residues, producing Man(5–6)GlcNAc2. Glycoproteins with these low-mannose N -glycans are retrotranslocated and degraded via the ERAD pathway 4 . ERAD facilitates the retrotranslocation of misfolded proteins into the cytoplasm through the SEC61 translocon, an energy-dependent process driven by the AAA + ATPase p97/valosin-containing protein (VCP), ultimately resulting in degradation by cytosolic proteasomes. ERLAD directs misfolded proteins into specialized ER subdomains that detach from the ER and fuse with lysosomes for degradation. This process is mediated by 9 ER-phagy receptors, including FAM134B/RETREG1, FAM134B-2/RETREG1-2, FAM134A/RETREG2, FAM134C/RETREG3, TEX264, RTN3L, ATL3, CCPG1, and SEC62. These receptors are embedded in the ER membrane and are characterized by at least one cytosolic LC3-interacting region (LIR). FAM134B and RTN3L also contain a reticulon-homology domain (RHD) within the ER membrane. Class I fusion proteins, such as human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), influenza A virus hemagglutinin (HA), Ebola virus glycoprotein (GP), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, play crucial roles in the entry of these viruses 5 . Although these proteins do not share sequence homology, they exhibit a common trimeric-hairpin structure with a central coiled coil covered by a dense glycan shield. Class I fusion proteins are expressed as type I transmembrane polypeptide precursors, folded in the ER, and cleaved by furin in the Golgi. We have previously shown that the expression of HIV-1-Env, EBOV-GP, and IFA-HA induces ER stress and undergoes MAN1B1-mediated degradation 6 – 11 . Here, we identify a protein degradation machinery across the ER membrane that specifically targets class I fusion proteins. This machinery is orchestrated by Membralin and includes MAN1B1 and p97/VCP, facilitating the degradation of these viral glycoproteins via ER-phagy. This process provides a crucial mechanism for cell-autonomous immunity within the ER. Results Human coronavirus spike proteins are MAN1B1 substrates. SARS-CoV-2 (SARS2) infection induces ER stress, significantly contributing to COVID-19 cytopathic and inflammatory pathogenesis 12 – 14 . During SARS-CoV (SARS1) infection, its spike protein plays a significant role in the induction of ER stress compared to other viral proteins 15 . Thus, we investigated how SARS2-S activates UPR and triggers its relevant signaling events, including SARS1 and Middle East respiratory syndrome coronavirus (MERS) spike proteins as controls. During ER stress, binding-immunoglobulin protein (BiP) is upregulated to serve as a master ER stress regulator, resulting in the activation of three ER transmembrane receptors: the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme type 1 (IRE1), and activating transcription factor 6 (ATF6). PERK activates a transcription factor ATF4, and IRE1 activates another transcription factor, X-box binding protein 1 (XBP1). As done previously, we used luciferase-based reporter assays to detect the upregulation and activation of BiP, XBP1, ATF4, and ATF6 6, 8 . As a positive control for ER stress, the terminally misfolded human Serpin A1 (SERPINA1)/alpha1-antitrypsin (AAT) variant, null (Hong Kong) (NHK), was used 16 . SARS1-S, SARS2-S, MERS-S, and NHK were expressed with these different reporters, and levels of ER stress were determined. SARS1-S, SARS2-S, and MERS-S were expressed at similar levels, as detected by western blotting (WB) (Fig. 1 A). As expected, NHK upregulated BiP and activated ATF6, XBP1, and ATF4 (Fig. 1 B). Like NHK, SARS2-S also did so at comparable levels. SARS1-S and MERS-S strongly activated ATF6, but their upregulation and activation of BiP, XBP1, and ATF4 were significantly lower than SARS2-S and NHK. Thus, these coronaviruses activate ER stress, but the SARS2 activity is much broader and more potent. Previously, we identified HIV-1-Env, IFA-HA, and EBOV-GP as MAN1B1 substrates for degradation 6 , 8 , 10 . When SARS1-S, SARS2-S, or MERS-S were expressed with MAN1B1, EDEM1, EDEM2, and EDEM3, MAN1B1 reduced all these viral protein expressions, whereas EDEM1, EDEM2, and EDEM3 had little effect (Fig. 1 C). We then knocked out MAN1B1 and EDEM2 (Fig. 1 D, Fig. 1 E). When SARS1-S, SARS2-S, and MERS-S were expressed in wild-type (WT) and these knockout (KO) cells, their expression was increased in MAN1B1 -KO, but not EDEM2 -KO cells. Next, ectopic MAN1B1 and EDEM2 were expressed with the individual spike proteins in KO cells. The MAN1B1 activity was restored, but EDEM2 had little activity (Fig. 1 F). MAN1B1 has three critical catalytic sites (E330, D463, and E599), which are conserved among class I α-mannosidases. When these residues were mutated to alanine, the MAN1B1 activity was disrupted, indicating that its enzymatic activity is required (Fig. 1 G). To demonstrate that this MAN1B1 activity is antiviral, HIV-1 luciferase-reporter pseudovirions (PVs) expressing SARS1-S, SARS2-S, and MERS-S were produced from HEK293T WT and MAN1B1 -KO cells in the presence of ectopic MAN1B1, and their infectivity was determined by infection of Huh7 cells. MAN1B1 strongly inhibited the infectivity of all three PVs up to 40-fold (Fig. 1 H). MAN1B1 targets coronavirus spike proteins to lysosomes. We investigated how MAN1B1 affects the subcellular localization of coronavirus spike proteins by confocal microscopy. MAN1B1 with a mCherry-tag was expressed with the ER marker calreticulin (CALR), or the Golgi marker trans -Golgi network integral membrane protein 2 (TGOLN2) with a GFP-tag. MAN1B1 exhibited cytoplasmic distribution, colocalizing with both CALR and TGOLN2 (Fig. 2 A). However, the Pearson's correlation coefficient (PCC) of MAN1B1 with CALR was approximately four times higher than that with TGOLN2, confirming that MAN1B1 predominantly localizes to the ER. When SARS1-S, SARS2-S, and MERS-S with a GFP-tag were expressed alone, they were predominantly found on the plasma membrane; however, when they were expressed with MAN1B1-mCherry, they were redistributed into cytoplasmic compartments and colocalized with MAN1B1 (Fig. 2 B). These spike proteins were then expressed with the lysosome marker, lysosomal-associated membrane protein 1 (LAMP1) with a mCherry-tag, and MAN1B1, followed by treatment with lysosomal/autophagy inhibitors NH 4 Cl, bafilomycin A1 (BafA1), concanamycin A (ConA), and 3-methyladenine (3-MA). These spike proteins predominantly localized to the plasma membrane but were redistributed into the cytoplasm by the presence of MAN1B1, showing colocalization with LAMP1 (Fig. 2 C). Notably, this colocalization was significantly promoted by treatments with these inhibitors (Fig. 2 D), suggesting that spike proteins are degraded by autophagy/lysosomes. Class I fusion proteins are degraded in lysosomes independently of polyubiquitination. To detect protein degradation, class I fusion proteins, including EBOV-GP, HIV-1-Env, IFA (H5N1)-HA, SARS2-S, SARS1-S, and MERS-S were expressed with MAN1B1 in HEK293T cells and treated with different types of inhibitors, including class I α-mannosidase inhibitor kifunensine (Kif), proteasomal inhibitors MG132 and lactacystin (Lac), lysosomal inhibitors NH 4 Cl and BafA1, and autophagy inhibitors ConA and 3-MA. As expected, Kif blocked the MAN1B1 activity (Fig. 3 A, lanes 3, 9, 15, 21, 28, 39), and neither MG132 nor Lac did so, indicating that they are not degraded in proteasomes. However, NH 4 Cl, BafA1, ConA, and 3-MA all blocked the MAN1B1 activity, indicating that they are degraded by autophagy/lysosomes. To further confirm the endogenous MAN1B1 activity, EBOV-GP was expressed again in WT and MAN1B1 -KO cells. EBOV-GP expression was greatly increased in KO cells, which was countered by ectopic MAN1B1 (Fig. 3 B). Next, we determined whether polyubiquitination gets involved in this degradation process. We reported that disulfide-isomerase A3 (PDIA3) targets EBOV-GP for ER-phagy in a polyubiquitination-dependent manner 8 , 17 . When SARS2-S and EBOV-GP were expressed with MAN1B1 and increasing amounts of WT ubiquitin (Ub WT ) or its lysine-free mutant (Ub KO ), neither influenced their downregulation (Fig. 3 C). SARS2-S and EBOV-GP were then expressed with MAN1B1 in the presence of Ub WT , and SARS2-S and EBOV-GP polyubiquitination were detected by immunoprecipitated (IP), including PDIA3 as a control. MAN1B1 and PDIA3 both decreased EBOV-GP expression (Fig. 3 D, lanes 3, 4, 9, 10), but only PDIA3 promoted EBOV-GP polyubiquitination, whereas MAN1B1 did not (Fig. 3 D, lanes 7, 8, 11, 12). Additionally, MAN1B1 did not induce SARS2-S polyubiquitination either (Fig. 3 D, lanes 5, 6). Thus, MAN1B1 does not trigger SARS2-S and EBOV-GP polyubiquitination. Membralin is required for class I fusion degradation. To identify the ER-phagy receptor, we knocked out FAM134B , RTN3L , ATL3 , SEC62 , CCPG1 , and TEX264 in HEK293T cells ( Fig. S1 A ). Since FAM134B-2 is an N-terminally truncated isoform of FAM134B, this short isoform was also knocked out in this FAM134B -KO cell line. However, we found that MAN1B1 still decreased class I fusion protein expression in these KO cells ( Fig. S1 B ). We also knocked out FAM134C , and excluded its involvement, too ( Fig. S1 C ). Thus, MAN1B1 does not seem to engage with these known ER-phagy receptors to initiate this destruction. Subsequently, we knocked out Mebralin in HEK293T cells (Fig. 4 A). When class I fusion proteins were expressed alongside MAN1B1, the reduction of their expression was all blocked in this KO cell line (Fig. 4 B, lanes 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24). However, PDIA3, CANX, and CALR continued to decrease EBOV-GP expression in these cells (Fig. 4 C). Next, we determined whether Membralin can target these viral proteins alone. Indeed, ectopic Membralin strongly decreased their expression (Fig. 4 D, lanes 1, 2, 9, 10, 17, 18, 25, 26, 33, 34, 41, 42). This decrease was blocked by BafA1, NH 4 Cl, and the p97/VCP inhibitor Eeyarestatin I (EerI) but not by MG132, Lac, and Kif (Fig. 4 D). To confirm the MAN1B1 independence, we expressed Membralin with class I fusion proteins in HEK293T WT and MAN1B1 -KO cells. We found that Membralin still decreased the class I protein expression in this KO cell line (Fig. 4 E). Because Membralin was implicated in ERAD, we wondered whether the SEC61 complex, which serves as the translocon for retro-translocation during ERAD, is required for the Membralin activity. The SEC61 complex comprises abg subunits that form transmembrane channels where proteins are translocated across the ER membrane 18 . The a subunit was silenced by siRNAs and the b subunit was knocked by CRISPR/Cas9 in HEK293T cells ( Fig. S2A , Fig. S2B ). When class I fusion proteins were expressed with MAN1B1 or Membralin, their expression was still inhibited in these silenced and KO cells ( Fig. S2A , Fig. S2C ). Thus, SEC61 translocon is not required for the MAN1B1 and Membralin activity. To demonstrate that the MAN1B1-Membralin axis has antiviral activity, PVs expressing EBOV-GP, SARS2-S, and HIV-1-Env were produced from HEK293T WT, MAN1B1 -KO, or Membralin -KO cells in the presence of ectopic MAN1B1 or Membralin expression. The infectivity of all these PVs was increased 3 to 4-fold when they were produced from MAN1B1 -KO or Membralin -KO cells (Fig. 4 F, compare Ctrl lanes in three cell lines). Conversely, their infectivity was decreased ~ 20-fold by ectopic MAN1B1 or Membralin (Fig. 4 F). We previously reported the potent MAN1B1 antiviral activity to authentic IFA and HIV-1 6, 9, 10 . Collectively, these results demonstrate the broad antiviral activity of the MAN1B1-Membralin axis by targeting class I fusion proteins. Furthermore, we used live-cell imaging to track the translocation of SARS2-S from the ER to lysosomes. SARS2-S-GFP, LAMP1-mCherry, and Membralin-BFP were expressed with MAN1B1 in HEK293T cells, and live cell images were captured by a super-resolution microscope every 5 min. Without MAN1B1, SARS2-S barely colocalized with Membralin and LAMP1 (Fig. 4 G, Fig. S3 ), indicating successful egress of native SARS2-S from the ER for expression. In the presence of MAN1B1, SARS2-S was restricted into Membralin-positive domains that migrated into LAMP1-positive compartments time-dependent. These results confirm that MAN1B1 senses and recruits SARS2-S to the Membralin-positive ER-subdomain for degradation. The ER location of Membralin is determined by its 1st transmembrane domain. Membralin consists of 620 amino acids (aa) and features four transmembrane (TM) domains, three cytoplasmic domains, and two luminal domains (Fig. 5 A). We initially created five Membralin deletion mutants by removing each TM domain individually (∆TM1, ∆TM2, ∆TM3, ∆TM4) and an additional mutant that removed all four TM domains (∆4TM). When expressed alongside class I fusion proteins, unlike the full-length (FL) Membralin, ∆TM2, ∆TM3, and ∆TM4, neither ∆TM1 nor ∆4TM reduced their expression (Fig. 5 B). Next, we compared the subcellular localization of these mutants using confocal microscopy, with CALR as the ER marker. We found that ∆TM2, ∆TM3, and ∆TM4 localized correctly within the ER, whereas ∆TM1 and ∆4TM did not (Fig. 5 C). These results demonstrate that TM1 is essential for the ER localization, and importantly, this localization is critical for its activity. The cytoplasmic tail of Membralin interacts with LC3/GABARAP family proteins. To screen for active motifs, we constructed additional Membralin deletion mutants. Mutant 1-466, which possesses the entire cytoplasmic tail deletion, localized to the ER (Fig. 5 C), but failed to reduce the expression of EBOV-GP and H5N1-HA (Fig. 5 D, lanes 3 and 15), indicating the functional importance of this cytoplasmic tail. We created six 24-aa-deletion mutants (∆467–490, ∆491–514, ∆515–538, ∆539–562, ∆563–586, and ∆587–610) and one 10-aa-deletion mutant (∆611–620) to screen the 467–620 region. When these seven mutants were expressed alongside EBOV-GP and H5N1-HA, only mutant ∆539–562 lost the activity (Fig. 5 D, lanes 7, 18). To further narrow down this 24-aa region, we constructed 11 overlapping 6-aa-deletion mutants, including ∆539–544, ∆541–546, ∆543–548, ∆545–550, ∆547–552, ∆549–554, ∆551–556, ∆553–558, ∆555–560, ∆557–562, and ∆560–565. When expressed with EBOV-GP and H5N1-HA, only mutant ∆549–554 lost the activity (Fig. 5 D, lanes 31, 45). The 549–554 region contains 6 residues TDASFL (Fig. 5 A). To gain further insight, we created mutant ∆550–554 by deleting the last five residues DASFL and found that it retained the activity (Fig. 5 D, lanes 11, 22). Because mutants ∆545–550, ∆547–552, and ∆551–556 contain partial deletions of these six residues but still exhibit activity, these results demonstrate that the complete deletion of 549 TDASFL 554 must occur to inactivate Membralin’s activity. Next, we tested whether this 549 TDASFL 554 domain functions as an LC3-interacting region (LIR), which is characteristic of ER-phagy receptors. The LIR is a short peptide sequence that serves as a binding site for LC3/GABARAP proteins, which are crucial for autophagy. We expressed Membralin and mutant 1-466 alongside LC3A, LC3B, LC3C, GABARAP, GABARAP1, and GABARAP2 to assess their interactions through immunoprecipitation. Membralin successfully pulled down all these autophagy proteins, whereas 1-466 did not (Fig. 5 E). We then expressed ∆539–562, ∆549–554, and ∆550–554 with LC3B and repeated this experiment. ∆550–554 was expressed at a similar level to the FL protein but showed significantly weaker binding to LC3B (Fig. 5 F, lanes 2, 10). On the other hand, 1-466, ∆539–562, and ∆549–554 were all expressed at similar lower levels, and none of them bound to LC3B (Fig. 5 F, lanes 4, 6, 8). Collectively, these results identify 549 TDASFL 554 as a critical motif for LC3-binding. Membralin recruits VCP via its cytoplasmic loop. The Membralin activity is blocked by the VCP inhibitor EerI (Fig. 4 D, lanes 4, 12, 20, 28, 36, 44), indicating an involvement of this AAA + ATPase. To further confirm this, we silenced VCP in HEK293T cells by siRNAs and tested the MAN1B1 and Membralin activity. The VCP siRNA