Figure 3.
Characterization of the cytosolic and intramembrane residues required for insertion by the EMC. (A) Displayed is an improved model of the human EMC determined using cryo-EM. View 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 WT or EMC3 Cys mutant EMC were incubated with purified CaM-SQS(L401C) complexes for disulfide crosslinking and analysis as in Fig. 2 B. (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 KO cells were transduced with lentivirus to express WT EMC7, or the indicated mutants of EMC7 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) Disulfide crosslinking, as described in Fig. 2 B, of SQS(L401C) with purified EMC complexes, containing cysteines either in H2 of EMC7 (M214S), loop 2 of EMC3 (T102C), or within the membrane (EMC3 N117C). Mw, molecular weight. (E) 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 as in C, normalized to WT, and is displayed according to the indicated legend. Source data are available for this figure: SourceData F3.

Characterization of the cytosolic and intramembrane residues required for insertion by the EMC. (A) Displayed is an improved model of the human EMC determined using cryo-EM. View 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 WT or EMC3 Cys mutant EMC were incubated with purified CaM-SQS(L401C) complexes for disulfide crosslinking and analysis as in Fig. 2 B. (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 KO cells were transduced with lentivirus to express WT EMC7, or the indicated mutants of EMC7 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) Disulfide crosslinking, as described in Fig. 2 B, of SQS(L401C) with purified EMC complexes, containing cysteines either in H2 of EMC7 (M214S), loop 2 of EMC3 (T102C), or within the membrane (EMC3 N117C). Mw, molecular weight. (E) 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 as in C, normalized to WT, and is displayed according to the indicated legend. Source data are available for this figure: SourceData F3.

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