Charge reversal in the hydrophilic vestibule alleviates charge repulsion. (A) K562 ER GFP1-10 cells were transduced with lentivirus to express either WT, R31A+R180A, or R31E+R180E EMC3. Cells were harvested, solubilized, and samples of the total lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Mw, molecular weight. (B) ER insertion of the indicated SQS charge mutants was measured in cells expressing either WT, R31A+R180A, or R31E+R180E EMC3 using the split GFP reporter system described in Fig. 1 B. (C) The indicated SQS mutants were prepared as in Fig. 4 E and incubated with hRMs from WT, R31A+R180A, or R31E+R180E EMC3 expressing cell lines. Successful ER insertion is monitored with a glycosylation (glyc) acceptor motif fused to the C-terminus of each substrate. The percent glycosylated is indicated below the gel. Expression of both EMC3 mutants does not impair the biogenesis of GET1/2-dependent VAMP2 or the secreted protein prolactin (Prl) that depends on the Sec61 complex (translocon). (D) WT (−5) or the indicated charge mutants of OPRK1 were fused to GFP and expressed with RFP as a translation normalization marker in RPE1 cells. Cells additionally expressed either BFP-tagged EMC3 WT, R31A+R180A, or R31E+R180E. Cells were analyzed by flow cytometry to derive the GFP:RFP ratio of BFP-positive cells. Source data are available for this figure: SourceData F6.