Cytosolic transmembrane helices enter the ER membrane through EMC6-driven electrostatic interactions

The endoplasmic reticulum membrane protein complex (EMC) facilitates transmembrane domain (TMD) insertion and translocation of small terminal domains. Here, we identify EMC as a critical determinant of epithelial sodium channel (ENaC) biosynthesis. EMC loss reduces ENaC expression without affecting surface trafficking, indicating a biosynthetic stabilizing role. We demonstrate that electrostatic interactions between the negatively charged distal segment of ENaC's TMD2 and a positively charged patch on EMC6 are essential for ENaC-EMC association. We propose a 'semi-insertase' mechanism wherein the Sec61 translocon partially inserts TMD2, leaving its hydrophilic distal segment exposed to the cytosol. EMC captures this segment through electrostatic attraction and completes membrane integration. Bioinformatic analysis identified a group of non-canonical EMC clients including >200 multipass proteins with similarly charged TMDs, suggesting that electrostatic capture may represent a solution for inserting unconventional hydrophilic transmembrane segments. Additionally, we demonstrate that EMC8 plays an essential role in stabilizing the EMC-a function that cannot be compensated by its homolog EMC9 due to lower expression and absence of compensatory upregulation in EMC8-deficient cells. These results reveal a multifaceted EMC mechanism coupling insertase activity with chaperone-like stabilization to facilitate biogenesis of multipass membrane proteins containing highly hydrophilic TMDs.