A molecular analysis of opsin integration at the endoplasmic reticulum

A major step in the biosynthesis of many membrane proteins is their insertion into themembrane of the endoplasmic reticulum (ER). The insertion of a multi-spanningmembrane protein is a complex process since several transmembrane (TM) domainshave to be correctly integrated in order to enable its correct assembly. At present it isunclear how the integration of multiple TM domains is co-ordinated by the ERtranslocon. The aim of this study was to analyse the molecular environment of the TMdomains of a model seven TM domain protein, opsin, so as to better understand themechanism by which integration occurs.For this purpose, stable ???integration intermediates??? of defined lengths representingdistinct stages of opsin biosynthesis were generated by in vitro translation of truncatedmRNA in the presence of semi-permeabilised cells. Cysteine-mediated, site-specificcross-linking and immunoprecipitation were employed to examine the environment ofthese integration intermediates. In addition, cysteine-specific modification reagents withdifferent physical properties were used to investigate the environment of opsin TM3during its insertion at the ER membrane.Opsin TM domains exhibit unique patterns of adduct formation with the ER transloconcomponents, Sec61?? and Sec61??. TM1 associates with the Sec61 complex at twodistinct stages during nascent chain extension, and this behaviour is dependent on thepresence of subsequent TM domains. The re-association of TM1 with the transloconmay well facilitate the co-ordinated integration of TMs 1-3 into the lipid bilayer. OpsinTM4 exits the Sec61 complex as soon as the subsequent TM domain is synthesised,while TM5, TM6 and TM7 remain associated with the ER translocon throughoutprotein synthesis, suggesting their concerted release upon chain termination. Evidenceis provided that opsin is integrated via a single Sec61 heterotrimer, despite the fact thatthe ER translocon appears to consist of multiple copies of the Sec61 complex. On thebasis of this work, a model is presented describing the complete integration of opsin atthe ER membrane.