Figure S4.
Substrate capture by EMC3’s hydrophobic loop 2 and EMC7’s hydrophobic helix H2. (A) HEK293 cells stably expressing RFP-SQS or -VAMP2 and cytosolic GFP as a normalization control were transduced with lentivirus to express the indicated EMC3 loop 2 mutants, along with BFP as a transduction marker. For each mutant, the RFP:GFP ratio of BFP-positive cells was derived via flow cytometry and is plotted as a histogram. ML2 refers to all four methionines in loop 2. (B) The indicated EMC3 loop 2 mutants were introduced into HEK293 cells via lentiviral transduction. Cells were harvested, solubilized and subjected to anti-FLAG IP. Eluates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Mw, molecular weight. (C) Alignment of EMC7 C-terminus sequences from various eukaryotes using Clustal Omega (Sievers et al., 2011). Two conserved sequence stretches are predicted by secondary structure algorithms to form α-helices, termed H1 and H2. Residues mutated in E are highlighted in blue. AlphaFold 2 models of H1 and H2 are shown. H1 is methionine-rich and H2 is predicted to form an amphipathic α-helix. (D) As in Fig. 3 C, but with the indicated mutants of H1 or the lumenal linker (link) between the EMC7’s β-sandwich and TMD. MH1 refers to all four methionines in H1. KKR→EEE denotes the combined mutation of K115E, K117E, and R119E. (E) WT or EMC7 knockout (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. (F) A BFP control, WT EMC7, 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 IP. Eluates were analyzed by SDS-PAGE and Western blotting with antibodies against EMC2 and 7. (G) 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 Fig. 2 B. (H) Coomassie stained SDS-PAGE gel of the disulfide crosslinking experiment shown in Fig. 3 D before analysis via autoradiography. The gel shows that equal amounts of EMC were used in the different crosslinking reactions. (I) Purified WT or EMC3 Cys mutant EMC were incubated with purified CaM–SQS(L401C) complexes with WT or positively charged (+4) C-terminal domain. Disulfide crosslinking and analysis was carried out as above. Source data are available for this figure: SourceData FS4.

Substrate capture by EMC3’s hydrophobic loop 2 and EMC7’s hydrophobic helix H2. (A) HEK293 cells stably expressing RFP-SQS or -VAMP2 and cytosolic GFP as a normalization control were transduced with lentivirus to express the indicated EMC3 loop 2 mutants, along with BFP as a transduction marker. For each mutant, the RFP:GFP ratio of BFP-positive cells was derived via flow cytometry and is plotted as a histogram. ML2 refers to all four methionines in loop 2. (B) The indicated EMC3 loop 2 mutants were introduced into HEK293 cells via lentiviral transduction. Cells were harvested, solubilized and subjected to anti-FLAG IP. Eluates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Mw, molecular weight. (C) Alignment of EMC7 C-terminus sequences from various eukaryotes using Clustal Omega (Sievers et al., 2011). Two conserved sequence stretches are predicted by secondary structure algorithms to form α-helices, termed H1 and H2. Residues mutated in E are highlighted in blue. AlphaFold 2 models of H1 and H2 are shown. H1 is methionine-rich and H2 is predicted to form an amphipathic α-helix. (D) As in Fig. 3 C, but with the indicated mutants of H1 or the lumenal linker (link) between the EMC7’s β-sandwich and TMD. MH1 refers to all four methionines in H1. KKR→EEE denotes the combined mutation of K115E, K117E, and R119E. (E) WT or EMC7 knockout (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. (F) A BFP control, WT EMC7, 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 IP. Eluates were analyzed by SDS-PAGE and Western blotting with antibodies against EMC2 and 7. (G) 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 Fig. 2 B. (H) Coomassie stained SDS-PAGE gel of the disulfide crosslinking experiment shown in Fig. 3 D before analysis via autoradiography. The gel shows that equal amounts of EMC were used in the different crosslinking reactions. (I) Purified WT or EMC3 Cys mutant EMC were incubated with purified CaM–SQS(L401C) complexes with WT or positively charged (+4) C-terminal domain. Disulfide crosslinking and analysis was carried out as above. Source data are available for this figure: SourceData FS4.

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