Journal: bioRxiv

Article Title: A selectivity filter in the EMC limits protein mislocalization to the ER

doi: 10.1101/2022.11.29.518402

Figure Lengend Snippet: ( A ) Displayed is an improved model of the human EMC determined using cryoelectron microscopy (cryo-EM), of the insertase core, composed of EMC3/6, enclosed by the three TMDs of EMC4, and the single TMDs of EMC7 and 10. ( B ) (Top) Schematic of the topology and domain organization of EMC3, highlighting three flexible cytosolic loops (L1-3) located beneath the hydrophilic vestibule of the EMC. (Bottom) Purified wildtype (WT) or EMC3 Cys mutant EMC were incubated with purified CaM-SQS(L401C) complexes for disulfide crosslinking and analysis as in . ( C ) (Top) Schematic of the topology and domain organization of EMC7. ss = signal sequence; Link = linker; H1 = helix 1; H2 = helix 2. (Bottom) HEK293 EMC7 knockout (KO) cells were transduced with lentivirus to express WT EMC7, or the indicated mutants of EMC7 helix 2 (H2). The effects of each mutant on biogenesis of SQS was determined using the ratiometric fluorescent reporter assay, normalized to WT and plotted as a bar chart. ( D ) View of the hydrophilic vestibule with EMC7 and 10 omitted for clarity. Residues indicated with spheres are colored according to the effects of individual alanine mutations at these positions in EMC3 and 4 on expression of SQS in HEK293T cells. The effect of each mutant was determined by flow cytometry using the ratiometric fluorescent reporter assay in (C), normalized to wildtype, and is displayed according to the indicated legend.

Article Snippet: The following antibodies were used in this study: EMC2 (25443-1-AP, Proteintech, USA); EMC3 (67205-1-Ig, Proteintech, USA); EMC4 (27708-1-AP, Proteintech, USA); EMC5 (A305-833, Bethyl Laboratories, USA); EMC7 (27550-1-AP, Proteintech, USA); EMC10 (ab180148, Abcam, UK); CAML (Synaptic Systems, Germany); Anti-HA-HRP (Millipore-Sigma, USA); Anti-FLAG-HRP (Millipore-Sigma, USA).

Techniques: Cryo-Electron Microscopy, Cryo-EM Sample Prep, Purification, Mutagenesis, Incubation, Sequencing, Knock-Out, Transduction, Reporter Assay, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: A selectivity filter in the EMC limits protein mislocalization to the ER

doi: 10.1101/2022.11.29.518402

Figure Lengend Snippet: ( A ) Updated model of the EMC, with views of the hydrophilic vestibule (left) and hydrophobic crevice side (right). ( B ) Low-pass filtered maps (5.5 Å) generated using volume tools in cryoSPARC V4.0. (Left) 9-subunit EMC complex map colored by the EMC subunits with the atomic model displayed as a superimposed cartoon. The EM density for the detergent micelle is displayed in gray. (Right) 8-subunit EMC complex (ΔEMC10) map. Due to the inherently flexible nature of EMC10’s TMD we could not unambiguously model its TMD, however, comparing +/Δ EMC10 maps gave insights into localization of its TMD because the ΔEMC10 map lacks additional density (colored in brown) enclosing the hydrophilic vestibule of the EMC. ( C ) Updated schematic of the topology of all nine EMC subunits. EMC8 and 9 are mutually exclusive paralogs. ( D ) EMC7 and EMC10 span the membrane. 35 S-methionine labeled EMC7 (top) or EMC10 (bottom) carrying an N-terminal signal sequence (ss) and 1xHA tag, as well as a C-terminal 3xFLAG tag were in vitro translated in rabbit reticulocyte lysate supplemented with canine pancreatic rough microsomes (cRMs). Nascent chains were released from the ribosome with puromycin, and non-incorporated as well as cytosolically accessible proteins were digested with proteinase K (PK) in the presence or absence of Triton-X-100 to solubilize the cRM membrane. The resulting protease protected fragments were subjected to denaturing anti-HA and anti-FLAG immunoprecipitations (IP). Note that only the N-terminal HA tags of EMC7 and EMC10 were protected (PF = protected fragment) from PK digestion, whereas the C-terminal 3xFLAG was PK-accessible, indicating a type I, single-spanning topology for both subunits. ( E ) EMC4 and EMC7, but not EMC10 are required for SQS biogenesis in human cells. WT or EMC4/7/10 KO HEK293 cells were transduced with lentivirus to express RFP-SQS or -VAMP2. The relative level of the RFP-fused TA to an internal GFP expression control was measured via flow cytometry and plotted as a histogram. ( F ) Purification of EMC complexes from HEK293 cells stably expressing GFP-EMC2 (WT), with or without additional knockout of EMC4, 7 or 10. Samples of total lysate and elution following an IP via GFP-EMC2 were analyzed by SDS-PAGE and western blotting with the indicated antibodies.

