A selectivity filter in the EMC limits mitochondrial TA protein misinsertion at the ER. (A) As in Fig. 6 B, but with the indicated mitochondrial TA proteins. Note the strong increase in ER mislocalization of RHOT1 in EMC3 R31E+R180E expressing cells. (B) As in Fig. 6 C but expressing the TMD and C-terminus of the indicated mitochondrial TA proteins in non-nucleased RRL. Mw, molecular weight. (C) Schematic of the split GFP reporter system used to selectively monitor TRAM2 insertion in the incorrect topology into the ER. GFP11-tagged TRAM2 is expressed in K562 cells constitutively expressing GFP1-10 in the ER lumen, along with a translation normalization marker (RFP). Successful integration of TRAM2 in the correct topology will result in no fluorescence. Insertion in the incorrect topology results in GFP complementation and fluorescence. (D) ER insertion of GFP11-TRAM2 was measured in cells expressing either WT, R31A+R180A, or R31E+R180E EMC3 with or without the p97 inhibitor CD-5083 using the split GFP reporter system described above. (E) Model for how the EMC distinguishes clients by polar domain charge. A TA protein TMD or the first TMD of a multipass membrane protein is initially captured by flexible hydrophobic loops in the cytosol, allowing their C- or N-terminal domain (CTD/NTD) to probe the net positively charged hydrophilic vestibule. In the absence of positive charge, the polar domain is translocated rapidly, enabling TMD insertion. Insertion of TA proteins with positively charged C-termini or multipass TMDs with positively charged N-termini is slowed by charge repulsion, which facilitates TMD dissociation (rejection). Charge repulsion can be alleviated by introducing negative charge into the hydrophilic vestibule, resulting in increased misinsertion of mitochondrial TA proteins into the ER membrane, as well as increased insertion of multipass proteins in the incorrect topology. Source data are available for this figure: SourceData F7.