Discrete cross-linking products identified during membrane protein biosynthesis.
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 Sec61alpha 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 Sec61beta and finally with Sec61alpha. 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.
Laird
V
V
School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT United Kingdom.
High
S
S
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D003432:Cross-Linking Reagents; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D017299:Rod Opsins / Q000235:genetics / Q000378:metabolism; D000069816:SEC Translocation Channels
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D012269:Ribosomal Proteins; D017299:Rod Opsins; D000069816:SEC Translocation Channels