Article Snippet: The following antibodies were used in this study: EMC2 (25443-1-AP, Proteintech, USA); EMC3 (67205-1-Ig, Proteintech, USA); EMC4 (27708-1-AP, Proteintech, USA); EMC5 (A305-833, Bethyl Laboratories, USA); EMC7 (27550-1-AP, Proteintech, USA); EMC10 (ab180148, Abcam, UK); CAML (Synaptic Systems, Germany); Anti-HA-HRP (Millipore-Sigma, USA); Anti-FLAG-HRP (Millipore-Sigma, USA).

Techniques: Generated, Membrane, Labeling, Sequencing, In Vitro, Transduction, Expressing, Control, Flow Cytometry, Purification, Stable Transfection, Knock-Out, SDS Page, Western Blot

Journal: bioRxiv

Article Title: A selectivity filter in the EMC limits protein mislocalization to the ER

doi: 10.1101/2022.11.29.518402

Figure Lengend Snippet: ( A ) Alignment of EMC7 C-terminus sequences from various eukaryotes using Clustal Omega . Two conserved sequence stretches are predicted by secondary structure algorithms to form α-helices, termed H1 and H2. Residues mutated in (C) are highlighted in blue. AlphaFold 2 models of EMC7’s H1 and H2 are shown below. H1 is methionine-rich and H2 is predicted to form an amphipathic α-helix. ( B ) As in , but with the indicated mutants of H1 or the lumenal linker (link) between the EMC7’s β-sandwich and TMD. M H1 refers to all four methionines in helix 1. ( C ) Wild type (WT) or EMC7 KO HEK293 cells were transduced with lentivirus to express either BFP alone or BFP plus EMC7(WT) or the indicated mutants. 48 h after rescue construct transduction, cells were transduced with lentivirus expressing either RFP-SQS or -VAMP2, as well as a cytosolic GFP normalization control. The RFP:GFP ratio was determined by flow cytometry and is plotted as a histogram. Note that deletion of H2 strongly impaired SQS insertion in cells. Mutation of hydrophobic residues F213, M214 and F218 on H2 to either alanine or glutamate, but not leucine, similarly impaired SQS, but not VAMP2 biogenesis. ( D ) A BFP control, wild type EMC (WT), or the indicated mutants of EMC7 were introduced into EMC7 KO HEK293 cells via lentiviral transduction. Cells were harvested, solubilized and subjected to anti-ALFA immunoprecipitation. Eluates were analyzed by SDS-PAGE and western blotting with antibodies against EMC2 and 7. ( E ) Purified EMC complexes containing either WT EMC7 or EMC7 with cysteines in H1 (R191C) or H2 (M214C) were incubated with purified CaM-SQS complexes with or without a TMD. The cysteine was placed either in the TMD (L401C) or the soluble linker (F58C), for the WT and ΔTMD SQS constructs, respectively. Disulfide crosslinking was carried out as in . ( F ) Disulfide crosslinking of SQS(L401C) with purified EMC complexes as above, containing cysteines either in H2 of EMC7 (M214S), loop 2 of EMC3 (T102C) or within the membrane (EMC3 N117C).