disrupted the MAN1B1 and Membralin activity class I fusion proteins, but a control siRNA did not (Fig. 6 A). The four TM domains of Membralin are linked by one large and one small luminal loop (aa 91–301, 367–425, Fig. 5 A, green) and a relatively shorter cytoplasmic loop (aa 323–345, Fig. 5 A, middle pink). To understand how VCP interacts with Membralin, we constructed another three Membralin deletion mutants to express aa 70–628, 1-322, or 1-345. Together with those five TM domain deletion mutants and mutant 1-466, they were expressed with VCP in HEK293T cells, and their interactions were determined by immunoprecipitation. The FL Membralin and its five TM deletion mutants pulled down VCP, whereas GFP did not, indicating that the Membralin-VCP binding does not depend on TM domains (Fig. 6 B, lanes 1 to 7). In addition, 70–628, 1-345, and 1-466 were also bound to VCP, whereas 1-322 did not (Fig. 6 B, lanes 10–13), indicating that Membralin recruits VCP via its cytoplasmic loop to destroy class I fusion proteins. Membralin recruits MAN1B1 via its large luminal loop. We used the same mutants to understand the Membralin-MAN1B1 interaction by immunoprecipitation. All these mutants successfully pulled down MAN1B1, except for ∆TM1 and ∆4TM (Fig. 6 C, lanes 3, 7). Since these two mutants do not localize to the ER, these results confirm that the MAN1B1-Membralin interaction must occur within the ER. Additionally, 1-466 was able to pull down MAN1B1, indicating that this interaction does not require the cytoplasmic tail of Membralin. Furthermore, the continued interaction of 1-322 and ∆TM2 with MAN1B1 further establishes that MAN1B1 specifically binds to the large luminal loop of Membralin (aa 90–302). Membralin is not required for degradation of domestic misfolded proteins. Mutations on human genes encoding AAT 19 , collagen α-1(II) chain (COL2A1) 20 , Niemann-Pick type C1 (NPC1) 21 , and dysferlin (DYSF) 22 , 23 can cause their misfolding in the ER, leading to degradation by ERQC and various genetic diseases. MAN1B1 targets AAT variant NHK to ERAD 16 , 24 , but targets variant Z (ATZ) to ERLAD 25 . NPC1 variant I1061T is targeted to ERAD 26 , or ERLAD, via the ER-phagy receptor FAM134B 27 . CANX also targets COL2A1 variant R989C/G1152D to ERLAD via FAM134B 28 . The DYSF variant L1341P is targeted to either ERAD or ERLAD 29 . Additionally, the autocrine motility factor receptor (AMFR) targets human CD3δ (CD3D) to ERAD 30 , and nicastrin (NCSTN) in the γ-secretase complex is also targeted to ERAD 2 . We collectively tested whether the MAN1B-Membralin axis could target these human proteins. First, we expressed AAT and its variants ATZ and NHK, NPC1 and its variant I1061T, COL2A1 and its variant R989C/G1152D, DYSF and its variant L1341P, CD3δ, and NCSTN with MAN1B1 in HEK293T cells. MAN1B1 effectively decreased the ATZ, NHK, 1061T, R989C/G1152D, L1341P, CD3δ, and NCSTN expression (Fig. 7 A, lanes 4, 6, 10, 14, 18, 20, 22), but much less effectively the WT AAT, NPC1, COL2A1, and DYSF expression (Fig. 7 A, lanes 2, 8, 12, 16). Second, we determined how these misfolded variants, CD3δ, and NCSTN, are degraded. All their decreases by MAN1B1 were blocked by Kif and EerI (Fig. 7 B, lanes 3, 4, 11, 12, 19, 20, 27, 28, 35, 36, 43, 44, 51, 52). The decrease of NHK, CD3δ, and NCSTN was blocked by Lac and MG132, indicating that they are targeted to ERAD (Fig. 7 B, lanes 13, 14, 45, 46, 53, 54). The decrease of ATZ, I1061T (NPC1), R989C/G1152D (COL2A1), and L1341P (DYSF) was blocked by BafA1 and NH 4 Cl, indicating that they are targeted to ERLAD (Fig. 7 B, lane 7, 8, 23, 24, 31, 32, 39, 40). Third, we determined whether Membralin is required. These proteins were expressed with MAN1B1 in HEK293T WT and Membralin -KO cells. Because FAM134B was required for R989C/G1152D (COL2A1) and I1061T (NPC1) degradation via ERLAD, the FAM134B -KO cell line was also included. Notably, none of their degradation was blocked in Membralin -KO and FAM134B -KO cells (Fig. 7 C). Selection of ER-phagy receptors. Surprisingly, FAM134B is not required for MAN1B1-mediated degradation of I1061T (NPC1) and R989C/G1152D (COL2A1) (Fig. 7 C, lanes 23, 24, 31, 32). To understand how ER-phagy receptors are selected, ATZ, I1061T (NPC1), R989C/G1152D (COL2A1), and L1341P (DYSF) were expressed with MAN1B1 in HEK293T WT cells and six ER-phagy receptor KO cell lines. MAN1B1 still effectively decreased their expression in these KO cells, indicating that these ER-phagy receptors are not required for the MAN1B1 activity ( Fig. S4 ). To further explore this mechanism, we investigated the degradation of COL2A1 and its variant R989C/G1152D in the context of CANX and FAM134B. CANX, CALR, and PDIA3 were also included because they degrade EBOV-GP via ERLAD. Consistently, CANX, CALR, and PDIA3 decreased EBOV-GP expression (Fig. 8 A, lanes 10, 20, 21), as previously reported 8 , 17 , and CANX decreased R989C/G1152D but not COL2A1 expression (Fig. 8 A, lanes 2, 6), as reported 28 . FAM134B did not decrease any COL2A1, R989C/G1152D, and EBOV-GP expression (Fig. 8 A, lanes 3, 7, 11), but FAM134B-2 decreased all these protein expression (Fig. 8 A, lanes 4, 8, 12). Additionally, CANX did not decrease R989C/G1152 expression in FAM134B -KO cells (Fig. 8 B, lanes 3, 4), confirming that FAM134B is the ER-phagy receptor for this degradation 28 . FAM134B-2 and MAN1B1 still decreased R989C/G1152D expression in CANX -KO cells, indicating that their activity is independent of CANX (Fig. 8 B, lanes 5–12). These results demonstrate that the selection of ER-phagy receptor is determined by both effector (MAN1B1, CANX, FAM134B-2) and client proteins (host and viral proteins), highlighting the specificity of the Membralin-MAN1B1 axis in class I fusion protein targeting. The Membralin-MAN1B1 axis senses dense glycans. In addition to the structural GP, EBOV expresses soluble GP (sGP) and small soluble GP (ssGP) via RNA editing 8 , 17 , all sharing the same N-terminal domain (Fig. 8 C). EBOV-GP is heavily glycosylated, particularly in the mucin-like domain (MLD) 31 . It has 17 N -glycosylation sites, 8 of which are in MLD. In addition, MLD contains over 80 O -glycosylation sites, resulting in higher GP 1 molecular weight than the precursor GP 0 (Fig. 3 A, lanes 1–6; Fig. 3 B, lanes 1–4). To understand how glycans contribute to GP degradation, we tested the degradation of GP deletion mutant GP∆MLD that does not express MLD, along with sGP and ssGP. MAN1B1 decreased their expression, which was blocked by Kif, BafA1, and NH 4 Cl, but not Lac and MG132 (Fig. 8 D). Thus, MAN1B1 could still target these less-glycosylated GP to lysosomes for degradation. However, when EBOV-GP, GP∆MLD, sGP, and ssGP were expressed with MAN1B1 in Membralin -KO cells, only the decrease of FL EBOV-GP was blocked, whereas the others were not (Fig. 8 E, lanes 3, 4). Thus, dense glycans are required for Membralin-MAN1B1 to target EBOV-GP for degradation. Discussion Membralin has been implicated as a critical component of the ERAD pathway. It forms a complex with the E3 ubiquitin ligase RNF185 and the UBL domain-containing proteins TMUB1 and TMUB2 on the ER membrane. This complex works in conjunction with the cytosolic ubiquitin ligase UBE3C and p97/VCP to facilitate the degradation of membrane substrates 32 . However, inconsistent findings have been reported regarding Membralin-mediated degradation of NCSTN, NHK, and CD3δ 2 , 32 . Our results also indicate that Membralin is not necessary for MAN1B1-mediated degradation of NHK, CD3δ, and NCSTN in proteasomes. Consequently, the role of Membralin in ERAD warrants further investigation. We find that Membralin is essential not only for MAN1B1-initiated ERLAD but also for directly initiating the degradation process. While both MAN1B1 and PDIA3 can trigger the degradation of EBOV-GP via ERLAD, the precise mechanisms appear to differ. For instance, MAN1B1 does not induce polyubiquitination of its substrate, whereas PDIA3 does, consistent with our previous findings that PDIA3’s activity is dependent on RNF185 17 . Therefore, Membralin's activity should not rely on RNF185. ERLAD clients are thought to be segregated into ER subdomains rather than being retrotranslocated across the ER membrane for engulfment 33 . In line with this, we find that the SEC61 translocon is not required for the degradation of class I fusion proteins. However, Membralin recruits p97/VCP through its cytoplasmic loop, and p97/VCP is essential for its activity. While p97/VCP serves as a critical energy provider for ERAD, it also performs diverse cellular functions, including membrane remodeling 34 , which we speculate facilitates the formation and detachment of ER subdomains. Like other known ER-phagy receptors, Membralin contains an LIR in its cytoplasmic tail, which should promote the degradation of class I fusion proteins through ER-phagy. We find that Membralin recruits MAN1B1 via its luminal loop to specifically target class I fusion proteins rather than known misfolded proteins or aggregates. During productive infection, viruses exploit host cells to produce abnormally high levels of class I fusion proteins, overwhelming the ER's folding machinery and inducing ER stress. Class I fusion proteins possess distinctive features that facilitate their recognition and clearance as foreign proteins. They are heavily glycosylated, primarily with N -glycans and fewer O -glycans, except for EBOV-GP, where O -glycans predominate. For instance, HIV-1 Env (gp120) undergoes extensive glycosylation, with approximately half of its molecular weight attributed to glycans 35 . While this dense glycan shield obscures key epitopes and aids immune evasion, it may also act as a non-host marker for being detected by MAN1B1. Our findings consistently demonstrate that significantly reduced glycosylation levels prevent EBOV-GP from being targeted by the Membralin-MAN1B1 axis. However, less glycosylated EBOV-GP can still undergo degradation via ERLAD through an alternative, unidentified mechanism. These results underscore glycosylation levels as a critical determinant for targeting by this protein complex. We confirm that, unlike NHK, the AAT polymeric variant ATZ is directed to ERLAD rather than ERAD, consistent with previous findings 36 . It has been proposed that misfolded large polypeptide aggregates resist ERAD due to the challenges of translocating through the SEC61 translocon and entering the proteasomes. In alignment with this, we observe that misfolded variants of NPC1, COL2A1, and DYSF are targeted to ERLAD but not ERAD. Class I fusion proteins, with their large molecular weights (exceeding 70 kDa) and trimeric structures, likely follow the same pattern, as their size and aggregation render them suitable candidates for ERLAD rather than ERAD. MAN1B1 is well-established in targeting the luminal substrate NHK to ERAD. Our findings further reveal that it also targets the membrane substrate CD3δ and another luminal substrate, NCSTN, through the same mechanism, demonstrating that MAN1B1 recognizes both ERAD luminal (ERAD-L) and ERAD membrane (ERAD-M) substrates. Moreover, MAN1B1 directs other misfolded human proteins, including AAT, NPC1, COL2A1, and DYSF variants (ATZ, I1061T, R989C/G1152D, and L1341P), to ERLAD. This degradation pathway operates independently of known ER-phagy receptors or Membralin, implying that MAN1B1 interacts with other unidentified ER-phagy receptor(s) for these host proteins. We also confirmed that CANX facilitates the degradation of the R989C/G1152D variant in a FAM134B-dependent manner 28 . These findings suggest that the selection of an ER-phagy receptor is dictated by both the effector (MAN1B1, CANX) and the specific client proteins. In summary, we find that Membralin orchestrates a protein degradation machinery across the ER membrane by recruiting MAN1B1 and p97/VCP, selectively targeting foreign glycoproteins via ER-phagy (Fig. 8 F). This mechanism represents a new arm of cell-autonomous immunity, playing a significant role in defending eukaryotic cells against viral infections. Materials and Methods Antibodies and inhibitors. Commercial reagents include: kifunensine, eeyarestatin I, MG132, Lactacystin (Lac), Concanamycin A (ConA), 3-Methyladenine (3-MA), and ammonium chloride (NH 4 Cl) (Sigma-Aldrich, K1140, E1286, C2211, L6785, C9705, M9281, A9434); Bafilomycin A1 (BafA1, Santa Cruz Biotechnology, sc-201550); mouse monoclonal anti-Myc (CST, 2276S); rabbit polyclonal anti-SQSTM1, mouse monoclonal anti-FLAG and anti-HA (Sigma-Aldrich, P0067, F3165, H3663); mouse monoclonal anti-ACTB/β-actin (CST, 3700S); mouse monoclonal anti-ERManI/MAN1B1 (Novus, NBP2-13167); rabbit polyclonal anti-FAM134B-2 and anti-Membralin (Gene Tex, GTX46621, GTX118618); rabbit polyclonal anti-ATL3, anti-FAM134B, anti-CCPG1, anti-TEX264, and anti-TOLLIP (Proteintech, 16921-1-AP, 21537-AP, 13861-1-AP, 25858-1-AP, 11315-1-AP); rabbit monoclonal anti-SEC62 and rabbit polyclonal anti-EDEM2 (Abcam, ab137022, ab181218); rabbit polyclonal anti-RTN3 (ThermoFisher Scientific, PA5-78316); mouse monoclonal anti-SERPINA1/AAT (Sigma-Aldrich, SAB4200198); rabbit polyclonal anti-Sec61A and Sec61B (Abcam, ab183046, ab15576); rabbit polyclonal anti-VCP (Huabio, ER30603); horseradish peroxidase (HRP)- conjugated goat anti-mouse IgG and anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 115-035-003, 111-035-003). Cell lines. Human embryonic kidney (HEK) 293 cell line transformed with SV40 large T antigen (HEK293T) and human cervical carcinoma cell line HeLa were purchased from American Type Culture Collection (ATCC, CRL-3216, CRM-CCL-2); human hepatoma cell line Huh7 was purchased from BioBW, China (BioBW, bio-73061); TZM-bI cells were from NIH HIV Reagent Program. All these cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, 11965092) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (pen-strep; Thermo Fisher Scientific, 10378016) and cultivated at 37°C in the humidified atmosphere in a 5% CO 2 incubator. CRISPR-Cas9 knockout. DNA oligos encoding small guide RNAs (sgRNAs) targeting MAN1B1 , EDEM2 , Membralin ( TMEM259) , FAM134B , RTN3L , ATL3 , SEC62 , CCPG1, TEX264 , and Sec61B are shown in supplemental Table 1, which were cloned into the pSpCas9(BB)-2A-GFP (PX458) vector via BbsI digestion. HEK293T cells were transfected with these vectors, and after 48 h, single clones of GFP-positive cells were isolated by fluorescence-activated cell sorting. Knockout clones were identified by western blotting (WB) and further validated by DNA sequencing. siRNA knockdown. SEC61A and VCP were knocked down by siRNAs using RNA oligo pairs 5’-CACUGAAAUGUCUACGUUUTT-3’/5’-AAACGUAGACAUUUCAGUGTT-3’, or 5’-GCAUGUGGGUGCUGACUUATT-3’/5’-UAAGUCAGCACCCACAUGCTT-3’. HEK293T cells were transfected with these siRNAs, and after 24 h, their expression was determined by WB. Plasmids . The pcDNA3.1-hERManI-HA-FLAG, pCMV6-hEDEM1-Myc-FLAG, pcDNA3.1-hEDEM2-HA-FLAG, and pCMV6-hEDEM3-Myc-FLAG vectors and ERManI catalytically inactive mutant expression vector were reported previously 6 . pcDNA3.1 vectors expressing ERManI/MAN1B1, EDEM1, EDEM2, and EDEM3 with a single C-terminal HA-tag were re-constructed in this study by homologous recombination after NheI/XhoI digestion. pNL-Luc-ΔEnv, pcDNA3.1-Flag-EBOV-GP, pcDNA3.1-FLAG-EBOV-GP∆MLD, pcDNA3.1-HiBiT-sGP, pcDNA3.1-HiBiT-ssGP, HIV-1 Env, and H5N1 HA expression vectors were reported previously 8 , 17 . pCAGGS-PDIA3-Myc, pCMV-CALR-Myc, pCMV6-CANX-Myc-FLAG, HSPA5/BiP promoter-luciferase reporter vector pLightSwitch-BiP, XBP1-activation reporter vector pXBP1u-FLuc, ATF4-expression reporter vector pATF4-UTR-Fluc, and ATF6-activation reporter vector p5xATF6-GL3 were reported previously 8 , 17 . pCMV-HA-Ub WT and pCMV-HA-Ub KO were reported previously 37 . pSpCas9 (BB)-2A-GFP (PX458) was obtained from Feng Zhang through Addgene (48138). pCMV6-Membralin-Myc-Flag was ordered from Comatebio (CoME). Membralin deletion mutants (∆TM1, ∆TM2, ∆TM3, ∆TM4, ∆4TM, 70–628, 1-322, 1-345, 1-466) were created by PCR and ASiSI/MluI digestion followed by homologous recombination. pCMV6-FAM134B-2-Myc-Flag was ordered from CoME. pCDNA3.1-FAM134B-Flag was