Discrete Cross-linking Products Identified during Membrane Protein Biosynthesis

doi:10.1074/jbc.272.3.1983

Discrete Cross-linking Products Identified during Membrane Protein Biosynthesis*

Vivienne Laird^‡ and
Stephen High^§

From the School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT United Kingdom

^§ Biotechnology and Biological Sciences Research Council Advanced Research Fellow. To whom correspondence should be addressed. Tel.: 0161-275-5070; Fax: 0161-275-5082.

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.

Footnotes

↵^‡ Biotechnology and Biological Sciences Research Council Research Studentship.
↵* This work was supported by funding from the Biotechnology and Biological Sciences Research Council, the Human Frontier Science Program Organisation, and the Royal Society. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

BMH

bismaleimidohexane

BMP

bismaleimidopropane

cko

cysteine knock out

Endo Hf

Endoglycosidase H, recombinant fusion protein

MBS

m-maleimidobenzoyl-N-hydroxysuccinimide ester

OP

opsin

S-MBS

m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester

SRP

signal recognition particle

TRAM

translocating chain-associating membrane protein

PCR

polymerase chain reaction.
↵²S. High, unpublished data.
- Received May 29, 1996.
- Revision received October 4, 1996.