Article Snippet: The following antibodies were used in this study: EMC2 (25443-1-AP, Proteintech, USA); EMC3 (67205-1-Ig, Proteintech, USA); EMC4 (27708-1-AP, Proteintech, USA); EMC5 (A305-833, Bethyl Laboratories, USA); EMC7 (27550-1-AP, Proteintech, USA); EMC10 (ab180148, Abcam, UK); CAML (Synaptic Systems, Germany); Anti-HA-HRP (Millipore-Sigma, USA); Anti-FLAG-HRP (Millipore-Sigma, USA).

Techniques: Sequencing, Transduction, Construct, Expressing, Control, Flow Cytometry, Mutagenesis, Immunoprecipitation, SDS Page, Western Blot, Purification, Incubation, Membrane

Journal: bioRxiv

Article Title: A selectivity filter in the EMC limits protein mislocalization to the ER

doi: 10.1101/2022.11.29.518402

Figure Lengend Snippet: ( A ) View of the insertase-competent side of the EMC. EMC7 and 10 were omitted for clarity. Residues of EMC4 mutated in (B) are highlighted. R31 and R180 are shown as blue sticks for reference. ( B ) HEK293 cells stably expressing RFP-SQS and cytosolic GFP as a normalization control were transduced with the indicated mutants of EMC4, along with BFP as a transduction marker. The RFP:GFP ratio of BFP-positive cells for each mutant was derived via flow cytometry and is plotted as a histogram. ( C ) The indicated EMC4 mutants from and (B) were introduced into HEK293 cells via lentiviral transduction. Cells were harvested, solubilized and subjected to anti-ALFA immunoprecipitation (IP). Eluates were analyzed by SDS-PAGE and Western blotting with antibodies against EMC2 and 4. ( D ) The N-terminus of EMC4 is required for TA biogenesis in cells. HEK293 WT or EMC4 KO cells were transduced with lentivirus to express either BFP alone or BFP plus EMC4(WT) or a ΔNT mutant (residues 57-end). 48 h after rescue construct transduction, cells were transduced with lentivirus expressing RFP-SQS, as well as a cytosolic GFP normalization control. The RFP:GFP ratio of BFP-positive cells was derived via flow cytometry and is plotted as a histogram. ( E ) A portion of the cells from (D) was harvested, solubilized and subjected to purification of EMC4 variants via their N-terminal ALFA tag using the ALFA nanobody. The eluate was analyzed by SDS-PAGE and Western blotting with HRP-coupled ALFA nanobody or the indicated antibodies. ( F ) HEK293 cells stably expressing RFP-SQS or -VAMP2 and cytosolic GFP as a normalization control were transduced with lentivirus to express the indicated mutants of EMC3, as well as BFP. The RFP:GFP ratio of BFP-positive cells for each mutant was derived via flow cytometry and is plotted as a histogram. ( G ) A portion of the cells from (F) was harvested, solubilized and subjected to purification of EMC3 variants via their C-terminal 3xFLAG tag. Incorporation of the single mutants was described before .

Article Snippet: The following antibodies were used in this study: EMC2 (25443-1-AP, Proteintech, USA); EMC3 (67205-1-Ig, Proteintech, USA); EMC4 (27708-1-AP, Proteintech, USA); EMC5 (A305-833, Bethyl Laboratories, USA); EMC7 (27550-1-AP, Proteintech, USA); EMC10 (ab180148, Abcam, UK); CAML (Synaptic Systems, Germany); Anti-HA-HRP (Millipore-Sigma, USA); Anti-FLAG-HRP (Millipore-Sigma, USA).