ordered from GenScript. pCDNA3.1-NPC1-3xFlag were reported previously 38 . pCDNA3.1-NHK was provided by Richard Sifers. pCMV6-SERPINA1-Myc-Flag, pCMV6-COL2A1-Myc-Flag, pCMV6-DYSF-Myc-Flag, pCMV6-CD3D-Myc-Flag, pCMV6-NCSTN-Myc-Flag, and pCMV6-VCP-Myc-FLAG were ordered from Origene (Cat# RC202082, RC218041, RC219485, RC210010, RC212646, RC211130). SERPINA1/AAT, NPC1, COL2A1, and DYSF mutants and pCMV6-VCP-His were created by PCR and ASiSI/MluI digestion followed by homologous recombination. All vectors constructed by us were confirmed by Sanger DNA sequencing. Detailed experimental procedures and primer sequences for the construction of these vectors are available upon request. Plasmids were prepared using Maxiprep kits (TIANGEN Biotech, DP117). To express S proteins from SARS2, SARS1 and MERS, their cDNAs with a N-terminal FLAG tag were commercially synthesized and cloned into pCAGGS, resulting in pCAGGS-FLAG-SARS2-S, pCAGGGS-FLAG-SARS1-S, and pCAGGS-FLAG-MERS-S (Comate Bioscience, China). A SARS2-S and SARS1-S expression vector with a N-terminal Flag tag and C-terminal 19 amino acids deletion, pCAGGS-FLAG-SRAS2-S-D19 and pCAGGS-FLAG-SARS1-S-D19, were re-created by EcoRI / XhoI digestion. A SARS1-S expression vector with a C-terminal FLAG, pCAGGS-SARS1-S-FLAG, was re-created by EcoRI / BspEI digestion. These two SARS2-S and SARS1-S with this deletion were used to produce HIV-1 pseudovirions for infectivity assay. To express S protein-EGFP fusion proteins, these cDNAs were cloned into pEGFP-HA-N1 vector that express a HA-tag in front of EGFP by XhoI / BspEI digestion, resulting in pEGFP-SARS2-S-HA, pEGFP-SARS1-S-HA, pEFGP-MERS-S-HA, which express SARS2-S-HA-EGFP, SARS1-S-HA-EGFP, and MERS-S-HA-EGFP fusion proteins. To express MAN1B1-mCherry fusion, MAN1B1 was subcloned from pcDNA3.1-hMAN1B1-HA into pCAGGS-mCherry by EcoRI digestion. pEGFP-N1 vectors expressing CALR and TGLON2 were constructed by PCR and XhoI / BspEI digestion. Transfection. HEK293T cells were cultured in 6-well plates and transfected with polyethyleneimine (PEI; Polysciences, 23966-2). HeLa cells were transfected using Lipofectamine 3000 according to the manufacturer’s protocol (Thermo Fisher Scientific, L3000015). The total indicated plasmids were diluted into 200µL serum-free Opti-MEM (Thermo Fisher Scientific, 31985062) and mixed with transfection reagents. After 20 min of incubation at room temperature, these transfection complexes were added directly into the supernatant of each well. Media were replaced after 6 h and cell lysate was collected at 48 h unless otherwise noted. Viruses released into the supernatants and proteins expressed in cells were analyzed (see below). Analysis of UPR. HEK293T cells were cultured in 24-well plates and transfected with the indicated plasmid and reporter vectors as we reported previously 6 , 8 . HSPA5/BiP activation was measured by expressing BiP-RLuc reporter construct and measuring the intracellular Renilla luciferase activities. XBP1, ATF4, and ATF6 activation were measured by expressing XBP1-Fluc, pATF4-UTR-Fluc or p5×ATF6-GL3 reporter construct and measuring the intracellular Firefly luciferase activities. Luciferase activities were analyzed by Dual-Luciferase® Reporter Assay System (Promega, E1910). Analysis of coronavirus spike protein-pseudotyped HIV-1 infectivity. SARS2-S, SARS1-S, and MERS-S pseudotyped HIV-1 virions were produced from HEK293T WT and MAN1B1 -KO cells as we reported previously 38 . These cells were transfected with pNL-Luc-ΔEnv and a S protein expression vector in the presence or absence of a MAN1B1 expression. After 48 h, viruses were collected from the culture supernatants and clarified by low-speed centrifugation. After being normalized by p24 Gag ELISA 39 , an equal amount of viruses were used to infect Huh7 cells. After 48 h of infection, cells were lysed, and viral infectivity was determined by measuring the intracellular luciferase activity using a firefly luciferase assay kit (US Everbright Inc, Cat# F6024). Western blotting (WB). Transfected cells were lysed in RIPA buffer (25 mM Tris, pH7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1%Nonidet P-40 [Sigma-Aldrich, R0278]) at 4°C. After centrifugation at 12,000 × g for 10 min at 4°C, cytosolic fractions were collected and boiled with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Solarbio Life Sciences, P1015). Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. These membranes were blocked with 5% nonfat milk powder in TBST (Tris-buffered saline [20 mM Tris, pH 7.4,150 mM NaCl] containing 0.1% Tween 20; Solarbio Life Sciences, T8220) for 1 h at room temperature, and probed by primary antibodies followed by HRP-conjugated secondary antibodies. Chemiluminescence signals were then measured by incubating the membrane with SuperSignal substrate (Thermo Fisher Scientific, 34580). Immunoprecipitation. After transfection of HEK293T cells cultured in a 6-cm dish, cells were lysed in 0.8 mL RIPA lysis buffer for 30 min on ice as we did previously 40 . After the removal of nuclei via low-speed centrifugation and collecting 100 µL as input, the remaining 700 µL lysate was incubated with anti-FLAG M2 Magnetic beads (Sigma-Aldrich, M8823) and rotated at 4°C overnight. After being washed 3 times with 1 mL pre-cooled RIPA lysis buffer, proteins were removed from beads after boiling in 40 µL RIPA lysis buffer plus 15 µL sample loading buffer (5×) and analyzed by WB. In vivo polyubiquitination assay. HEK293T cells were seeded in 6-cm dishes and transfected with vectors expressing SARS2-S and MAN1B1, or EBOV-GP and PDIA3, in the presence of ubiquitin expression vector as we did previously 37 . After 48 h, cells in each dish were lysed in 600 µL RIPA buffer at 4°C for 30 min. After the removal of nuclei by low-speed centrifugation, 100 µL was collected as input, and the remaining 500 µL was incubated with anti-FLAG beads (Sigma-Aldrich, M8823) at 4°C overnight. Beads were washed three times with phosphate-buffered saline (PBS), then boiled in SDS-PAGE loading buffer and analyzed by WB. Confocal microscopy. HeLa cells were seeded on a glass bottom cell culture dish (NEST Biotechnology, 801001) and transfected with various vectors using Lipofectamine 3000, as we did previously 41 . After 30 h, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 (Solarbio Life Sciences, T8200) for 10 min at room temperature, and then blocked with 5% bovine serum albumin (BSA; APPLYGEN, P1622) solution overnight at 4°C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 3–5 min and washed with PBS, and cells were observed and imaged under a confocal microscope (LSM880, Zeiss, White Plains, New York). Quantitative colocalization measurements were performed using ImageJ software. Pearson’s correlation coefficient (PCC) was calculated to describe the colocalization correlation of the intensity distributions between two channels. Live-cell imaging. Approximately 1.0 ´ 10 5 HEK293T cells were seeded on a glass bottom cell culture dish (NEST Biotechnology, 801001) and transfected with various vectors using PEI. After 14 h, Live-cell imaging was performed using LSM800 (Zeiss, White Plains, New York). During live-cell imaging, the dish was mounted in a chamber to maintain the incubation conditions at 37°C and 5% CO 2 . Statistical analysis. All experiments were performed independently at least three times, with representative experiment being shown. GraphPad Prism (Graph Pad Software Inc., San Diego, CA, USA) was used for the data analysis. Data were presented as means ± standard error of measurements (SEMs) and represented by error bars. The significance of differences between samples was assessed by an unpaired two-tailed Student’s t test. A p value < 0.05 ( p < 0.05) was statistically significant (*p < 0.05, ** p < 0.01, *** p 0.05 was not significant (ns). Declarations Conflict of interest The authors declare no conflict of interest. Author contributions J.Z., X. L., S. L., T. W., and I.A. conducted experiments; J.Z. and Y.H.Z. designed experiments; Y.H.Z. wrote the manuscript with input from all authors. Acknowledgment We thank Feng Zhang for the reagent. J.Z. is supported by the National Natural Science Foundation of China (32300129), the Youth Innovation Program of the Chinese Academy of Agricultural Sciences (Y2025QC19), and the China Postdoctoral Science Foundation (2023T160700). S.L. is supported by the National Natural Science Foundation of China (32172836). Y.H.Z. is supported by the National Institutes of Health (AI175008). References Molinari, M. ER-phagy responses in yeast, plants, and mammalian cells and their crosstalk with UPR and ERAD. Dev Cell 56, 949–966 (2021). Zhu, B. et al. ER-associated degradation regulates Alzheimer's amyloid pathology and memory function by modulating gamma-secretase activity. Nat Commun 8, 1472 (2017). Yang, B. et al. The critical role of membralin in postnatal motor neuron survival and disease. Elife 4 (2015). Ninagawa, S., George, G. & Mori, K. Mechanisms of productive folding and endoplasmic reticulum-associated degradation of glycoproteins and non-glycoproteins. Biochim Biophys Acta Gen Subj 1865, 129812 (2021). Frabutt, D.A. & Zheng, Y.H. Arms Race between Enveloped Viruses and the Host ERAD Machinery. Viruses 8 (2016). Frabutt, D.A., Wang, B., Riaz, S., Schwartz, R.C. & Zheng, Y.H. Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein Degradation. J Virol 92, e01690-01617 (2018). Han, Y., Wang, X., Dang, Y. & Zheng, Y.H. Demonstration of a novel HIV-1 restriction phenotype from a human T cell line. PLoS One 3, e2796 (2008). Wang, B. et al. Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway. Autophagy 18, 2350–2367 (2022). Zhou, T., Dang, Y. & Zheng, Y.H. The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway. J Virol 88, 3474–3484 (2014). Zhou, T., Frabutt, D.A., Moremen, K.W. & Zheng, Y.H. ERManI (Endoplasmic Reticulum Class I alpha-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway. J Biol Chem 290, 22184–22192 (2015). Zhou, T., Han, Y., Dang, Y., Wang, X. & Zheng, Y.H. A novel HIV-1 restriction factor that is biologically distinct from APOBEC3 cytidine deaminases in a human T cell line CEM.NKR. Retrovirology 6, 31 (2009). Bartolini, D. et al. Endoplasmic reticulum stress and NF-kB activation in SARS-CoV-2 infected cells and their response to antiviral therapy. IUBMB Life 74, 93–100 (2022). Chaudhry, Z.L., Gamal, M., Ferhati, I., Warda, M. & Ahmed, B.Y. ER Stress in COVID-19 and Parkinson's Disease: In Vitro and In Silico Evidences. Brain Sci 12 (2022). Rosa-Fernandes, L. et al. SARS-CoV-2 activates ER stress and Unfolded protein response. bioRxiv , 2021.2006.2021.449284 (2021). Chan, C.P. et al. Modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein. J Virol 80, 9279–9287 (2006). Wu, Y., Swulius, M.T., Moremen, K.W. & Sifers, R.N. Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation. Proc Natl Acad Sci U S A 100, 8229–8234 (2003). Zhang, J. et al. RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy. Nat Commun 13, 6007 (2022). Greenfield, J.J. & High, S. The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment. J Cell Sci 112 (Pt 10), 1477–1486 (1999). Greene, C.M. et al. alpha1-Antitrypsin deficiency. Nat Rev Dis Primers 2, 16051 (2016). Murray, L.W., Bautista, J., James, P.L. & Rimoin, D.L. Type II collagen defects in the chondrodysplasias. I. Spondyloepiphyseal dysplasias. Am J Hum Genet 45, 5–15 (1989). Guatibonza Moreno, P. et al. At a glance: the largest Niemann-Pick type C1 cohort with 602 patients diagnosed over 15 years. Eur J Hum Genet 31, 1108–1116 (2023). Bashir, R. et al. A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B. Nat Genet 20, 37–42 (1998). Liu, J. et al. Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 20, 31–36 (1998). Hosokawa, N. et al. Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I. J Biol Chem 278, 26287–26294 (2003). Teckman, J.H. & Perlmutter, D.H. Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response. Am J Physiol Gastrointest Liver Physiol 279, G961-974 (2000). Gelsthorpe, M.E. et al. Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding. J Biol Chem 283, 8229–8236 (2008). Schultz, M.L. et al. Coordinate regulation of mutant NPC1 degradation by selective ER autophagy and MARCH6-dependent ERAD. Nat Commun 9, 3671 (2018). Forrester, A. et al. A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex. EMBO J 38 (2019). Fujita, E. et al. Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Hum Mol Genet 16, 618–629 (2007). Fang, S. et al. The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum. Proc Natl Acad Sci U S A 98, 14422–14427 (2001). Wang, B. et al. Mechanistic understanding of N-glycosylation in Ebola virus glycoprotein maturation and function. J Biol Chem 292, 5860–5870 (2017). van de Weijer, M.L. et al. Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex. Mol Cell 79, 768–781 e767 (2020). Rudinskiy, M. & Molinari, M. ER-to-lysosome-associated degradation in a nutshell: mammalian, yeast, and plant ER-phagy as induced by misfolded proteins. FEBS Lett 597, 1928–1945 (2023). Braxton, J.R. & Southworth, D.R. Structural insights of the p97/VCP AAA + ATPase: How adapter interactions coordinate diverse cellular functionality. J Biol Chem 299, 105182 (2023). Leonard, C.K. et al. Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J Biol Chem 265, 10373–10382 (1990). Fregno, I. et al. ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport. EMBO J 37 (2018). Su, W. et al. Furin Egress from the TGN is Regulated by Membrane-Associated RING-CH Finger (MARCHF) Proteins and Ubiquitin-Specific Protease 32 (USP32) via Nondegradable K33-Polyubiquitination. Adv Sci (Weinh) 11, e2403732 (2024). Khan, I. et al. Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1. Nat Commun 15, 162 (2024). Dang, Y. et al. Identification of a single amino acid required for APOBEC3 antiretroviral cytidine deaminase activity. J Virol 85, 5691–5695 (2011). Ahmad, I. et al. The retroviral accessory proteins S2, Nef, and glycoMA use similar mechanisms for antagonizing the host restriction factor SERINC5. J Biol Chem 294, 7013–7024 (2019). Han, Y., Wang, X., Dang, Y. & Zheng, Y.H. APOBEC3G and APOBEC3F require an endogenous cofactor to block HIV-1 replication. PLoS Pathog 4, e1000095 (2008). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalTableandFigures.docx Supplemental Table and Figures Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6498082","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471030587,"identity":"a7fb1576-b504-4d6f-8666-3ba78a458add","order_by":0,"name":"Yong-Hui Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYDACCTB5QI6BGUSzkaDFmHQtiQ0MxGqRn9187OHXtjvpG44zP2D4UHaYsBbGOcfSjWXbnuVuOMxmwDjjHBFamCVyzKQl2w4DtfAwMPO2EaGFTSL/G0hLugFIy19itPBI5LBJfmw7nADWwkiMFgmJNDNphnOHDWcC/XKw51w6YS3yM5KfSf4oOyzPd/7wwwc/yqwJawEBZl5odBwgTj0QMP74Q7TaUTAKRsEoGIkAALlZOnwDA0WhAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1098-7385","institution":"University of Illinois Chicago","correspondingAuthor":true,"prefix":"","firstName":"Yong-Hui","middleName":"","lastName":"Zheng","suffix":""},{"id":471030588,"identity":"85dcd198-7456-4455-89f9-43bfb880a152","order_by":1,"name":"Jing Zhang","email":"","orcid":"","institution":"Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":471030589,"identity":"a050072d-19b4-4ea3-a162-36cb43fc6630","order_by":2,"name":"Xiaoran Lu","email":"","orcid":"","institution":"Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaoran","middleName":"","lastName":"Lu","suffix":""},{"id":471030590,"identity":"29284b53-0536-4342-b1cc-f9d7a536086e","order_by":3,"name":"Sunan Li","email":"","orcid":"","institution":"Harbin Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sunan","middleName":"","lastName":"Li","suffix":""},{"id":471030591,"identity":"7cb08cbe-d7b0-4d14-a4e9-8d99b2d15181","order_by":4,"name":"Tao Wang","email":"","orcid":"","institution":"Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Wang","suffix":""},{"id":471030592,"identity":"2f8aac37-2610-4333-bbe8-7405b16568b8","order_by":5,"name":"Iqbal Ahmad","email":"","orcid":"","institution":"Harbin Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Iqbal","middleName":"","lastName":"Ahmad","suffix":""}],"badges":[],"createdAt":"2025-04-21 17:55:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6498082/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6498082/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85211251,"identity":"a8f0fdb3-4e15-4951-a2e4-73e880b33df6","added_by":"auto","created_at":"2025-06-23 12:27:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":590234,"visible":true,"origin":"","legend":"\u003cp\u003eError bars in (\u003cstrong\u003eB\u003c/strong\u003e) and (\u003cstrong\u003eH\u003c/strong\u003e) represent the standard error of measurements (SEMs) calculated from three experiments (n=3). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, ns (not significant, p\u0026gt;0.05). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman coronavirus spike proteins are MAN1B1 substrates.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) SARS1-S, SARS2-S, and MERS-S proteins with a FLAG-tag were expressed in HEK293T cells and detected by WB. The unprocessed S\u003csub\u003e0\u003c/sub\u003e precursor and processed S\u003csub\u003e1\u003c/sub\u003e subunit are indicated. \u0026nbsp;b-actin (ACTB) was used as a loading control.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Coronavirus spike and NHK proteins were expressed with ER-stress luciferase-reporter vector pLightSwitch-BiP, p5´ATF6-GL3, pXBP1u-FLuc, or pATF4-UTR-Fluc in HEK293T cells. Luciferase activity was measured and are presented as a relative value, with the activity in the presence of a control vector (Ctrl) set as 1.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Coronavirus spike proteins were expressed with MAN1B1, EDEM1, EDEM2, or EDEM3 with a HA-tag in HEK293T cells, and their expression was detected by WB.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) Coronavirus spike proteins were expressed in HEK293T wild-type (WT) and \u003cem\u003eMAN1B1\u003c/em\u003e-knockout (KO) cells, and their expression was compared by WB.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE\u003c/strong\u003e) Coronavirus spike proteins were expressed in HEK293T WT and \u003cem\u003eEDEM2\u003c/em\u003e-KO cells, and their expression was compared by WB.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) Coronavirus spike proteins were expressed with MAN1B1 and EDEM2 in HEK293T \u003cem\u003eMAN1B1\u003c/em\u003e-KO or \u003cem\u003eEDEM2\u003c/em\u003e-KO cells, and their expression was compared by WB.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eG\u003c/strong\u003e) S proteins were expressed with MAN1B1 and its catalytic site-deficient mutants (E330A, D463A, E599A) in \u003cem\u003eMAN1B1\u003c/em\u003e-KO cells, and their expression was analyzed by WB.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eH\u003c/strong\u003e) HIV-1 firefly luciferase reporter pseudovirions (PVs) expressing coronavirus spike proteins were produced in the presence of ectopic MAN1B1 from HEK293T WT or \u003cem\u003eMAN1B1\u003c/em\u003e-KO cells. After infecting Huh7 cells with an equal number of PVs, viral infection was determined by measuring intracellular luciferase activities. \u0026nbsp;Results are presented as relative values, with the activities in the presence of a control vector set as 100.\u003c/p\u003e","description":"","filename":"Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/9f1db35da7b948184bfb93f9.png"},{"id":85212758,"identity":"c5f80202-f207-48ea-85b5-ba01e21c6d6a","added_by":"auto","created_at":"2025-06-23 12:43:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":988900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAN1B1 targets coronavirus spike proteins to lysosomes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) MAN1B1 with a mCherry-tag and CALR or TGOLN2 with a GFP-tag were expressed in HeLa cells, and their colocalization was investigated by confocal microscopy (scale bar 5 µm). PCC, Pearson's correlation coefficient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Coronavirus spike proteins with a GFP-tag were expressed with MAN1B1-mCherry in HeLa cells and their subcellular localization was determined by confocal microscopy (scale bar 5 μm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Coronavirus spike-GFP proteins were expressed with LAMP1-mCherry and MAN1B1 in HeLa cells. Cells were treated with 100 nM Bafilomycin A1 (BafA1), 20 mM NH\u003csub\u003e4\u003c/sub\u003eCl, 20 nM concanamycin A (ConA), 10 mM 3-methyladenine (3-MA), or DMSO only. The co-localization of these spike proteins with LAMP1 was determined by confocal microscopy (scale bar 5 μm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e PCC values from (C) are calculated and shown.\u0026nbsp; Error bars represent SEMs calculated from three experiments (n=3). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001, ns (not significant, p\u0026gt;0.05).\u003c/p\u003e","description":"","filename":"Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/472d6a2e0abb8f5ce57e051a.png"},{"id":85211252,"identity":"a754cebf-2415-4d77-b6ac-79920fad0ad5","added_by":"auto","created_at":"2025-06-23 12:27:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":700541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAN1B1 targets class I fusion proteins for lysosomal degradation independently of polyubiquitination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e EBOV-GP, HIV-1-Env, H5N1-HA, SARS2-S, SARS1-S, and MERS-S were expressed with MAN1B1 in HEK293T cells and treated with 50 mM kifunensine (Kif), 20 mM MG132, 20 mM lactacystin (Lac), 100 nM BafA1, 20 mM NH\u003csub\u003e4\u003c/sub\u003eCl, 20 nM ConA, or 10 mM 3-MA. Protein expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e EBOV-GP was expressed in HEK293T WT and \u003cem\u003eMAN1B1\u003c/em\u003e-KO cells, or, EBOV-GP was expressed in HEK293T MAN1B1-KO cells in the presence or absence of ectopic MAN1B1 expression. EBOV-GP expression was compared by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e SARS2-S and EBOV-GP were expressed with MAN1B1 in HEK293T cells in the presence of increasing amounts of WT ubiquitin (Ub\u003csub\u003eWT\u003c/sub\u003e) or its mutant that does not express any lysine residues (Ub\u003csub\u003eKO\u003c/sub\u003e). Protein expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e SARS2-S and EBOV-GP with a FLAG-tag were expressed with Ub\u003csub\u003eWT\u003c/sub\u003e and MAN1B1 or PDIA3 in HEK293T cells. Proteins were immunoprecipitated (IP) with anti-FLAG and analyzed by WB.\u003c/p\u003e","description":"","filename":"Figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/518c153c42cb56fb045cfcbf.png"},{"id":85211255,"identity":"48c52c30-d8f0-4b6e-acb7-84176a356d1d","added_by":"auto","created_at":"2025-06-23 12:27:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":883673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMembralin is required for class I fusion protein degradation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Membralin was knocked out in HEK293T cells by CRISPR/Cas9, which is confirmed by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Class I fusion proteins were expressed with MAN1B1 in HEK293T WT and \u003cem\u003eMembralin\u003c/em\u003e-KO cells, and their expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e EBOV-GP was expressed with indicated ER chaperones in HEK293T WT \u003cem\u003eMembralin\u003c/em\u003e-KO cells, and their expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eClass I fusion proteins were expressed with Membralin in HEK293T cells, and treated with 50 mM Kif, 10 mM Eerl, 20 mM Lac, 20 mM MG132, 100 nM BafA1, or 20 mM NH\u003csub\u003e4\u003c/sub\u003eCl. Protein expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Class I fusion proteins were expressed with Membralin in HEK293T WT and \u003cem\u003eMAN1B1\u003c/em\u003e-KO cells, and their expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e PVs expressing HIV-1-Env, EBOV-GP, or SARS2-S were produced from HEK293T WT, \u003cem\u003eMAN1B1\u003c/em\u003e-KO, and \u003cem\u003eMembralin\u003c/em\u003e-KO cells in the presence of ectopic MAN1B1 or TMEM239 expression. Viral infectivity was determined in TZM-bI cells for PVs expressing HIV-1 Env, or in Huh7 cells for PVs expressing EBOV-GP or SARS2-S. \u0026nbsp;Results are presented as relative values, with the activity in the presence of a control vector (Ctrl) set as 100. Error bars represent the SEMs calculated from three experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eSARS2-S-GFP, LAMP1-mCherry, and Membralin-BFP were expressed in HEK293T cells in the absence or presence of ectopic MAN1B1. Images were captured by Airyscan super-resolution microscopy every 5 min for 12.8 or 23.3 hours and are shown at indicated times. Scale bar, 5 mm.\u003c/p\u003e","description":"","filename":"Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/afde93db25b5cfdbeca54e82.png"},{"id":85211590,"identity":"70408535-d9f3-422d-b135-d781424cac22","added_by":"auto","created_at":"2025-06-23 12:35:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1152926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMembralin relies on different regions for ER-localization and functionality.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e A schematic representation of Membralin is shown, featuring four transmembrane (TM) domains (grey), three cytoplasmic domains (pink), and two luminal domains (green), all indicated by their corresponding amino acid numbers. The sequence of the cytoplasmic tail (amino acids 467-620) is also displayed, with the region spanning amino acids 539-562 underlined and the \u003csup\u003e549\u003c/sup\u003eTDASFL\u003csup\u003e554\u003c/sup\u003e motif highlighted in red.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e The indicated Membralin mutants were expressed with class I fusion proteins in HEK293T cells, and their expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e CALR-GFP was expressed with indicated Membralin mutants with a FLAG-tag in HeLa cells. Cells were stained with anti-FLAG followed by Alexa Fluor 647-conjugated goat anti-mouse IgG, and their colocalization was determined by confocal microscopy (scale bar 5 µm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e The indicated Membralin mutants were expressed with EBOV-GP and H5N1-HA in HEK293T cells, and their expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eMembralin\u003cstrong\u003e \u003c/strong\u003eand mutant 1-466 with a FLAG-tag were expressed with LC3/GABARAP family proteins with a HA-tag in HEK293T cells. Proteins were pulled down by anti-FLAG and analyzed by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e The indicated Membralin mutants with a FLAG-tag were expressed with LC3B with a HA-tag in HEK293T cells. Proteins were pulled down by anti-FLAG and analyzed by WB.\u003c/p\u003e","description":"","filename":"Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/20a9f68b26a3def55ddc1fec.png"},{"id":85211259,"identity":"105ac3a9-bdba-43bb-907b-186f9493a3bb","added_by":"auto","created_at":"2025-06-23 12:27:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":738419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMembralin recruits VCP and MAN1B1 via different regions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eClass I fusion proteins were expressed alongside MAN1B1 or Membralin in HEK293T cells in the presence of siRNAs targeting \u003cem\u003eVCP\u003c/em\u003e or a control (Ctrl). Protein expression levels were assessed by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eVCP-HA was co-expressed with the indicated Membralin deletion mutants or GFP as a control in HEK293T cells. Proteins were pulled down by anti-FLAG beads. Proteins in the pulldown samples (IP) and cell lysate (Input) were detected by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e MAN1B1-HA was expressed with the indicated Membralin mutants and GFP with a FLAG-tag in HEK293T cells. Proteins were pulled down by anti-FLAG beads. Proteins in the pulldown samples (IP) and cell lysate (Input) were detected by WB.\u003c/p\u003e","description":"","filename":"Figures6.png","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/eb71614942480706085c3f00.png"},{"id":85211258,"identity":"276f5bc4-c76c-4f91-8930-db346ac56beb","added_by":"auto","created_at":"2025-06-23 12:27:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":674595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Membralin-MAN1B1 axis does not target well-defined human misfolded or aggregated proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e AAT and its variants ATZ and NHK, NPC1 and its variant I1061T, collagen alpha-1(II) chain (CO2A1) and its variant R989C/G1152D, dysferin (DYSF) and its variant L1341P, CD3d (CD3D), and nicastrin (NCSTN) were expressed with MAN1B1 in HEK293T cells and their expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eThe indicated misfolded and aggregated proteins were expressed with MAN1B1 in HEK293T cells.\u0026nbsp; Cells were treated with 50 mM Kif, 10 mM EerI, 20 mM Lac, 20 mM MG132, 100 nM BafA1, or 20 mM NH\u003csub\u003e4\u003c/sub\u003eCl. Protein expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e The indicated misfolded and aggregated proteins were expressed with MAN1B1 in HEK293T WT, \u003cem\u003eMembralin\u003c/em\u003e-KO, and \u003cem\u003eFAM134B\u003c/em\u003e-KO cells. Protein expression was determined by WB.\u003c/p\u003e","description":"","filename":"Figures7.png","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/2ff2676c20d2b5021d81e347.png"},{"id":85212761,"identity":"d1b83798-1ab4-48ba-b803-2c2809388b92","added_by":"auto","created_at":"2025-06-23 12:43:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":536455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultiple factors determine the specific degradation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e COL2A1, its variants R989C/G1152D, and EBOV glycoprotein (GP) were co-expressed with calnexin (CANX), protein disulfide isomerase A3 (PDIA3), calreticulin (CALR), FAM134B, and FAM134B-2 in HEK293T cells. Protein expression levels were assessed by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e The R989C/G1152D variant was expressed with CANX, FAM134B-2, or MAN1B1 in HEK293T wild-type (WT), \u003cem\u003eFAM134B\u003c/em\u003e-KO, or \u003cem\u003eCANX\u003c/em\u003e-KO cells. Protein expression was again evaluated by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eA schematic representation of EBOV glycoproteins is shown, including the structural glycoprotein (GP), secreted glycoprotein (sGP), and soluble glycoprotein (ssGP). The structural glycoprotein is processed into GP\u003csub\u003e1\u003c/sub\u003e and \u003csub\u003eGP2\u003c/sub\u003e by furin and linked by a disulfide bond. The letter \"Y\" indicates \u003cem\u003eN\u003c/em\u003e-glycosylation sites, while ⚲ indicates \u003cem\u003eO\u003c/em\u003e-glycosylation sites. MLD refers to the mucin-like domain, and TM indicates the transmembrane domain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eThe indicated EBOV glycoproteins were expressed alongside MAN1B1 in HEK293T cells and treated with the specified inhibitors. Protein expression levels were determined by WB.\u003c/p\u003e\n\u003cp\u003eIndicated EBOV glycoproteins were expressed with MAN1B1 in HEK293T cells and treated with indicated inhibitors. Protein expression was determined by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e The indicated EBOV glycoproteins were expressed with MAN1B1 in HEK293T WT and \u003cem\u003eMembralin\u003c/em\u003e-KO cells, and protein expression was assessed by WB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e A proposed model for the degradation of class I fusion proteins by Membralin. During productive viral infection, heavily glycosylated class I fusion proteins are recognized by MAN1B1, which catalyzes their extensive demannosylation in the ER. The Membralin-MAN1B1 complex then targets these low-mannose viral glycoproteins to an ER subdomain, from which they are detached via p97/VCP and engulfed in lysosomes via Membralin LIR for ER-phagy.