Specific transmembrane segments are selectively delayed at the ER translocon during opsin biogenesis
Résumé
A site-specific cross-linking approach was used to study the integration of transmembrane (TM) segments 4-7 of the polytopic membrane protein, opsin, at the endoplasmic reticulum (ER). We find that whilst TM4 exits the ER translocon rapidly, TM segments 5, 6 and 7 are all retained at the translocon until opsin biosynthesis is terminated. Furthermore, although artificial extension of the nascent chain is not sufficient to release the C-terminal region of opsin from the translocon, substitution of the native TM segment 7 with a more hydrophobic TM segment results in its rapid lateral exit into the lipid bilayer. We conclude that the intrinsic properties of a TM segment determine the timing of its membrane integration rather than its relative location within the polypeptide chain. A pronounced and prolonged association of opsin TM5 with the translocon associated component PAT-10 was also observed, suggesting that PAT-10 may facilitate the assembly of distinct opsin sub-domains during membrane integration. Our data strongly support a model in which the ER translocon co-ordinates the integration of selected TM segments in response to the specific requirements of the precursor being synthesised.
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