Techniques: Stable Transfection, Expressing, Control, Transduction, Marker, Mutagenesis, Derivative Assay, Flow Cytometry, Immunoprecipitation, SDS Page, Western Blot, Construct, Purification

Journal: bioRxiv

Article Title: Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins

doi: 10.1101/2023.11.28.569054

Figure Lengend Snippet: (A) Topology of the nine-subunit human EMC. In mammals, the EMC is composed of seven membrane-spanning and two soluble subunits. The central insertase is composed of the two central subunits EMC3 (a homolog of YidC and member of the Oxa1 superfamily of insertases) and EMC6 ( ; ; ). In both yeast and humans, the EMC also contains a globular cytosolic domain (scaffolded around EMC2 and in mammals containing the redundant paralogs EMC8 or 9) and a large lumenal domain (composed of EMC1, 7, 10, and a single β-strand of EMC4). The function of the lumenal domain of the EMC is not known, but β -propellers, like those conserved in EMC1, are typically considered protein-protein interaction motifs. (B) Topology of the heterotrimeric BOS complex. NOMO contains 12 lumenal IgG repeats and a single C-terminal TMD. The single-spanning protein NCLN also has a large lumenal domain, and is homologous to nicastrin, a component of the γ-secretase complex . Finally, TMEM147 contains 7 TMDs and is homologous to APH-1 in the γ-secretase complex . (C) Flow cytometry analysis of the ratiometric ADRA1A protein reporter with or without the N-terminal fusion of the signal sequence (ss) of Pre-Prolactin followed by T4 Lysozyme (T4L), as described in . (D) Flow cytometry analysis of the ratiometric AGTR2 protein reporter with or without an N-terminal fusion of the first TMD of MAN1A1, a membrane protein of N cyt topology. Note that both the N-terminal fusion of MAN1A1 and of (ss)-T4 lysozyme behave similarly and rescue the NOMO kd and EMC5 kd phenotypes.

Article Snippet: The following antibodies were used in this study: EMC2 (25443-1-AP, Proteintech, USA); EMC3 (67205-1-Ig, Proteintech, USA); EMC4 (27708-1-AP, Proteintech, USA); EMC5 (A305-833, Bethyl Laboratories, USA); EMC7 (27550-1-AP, Proteintech, USA); EMC10 (ab180148, Abcam, UK); TMCO1 (27757-1-AP, Proteintech, USA); TMEM147 (PA5-95876, Invitrogen, USA); NCLN (A305-623A-M, Bethyl, USA); NOMO1 (PA5-47534, Invitrogen, USA); NOMO2 (PA5-95819, Invitrogen, USA); CCDC47 (A305-100A-M, Bethyl, USA); GET2 (Synaptic Systems, Germany); SYVN1 (13473-1-AP, Proteintech, USA); FAF2 (16251-1-AP, Proteintech, USA); UGGT1 (sc-374565, Santa Cruz Biotechnology, USA); Asterix (HPA06568S, Sigma Aldrich); Calnexin (10427-2-AP, Proteintech); BIP (#610979, BD Biosciences); PDI (ADI-SPA-891-D, Enzo, USA); alpha tubulin (T9026, Millipore Sigma, USA); Anti-HA-HRP (Millipore-Sigma, USA); Anti-FLAG-HRP (Millipore-Sigma, USA).

