Figure S5.
Biophysical properties of the hydrophilic vestibule. (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 of EMC3 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 Fig. 3 D and B were introduced into 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 4. (D) The N-terminus of EMC4 is required for TA protein 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. Mw, molecular weight. (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 (Pleiner et al., 2020). (H) Expi293 suspension cells stably expressing EMC3-GFP WT or R31E+R180E were solubilized and subjected to anti-GFP nanobody purification. The eluate was normalized by GFP fluorescence and analyzed by SDS-PAGE followed by Sypro Ruby staining. Note that both EMC3 WT and R31E+R180E mutant incorporate into EMCs with similar efficiency as they co-purify with all other EMC subunits. (I) Same assay as in Fig. 7 A but in cells transduced with either a non-targeting (control) or MTCH2 knockdown sgRNA. (J) Same assay as in Fig. 7 D measuring the ER insertion of GFP11-TRAM2, but showing only WT EMC3 −/+ p97 inhibitor CD-5083. Source data are available for this figure: SourceData FS5.

Biophysical properties of the hydrophilic vestibule. (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 of EMC3 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 Fig. 3 D and B were introduced into 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 4. (D) The N-terminus of EMC4 is required for TA protein 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. Mw, molecular weight. (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 (Pleiner et al., 2020). (H) Expi293 suspension cells stably expressing EMC3-GFP WT or R31E+R180E were solubilized and subjected to anti-GFP nanobody purification. The eluate was normalized by GFP fluorescence and analyzed by SDS-PAGE followed by Sypro Ruby staining. Note that both EMC3 WT and R31E+R180E mutant incorporate into EMCs with similar efficiency as they co-purify with all other EMC subunits. (I) Same assay as in Fig. 7 A but in cells transduced with either a non-targeting (control) or MTCH2 knockdown sgRNA. (J) Same assay as in Fig. 7 D measuring the ER insertion of GFP11-TRAM2, but showing only WT EMC3 −/+ p97 inhibitor CD-5083. Source data are available for this figure: SourceData FS5.

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