\u003c/p\u003e","description":"","filename":"Figures8.png","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/a003bffd0bb8d08a75ac7f6e.png"},{"id":85213126,"identity":"87ea4314-718c-4c0a-ad21-841076a25074","added_by":"auto","created_at":"2025-06-23 12:51:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7381247,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/0ed1ea73-d64a-4f94-a264-272a87ebfbd0.pdf"},{"id":85211263,"identity":"db189627-94ca-4615-9f1f-3ee212cfbcff","added_by":"auto","created_at":"2025-06-23 12:27:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5015279,"visible":true,"origin":"","legend":"Supplemental Table and Figures","description":"","filename":"SupplementalTableandFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6498082/v1/fe73472f5d387e1ea8326088.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Membralin Selects Foreign Glycoproteins from the Endoplasmic Reticulum to Lysosomes for Degradation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe endoplasmic reticulum (ER) is essential for protein synthesis, producing both secreted and integral membrane proteins. It plays a critical role in ensuring that newly synthesized proteins are properly folded into their native conformations. When terminally misfolded proteins accumulate, the ER experiences stress, which can be mitigated by the ER quality control systems, including ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMembralin/TMEM259, encoded by \u003cem\u003eTMEM259\u003c/em\u003e, is crucial for motor neuron survival as it maintains optimal expression levels of nicastrin, a core component of the γ-secretase complex \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It is a multi-pass membrane protein in the ER and implicated in alleviating ER stress by interacting with proteins in the ERAD network \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMannosyl-oligosaccharide 1,2-α-mannosidase (MAN1B1) is a key enzyme in the ERAD process that specifically targets glycoproteins by co-opting with three ER degradation-enhancing α -mannosidase-like (EDEM) proteins. During glycoprotein folding in the ER, MAN1B1 primarily removes a single 1,2-α-linked mannose residue from Man9GlcNAc2 on \u003cem\u003eN\u003c/em\u003e-glycan precursors, yielding Man8GlcNAc2. For terminally misfolded glycoproteins, MAN1B1 and EDEM proteins further trim two or three additional 1,2-α-linked mannose residues, producing Man(5\u0026ndash;6)GlcNAc2. Glycoproteins with these low-mannose \u003cem\u003eN\u003c/em\u003e-glycans are retrotranslocated and degraded via the ERAD pathway \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eERAD facilitates the retrotranslocation of misfolded proteins into the cytoplasm through the SEC61 translocon, an energy-dependent process driven by the AAA\u0026thinsp;+\u0026thinsp;ATPase p97/valosin-containing protein (VCP), ultimately resulting in degradation by cytosolic proteasomes. ERLAD directs misfolded proteins into specialized ER subdomains that detach from the ER and fuse with lysosomes for degradation. This process is mediated by 9 ER-phagy receptors, including FAM134B/RETREG1, FAM134B-2/RETREG1-2, FAM134A/RETREG2, FAM134C/RETREG3, TEX264, RTN3L, ATL3, CCPG1, and SEC62. These receptors are embedded in the ER membrane and are characterized by at least one cytosolic LC3-interacting region (LIR). FAM134B and RTN3L also contain a reticulon-homology domain (RHD) within the ER membrane.\u003c/p\u003e \u003cp\u003eClass I fusion proteins, such as human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), influenza A virus hemagglutinin (HA), Ebola virus glycoprotein (GP), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, play crucial roles in the entry of these viruses \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although these proteins do not share sequence homology, they exhibit a common trimeric-hairpin structure with a central coiled coil covered by a dense glycan shield. Class I fusion proteins are expressed as type I transmembrane polypeptide precursors, folded in the ER, and cleaved by furin in the Golgi. We have previously shown that the expression of HIV-1-Env, EBOV-GP, and IFA-HA induces ER stress and undergoes MAN1B1-mediated degradation \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we identify a protein degradation machinery across the ER membrane that specifically targets class I fusion proteins. This machinery is orchestrated by Membralin and includes MAN1B1 and p97/VCP, facilitating the degradation of these viral glycoproteins via ER-phagy. This process provides a crucial mechanism for cell-autonomous immunity within the ER.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eHuman coronavirus spike proteins are MAN1B1 substrates.\u003c/b\u003e SARS-CoV-2 (SARS2) infection induces ER stress, significantly contributing to COVID-19 cytopathic and inflammatory pathogenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. During SARS-CoV (SARS1) infection, its spike protein plays a significant role in the induction of ER stress compared to other viral proteins \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Thus, we investigated how SARS2-S activates UPR and triggers its relevant signaling events, including SARS1 and Middle East respiratory syndrome coronavirus (MERS) spike proteins as controls.\u003c/p\u003e \u003cp\u003eDuring ER stress, binding-immunoglobulin protein (BiP) is upregulated to serve as a master ER stress regulator, resulting in the activation of three ER transmembrane receptors: the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme type 1 (IRE1), and activating transcription factor 6 (ATF6). PERK activates a transcription factor ATF4, and IRE1 activates another transcription factor, X-box binding protein 1 (XBP1). As done previously, we used luciferase-based reporter assays to detect the upregulation and activation of BiP, XBP1, ATF4, and ATF6 \u003csup\u003e6, 8\u003c/sup\u003e. As a positive control for ER stress, the terminally misfolded human Serpin A1 (SERPINA1)/alpha1-antitrypsin (AAT) variant, null (Hong Kong) (NHK), was used \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. SARS1-S, SARS2-S, MERS-S, and NHK were expressed with these different reporters, and levels of ER stress were determined. SARS1-S, SARS2-S, and MERS-S were expressed at similar levels, as detected by western blotting (WB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). As expected, NHK upregulated BiP and activated ATF6, XBP1, and ATF4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Like NHK, SARS2-S also did so at comparable levels. SARS1-S and MERS-S strongly activated ATF6, but their upregulation and activation of BiP, XBP1, and ATF4 were significantly lower than SARS2-S and NHK. Thus, these coronaviruses activate ER stress, but the SARS2 activity is much broader and more potent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePreviously, we identified HIV-1-Env, IFA-HA, and EBOV-GP as MAN1B1 substrates for degradation \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. When SARS1-S, SARS2-S, or MERS-S were expressed with MAN1B1, EDEM1, EDEM2, and EDEM3, MAN1B1 reduced all these viral protein expressions, whereas EDEM1, EDEM2, and EDEM3 had little effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We then knocked out \u003cem\u003eMAN1B1\u003c/em\u003e and \u003cem\u003eEDEM2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). When SARS1-S, SARS2-S, and MERS-S were expressed in wild-type (WT) and these knockout (KO) cells, their expression was increased in \u003cem\u003eMAN1B1\u003c/em\u003e-KO, but not \u003cem\u003eEDEM2\u003c/em\u003e-KO cells. Next, ectopic MAN1B1 and EDEM2 were expressed with the individual spike proteins in KO cells. The MAN1B1 activity was restored, but EDEM2 had little activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eMAN1B1 has three critical catalytic sites (E330, D463, and E599), which are conserved among class I α-mannosidases. When these residues were mutated to alanine, the MAN1B1 activity was disrupted, indicating that its enzymatic activity is required (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eTo demonstrate that this MAN1B1 activity is antiviral, HIV-1 luciferase-reporter pseudovirions (PVs) expressing SARS1-S, SARS2-S, and MERS-S were produced from HEK293T WT and \u003cem\u003eMAN1B1\u003c/em\u003e-KO cells in the presence of ectopic MAN1B1, and their infectivity was determined by infection of Huh7 cells. MAN1B1 strongly inhibited the infectivity of all three PVs up to 40-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMAN1B1 targets coronavirus spike proteins to lysosomes.\u003c/b\u003e We investigated how MAN1B1 affects the subcellular localization of coronavirus spike proteins by confocal microscopy.\u003c/p\u003e \u003cp\u003eMAN1B1 with a mCherry-tag was expressed with the ER marker calreticulin (CALR), or the Golgi marker \u003cem\u003etrans\u003c/em\u003e-Golgi network integral membrane protein 2 (TGOLN2) with a GFP-tag. MAN1B1 exhibited cytoplasmic distribution, colocalizing with both CALR and TGOLN2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, the Pearson's correlation coefficient (PCC) of MAN1B1 with CALR was approximately four times higher than that with TGOLN2, confirming that MAN1B1 predominantly localizes to the ER.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen SARS1-S, SARS2-S, and MERS-S with a GFP-tag were expressed alone, they were predominantly found on the plasma membrane; however, when they were expressed with MAN1B1-mCherry, they were redistributed into cytoplasmic compartments and colocalized with MAN1B1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThese spike proteins were then expressed with the lysosome marker, lysosomal-associated membrane protein 1 (LAMP1) with a mCherry-tag, and MAN1B1, followed by treatment with lysosomal/autophagy inhibitors NH\u003csub\u003e4\u003c/sub\u003eCl, bafilomycin A1 (BafA1), concanamycin A (ConA), and 3-methyladenine (3-MA). These spike proteins predominantly localized to the plasma membrane but were redistributed into the cytoplasm by the presence of MAN1B1, showing colocalization with LAMP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Notably, this colocalization was significantly promoted by treatments with these inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting that spike proteins are degraded by autophagy/lysosomes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eClass I fusion proteins are degraded in lysosomes independently of polyubiquitination.\u003c/b\u003e To detect protein degradation, class I fusion proteins, including EBOV-GP, HIV-1-Env, IFA (H5N1)-HA, SARS2-S, SARS1-S, and MERS-S were expressed with MAN1B1 in HEK293T cells and treated with different types of inhibitors, including class I α-mannosidase inhibitor kifunensine (Kif), proteasomal inhibitors MG132 and lactacystin (Lac), lysosomal inhibitors NH\u003csub\u003e4\u003c/sub\u003eCl and BafA1, and autophagy inhibitors ConA and 3-MA. As expected, Kif blocked the MAN1B1 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, lanes 3, 9, 15, 21, 28, 39), and neither MG132 nor Lac did so, indicating that they are not degraded in proteasomes. However, NH\u003csub\u003e4\u003c/sub\u003eCl, BafA1, ConA, and 3-MA all blocked the MAN1B1 activity, indicating that they are degraded by autophagy/lysosomes. To further confirm the endogenous MAN1B1 activity, EBOV-GP was expressed again in WT and \u003cem\u003eMAN1B1\u003c/em\u003e-KO cells. EBOV-GP expression was greatly increased in KO cells, which was countered by ectopic MAN1B1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we determined whether polyubiquitination gets involved in this degradation process. We reported that disulfide-isomerase A3 (PDIA3) targets EBOV-GP for ER-phagy in a polyubiquitination-dependent manner \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. When SARS2-S and EBOV-GP were expressed with MAN1B1 and increasing amounts of WT ubiquitin (Ub\u003csub\u003eWT\u003c/sub\u003e) or its lysine-free mutant (Ub\u003csub\u003eKO\u003c/sub\u003e), neither influenced their downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). SARS2-S and EBOV-GP were then expressed with MAN1B1 in the presence of Ub\u003csub\u003eWT\u003c/sub\u003e, and SARS2-S and EBOV-GP polyubiquitination were detected by immunoprecipitated (IP), including PDIA3 as a control. MAN1B1 and PDIA3 both decreased EBOV-GP expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, lanes 3, 4, 9, 10), but only PDIA3 promoted EBOV-GP polyubiquitination, whereas MAN1B1 did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, lanes 7, 8, 11, 12). Additionally, MAN1B1 did not induce SARS2-S polyubiquitination either (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, lanes 5, 6). Thus, MAN1B1 does not trigger SARS2-S and EBOV-GP polyubiquitination.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMembralin is required for class I fusion degradation.\u003c/b\u003e To identify the ER-phagy receptor, we knocked out \u003cem\u003eFAM134B\u003c/em\u003e, \u003cem\u003eRTN3L\u003c/em\u003e, \u003cem\u003eATL3\u003c/em\u003e, \u003cem\u003eSEC62\u003c/em\u003e, \u003cem\u003eCCPG1\u003c/em\u003e, and \u003cem\u003eTEX264\u003c/em\u003e in HEK293T cells (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). Since FAM134B-2 is an N-terminally truncated isoform of FAM134B, this short isoform was also knocked out in this \u003cem\u003eFAM134B\u003c/em\u003e-KO cell line. However, we found that MAN1B1 still decreased class I fusion protein expression in these KO cells (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). We also knocked out \u003cem\u003eFAM134C\u003c/em\u003e, and excluded its involvement, too (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e). Thus, MAN1B1 does not seem to engage with these known ER-phagy receptors to initiate this destruction.\u003c/p\u003e \u003cp\u003eSubsequently, we knocked out \u003cem\u003eMebralin\u003c/em\u003e in HEK293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). When class I fusion proteins were expressed alongside MAN1B1, the reduction of their expression was all blocked in this KO cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, lanes 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24). However, PDIA3, CANX, and CALR continued to decrease EBOV-GP expression in these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we determined whether Membralin can target these viral proteins alone. Indeed, ectopic Membralin strongly decreased their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, lanes 1, 2, 9, 10, 17, 18, 25, 26, 33, 34, 41, 42). This decrease was blocked by BafA1, NH\u003csub\u003e4\u003c/sub\u003eCl, and the p97/VCP inhibitor Eeyarestatin I (EerI) but not by MG132, Lac, and Kif (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). To confirm the MAN1B1 independence, we expressed Membralin with class I fusion proteins in HEK293T WT and \u003cem\u003eMAN1B1\u003c/em\u003e-KO cells. We found that Membralin still decreased the class I protein expression in this KO cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eBecause Membralin was implicated in ERAD, we wondered whether the SEC61 complex, which serves as the translocon for retro-translocation during ERAD, is required for the Membralin activity. The SEC61 complex comprises abg subunits that form transmembrane channels where proteins are translocated across the ER membrane \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The a subunit was silenced by siRNAs and the b subunit was knocked by CRISPR/Cas9 in HEK293T cells (\u003cb\u003eFig. S2A\u003c/b\u003e, \u003cb\u003eFig. S2B\u003c/b\u003e). When class I fusion proteins were expressed with MAN1B1 or Membralin, their expression was still inhibited in these silenced and KO cells (\u003cb\u003eFig. S2A\u003c/b\u003e, \u003cb\u003eFig. S2C\u003c/b\u003e). Thus, SEC61 translocon is not required for the MAN1B1 and Membralin activity.