Techniques: Membrane, Flow Cytometry, Sequencing

Journal: Journal of cell science

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis.

doi: 10.1242/jcs.223453

Figure Lengend Snippet: Fig. 1. EMC5 and EMC6 are essential for EMC maturation. (A) Schematic representation of the primary structure of all EMC subunits (EMC1–EMC10). Domains, boundary residue numbers and predicted glycosylation sites are indicated. Pyrrolo-quinoline quinone (PQQ) and tetratricopeptide repeats (TPR) are shown. (B) siRNA-mediated depletion of EMC1–EMC10 and non-targeting control (NTC) for 72 h in U2OS Flp-In™T-Rex™cells. Whole-cell lysates (WCL) of individually depleted cells were separated by SDS-PAGE and resulting western blots probed for each subunit and tubulin (TUB) as indicated. The asterisk (*) denotes a nonspecific band. (C) U2OS Flp-In™T-Rex™cells modified by CRISPR/Cas9 to knockout EMC6 (Δ6) were reconstituted by inducing expression of an empty vector control (EV), EMC5 or EMC6 (DOX 1 ng/ml, 72 h). MG132 was added to cells where indicated (5 µg/ml, 8 h). Samples were prepared as in B. TUB, tubulin; Ub, ubiquitin.

Article Snippet: The following antibodies were used in this study: ACC1 (Cell Signaling, Danvers, MA, rabbit pAb, #4190), 1:1000; AlexaFluor R488 anti-mouse IgG (H+L) (Invitrogen, donkey pAb, #A21202), 1:4000 [immunoblotting (IB)], 1:400 [immunofluorescence (IF)]; AlexaFluor R488 anti-rabbit IgG (H +L) (Invitrogen, goat pAb, #A11008), 1:4000 (IB), 1:400 (IF); anti-mouse IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2005), 1:10,000; anti-rabbit IgG-HRP (Santa Cruz Biotechnology, goat, #sc-2030), 1:10,000; EMC1 (rabbit pAb, kind gift of Enilza Espreafico, Sao Paulo, Brazil), 1:1000; EMC2 (Proteintech, Rosemont, IL, rabbit pAb, #25443-1-AP), 1:2000; EMC3 (Abcam, Cambridge, UK, rabbit pAb, #ab175537), 1:500; EMC4 (Abcam, rabbit pAb, #ab123719), 1:1000; EMC5/MMGT1 (Abcam, rabbit pAb, #ab174366), 1:1000; EMC6 (Abcam, rabbit pAb, #ab84902), 1:1000; EMC7 (Abgent, San Diego, CA, rabbit pAb, #AP11145c), 1:500; EMC8 (Abcam, rabbit pAb, #ab180065), 1:500; EMC9 (Abgent, rabbit pAb, #AP5632b), 1:500; EMC10 (Abcam, rabbit pAb, #ab180148), 1:1000; HA (purified from hybridoma, mouse mAb, clone 12CA5), 1:1000; histone H3 (Abcam, rabbit pAb, ab1791), 1:5000; HMGCR (Atlas Antibodies, Bromma, Sweden, mouse mAb, clone CL0260), 1:1000; Hsp70 (Santa Cruz Biotechnology, goat pAb, clone K-20, #sc-1060), 1:1000; LDLR (R&D Systems, Minneapolis, MN, goat pAb, #AF2148), 1:1000; Rap1a (Santa Cruz Biotechnology, rabbit pAb, clone C-17, #sc-1482), 1:1000; SCD1 (Abcam, mouse mAb, clone CD.E10, #ab19862), 1:1000; SM (Proteintech, rabbit pAb, #12544-1-AP), 1:1000; SOAT1 (Santa Cruz Biotechnology, mouse mAb, clone A-11, #sc-136959), 1:1000; SQS (Abcam, rabbit mAb, #ab109723), 1:2000; SQS (Abcam, rabbit mAb, #ab195046), 1:200 (IF), 1:2000 (WB); SREBP2 (R&D Systems, goat pAb, #AF7119), 1:1000; tubulin (Sigma-Aldrich, mouse mAb, #T5168/b-5-1-2), 1:1000; and ubiquitin (Cell Signaling Technologies, rabbit pAb, #3933S), 1:1000.

Techniques: Residue, Glycoproteomics, Control, SDS Page, Western Blot, Modification, CRISPR, Knock-Out, Expressing, Plasmid Preparation, Ubiquitin Proteomics