Figure S3.
Architecture of the insertase-competent region of the EMC. (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: Nine-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: Eight-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 ΕΜC10 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. 35S-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 RRL supplemented with 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 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. Mw, molecular weight. (E) EMC4 and EMC7, but not EMC10, are required for SQS biogenesis in human cells. WT or EMC4/7/10 knockout (KO) HEK293 cells were transduced with lentivirus to express RFP-SQS or -VAMP2. The relative level of the RFP-fused TA protein 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. Source data are available for this figure: SourceData FS3.

Architecture of the insertase-competent region of the EMC. (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: Nine-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: Eight-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 ΕΜC10 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. 35S-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 RRL supplemented with 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 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. Mw, molecular weight. (E) EMC4 and EMC7, but not EMC10, are required for SQS biogenesis in human cells. WT or EMC4/7/10 knockout (KO) HEK293 cells were transduced with lentivirus to express RFP-SQS or -VAMP2. The relative level of the RFP-fused TA protein 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. Source data are available for this figure: SourceData FS3.

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