EMC rectifies the topology of multipass membrane proteins

Abstract

Most eukaryotic multipass membrane proteins are inserted into the membrane of the endoplasmic reticulum. Their transmembrane domains (TMDs) are thought to be inserted co-translationally as they emerge from a membrane-bound ribosome. Here we find that TMDs near the carboxyl terminus of mammalian multipass proteins are inserted post-translationally by the endoplasmic reticulum membrane protein complex (EMC). Site-specific crosslinking shows that the EMC’s cytosol-facing hydrophilic vestibule is adjacent to a pre-translocated C-terminal tail. EMC-mediated insertion is mostly agnostic to TMD hydrophobicity, favored for short uncharged C-tails and stimulated by a preceding unassembled TMD bundle. Thus, multipass membrane proteins can be released by the ribosome–translocon complex in an incompletely inserted state, requiring a separate EMC-mediated post-translational insertion step to rectify their topology, complete biogenesis and evade quality control. This sequential co-translational and post-translational mechanism may apply to ~250 diverse multipass proteins, including subunits of the pentameric ion channel family that are crucial for neurotransmission.

Structural analysis of the dynamic ribosome-translocon complex

“…S. cerevisiae has no annotated TRAP genes, and indeed it has been suggested that most fungi lack TRAP 104 . However, we find that S. cerevisiae contains a previously unidentified gene for TRAPα (Irc22, HHpred E = 10 -35 ). This suggests TRAP’s prevalence in fungi has been underestimated.…”

“…At least one main function of EMC is to facilitate insertion of TMDs close to the N- or C-terminus. This includes post-translational insertion of tail-anchored proteins 34 , post-translational insertion of the final TMD of some multipass membrane proteins 35 , and co-translational insertion of SAs in the N exo topology (N-terminus facing the exoplasmic side of the membrane) 23,36 . This insertase function is thought to involve its core EMC3 subunit 24,35,37–40 , which is a member of the Oxa1 family of insertases 22 .…”

“…This includes post-translational insertion of tail-anchored proteins 34 , post-translational insertion of the final TMD of some multipass membrane proteins 35 , and co-translational insertion of SAs in the N exo topology (N-terminus facing the exoplasmic side of the membrane) 23,36 . This insertase function is thought to involve its core EMC3 subunit 24,35,37–40 , which is a member of the Oxa1 family of insertases 22 . Although EMC has been implicated in co-translational membrane protein insertion, it has thus far not been observed at RTCs in cryo-electron tomography studies 29,41 .…”

ER membrane complex (EMC): Structure, functions, and roles in diseases

“…The gap between the vestibule and the intramembrane sulcus provides a possible pathway for matrix outflow from EMC. These findings shed light on how EMC facilitates energy‐independent membrane insertion of TMDs, elucidating why only short‐lumen domains are transported by EMC and supporting the proposed companion function model of EMC 41,42 . Next, we will focus on cryo‐electron microscopy (cryo‐EM) structures of human EMC.…”

“…These findings shed light on how EMC facilitates energyindependent membrane insertion of TMDs, elucidating why only short-lumen domains are transported by EMC and supporting the proposed companion function model of EMC. 41,42 Next, we will focus on cryo-electron microscopy (cryo-EM) structures of human EMC. When it was first discovered, the structure of EMC was not clear, but research teams continued to contribute to the analysis of the EMC structure, and the precise structure of EMC was outlined step by step (Table 2).…”