Positively charged N-terminal domains of GPCRs impair EMC insertion. (A) Distribution of charge within the soluble N-terminal domain of the 709 human GPCR sequences annotated in the Uniprot database. Only those GPCRs lacking a signal sequence (i.e., signal anchored) were included, because these represent substrates that could potentially rely on the EMC for insertion of their first TMD in an N_exo topology (Chitwood et al., 2018). (B) WT (total N-terminal charge of −5) or the indicated N-terminal domain (NTD) charge mutants of the GPCR OPRK1 GFP fusions were expressed along with an RFP normalization marker in RPE1 cells. Cells were analyzed by flow cytometry, and the GFP:RFP ratio is displayed as a histogram. Bypassing insertion by the EMC by fusion to a cleavable signal sequence (ss) enhances ER integration of the OPRK1(+5) charge mutant. (C) As in B, but cells were treated with scrambled (control) or EMC5 knockdown (kd) siRNAs and analyzed by flow cytometry. Note that though the stability of the positively charged NTD variants is reduced, they remain EMC dependent for their insertion.