Abstract
The Sec61 complex is a central component of the endoplasmic reticulum (ER) translocation site. The complex consists of three subunits: Sec61alpha, Sec61beta and Sec61gamma, at least two of which (alpha and beta) are adjacent to nascent proteins during membrane insertion. Another component of the translocation machinery is the translocating chain-associating membrane (TRAM) protein, which is also adjacent to many nascent proteins during membrane insertion. Sec61alpha functions as the major component of a transmembrane channel formed by oligomers of the Sec61 complex. This channel is the site of secretory protein translocation and membrane protein integration at the ER membrane. Sec61alpha is a polytopic integral membrane protein, and we have studied its biosynthesis and membrane integration in vitro. Using a cross-linking approach to analyse the environment of a series of discrete Sec61alpha membrane-integration intermediates, we find: (i) newly synthesized Sec61alpha is adjacent to known components of the ER membrane-insertion site, namely Sec61alpha, Sec61beta and TRAM, and thus the integration of Sec61alpha appears to require a pre-existing Sec61 complex; (ii) a site-specific cross-linking analysis indicates that the first transmembrane domain of Sec61alpha remains adjacent to protein components of the ER-insertion site (specifically TRAM and Sec61beta) during the insertion of at least three subsequent transmembrane domains; and (iii) the membrane integration of Sec61alpha requires ER targeting by the signal-recognition particle.
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- Borel A. C., Simon S. M. Biogenesis of polytopic membrane proteins: membrane segments assemble within translocation channels prior to membrane integration. Cell. 1996 May 3;85(3):379–389. doi: 10.1016/s0092-8674(00)81116-2. [DOI] [PubMed] [Google Scholar]
- Do H., Falcone D., Lin J., Andrews D. W., Johnson A. E. The cotranslational integration of membrane proteins into the phospholipid bilayer is a multistep process. Cell. 1996 May 3;85(3):369–378. doi: 10.1016/s0092-8674(00)81115-0. [DOI] [PubMed] [Google Scholar]
- Fujiki Y., Fowler S., Shio H., Hubbard A. L., Lazarow P. B. Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes. J Cell Biol. 1982 Apr;93(1):103–110. doi: 10.1083/jcb.93.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gilmore R., Collins P., Johnson J., Kellaris K., Rapiejko P. Transcription of full-length and truncated mRNA transcripts to study protein translocation across the endoplasmic reticulum. Methods Cell Biol. 1991;34:223–239. doi: 10.1016/s0091-679x(08)61683-0. [DOI] [PubMed] [Google Scholar]
- Görlich D., Prehn S., Hartmann E., Kalies K. U., Rapoport T. A. A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation. Cell. 1992 Oct 30;71(3):489–503. doi: 10.1016/0092-8674(92)90517-g. [DOI] [PubMed] [Google Scholar]
- Görlich D., Rapoport T. A. Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell. 1993 Nov 19;75(4):615–630. doi: 10.1016/0092-8674(93)90483-7. [DOI] [PubMed] [Google Scholar]
- Hamman B. D., Chen J. C., Johnson E. E., Johnson A. E. The aqueous pore through the translocon has a diameter of 40-60 A during cotranslational protein translocation at the ER membrane. Cell. 1997 May 16;89(4):535–544. doi: 10.1016/s0092-8674(00)80235-4. [DOI] [PubMed] [Google Scholar]
- Hanein D., Matlack K. E., Jungnickel B., Plath K., Kalies K. U., Miller K. R., Rapoport T. A., Akey C. W. Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation. Cell. 1996 Nov 15;87(4):721–732. doi: 10.1016/s0092-8674(00)81391-4. [DOI] [PubMed] [Google Scholar]
- Hartmann E., Sommer T., Prehn S., Görlich D., Jentsch S., Rapoport T. A. Evolutionary conservation of components of the protein translocation complex. Nature. 1994 Feb 17;367(6464):654–657. doi: 10.1038/367654a0. [DOI] [PubMed] [Google Scholar]
- High S., Andersen S. S., Görlich D., Hartmann E., Prehn S., Rapoport T. A., Dobberstein B. Sec61p is adjacent to nascent type I and type II signal-anchor proteins during their membrane insertion. J Cell Biol. 1993 May;121(4):743–750. doi: 10.1083/jcb.121.4.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
- High S., Andersen S. S., Görlich D., Hartmann E., Prehn S., Rapoport T. A., Dobberstein B. Sec61p is adjacent to nascent type I and type II signal-anchor proteins during their membrane insertion. J Cell Biol. 1993 May;121(4):743–750. doi: 10.1083/jcb.121.4.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
- High S., Flint N., Dobberstein B. Requirements for the membrane insertion of signal-anchor type proteins. J Cell Biol. 1991 Apr;113(1):25–34. doi: 10.1083/jcb.113.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
- High S., Henry R., Mould R. M., Valent Q., Meacock S., Cline K., Gray J. C., Luirink J. Chloroplast SRP54 interacts with a specific subset of thylakoid precursor proteins. J Biol Chem. 1997 Apr 25;272(17):11622–11628. doi: 10.1074/jbc.272.17.11622. [DOI] [PubMed] [Google Scholar]