\u003c/p\u003e \u003cp\u003eTo demonstrate that the MAN1B1-Membralin axis has antiviral activity, PVs expressing EBOV-GP, SARS2-S, and HIV-1-Env were produced from HEK293T WT, \u003cem\u003eMAN1B1\u003c/em\u003e-KO, or \u003cem\u003eMembralin\u003c/em\u003e-KO cells in the presence of ectopic MAN1B1 or Membralin expression. The infectivity of all these PVs was increased 3 to 4-fold when they were produced from \u003cem\u003eMAN1B1\u003c/em\u003e-KO or \u003cem\u003eMembralin\u003c/em\u003e-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, compare Ctrl lanes in three cell lines). Conversely, their infectivity was decreased\u0026thinsp;~\u0026thinsp;20-fold by ectopic MAN1B1 or Membralin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). We previously reported the potent MAN1B1 antiviral activity to authentic IFA and HIV-1 \u003csup\u003e6, 9, 10\u003c/sup\u003e. Collectively, these results demonstrate the broad antiviral activity of the MAN1B1-Membralin axis by targeting class I fusion proteins.\u003c/p\u003e \u003cp\u003eFurthermore, we used live-cell imaging to track the translocation of SARS2-S from the ER to lysosomes. SARS2-S-GFP, LAMP1-mCherry, and Membralin-BFP were expressed with MAN1B1 in HEK293T cells, and live cell images were captured by a super-resolution microscope every 5 min. Without MAN1B1, SARS2-S barely colocalized with Membralin and LAMP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, \u003cb\u003eFig. S3\u003c/b\u003e), indicating successful egress of native SARS2-S from the ER for expression. In the presence of MAN1B1, SARS2-S was restricted into Membralin-positive domains that migrated into LAMP1-positive compartments time-dependent. These results confirm that MAN1B1 senses and recruits SARS2-S to the Membralin-positive ER-subdomain for degradation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe ER location of Membralin is determined by its 1st transmembrane domain.\u003c/b\u003e Membralin consists of 620 amino acids (aa) and features four transmembrane (TM) domains, three cytoplasmic domains, and two luminal domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We initially created five Membralin deletion mutants by removing each TM domain individually (∆TM1, ∆TM2, ∆TM3, ∆TM4) and an additional mutant that removed all four TM domains (∆4TM). When expressed alongside class I fusion proteins, unlike the full-length (FL) Membralin, ∆TM2, ∆TM3, and ∆TM4, neither ∆TM1 nor ∆4TM reduced their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we compared the subcellular localization of these mutants using confocal microscopy, with CALR as the ER marker. We found that ∆TM2, ∆TM3, and ∆TM4 localized correctly within the ER, whereas ∆TM1 and ∆4TM did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results demonstrate that TM1 is essential for the ER localization, and importantly, this localization is critical for its activity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe cytoplasmic tail of Membralin interacts with LC3/GABARAP family proteins.\u003c/b\u003e To screen for active motifs, we constructed additional Membralin deletion mutants. Mutant 1-466, which possesses the entire cytoplasmic tail deletion, localized to the ER (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), but failed to reduce the expression of EBOV-GP and H5N1-HA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, lanes 3 and 15), indicating the functional importance of this cytoplasmic tail.\u003c/p\u003e \u003cp\u003eWe created six 24-aa-deletion mutants (∆467\u0026ndash;490, ∆491\u0026ndash;514, ∆515\u0026ndash;538, ∆539\u0026ndash;562, ∆563\u0026ndash;586, and ∆587\u0026ndash;610) and one 10-aa-deletion mutant (∆611\u0026ndash;620) to screen the 467\u0026ndash;620 region. When these seven mutants were expressed alongside EBOV-GP and H5N1-HA, only mutant ∆539\u0026ndash;562 lost the activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, lanes 7, 18). To further narrow down this 24-aa region, we constructed 11 overlapping 6-aa-deletion mutants, including ∆539\u0026ndash;544, ∆541\u0026ndash;546, ∆543\u0026ndash;548, ∆545\u0026ndash;550, ∆547\u0026ndash;552, ∆549\u0026ndash;554, ∆551\u0026ndash;556, ∆553\u0026ndash;558, ∆555\u0026ndash;560, ∆557\u0026ndash;562, and ∆560\u0026ndash;565. When expressed with EBOV-GP and H5N1-HA, only mutant ∆549\u0026ndash;554 lost the activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, lanes 31, 45).\u003c/p\u003e \u003cp\u003eThe 549\u0026ndash;554 region contains 6 residues TDASFL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To gain further insight, we created mutant ∆550\u0026ndash;554 by deleting the last five residues DASFL and found that it retained the activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, lanes 11, 22). Because mutants ∆545\u0026ndash;550, ∆547\u0026ndash;552, and ∆551\u0026ndash;556 contain partial deletions of these six residues but still exhibit activity, these results demonstrate that the complete deletion of \u003csup\u003e549\u003c/sup\u003eTDASFL\u003csup\u003e554\u003c/sup\u003e must occur to inactivate Membralin\u0026rsquo;s activity.\u003c/p\u003e \u003cp\u003eNext, we tested whether this \u003csup\u003e549\u003c/sup\u003eTDASFL\u003csup\u003e554\u003c/sup\u003e domain functions as an LC3-interacting region (LIR), which is characteristic of ER-phagy receptors. The LIR is a short peptide sequence that serves as a binding site for LC3/GABARAP proteins, which are crucial for autophagy. We expressed Membralin and mutant 1-466 alongside LC3A, LC3B, LC3C, GABARAP, GABARAP1, and GABARAP2 to assess their interactions through immunoprecipitation. Membralin successfully pulled down all these autophagy proteins, whereas 1-466 did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). We then expressed ∆539\u0026ndash;562, ∆549\u0026ndash;554, and ∆550\u0026ndash;554 with LC3B and repeated this experiment. ∆550\u0026ndash;554 was expressed at a similar level to the FL protein but showed significantly weaker binding to LC3B (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, lanes 2, 10). On the other hand, 1-466, ∆539\u0026ndash;562, and ∆549\u0026ndash;554 were all expressed at similar lower levels, and none of them bound to LC3B (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, lanes 4, 6, 8). Collectively, these results identify \u003csup\u003e549\u003c/sup\u003eTDASFL\u003csup\u003e554\u003c/sup\u003e as a critical motif for LC3-binding.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMembralin recruits VCP via its cytoplasmic loop.\u003c/b\u003e The Membralin activity is blocked by the VCP inhibitor EerI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, lanes 4, 12, 20, 28, 36, 44), indicating an involvement of this AAA\u0026thinsp;+\u0026thinsp;ATPase. To further confirm this, we silenced \u003cem\u003eVCP\u003c/em\u003e in HEK293T cells by siRNAs and tested the MAN1B1 and Membralin activity. The \u003cem\u003eVCP\u003c/em\u003e siRNA disrupted the MAN1B1 and Membralin activity class I fusion proteins, but a control siRNA did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe four TM domains of Membralin are linked by one large and one small luminal loop (aa 91\u0026ndash;301, 367\u0026ndash;425, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, green) and a relatively shorter cytoplasmic loop (aa 323\u0026ndash;345, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, middle pink). To understand how VCP interacts with Membralin, we constructed another three Membralin deletion mutants to express aa 70\u0026ndash;628, 1-322, or 1-345. Together with those five TM domain deletion mutants and mutant 1-466, they were expressed with VCP in HEK293T cells, and their interactions were determined by immunoprecipitation. The FL Membralin and its five TM deletion mutants pulled down VCP, whereas GFP did not, indicating that the Membralin-VCP binding does not depend on TM domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, lanes 1 to 7). In addition, 70\u0026ndash;628, 1-345, and 1-466 were also bound to VCP, whereas 1-322 did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, lanes 10\u0026ndash;13), indicating that Membralin recruits VCP via its cytoplasmic loop to destroy class I fusion proteins.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMembralin recruits MAN1B1 via its large luminal loop.\u003c/b\u003e We used the same mutants to understand the Membralin-MAN1B1 interaction by immunoprecipitation. All these mutants successfully pulled down MAN1B1, except for ∆TM1 and ∆4TM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, lanes 3, 7). Since these two mutants do not localize to the ER, these results confirm that the MAN1B1-Membralin interaction must occur within the ER. Additionally, 1-466 was able to pull down MAN1B1, indicating that this interaction does not require the cytoplasmic tail of Membralin. Furthermore, the continued interaction of 1-322 and ∆TM2 with MAN1B1 further establishes that MAN1B1 specifically binds to the large luminal loop of Membralin (aa 90\u0026ndash;302).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMembralin is not required for degradation of domestic misfolded proteins.\u003c/b\u003e Mutations on human genes encoding AAT \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, collagen α-1(II) chain (COL2A1) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, Niemann-Pick type C1 (NPC1) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and dysferlin (DYSF) \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e can cause their misfolding in the ER, leading to degradation by ERQC and various genetic diseases.\u003c/p\u003e \u003cp\u003eMAN1B1 targets AAT variant NHK to ERAD \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, but targets variant Z (ATZ) to ERLAD \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. NPC1 variant I1061T is targeted to ERAD \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, or ERLAD, via the ER-phagy receptor FAM134B \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. CANX also targets COL2A1 variant R989C/G1152D to ERLAD via FAM134B \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The DYSF variant L1341P is targeted to either ERAD or ERLAD \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Additionally, the autocrine motility factor receptor (AMFR) targets human CD3δ (CD3D) to ERAD \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and nicastrin (NCSTN) in the γ-secretase complex is also targeted to ERAD \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. We collectively tested whether the MAN1B-Membralin axis could target these human proteins.\u003c/p\u003e \u003cp\u003eFirst, we expressed AAT and its variants ATZ and NHK, NPC1 and its variant I1061T, COL2A1 and its variant R989C/G1152D, DYSF and its variant L1341P, CD3δ, and NCSTN with MAN1B1 in HEK293T cells. MAN1B1 effectively decreased the ATZ, NHK, 1061T, R989C/G1152D, L1341P, CD3δ, and NCSTN expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, lanes 4, 6, 10, 14, 18, 20, 22), but much less effectively the WT AAT, NPC1, COL2A1, and DYSF expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, lanes 2, 8, 12, 16).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecond, we determined how these misfolded variants, CD3δ, and NCSTN, are degraded. All their decreases by MAN1B1 were blocked by Kif and EerI (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, lanes 3, 4, 11, 12, 19, 20, 27, 28, 35, 36, 43, 44, 51, 52). The decrease of NHK, CD3δ, and NCSTN was blocked by Lac and MG132, indicating that they are targeted to ERAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, lanes 13, 14, 45, 46, 53, 54). The decrease of ATZ, I1061T (NPC1), R989C/G1152D (COL2A1), and L1341P (DYSF) was blocked by BafA1 and NH\u003csub\u003e4\u003c/sub\u003eCl, indicating that they are targeted to ERLAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, lane 7, 8, 23, 24, 31, 32, 39, 40).\u003c/p\u003e \u003cp\u003eThird, we determined whether Membralin is required. These proteins were expressed with MAN1B1 in HEK293T WT and \u003cem\u003eMembralin\u003c/em\u003e-KO cells. Because FAM134B was required for R989C/G1152D (COL2A1) and I1061T (NPC1) degradation via ERLAD, the \u003cem\u003eFAM134B\u003c/em\u003e-KO cell line was also included. Notably, none of their degradation was blocked in \u003cem\u003eMembralin\u003c/em\u003e-KO and \u003cem\u003eFAM134B\u003c/em\u003e-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelection of ER-phagy receptors.\u003c/b\u003e Surprisingly, FAM134B is not required for MAN1B1-mediated degradation of I1061T (NPC1) and R989C/G1152D (COL2A1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, lanes 23, 24, 31, 32). To understand how ER-phagy receptors are selected, ATZ, I1061T (NPC1), R989C/G1152D (COL2A1), and L1341P (DYSF) were expressed with MAN1B1 in HEK293T WT cells and six ER-phagy receptor KO cell lines. MAN1B1 still effectively decreased their expression in these KO cells, indicating that these ER-phagy receptors are not required for the MAN1B1 activity (\u003cb\u003eFig. S4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo further explore this mechanism, we investigated the degradation of COL2A1 and its variant R989C/G1152D in the context of CANX and FAM134B. CANX, CALR, and PDIA3 were also included because they degrade EBOV-GP via ERLAD. Consistently, CANX, CALR, and PDIA3 decreased EBOV-GP expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, lanes 10, 20, 21), as previously reported \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and CANX decreased R989C/G1152D but not COL2A1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, lanes 2, 6), as reported \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. FAM134B did not decrease any COL2A1, R989C/G1152D, and EBOV-GP expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, lanes 3, 7, 11), but FAM134B-2 decreased all these protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, lanes 4, 8, 12).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, CANX did not decrease R989C/G1152 expression in \u003cem\u003eFAM134B\u003c/em\u003e-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, lanes 3, 4), confirming that FAM134B is the ER-phagy receptor for this degradation \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. FAM134B-2 and MAN1B1 still decreased R989C/G1152D expression in \u003cem\u003eCANX\u003c/em\u003e-KO cells, indicating that their activity is independent of CANX (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, lanes 5\u0026ndash;12). These results demonstrate that the selection of ER-phagy receptor is determined by both effector (MAN1B1, CANX, FAM134B-2) and client proteins (host and viral proteins), highlighting the specificity of the Membralin-MAN1B1 axis in class I fusion protein targeting.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Membralin-MAN1B1 axis senses dense glycans.\u003c/b\u003e In addition to the structural GP, EBOV expresses soluble GP (sGP) and small soluble GP (ssGP) via RNA editing \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, all sharing the same N-terminal domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). EBOV-GP is heavily glycosylated, particularly in the mucin-like domain (MLD) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It has 17 \u003cem\u003eN\u003c/em\u003e-glycosylation sites, 8 of which are in MLD. In addition, MLD contains over 80 \u003cem\u003eO\u003c/em\u003e-glycosylation sites, resulting in higher GP\u003csub\u003e1\u003c/sub\u003e molecular weight than the precursor GP\u003csub\u003e0\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, lanes 1\u0026ndash;6; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, lanes 1\u0026ndash;4). To understand how glycans contribute to GP degradation, we tested the degradation of GP deletion mutant GP∆MLD that does not express MLD, along with sGP and ssGP. MAN1B1 decreased their expression, which was blocked by Kif, BafA1, and NH\u003csub\u003e4\u003c/sub\u003eCl, but not Lac and MG132 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Thus, MAN1B1 could still target these less-glycosylated GP to lysosomes for degradation. However, when EBOV-GP, GP∆MLD, sGP, and ssGP were expressed with MAN1B1 in \u003cem\u003eMembralin\u003c/em\u003e-KO cells, only the decrease of FL EBOV-GP was blocked, whereas the others were not (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, lanes 3, 4). Thus, dense glycans are required for Membralin-MAN1B1 to target EBOV-GP for degradation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMembralin has been implicated as a critical component of the ERAD pathway. It forms a complex with the E3 ubiquitin ligase RNF185 and the UBL domain-containing proteins TMUB1 and TMUB2 on the ER membrane. This complex works in conjunction with the cytosolic ubiquitin ligase UBE3C and p97/VCP to facilitate the degradation of membrane substrates \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, inconsistent findings have been reported regarding Membralin-mediated degradation of NCSTN, NHK, and CD3δ \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Our results also indicate that Membralin is not necessary for MAN1B1-mediated degradation of NHK, CD3δ, and NCSTN in proteasomes. Consequently, the role of Membralin in ERAD warrants further investigation.