Discrete cross-linking products identified during membrane protein biosynthesis

Abstract

We have investigated the molecular details of the membrane insertion of the multiple-spanning membrane protein opsin. Using heterobifunctional cross- linking reagents the endoplasmic reticulum (ER) proteins adjacent to a series of defined translocation intermediates were determined. Once the nascent opsin chain reaches a critical minimum length Sec61α is the major ER component adjacent to the polypeptide. Using a homobifunctional reagent, the cross-linking partners from a single cysteine residue in the nascent chain were analyzed. This approach identified chain length-dependent cross-linking products between nascent opsin and a 21-kDa ribosomal protein, followed by Sec61β and finally with Sec61α. Our data support a model where the sequential transmembrane domains of a multiple-spanning membrane protein are integrated at an ER insertion site similar to that mediating the insertion of single-spanning membrane proteins.