Selectivity at the ER membrane limits misinsertion of mitochondrial TA proteins by the EMC. (A) Top: Topology of a TA protein. Bottom: ³⁵S-methionine–labeled TA protein with the indicated TMDs and C-terminal domains (CTDs) were expressed in the PURE system and purified as complexes with the cytosolic chaperone calmodulin. Glycosylation (glyc) of the CTD upon incubation with hRMs indicates successful insertion. Samples were analyzed by SDS-PAGE followed by autoradiography. (B) Schematic of the split GFP reporter system used to selectively monitor TA protein insertion into the ER. TA proteins fused to GFP11 are expressed in K562 cells constitutively expressing GFP1-10 in the ER lumen, along with a translation normalization marker (RFP). Successful integration into the ER results in GFP complementation and fluorescence. (C) Top: ER insertion pathways. Bottom: ER insertion of the indicated ER (SQS, VAMP2, ASGR1) and mitochondrial (RHOT2, RHOT1, MAOA, MAOB, Fis1) TA proteins, using the split GFP system as described in B, was assessed in cells transduced with either a non-targeting (control), EMC2, or GET2 knockdown (kd) sgRNA. GFP fluorescence relative to the normalization marker RFP was determined by flow cytometry and displayed as a histogram. (D) Cells from C were harvested and samples of total cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against EMC2, GET2, and BAG6, a non-targeted control protein. Mw, molecular weight. Source data are available for this figure: SourceData F1.