Positively charged C-terminal domains of TA proteins impair insertion by the EMC. (A) Left: Model of the TMDs of EMC3 and 6 that constitute the central insertase of the EMC. Right: Surface representation of the electrostatic potential of the insertase core ranging from −3 to +3 kT/e. EMC4, 7, and 10 were omitted for clarity. (B) Schematic of the SQS C-terminal domain (CTD) charge series. The C-terminus of SQS was mutated to introduce positively charged residues at the indicated positions. (C) Integration of the indicated SQS mutants into the ER was determined using the split GFP reporter system described in Fig. 1 B. (D) Same assay as in C, but with cells expressing either a non-targeting (control) or EMC2 knockdown (kd) sgRNA. (E) The indicated ³⁵S-methionine–labeled SQS charge mutants were expressed in RRL and incubated with hRMs prepared from HEK293 WT or EMC6 knockout (KO) cells. ER insertion is monitored by glycosylation (glyc) of an acceptor motif fused to the C-terminus of the TA protein substrates. Source data are available for this figure: SourceData F4.