![]() ![]() The population of polypeptides inserted into the membrane is protected from PK. The translation products were then digested with proteinase K (PK). b, 35S-methionine labelled Rho ext RNC of the indicated length, lacking or containing the glycosylation site, was synthesised in RRL containing or lacking RMs. The pattern of PAT complex recruitment for Rho ext construct is similar to the non-extended Rho construct construct described previously. The crosslink to Asterix (indicated by x-Asx) was verified by immunoprecipitation under denaturing conditions using anti-Asterix antibody (bottom panel). The positions of the glycosylated (+glyc.) and non-glycosylated (-glyc.) translation products are indicated. Where indicated, RNCs were chemically crosslinked using bismaleimidohexane (BMH). The Author(s), under exclusive licence to Springer Nature Limited.Ī, 35S-methionine labelled Rho ext ribosome nascent chain complexes (RNCs) of indicated length were synthesised in rabbit reticulocyte lysate (RRL) in the presence of canine pancreas-derived rough microsomes (RMs). These findings elucidate the mechanism of an intramembrane chaperone and suggest a new framework for multipass membrane protein biogenesis at the endoplasmic reticulum. Accordingly, biogenesis of several multipass proteins was unimpeded by inhibitors of the Sec61 lateral gate. Detection of multiple TMDs in this cavity after their emergence from the ribosome suggests that multipass proteins insert and fold behind Sec61. Instead, Asterix binds to and redirects the substrate to a location behind Sec61, where the PAT complex contributes to a multipass translocon surrounding a semi-enclosed, lipid-filled cavity 11. ![]() Here, biochemical and structural analysis of intermediates during multipass protein biogenesis showed that the nascent chain is not engaged with Sec61, which is occluded and latched closed by CCDC47. The PAT complex, an intramembrane chaperone comprising Asterix and CCDC47, engages early TMDs of multipass proteins to promote their biogenesis by an unknown mechanism 10. The prevailing model posits that each transmembrane domain (TMD) of a multipass protein successively passes into the lipid bilayer through a front-side lateral gate of the Sec61 protein translocation channel 3-9. How multipass proteins are co-translationally inserted and folded at the endoplasmic reticulum is not well understood 2. Multipass membrane proteins play numerous roles in biology and include receptors, transporters, ion channels and enzymes 1,2. ![]()
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