Exploring the molecular composition of the multipass translocon in its native membrane environment

Abstract

AbstractMultispanning membrane proteins are inserted into the endoplasmic reticulum membrane by the ribosome-bound multipass translocon machinery. Based on cryo-electron tomography and extensive subtomogram analysis, we reveal the composition and arrangement of multipass translocon components in their native membrane environment. The intramembrane chaperone complex PAT and the translocon associated protein (TRAP) complex associate substoichiometrically with the multipass translocon in a translation-dependent manner. While PAT is preferentially recruited to active complexes, TRAP primarily associates with inactive translocons. The subtomogram average of the TRAP-multipass translocon reveals intermolecular contacts between the luminal domains of TRAP and an unknown subunit of the BOS complex. AlphaFold modeling suggests this protein is NOMO, bridging the luminal domains of nicalin and TRAPα. Collectively, our results visualize the interplay of accessory factors associated with multipass membrane protein biogenesis under near-native conditions.