\u003c/p\u003e \u003cp\u003eWe find that Membralin is essential not only for MAN1B1-initiated ERLAD but also for directly initiating the degradation process. While both MAN1B1 and PDIA3 can trigger the degradation of EBOV-GP via ERLAD, the precise mechanisms appear to differ. For instance, MAN1B1 does not induce polyubiquitination of its substrate, whereas PDIA3 does, consistent with our previous findings that PDIA3\u0026rsquo;s activity is dependent on RNF185 \u003csup\u003e17\u003c/sup\u003e. Therefore, Membralin's activity should not rely on RNF185. ERLAD clients are thought to be segregated into ER subdomains rather than being retrotranslocated across the ER membrane for engulfment \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In line with this, we find that the SEC61 translocon is not required for the degradation of class I fusion proteins. However, Membralin recruits p97/VCP through its cytoplasmic loop, and p97/VCP is essential for its activity. While p97/VCP serves as a critical energy provider for ERAD, it also performs diverse cellular functions, including membrane remodeling \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, which we speculate facilitates the formation and detachment of ER subdomains. Like other known ER-phagy receptors, Membralin contains an LIR in its cytoplasmic tail, which should promote the degradation of class I fusion proteins through ER-phagy.\u003c/p\u003e \u003cp\u003eWe find that Membralin recruits MAN1B1 via its luminal loop to specifically target class I fusion proteins rather than known misfolded proteins or aggregates. During productive infection, viruses exploit host cells to produce abnormally high levels of class I fusion proteins, overwhelming the ER's folding machinery and inducing ER stress. Class I fusion proteins possess distinctive features that facilitate their recognition and clearance as foreign proteins. They are heavily glycosylated, primarily with \u003cem\u003eN\u003c/em\u003e-glycans and fewer \u003cem\u003eO\u003c/em\u003e-glycans, except for EBOV-GP, where \u003cem\u003eO\u003c/em\u003e-glycans predominate. For instance, HIV-1 Env (gp120) undergoes extensive glycosylation, with approximately half of its molecular weight attributed to glycans \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. While this dense glycan shield obscures key epitopes and aids immune evasion, it may also act as a non-host marker for being detected by MAN1B1. Our findings consistently demonstrate that significantly reduced glycosylation levels prevent EBOV-GP from being targeted by the Membralin-MAN1B1 axis. However, less glycosylated EBOV-GP can still undergo degradation via ERLAD through an alternative, unidentified mechanism. These results underscore glycosylation levels as a critical determinant for targeting by this protein complex.\u003c/p\u003e \u003cp\u003eWe confirm that, unlike NHK, the AAT polymeric variant ATZ is directed to ERLAD rather than ERAD, consistent with previous findings \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. It has been proposed that misfolded large polypeptide aggregates resist ERAD due to the challenges of translocating through the SEC61 translocon and entering the proteasomes. In alignment with this, we observe that misfolded variants of NPC1, COL2A1, and DYSF are targeted to ERLAD but not ERAD. Class I fusion proteins, with their large molecular weights (exceeding 70 kDa) and trimeric structures, likely follow the same pattern, as their size and aggregation render them suitable candidates for ERLAD rather than ERAD.\u003c/p\u003e \u003cp\u003eMAN1B1 is well-established in targeting the luminal substrate NHK to ERAD. Our findings further reveal that it also targets the membrane substrate CD3δ and another luminal substrate, NCSTN, through the same mechanism, demonstrating that MAN1B1 recognizes both ERAD luminal (ERAD-L) and ERAD membrane (ERAD-M) substrates. Moreover, MAN1B1 directs other misfolded human proteins, including AAT, NPC1, COL2A1, and DYSF variants (ATZ, I1061T, R989C/G1152D, and L1341P), to ERLAD. This degradation pathway operates independently of known ER-phagy receptors or Membralin, implying that MAN1B1 interacts with other unidentified ER-phagy receptor(s) for these host proteins. We also confirmed that CANX facilitates the degradation of the R989C/G1152D variant in a FAM134B-dependent manner \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. These findings suggest that the selection of an ER-phagy receptor is dictated by both the effector (MAN1B1, CANX) and the specific client proteins.\u003c/p\u003e \u003cp\u003eIn summary, we find that Membralin orchestrates a protein degradation machinery across the ER membrane by recruiting MAN1B1 and p97/VCP, selectively targeting foreign glycoproteins via ER-phagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). This mechanism represents a new arm of cell-autonomous immunity, playing a significant role in defending eukaryotic cells against viral infections.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eAntibodies and inhibitors.\u003c/b\u003e Commercial reagents include: kifunensine, eeyarestatin I, MG132, Lactacystin (Lac), Concanamycin A (ConA), 3-Methyladenine (3-MA), and ammonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl) (Sigma-Aldrich, K1140, E1286, C2211, L6785, C9705, M9281, A9434); Bafilomycin A1 (BafA1, Santa Cruz Biotechnology, sc-201550); mouse monoclonal anti-Myc (CST, 2276S); rabbit polyclonal anti-SQSTM1, mouse monoclonal anti-FLAG and anti-HA (Sigma-Aldrich, P0067, F3165, H3663); mouse monoclonal anti-ACTB/β-actin (CST, 3700S); mouse monoclonal anti-ERManI/MAN1B1 (Novus, NBP2-13167); rabbit polyclonal anti-FAM134B-2 and anti-Membralin (Gene Tex, GTX46621, GTX118618); rabbit polyclonal anti-ATL3, anti-FAM134B, anti-CCPG1, anti-TEX264, and anti-TOLLIP (Proteintech, 16921-1-AP, 21537-AP, 13861-1-AP, 25858-1-AP, 11315-1-AP); rabbit monoclonal anti-SEC62 and rabbit polyclonal anti-EDEM2 (Abcam, ab137022, ab181218); rabbit polyclonal anti-RTN3 (ThermoFisher Scientific, PA5-78316); mouse monoclonal anti-SERPINA1/AAT (Sigma-Aldrich, SAB4200198); rabbit polyclonal anti-Sec61A and Sec61B (Abcam, ab183046, ab15576); rabbit polyclonal anti-VCP (Huabio, ER30603); horseradish peroxidase (HRP)- conjugated goat anti-mouse IgG and anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 115-035-003, 111-035-003).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell lines.\u003c/b\u003e Human embryonic kidney (HEK) 293 cell line transformed with SV40 large T antigen (HEK293T) and human cervical carcinoma cell line HeLa were purchased from American Type Culture Collection (ATCC, CRL-3216, CRM-CCL-2); human hepatoma cell line Huh7 was purchased from BioBW, China (BioBW, bio-73061); TZM-bI cells were from NIH HIV Reagent Program. All these cells were maintained in Dulbecco\u0026rsquo;s modified Eagle medium (DMEM; Thermo Fisher Scientific, 11965092) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (pen-strep; Thermo Fisher Scientific, 10378016) and cultivated at 37\u0026deg;C in the humidified atmosphere in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCRISPR-Cas9 knockout.\u003c/b\u003e DNA oligos encoding small guide RNAs (sgRNAs) targeting \u003cem\u003eMAN1B1\u003c/em\u003e, \u003cem\u003eEDEM2\u003c/em\u003e, \u003cem\u003eMembralin\u003c/em\u003e (\u003cem\u003eTMEM259)\u003c/em\u003e, \u003cem\u003eFAM134B\u003c/em\u003e, \u003cem\u003eRTN3L\u003c/em\u003e, \u003cem\u003eATL3\u003c/em\u003e, \u003cem\u003eSEC62\u003c/em\u003e, \u003cem\u003eCCPG1, TEX264\u003c/em\u003e, and \u003cem\u003eSec61B\u003c/em\u003e are shown in supplemental Table\u0026nbsp;1, which were cloned into the pSpCas9(BB)-2A-GFP (PX458) vector via BbsI digestion. HEK293T cells were transfected with these vectors, and after 48 h, single clones of GFP-positive cells were isolated by fluorescence-activated cell sorting. Knockout clones were identified by western blotting (WB) and further validated by DNA sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003esiRNA knockdown.\u003c/b\u003e \u003cem\u003eSEC61A\u003c/em\u003e and \u003cem\u003eVCP\u003c/em\u003e were knocked down by siRNAs using RNA oligo pairs 5\u0026rsquo;-CACUGAAAUGUCUACGUUUTT-3\u0026rsquo;/5\u0026rsquo;-AAACGUAGACAUUUCAGUGTT-3\u0026rsquo;, or 5\u0026rsquo;-GCAUGUGGGUGCUGACUUATT-3\u0026rsquo;/5\u0026rsquo;-UAAGUCAGCACCCACAUGCTT-3\u0026rsquo;. HEK293T cells were transfected with these siRNAs, and after 24 h, their expression was determined by WB.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmids\u003c/b\u003e. The pcDNA3.1-hERManI-HA-FLAG, pCMV6-hEDEM1-Myc-FLAG, pcDNA3.1-hEDEM2-HA-FLAG, and pCMV6-hEDEM3-Myc-FLAG vectors and ERManI catalytically inactive mutant expression vector were reported previously \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. pcDNA3.1 vectors expressing ERManI/MAN1B1, EDEM1, EDEM2, and EDEM3 with a single C-terminal HA-tag were re-constructed in this study by homologous recombination after NheI/XhoI digestion. pNL-Luc-ΔEnv, pcDNA3.1-Flag-EBOV-GP, pcDNA3.1-FLAG-EBOV-GP∆MLD, pcDNA3.1-HiBiT-sGP, pcDNA3.1-HiBiT-ssGP, HIV-1 Env, and H5N1 HA expression vectors were reported previously \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. pCAGGS-PDIA3-Myc, pCMV-CALR-Myc, pCMV6-CANX-Myc-FLAG, HSPA5/BiP promoter-luciferase reporter vector pLightSwitch-BiP, XBP1-activation reporter vector pXBP1u-FLuc, ATF4-expression reporter vector pATF4-UTR-Fluc, and ATF6-activation reporter vector p5xATF6-GL3 were reported previously \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. pCMV-HA-Ub\u003csub\u003eWT\u003c/sub\u003e and pCMV-HA-Ub\u003csub\u003eKO\u003c/sub\u003e were reported previously \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. pSpCas9 (BB)-2A-GFP (PX458) was obtained from Feng Zhang through Addgene (48138). pCMV6-Membralin-Myc-Flag was ordered from Comatebio (CoME). Membralin deletion mutants (∆TM1, ∆TM2, ∆TM3, ∆TM4, ∆4TM, 70\u0026ndash;628, 1-322, 1-345, 1-466) were created by PCR and ASiSI/MluI digestion followed by homologous recombination. pCMV6-FAM134B-2-Myc-Flag was ordered from CoME. pCDNA3.1-FAM134B-Flag was ordered from GenScript. pCDNA3.1-NPC1-3xFlag were reported previously \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. pCDNA3.1-NHK was provided by Richard Sifers. pCMV6-SERPINA1-Myc-Flag, pCMV6-COL2A1-Myc-Flag, pCMV6-DYSF-Myc-Flag, pCMV6-CD3D-Myc-Flag, pCMV6-NCSTN-Myc-Flag, and pCMV6-VCP-Myc-FLAG were ordered from Origene (Cat# RC202082, RC218041, RC219485, RC210010, RC212646, RC211130). SERPINA1/AAT, NPC1, COL2A1, and DYSF mutants and pCMV6-VCP-His were created by PCR and ASiSI/MluI digestion followed by homologous recombination. All vectors constructed by us were confirmed by Sanger DNA sequencing. Detailed experimental procedures and primer sequences for the construction of these vectors are available upon request. Plasmids were prepared using Maxiprep kits (TIANGEN Biotech, DP117).\u003c/p\u003e \u003cp\u003eTo express S proteins from SARS2, SARS1 and MERS, their cDNAs with a N-terminal FLAG tag were commercially synthesized and cloned into pCAGGS, resulting in pCAGGS-FLAG-SARS2-S, pCAGGGS-FLAG-SARS1-S, and pCAGGS-FLAG-MERS-S (Comate Bioscience, China). A SARS2-S and SARS1-S expression vector with a N-terminal Flag tag and C-terminal 19 amino acids deletion, pCAGGS-FLAG-SRAS2-S-D19 and pCAGGS-FLAG-SARS1-S-D19, were re-created by \u003cem\u003eEcoRI\u003c/em\u003e/\u003cem\u003eXhoI\u003c/em\u003e digestion. A SARS1-S expression vector with a C-terminal FLAG, pCAGGS-SARS1-S-FLAG, was re-created by \u003cem\u003eEcoRI\u003c/em\u003e/\u003cem\u003eBspEI\u003c/em\u003e digestion. These two SARS2-S and SARS1-S with this deletion were used to produce HIV-1 pseudovirions for infectivity assay. To express S protein-EGFP fusion proteins, these cDNAs were cloned into pEGFP-HA-N1 vector that express a HA-tag in front of EGFP by \u003cem\u003eXhoI\u003c/em\u003e/\u003cem\u003eBspEI\u003c/em\u003e digestion, resulting in pEGFP-SARS2-S-HA, pEGFP-SARS1-S-HA, pEFGP-MERS-S-HA, which express SARS2-S-HA-EGFP, SARS1-S-HA-EGFP, and MERS-S-HA-EGFP fusion proteins. To express MAN1B1-mCherry fusion, MAN1B1 was subcloned from pcDNA3.1-hMAN1B1-HA into pCAGGS-mCherry by \u003cem\u003eEcoRI\u003c/em\u003e digestion. pEGFP-N1 vectors expressing CALR and TGLON2 were constructed by PCR and \u003cem\u003eXhoI\u003c/em\u003e/\u003cem\u003eBspEI\u003c/em\u003e digestion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransfection.\u003c/b\u003e HEK293T cells were cultured in 6-well plates and transfected with polyethyleneimine (PEI; Polysciences, 23966-2). HeLa cells were transfected using Lipofectamine 3000 according to the manufacturer\u0026rsquo;s protocol (Thermo Fisher Scientific, L3000015). The total indicated plasmids were diluted into 200\u0026micro;L serum-free Opti-MEM (Thermo Fisher Scientific, 31985062) and mixed with transfection reagents. After 20 min of incubation at room temperature, these transfection complexes were added directly into the supernatant of each well. Media were replaced after 6 h and cell lysate was collected at 48 h unless otherwise noted. Viruses released into the supernatants and proteins expressed in cells were analyzed (see below).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of UPR.\u003c/b\u003e HEK293T cells were cultured in 24-well plates and transfected with the indicated plasmid and reporter vectors as we reported previously \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. HSPA5/BiP activation was measured by expressing BiP-RLuc reporter construct and measuring the intracellular Renilla luciferase activities. XBP1, ATF4, and ATF6 activation were measured by expressing XBP1-Fluc, pATF4-UTR-Fluc or p5\u0026times;ATF6-GL3 reporter construct and measuring the intracellular Firefly luciferase activities. Luciferase activities were analyzed by Dual-Luciferase\u0026reg; Reporter Assay System (Promega, E1910).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of coronavirus spike protein-pseudotyped HIV-1 infectivity.\u003c/b\u003e SARS2-S, SARS1-S, and MERS-S pseudotyped HIV-1 virions were produced from HEK293T WT and \u003cem\u003eMAN1B1\u003c/em\u003e-KO cells as we reported previously \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. These cells were transfected with pNL-Luc-ΔEnv and a S protein expression vector in the presence or absence of a MAN1B1 expression. After 48 h, viruses were collected from the culture supernatants and clarified by low-speed centrifugation. After being normalized by p24\u003csup\u003eGag\u003c/sup\u003e ELISA \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, an equal amount of viruses were used to infect Huh7 cells. After 48 h of infection, cells were lysed, and viral infectivity was determined by measuring the intracellular luciferase activity using a firefly luciferase assay kit (US Everbright Inc, Cat# F6024).\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting (WB).