Defining the hydrophilic vestibule as the insertase-competent side. (A) Schematic depiction of the site-specific photocrosslinking approach. The ³⁵S-methionine–labeled TA protein substrate SEC61β, with a BpA photocrosslinker incorporated into its TMD, was produced as a complex with CaM in the PURE in vitro translation system. It was then incubated with EMC solubilized and purified in the detergent LMNG. Except for the −UV controls, all reactions were irradiated with UV light after substrate release from CaM with EGTA and then analyzed by SDS-PAGE and autoradiography. Crosslinks to EMC3 and EMC4 were identified by IP with anti-EMC3 and -EMC4 antibodies. The asterisk indicates the crosslinked TA protein dimer band. (B) Coomassie stained SDS-PAGE gel of the disulfide crosslinking experiments with purified EMC shown in Fig. 2 B before analysis via autoradiography. The gel shows that equal amounts of EMC were used in the different crosslinking reactions. Mw, molecular weight. (C) Disulfide crosslinking with purified EMC as in Fig. 2 B, but with cysteines positioned around a turn of the SQS TMD, showing that the observed crosslinking bias to residues on the hydrophilic vestibule (in blue) is independent of cysteine position. All crosslinking reactions were performed in parallel, and gels were exposed to the same film. (D) Purified EMC complexes containing the unnatural amino acid and photocrosslinker AbK incorporated into EMC3 at the indicated positions were mixed with SQS(WT)–CaM complexes prepared in the PURE system and irradiated with UV light after substrate release from CaM with EGTA. Samples were analyzed by SDS-PAGE and autoradiography. (E) Insertion activity of hRMs prepared from EMC3 WT or Cys mutant cell lines. Two well-characterized EMC substrates, SQS and TMD1 of the β-adrenergic receptor 1 (βADR1; Chitwood et al., 2018; Guna et al., 2018), were translated in rabbit reticulocyte lysate in the presence of the indicated hRMs. Successful ER insertion results in the glycosylation (glyc) of the fused Opsin tag. cRMs were used as a control. (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, 5, and 6 in the hydrophobic crevice. The RFP:GFP ratio for each mutant was determined using flow cytometry and is plotted as a histogram. (G) Side view of the membrane-spanning region of the EMC, focusing on the large cleft-like hydrophobic crevice. Residues on EMC3, 5, and 6 that were mutated in F line the cleft and are highlighted. (H) Incorporation of EMC subunit mutants into intact EMCs. A fraction of cells from F were harvested, solubilized, and subjected to anti-HA or anti-FLAG IP. Co-purification with the soluble subunit EMC2 indicates successful incorporation of WT and mutant EMC3, 5, and 6 variants, suggesting that all of the mutant subunits are assembled into the mature EMC. Source data are available for this figure: SourceData FS1.