- High S., Martoglio B., Görlich D., Andersen S. S., Ashford A. J., Giner A., Hartmann E., Prehn S., Rapoport T. A., Dobberstein B. Site-specific photocross-linking reveals that Sec61p and TRAM contact different regions of a membrane-inserted signal sequence. J Biol Chem. 1993 Dec 15;268(35):26745–26751. [PubMed] [Google Scholar]
- Kaderbhai M. A., Harding V. J., Karim A., Austen B. M., Kaderbhai N. N. Sheep pancreatic microsomes as an alternative to the dog source for studying protein translocation. Biochem J. 1995 Feb 15;306(Pt 1):57–61. doi: 10.1042/bj3060057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laird V., High S. Discrete cross-linking products identified during membrane protein biosynthesis. J Biol Chem. 1997 Jan 17;272(3):1983–1989. doi: 10.1074/jbc.272.3.1983. [DOI] [PubMed] [Google Scholar]
- Martoglio B., Hofmann M. W., Brunner J., Dobberstein B. The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer. Cell. 1995 Apr 21;81(2):207–214. doi: 10.1016/0092-8674(95)90330-5. [DOI] [PubMed] [Google Scholar]
- Membrane protein biosynthesis - all sewn up? Trends Cell Biol. 1997 May;7(5):206–210. doi: 10.1016/S0962-8924(97)01035-0. [DOI] [PubMed] [Google Scholar]
- Mothes W., Heinrich S. U., Graf R., Nilsson I., von Heijne G., Brunner J., Rapoport T. A. Molecular mechanism of membrane protein integration into the endoplasmic reticulum. Cell. 1997 May 16;89(4):523–533. doi: 10.1016/s0092-8674(00)80234-2. [DOI] [PubMed] [Google Scholar]
- Mothes W., Prehn S., Rapoport T. A. Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane. EMBO J. 1994 Sep 1;13(17):3973–3982. doi: 10.1002/j.1460-2075.1994.tb06713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ng D. T., Brown J. D., Walter P. Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J Cell Biol. 1996 Jul;134(2):269–278. doi: 10.1083/jcb.134.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nilsson I., Whitley P., von Heijne G. The COOH-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase. J Cell Biol. 1994 Sep;126(5):1127–1132. doi: 10.1083/jcb.126.5.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oliver J. D., Hresko R. C., Mueckler M., High S. The glut 1 glucose transporter interacts with calnexin and calreticulin. J Biol Chem. 1996 Jun 7;271(23):13691–13696. doi: 10.1074/jbc.271.23.13691. [DOI] [PubMed] [Google Scholar]
- Oliver J., Jungnickel B., Görlich D., Rapoport T., High S. The Sec61 complex is essential for the insertion of proteins into the membrane of the endoplasmic reticulum. FEBS Lett. 1995 Apr 3;362(2):126–130. doi: 10.1016/0014-5793(95)00223-v. [DOI] [PubMed] [Google Scholar]
- Rapoport T. A., Jungnickel B., Kutay U. Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes. Annu Rev Biochem. 1996;65:271–303. doi: 10.1146/annurev.bi.65.070196.001415. [DOI] [PubMed] [Google Scholar]
- Stirling C. J., Rothblatt J., Hosobuchi M., Deshaies R., Schekman R. Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum. Mol Biol Cell. 1992 Feb;3(2):129–142. doi: 10.1091/mbc.3.2.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stueber D., Ibrahimi I., Cutler D., Dobberstein B., Bujard H. A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences. EMBO J. 1984 Dec 20;3(13):3143–3148. doi: 10.1002/j.1460-2075.1984.tb02271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ulbrandt N. D., Newitt J. A., Bernstein H. D. The E. coli signal recognition particle is required for the insertion of a subset of inner membrane proteins. Cell. 1997 Jan 24;88(2):187–196. doi: 10.1016/s0092-8674(00)81839-5. [DOI] [PubMed] [Google Scholar]
- Valent Q. A., Kendall D. A., High S., Kusters R., Oudega B., Luirink J. Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides. EMBO J. 1995 Nov 15;14(22):5494–5505. doi: 10.1002/j.1460-2075.1995.tb00236.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Voigt S., Jungnickel B., Hartmann E., Rapoport T. A. Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane. J Cell Biol. 1996 Jul;134(1):25–35. doi: 10.1083/jcb.134.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walter P., Johnson A. E. Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu Rev Cell Biol. 1994;10:87–119. doi: 10.1146/annurev.cb.10.110194.000511. [DOI] [PubMed] [Google Scholar]
- Warren G., Wickner W. Organelle inheritance. Cell. 1996 Feb 9;84(3):395–400. doi: 10.1016/s0092-8674(00)81284-2. [DOI] [PubMed] [Google Scholar]
- Wilkinson B. M., Critchley A. J., Stirling C. J. Determination of the transmembrane topology of yeast Sec61p, an essential component of the endoplasmic reticulum translocation complex. J Biol Chem. 1996 Oct 11;271(41):25590–25597. doi: 10.1074/jbc.271.41.25590. [DOI] [PubMed] [Google Scholar]
- de Gier J. W., Mansournia P., Valent Q. A., Phillips G. J., Luirink J., von Heijne G. Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle. FEBS Lett. 1996 Dec 16;399(3):307–309. doi: 10.1016/s0014-5793(96)01354-3. [DOI] [PubMed] [Google Scholar]