\u003c/b\u003e Transfected cells were lysed in RIPA buffer (25 mM Tris, pH7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1%Nonidet P-40 [Sigma-Aldrich, R0278]) at 4\u0026deg;C. After centrifugation at 12,000 \u0026times; g for 10 min at 4\u0026deg;C, cytosolic fractions were collected and boiled with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Solarbio Life Sciences, P1015). Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. These membranes were blocked with 5% nonfat milk powder in TBST (Tris-buffered saline [20 mM Tris, pH 7.4,150 mM NaCl] containing 0.1% Tween 20; Solarbio Life Sciences, T8220) for 1 h at room temperature, and probed by primary antibodies followed by HRP-conjugated secondary antibodies. Chemiluminescence signals were then measured by incubating the membrane with SuperSignal substrate (Thermo Fisher Scientific, 34580).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoprecipitation.\u003c/b\u003e After transfection of HEK293T cells cultured in a 6-cm dish, cells were lysed in 0.8 mL RIPA lysis buffer for 30 min on ice as we did previously \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. After the removal of nuclei via low-speed centrifugation and collecting 100 \u0026micro;L as input, the remaining 700 \u0026micro;L lysate was incubated with anti-FLAG M2 Magnetic beads (Sigma-Aldrich, M8823) and rotated at 4\u0026deg;C overnight. After being washed 3 times with 1 mL pre-cooled RIPA lysis buffer, proteins were removed from beads after boiling in 40 \u0026micro;L RIPA lysis buffer plus 15 \u0026micro;L sample loading buffer (5\u0026times;) and analyzed by WB.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo polyubiquitination assay.\u003c/b\u003e HEK293T cells were seeded in 6-cm dishes and transfected with vectors expressing SARS2-S and MAN1B1, or EBOV-GP and PDIA3, in the presence of ubiquitin expression vector as we did previously \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. After 48 h, cells in each dish were lysed in 600 \u0026micro;L RIPA buffer at 4\u0026deg;C for 30 min. After the removal of nuclei by low-speed centrifugation, 100 \u0026micro;L was collected as input, and the remaining 500 \u0026micro;L was incubated with anti-FLAG beads (Sigma-Aldrich, M8823) at 4\u0026deg;C overnight. Beads were washed three times with phosphate-buffered saline (PBS), then boiled in SDS-PAGE loading buffer and analyzed by WB.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConfocal microscopy.\u003c/b\u003e HeLa cells were seeded on a glass bottom cell culture dish (NEST Biotechnology, 801001) and transfected with various vectors using Lipofectamine 3000, as we did previously \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. After 30 h, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 (Solarbio Life Sciences, T8200) for 10 min at room temperature, and then blocked with 5% bovine serum albumin (BSA; APPLYGEN, P1622) solution overnight at 4\u0026deg;C. Nuclei were stained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) for 3\u0026ndash;5 min and washed with PBS, and cells were observed and imaged under a confocal microscope (LSM880, Zeiss, White Plains, New York). Quantitative colocalization measurements were performed using ImageJ software. Pearson\u0026rsquo;s correlation coefficient (PCC) was calculated to describe the colocalization correlation of the intensity distributions between two channels.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLive-cell imaging.\u003c/b\u003e Approximately 1.0 \u0026acute; 10\u003csup\u003e5\u003c/sup\u003e HEK293T cells were seeded on a glass bottom cell culture dish (NEST Biotechnology, 801001) and transfected with various vectors using PEI. After 14 h, Live-cell imaging was performed using LSM800 (Zeiss, White Plains, New York). During live-cell imaging, the dish was mounted in a chamber to maintain the incubation conditions at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e All experiments were performed independently at least three times, with representative experiment being shown. GraphPad Prism (Graph Pad Software Inc., San Diego, CA, USA) was used for the data analysis. Data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of measurements (SEMs) and represented by error bars. The significance of differences between samples was assessed by an unpaired two-tailed Student\u0026rsquo;s t test. A \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was statistically significant (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 was not significant (ns).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eJ.Z., X. L., S. L., T. W., and I.A. conducted experiments; J.Z. and Y.H.Z. designed experiments; Y.H.Z. wrote the manuscript with input from all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eWe thank Feng Zhang for the reagent. J.Z. is supported by the National Natural Science Foundation of China (32300129), the Youth Innovation Program of the Chinese Academy of Agricultural Sciences (Y2025QC19), and the China Postdoctoral Science Foundation (2023T160700). S.L. is supported by the National Natural Science Foundation of China (32172836). Y.H.Z. is supported by the National Institutes of Health (AI175008).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMolinari, M. ER-phagy responses in yeast, plants, and mammalian cells and their crosstalk with UPR and ERAD. Dev Cell 56, 949\u0026ndash;966 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, B. \u003cem\u003eet al.\u003c/em\u003e ER-associated degradation regulates Alzheimer's amyloid pathology and memory function by modulating gamma-secretase activity. Nat Commun 8, 1472 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, B. \u003cem\u003eet al.\u003c/em\u003e The critical role of membralin in postnatal motor neuron survival and disease. Elife 4 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNinagawa, S., George, G. \u0026amp; Mori, K. Mechanisms of productive folding and endoplasmic reticulum-associated degradation of glycoproteins and non-glycoproteins. Biochim Biophys Acta Gen Subj 1865, 129812 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrabutt, D.A. \u0026amp; Zheng, Y.H. Arms Race between Enveloped Viruses and the Host ERAD Machinery. Viruses 8 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrabutt, D.A., Wang, B., Riaz, S., Schwartz, R.C. \u0026amp; Zheng, Y.H. Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein Degradation. J Virol 92, e01690-01617 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan, Y., Wang, X., Dang, Y. \u0026amp; Zheng, Y.H. Demonstration of a novel HIV-1 restriction phenotype from a human T cell line. PLoS One 3, e2796 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, B. \u003cem\u003eet al.\u003c/em\u003e Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway. Autophagy 18, 2350\u0026ndash;2367 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, T., Dang, Y. \u0026amp; Zheng, Y.H. The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway. J Virol 88, 3474\u0026ndash;3484 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, T., Frabutt, D.A., Moremen, K.W. \u0026amp; Zheng, Y.H. ERManI (Endoplasmic Reticulum Class I alpha-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway. J Biol Chem 290, 22184\u0026ndash;22192 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, T., Han, Y., Dang, Y., Wang, X. \u0026amp; Zheng, Y.H. A novel HIV-1 restriction factor that is biologically distinct from APOBEC3 cytidine deaminases in a human T cell line CEM.NKR. Retrovirology 6, 31 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBartolini, D. \u003cem\u003eet al.\u003c/em\u003e Endoplasmic reticulum stress and NF-kB activation in SARS-CoV-2 infected cells and their response to antiviral therapy. IUBMB Life 74, 93\u0026ndash;100 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaudhry, Z.L., Gamal, M., Ferhati, I., Warda, M. \u0026amp; Ahmed, B.Y. ER Stress in COVID-19 and Parkinson's Disease: In Vitro and In Silico Evidences. Brain Sci 12 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosa-Fernandes, L. \u003cem\u003eet al.\u003c/em\u003e SARS-CoV-2 activates ER stress and Unfolded protein response. \u003cem\u003ebioRxiv\u003c/em\u003e, 2021.2006.2021.449284 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan, C.P. \u003cem\u003eet al.\u003c/em\u003e Modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein. J Virol 80, 9279\u0026ndash;9287 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, Y., Swulius, M.T., Moremen, K.W. \u0026amp; Sifers, R.N. Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation. Proc Natl Acad Sci U S A 100, 8229\u0026ndash;8234 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, J. \u003cem\u003eet al.\u003c/em\u003e RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy. Nat Commun 13, 6007 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreenfield, J.J. \u0026amp; High, S. The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment. J Cell Sci 112 (Pt 10), 1477\u0026ndash;1486 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreene, C.M. \u003cem\u003eet al.\u003c/em\u003e alpha1-Antitrypsin deficiency. Nat Rev Dis Primers 2, 16051 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurray, L.W., Bautista, J., James, P.L. \u0026amp; Rimoin, D.L. Type II collagen defects in the chondrodysplasias. I. Spondyloepiphyseal dysplasias. Am J Hum Genet 45, 5\u0026ndash;15 (1989).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuatibonza Moreno, P. \u003cem\u003eet al.\u003c/em\u003e At a glance: the largest Niemann-Pick type C1 cohort with 602 patients diagnosed over 15 years. Eur J Hum Genet 31, 1108\u0026ndash;1116 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBashir, R. \u003cem\u003eet al.\u003c/em\u003e A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B. Nat Genet 20, 37\u0026ndash;42 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, J. \u003cem\u003eet al.\u003c/em\u003e Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 20, 31\u0026ndash;36 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHosokawa, N. \u003cem\u003eet al.\u003c/em\u003e Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I. J Biol Chem 278, 26287\u0026ndash;26294 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeckman, J.H. \u0026amp; Perlmutter, D.H. Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response. Am J Physiol Gastrointest Liver Physiol 279, G961-974 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGelsthorpe, M.E. \u003cem\u003eet al.\u003c/em\u003e Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding. J Biol Chem 283, 8229\u0026ndash;8236 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchultz, M.L. \u003cem\u003eet al.\u003c/em\u003e Coordinate regulation of mutant NPC1 degradation by selective ER autophagy and MARCH6-dependent ERAD. Nat Commun 9, 3671 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForrester, A. \u003cem\u003eet al.\u003c/em\u003e A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex. EMBO J 38 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujita, E. \u003cem\u003eet al.\u003c/em\u003e Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Hum Mol Genet 16, 618\u0026ndash;629 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang, S. \u003cem\u003eet al.\u003c/em\u003e The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum. Proc Natl Acad Sci U S A 98, 14422\u0026ndash;14427 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, B. \u003cem\u003eet al.\u003c/em\u003e Mechanistic understanding of N-glycosylation in Ebola virus glycoprotein maturation and function. J Biol Chem 292, 5860\u0026ndash;5870 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan de Weijer, M.L. \u003cem\u003eet al.\u003c/em\u003e Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex. Mol Cell 79, 768\u0026ndash;781 e767 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRudinskiy, M. \u0026amp; Molinari, M. ER-to-lysosome-associated degradation in a nutshell: mammalian, yeast, and plant ER-phagy as induced by misfolded proteins. FEBS Lett 597, 1928\u0026ndash;1945 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBraxton, J.R. \u0026amp; Southworth, D.R. Structural insights of the p97/VCP AAA\u0026thinsp;+\u0026thinsp;ATPase: How adapter interactions coordinate diverse cellular functionality. J Biol Chem 299, 105182 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeonard, C.K. \u003cem\u003eet al.\u003c/em\u003e Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J Biol Chem 265, 10373\u0026ndash;10382 (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFregno, I. \u003cem\u003eet al.\u003c/em\u003e ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport. EMBO J 37 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu, W. \u003cem\u003eet al.\u003c/em\u003e Furin Egress from the TGN is Regulated by Membrane-Associated RING-CH Finger (MARCHF) Proteins and Ubiquitin-Specific Protease 32 (USP32) via Nondegradable K33-Polyubiquitination. Adv Sci (Weinh) 11, e2403732 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan, I. \u003cem\u003eet al.\u003c/em\u003e Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1. Nat Commun 15, 162 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDang, Y. \u003cem\u003eet al.\u003c/em\u003e Identification of a single amino acid required for APOBEC3 antiretroviral cytidine deaminase activity. J Virol 85, 5691\u0026ndash;5695 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad, I. \u003cem\u003eet al.\u003c/em\u003e The retroviral accessory proteins S2, Nef, and glycoMA use similar mechanisms for antagonizing the host restriction factor SERINC5. J Biol Chem 294, 7013\u0026ndash;7024 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan, Y., Wang, X., Dang, Y. \u0026amp; Zheng, Y.H. APOBEC3G and APOBEC3F require an endogenous cofactor to block HIV-1 replication. PLoS Pathog 4, e1000095 (2008).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Membralin, ERLAD, reticulophagy, class I fusion proteins, MAN1B1, TMEM259","lastPublishedDoi":"10.21203/rs.3.rs-6498082/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6498082/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe endoplasmic reticulum (ER) plays a central role in protein synthesis and folding. Membralin is a multi-pass membrane protein involved in ER-associated degradation (ERAD). Here, we demonstrate that Membralin assembles a protein degradation machinery across the ER membrane, specifically targeting class I fusion proteins expressed by major human viruses. Membralin interacts with MAN1B1 and p97/VCP through its luminal and cytoplasmic loops, respectively. Importantly, Membralin also contains an LC3-interacting region (LIR) in its cytoplasmic tail. The expression of these viral glycoproteins induces ER stress, prompting MAN1B1 to trim mannose residues extensively. Subsequently, Membralin recruits p97/VCP and initiate ER-phagy via its LIR, leading to degradation. This pathway specifically recognizes dense \u003cem\u003eN\u003c/em\u003e-glycans and is selective, as it does not degrade misfolded domestic proteins. Collectively, our study reveals a cell-autonomous immunity inside the ER orchestrated by Membralin, underscoring its important role in the clearance of foreign glycoproteins to maintain cellular homeostasis.\u003c/p\u003e","manuscriptTitle":"Membralin Selects Foreign Glycoproteins from the Endoplasmic Reticulum to Lysosomes for Degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 12:27:22","doi":"10.21203/rs.3.rs-6498082/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7e703dab-7659-4efd-9b0b-dd91f241002f","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":50026057,"name":"Biological sciences/Cell biology/Organelles/Endoplasmic reticulum"},{"id":50026058,"name":"Biological sciences/Microbiology/Virology/SARS-CoV-2"}],"tags":[],"updatedAt":"2025-06-23T12:27:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-23 12:27:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6498082","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6498082","identity":"rs-6498082","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}