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

Abstract

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.

Post-translational integration of tail-anchored proteins is facilitated by defined molecular chaperones

“…Of the membrane-associated products recovered, 8% were Nglycosylated for the control sample (lane 4). Lower molecular weight forms of non-glycosylated Sec61␤ were more prevalent after RNC preparation (lanes 1-6, product 0g and below), most likely as a result of ribosome stacking (Ismail et al, 2006). We confirmed that EDTA treatment does not prevent N-glycosylation per se (data not shown), hence, a lack of glycosylated Sec61␤ reflects a lack of integration.…”

The Translocation Inhibitor CAM741 Interferes with Vascular Cell Adhesion Molecule 1 Signal Peptide Insertion at the Translocon

“…It has been shown by chemical cross-linking that a close proximity to Sec61␤ is one of the early events during TM domain integration (40,41). The cross-links with Sec61␤ disappear once the TM domain moves laterally into the membrane, as evident from enhanced lipid cross-links and glycosylation of a tag N-terminal to the TM domain (38,42).…”

Dissecting the physiological role of selective transmembrane-segment retention at the ER translocon

“…Although a single heterotrimer is suggested to form the proteinconducting channel through the membrane, it is likely that several Sec61 heterotrimers assemble in the active ER translocon together with multiple accessory components, including TRAM (translocating chain-associated membrane protein), the TRAP complex and PAT-10, during membrane protein integration (Hegde and Kang, 2008). Such an arrangement might contribute to the translocon architecture required to allow the simultaneous accommodation of multiple TM segments during membrane protein biosynthesis (Ismail et al, 2006;Kida et al, 2005;Sadlish et al, 2005).…”