Active and passive displacement of transmembrane domains both occur during opsin biogenesis at the Sec61 translocon

We used a site-specific crosslinking approach to study the membrane integration of the polytopic protein opsin at the endoplasmic reticulum. We show that transmembrane domain 1 occupies two distinct Sec61-based environments during its integration. However, transmembrane domains 2 and 3 exit the Sec61 translocon more rapidly in a process that suggests a displacement model for their integration where the biosynthesis of one transmembrane domain would facilitate the exit of another. In order to investigate this hypothesis further, we studied the integration of the first and third transmembrane domains of opsin in the absence of any additional C-terminal transmembrane domains. In the case of transmembrane domain 1, we found that its lateral exit from the translocon is clearly dependent upon the synthesis of subsequent transmembrane domains. By contrast, the lateral exit of the third transmembrane domain occurred independently of any such requirement. Thus, even within a single polypeptide chain, distinct transmembrane domains display different requirements for their integration through the endoplasmic reticulum translocon, and the displacement of one transmembrane domain by another is not a global requirement for membrane integration.