Roughly one-fourth of all genes code for integral membrane proteins. Most membrane proteins pass back and forth across the lipid bilayer multiple times. Multipass membrane proteins, such as ion channels and receptors, are essential for nearly all transport and communication across the plasma membrane. In eukaryotes, nascent multipass membrane proteins are initially inserted into the endoplasmic reticulum (ER) membrane, where they fold and assemble before being trafficked to their final destination. Membrane proteins typically insert co-translationally as the nascent multipass protein emerges from an ER-associated ribosome. The long-standing model has been that each transmembrane domain (TMD) inserts into the membrane sequentially via the Sec61 translocation channel. This framework was primarily extrapolated from mechanistic studies of a few single-pass membrane proteins, but direct analyses of multipass membrane proteins were limited. This thesis reports efforts to understand the sequence of events, machinery, and mechanisms of multipass membrane protein biogenesis.

Biochemical reconstitution combined with genetic and chemical perturbations showed that the first two TMDs of most multipass proteins with a cytosol-facing N-terminus are inserted by a Sec61-independent mechanism. Instead, this insertion relies, at least in part, on the ER membrane complex (EMC), a TMD insertase recently appreciated to be a member of the universally conserved Oxa1 family of insertases. Strict dependence on Sec61 was only seen for TMD pairs separated by a translocated region longer than ≈100 amino acids. Purification and analysis of later multipass insertion intermediates revealed a ribosome-associated machinery comprised of four complexes: the Sec61 translocation channel, an intramembrane chaperone called PAT, an Oxa1 insertase family member called GEL, and a complex of unknown function termed BOS. Structural and mutational analysis showed that the PAT complex contains a membrane-exposed amphipathic surface that binds nascent TMDs and a ‘latch’ domain that prevents opening of the Sec61 channel. These activities re-direct the nascent protein away from Sec61 and towards GEL, which is seen adjacent to subsequently inserted TMD pairs. Crosslinking experiments and structural work show that newly inserted TMDs accumulate in a semi-circular lipid-filled cavity formed by Sec61, PAT, GEL and BOS, known as the multipass translocon. These findings lead to a model for multipass protein biogenesis in which most TMDs are inserted by Oxa1 family members; only TMDs flanked by long translocated domains rely strictly on the Sec61 channel. The nascent TMDs of a multipass protein progressively accumulate in the semi-protected environment provided by the multipass translocon, where they can fold to complete their biogenesis.