emc4  (Proteintech)

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: Nature Structural & Molecular Biology

Article Title: EMC rectifies the topology of multipass membrane proteins

doi: 10.1038/s41594-023-01120-6

Figure Lengend Snippet: a , Stable cell lines expressing the indicated inducible constructs were treated with non-targeting or EMC4-targeting siRNAs for 3 days, induced for 6 h and analyzed by flow cytometry for surface GABA A receptor levels using phycoerythrin (PE) labeled antibody (left), total levels of RFP-SQS (middle) or total levels of RFP-ASGR1 (right). SQS and ASGR1 levels are normalized to GFP, an internal expression control that is separated from the reporter by a ribosome-skipping viral P2A sequence. KD indicates knockdown. b , Total levels of GABRA1 and EMC4 were analyzed by blotting in cells treated with control or EMC4 siRNA. β-actin was used as a loading control. c , Topology diagram of human GABRA1; the predicted fragments resulting from proteinase K (PK) digestion are shown on top. Tail and loop lengths are indicated. NPF, N-terminal protected fragment; CPF, C-terminal protected fragment. The bottom panel shows the topology analysis of GABRA1 in the endoplasmic reticulum of wild-type (WT) and EMC6-knockout (ΔE) 293 cells. 35 S-methionine-labeled GABRA1 was translated in rabbit reticulocyte lysate in the absence (Ø) or presence of SPCs derived from WT or ΔE 293 cells. After translation, the SPCs were recovered by centrifugation and analyzed directly (−PK) by SDS–PAGE and autoradiography or subjected to PK digestion (+PK). Reactions lacking SPCs were analyzed similarly without centrifugation. Aliquots of both −PK and +PK samples were subjected to immunoprecipitation via an antibody against the C terminus of GABRA1 (antigen labeled in purple). The glycosylated (+glyc) and non-glycosylated (−glyc) products are indicated. NPF and CPF are indicated with green and purple arrowheads, respectively. d , 35 S-methionine-labeled SQS and GABRA1 each with a C-terminal glycosylation site were translated in the presence of SPCs derived from ΔEMC cells or cells stably overexpressing either wild-type EMC3-FLAG or insertase-deficient mutants of EMC3-FLAG variants (M cyt-1 -S and R31A). SS indicates a cleavable signal sequence. e , Quantification of three independent experiments as shown in d , with mean ± s.d. plotted. C-tail translocation was quantified by plotting per cent glycosylation (for SQS) or the per cent of glycosylated products that contain two glycans (For GABRA1). C-term, C-terminal.

Article Snippet: The following antibodies and dilutions were used for blotting: CCDC47 (Bethyl Laboratories, A305-100A; 1:5,000); EMC3 (Invitrogen, 711771; 1:5,000); EMC6 (Abcam, ab84902; 1:1,000); Calnexin (Enzo, ADI-SPA-865; 1:5,000); Sec61α (ref. ; 1:5,000); TMCO1 (Invitrogen, PA5-43350; 1:500); Sec61β (ref. ; 1:5,000); CAML (Cell Signaling Technology, 13913S; 1:1,000); FLAG M2-HRP (Sigma, A8592; 1:5,000); EMC4 (Abcam, ab123719; 1:2,000); and β-Actin-HRP (Sigma, A3854; 1:10,000).

Techniques: Stable Transfection, Expressing, Construct, Flow Cytometry, Labeling, Sequencing, Knock-Out, Derivative Assay, Centrifugation, SDS Page, Autoradiography, Immunoprecipitation, Translocation Assay

Journal: Nature Structural & Molecular Biology

Article Title: EMC rectifies the topology of multipass membrane proteins

doi: 10.1038/s41594-023-01120-6

Figure Lengend Snippet: a , Stable cell lines expressing the indicated inducible constructs were treated with non-targeting or EMC4-targeting siRNAs for 3 days, induced for 6 h and analyzed by flow cytometry for surface GABA A receptor levels using phycoerythrin (PE) labeled antibody (left), total levels of RFP-SQS (middle) or total levels of RFP-ASGR1 (right). SQS and ASGR1 levels are normalized to GFP, an internal expression control that is separated from the reporter by a ribosome-skipping viral P2A sequence. KD indicates knockdown. b , Total levels of GABRA1 and EMC4 were analyzed by blotting in cells treated with control or EMC4 siRNA. β-actin was used as a loading control. c , Topology diagram of human GABRA1; the predicted fragments resulting from proteinase K (PK) digestion are shown on top. Tail and loop lengths are indicated. NPF, N-terminal protected fragment; CPF, C-terminal protected fragment. The bottom panel shows the topology analysis of GABRA1 in the endoplasmic reticulum of wild-type (WT) and EMC6-knockout (ΔE) 293 cells. 35 S-methionine-labeled GABRA1 was translated in rabbit reticulocyte lysate in the absence (Ø) or presence of SPCs derived from WT or ΔE 293 cells. After translation, the SPCs were recovered by centrifugation and analyzed directly (−PK) by SDS–PAGE and autoradiography or subjected to PK digestion (+PK). Reactions lacking SPCs were analyzed similarly without centrifugation. Aliquots of both −PK and +PK samples were subjected to immunoprecipitation via an antibody against the C terminus of GABRA1 (antigen labeled in purple). The glycosylated (+glyc) and non-glycosylated (−glyc) products are indicated. NPF and CPF are indicated with green and purple arrowheads, respectively. d , 35 S-methionine-labeled SQS and GABRA1 each with a C-terminal glycosylation site were translated in the presence of SPCs derived from ΔEMC cells or cells stably overexpressing either wild-type EMC3-FLAG or insertase-deficient mutants of EMC3-FLAG variants (M cyt-1 -S and R31A). SS indicates a cleavable signal sequence. e , Quantification of three independent experiments as shown in d , with mean ± s.d. plotted. C-tail translocation was quantified by plotting per cent glycosylation (for SQS) or the per cent of glycosylated products that contain two glycans (For GABRA1). C-term, C-terminal.

Article Snippet: For monitoring the surface expression of GABA A receptors, either negative control small interfering RNA (siRNA) (Invitrogen, 4390843) or EMC4 siRNA (Ambion, s27733) was transfected into the 293 cell line expressing the human GABA A receptor using Lipofectamine RNAiMAX according to the manufacturer’s protocol.

Techniques: Stable Transfection, Expressing, Construct, Flow Cytometry, Labeling, Sequencing, Knock-Out, Derivative Assay, Centrifugation, SDS Page, Autoradiography, Immunoprecipitation, Translocation Assay