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

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.

Keywords: ERLAD; MAN1B1; Membralin; TMEM259; class I fusion proteins; reticulophagy.

Figure 1

Error bars in (B) and (H) represent the standard error of measurements (SEMs) calculated from three experiments (n=3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns (not significant, p>0.05). Human coronavirus spike proteins are MAN1B1 substrates. (A) SARS1-S, SARS2-S, and MERS-S proteins with a FLAG-tag were expressed in HEK293T cells and detected by WB. The unprocessed S₀ precursor and processed S₁ subunit are indicated. b-actin (ACTB) was used as a loading control. (B) Coronavirus spike and NHK proteins were expressed with ER-stress luciferase-reporter vector pLightSwitch-BiP, p5ÁTF6-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. (C) 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. (D) Coronavirus spike proteins were expressed in HEK293T wild-type (WT) and MAN1B1-knockout (KO) cells, and their expression was compared by WB. (E) Coronavirus spike proteins were expressed in HEK293T WT and EDEM2-KO cells, and their expression was compared by WB. (F) Coronavirus spike proteins were expressed with MAN1B1 and EDEM2 in HEK293T MAN1B1-KO or EDEM2-KO cells, and their expression was compared by WB. (G) S proteins were expressed with MAN1B1 and its catalytic site-de cient mutants (E330A, D463A, E599A) in MAN1B1-KO cells, and their expression was analyzed by WB. (H) HIV-1 firefly luciferase reporter pseudovirions (PVs) expressing coronavirus spike proteins were produced in the presence of ectopic MAN1B1 from HEK293T WT or MAN1B1-KO cells. After infecting Huh7 cells with an equal number of PVs, viral infection was determined by measuring intracellular luciferase activities. Results are presented as relative values, with the activities in the presence of a control vector set as 100.

Figure 2. MAN1B1 targets coronavirus spike proteins to lysosomes.

(A) 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. (B) 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). (C) 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 NH₄Cl, 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). (D) PCC values from (C) are calculated and shown. Error bars represent SEMs calculated from three experiments (n=3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns (not significant, p>0.05).

Figure 3. MAN1B1 targets class I fusion proteins for lysosomal degradation independently of polyubiquitination.

(A) 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 NH₄Cl, 20 nM ConA, or 10 mM 3-MA. Protein expression was determined by WB. (B) EBOV-GP was expressed in HEK293T WT and MAN1B1-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. (C) SARS2-S and EBOV-GP were expressed with MAN1B1 in HEK293T cells in the presence of increasing amounts of WT ubiquitin (Ub_WT) or its mutant that does not express any lysine residues (Ub_KO). Protein expression was determined by WB. (D) SARS2-S and EBOV-GP with a FLAG-tag were expressed with Ub_WT and MAN1B1 or PDIA3 in HEK293T cells. Proteins were immunoprecipitated (IP) with anti-FLAG and analyzed by WB.

Figure 4. Membralin is required for class I fusion protein degradation.

(A) Membralin was knocked out in HEK293T cells by CRISPR/Cas9, which is confirmed by WB. (B) Class I fusion proteins were expressed with MAN1B1 in HEK293T WT and Membralin-KO cells, and their expression was determined by WB. (C) EBOV-GP was expressed with indicated ER chaperones in HEK293T WT Membralin-KO cells, and their expression was determined by WB. (D) Class 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 NH₄Cl. Protein expression was determined by WB. (E) Class I fusion proteins were expressed with Membralin in HEK293T WT and MAN1B1-KO cells, and their expression was determined by WB. (F) PVs expressing HIV-1-Env, EBOV-GP, or SARS2-S were produced from HEK293T WT, MAN1B1-KO, and Membralin-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. 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. (G) SARS2-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.

Figure 5. Membralin relies on different regions for ER-localization and functionality.

(A) 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 ⁵⁴⁹TDASFL⁵⁵⁴ motif highlighted in red. (B) The indicated Membralin mutants were expressed with class I fusion proteins in HEK293T cells, and their expression was determined by WB. (C) 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). (D) The indicated Membralin mutants were expressed with EBOV-GP and H5N1-HA in HEK293T cells, and their expression was determined by WB. (E) Membralin and 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. (F) 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.

Figure 6. Membralin recruits VCP and MAN1B1 via different regions.

(A) Class I fusion proteins were expressed alongside MAN1B1 or Membralin in HEK293T cells in the presence of siRNAs targeting VCP or a control (Ctrl). Protein expression levels were assessed by WB. (B) VCP-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. (C) 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.

Figure 7. The Membralin-MAN1B1 axis does not target well-defined human misfolded or aggregated proteins.

(A) 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. (B) The indicated misfolded and aggregated proteins were expressed with MAN1B1 in HEK293T cells. Cells were treated with 50 mM Kif, 10 mM EerI, 20 mM Lac, 20 mM MG132, 100 nM BafA1, or 20 mM NH₄Cl. Protein expression was determined by WB. (C) The indicated misfolded and aggregated proteins were expressed with MAN1B1 in HEK293T WT, Membralin-KO, and FAM134B-KO cells. Protein expression was determined by WB.

Figure 8. Multiple factors determine the specific degradation.

(A) 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. (B) The R989C/G1152D variant was expressed with CANX, FAM134B-2, or MAN1B1 in HEK293T wild-type (WT), FAM134B-KO, or CANX-KO cells. Protein expression was again evaluated by WB. (C) A 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 GP₁ and _GP2 by furin and linked by a disulfide bond. The letter “Y” indicates N-glycosylation sites, while indicates O-glycosylation sites. MLD refers to the mucin-like domain, and TM indicates the transmembrane domain. (D) The indicated EBOV glycoproteins were expressed alongside MAN1B1 in HEK293T cells and treated with the specified inhibitors. Protein expression levels were determined by WB. Indicated EBOV glycoproteins were expressed with MAN1B1 in HEK293T cells and treated with indicated inhibitors. Protein expression was determined by WB. (E) The indicated EBOV glycoproteins were expressed with MAN1B1 in HEK293T WT and Membralin-KO cells, and protein expression was assessed by WB. (F) 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.