Determination of the Transmembrane Topology of Yeast Sec61p, an Essential Component of the Endoplasmic Reticulum Translocation Complex*
- From the School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom
- ‡ A Lister Institute Jenner Research Fellow. To whom correspondence should be addressed. Tel.: 0161-275-5104; Fax: 0161-275-5082; E-mail: stirling{at}man.ac.uk
Abstract
Sec61p is a highly conserved integral membrane protein that plays a role in the formation of a protein-conducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum. As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites. Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity. Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis. The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains.
Footnotes
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↵* This work was supported by the Biotechnology and Biological Sciences Research Council. 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.
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↵1 The abbreviations used are:
- ER
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endoplasmic reticulum
- PAGE
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polyacrylamide gel electrophoresis
- 5-FOA
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5-fluoro-orotic acid
- Suc2p
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invertase
- PMSF
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phenylmethanesulfonyl fluoride
- fXa
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factor Xa
- Endo H
-
endoglycosidase H
- HS
-
hydrophobic segment
- DTT
-
dithiothreitol
- kbp
-
kilobase pair(s).
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↵2 B. M. Wilkinson, Y. Esnault, F. Kepes, and C. J. Stirling, manuscript in preparation.
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↵3 B. M. Wilkinson and C. J. Stirling, unpublished observations.
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↵4 J. Broughton and C. J. Stirling, manuscript in preparation.
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- Received April 29, 1996.
- Revision received July 26, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
