Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Nov 1;109(5):2013–2022. doi: 10.1083/jcb.109.5.2013

Structural requirements for membrane assembly of proteins spanning the membrane several times

PMCID: PMC2115853  PMID: 2808519

Abstract

We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the opposite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Audigier Y., Friedlander M., Blobel G. Multiple topogenic sequences in bovine opsin. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5783–5787. doi: 10.1073/pnas.84.16.5783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bause E. Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem J. 1983 Feb 1;209(2):331–336. doi: 10.1042/bj2090331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blanck A., Oesterhelt D. The halo-opsin gene. II. Sequence, primary structure of halorhodopsin and comparison with bacteriorhodopsin. EMBO J. 1987 Jan;6(1):265–273. doi: 10.1002/j.1460-2075.1987.tb04749.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boyd D., Manoil C., Beckwith J. Determinants of membrane protein topology. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8525–8529. doi: 10.1073/pnas.84.23.8525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brégégère F., Abastado J. P., Kvist S., Rask L., Lalanne J. L., Garoff H., Cami B., Wiman K., Larhammar D., Peterson P. A. Structure of C-terminal half of two H-2 antigens from cloned mRNA. Nature. 1981 Jul 2;292(5818):78–81. doi: 10.1038/292078a0. [DOI] [PubMed] [Google Scholar]
  7. Claesson L., Larhammar D., Rask L., Peterson P. A. cDNA clone for the human invariant gamma chain of class II histocompatibility antigens and its implications for the protein structure. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7395–7399. doi: 10.1073/pnas.80.24.7395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coleman J., Inukai M., Inouye M. Dual functions of the signal peptide in protein transfer across the membrane. Cell. 1985 Nov;43(1):351–360. doi: 10.1016/0092-8674(85)90040-6. [DOI] [PubMed] [Google Scholar]
  9. Cutler D. F., Melancon P., Garoff H. Mutants of the membrane-binding region of Semliki Forest virus E2 protein. II. Topology and membrane binding. J Cell Biol. 1986 Mar;102(3):902–910. doi: 10.1083/jcb.102.3.902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dobberstein B., Kvist S., Roberts L. Structure and biosynthesis of histocompatibility antigens (H-2, HLA). Philos Trans R Soc Lond B Biol Sci. 1982 Dec 24;300(1099):161–172. doi: 10.1098/rstb.1982.0163. [DOI] [PubMed] [Google Scholar]
  11. Friedlander M., Blobel G. Bovine opsin has more than one signal sequence. 1985 Nov 28-Dec 4Nature. 318(6044):338–343. doi: 10.1038/318338a0. [DOI] [PubMed] [Google Scholar]
  12. Garcia P. D., Ou J. H., Rutter W. J., Walter P. Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm. J Cell Biol. 1988 Apr;106(4):1093–1104. doi: 10.1083/jcb.106.4.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haeuptle M. T., Flint N., Gough N. M., Dobberstein B. A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane. J Cell Biol. 1989 Apr;108(4):1227–1236. doi: 10.1083/jcb.108.4.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haeuptle M. T., Frank R., Dobberstein B. Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length. Nucleic Acids Res. 1986 Feb 11;14(3):1427–1448. doi: 10.1093/nar/14.3.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaufman J. F., Strominger J. L. HLA-DR light chain has a polymorphic N-terminal region and a conserved immunoglobulin-like C-terminal region. Nature. 1982 Jun 24;297(5868):694–697. doi: 10.1038/297694a0. [DOI] [PubMed] [Google Scholar]
  16. Kopito R. R., Lodish H. F. Primary structure and transmembrane orientation of the murine anion exchange protein. Nature. 1985 Jul 18;316(6025):234–238. doi: 10.1038/316234a0. [DOI] [PubMed] [Google Scholar]
  17. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lau J. T., Welply J. K., Shenbagamurthi P., Naider F., Lennarz W. J. Substrate recognition by oligosaccharyl transferase. Inhibition of co-translational glycosylation by acceptor peptides. J Biol Chem. 1983 Dec 25;258(24):15255–15260. [PubMed] [Google Scholar]
  20. Lipp J., Dobberstein B. Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT. J Cell Biol. 1988 Jun;106(6):1813–1820. doi: 10.1083/jcb.106.6.1813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lipp J., Dobberstein B. The membrane-spanning segment of invariant chain (I gamma) contains a potentially cleavable signal sequence. Cell. 1986 Sep 26;46(7):1103–1112. doi: 10.1016/0092-8674(86)90710-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mize N. K., Andrews D. W., Lingappa V. R. A stop transfer sequence recognizes receptors for nascent chain translocation across the endoplasmic reticulum membrane. Cell. 1986 Dec 5;47(5):711–719. doi: 10.1016/0092-8674(86)90514-3. [DOI] [PubMed] [Google Scholar]
  23. Nathans J., Thomas D., Hogness D. S. Molecular genetics of human color vision: the genes encoding blue, green, and red pigments. Science. 1986 Apr 11;232(4747):193–202. doi: 10.1126/science.2937147. [DOI] [PubMed] [Google Scholar]
  24. Palmiter R. D., Gagnon J., Walsh K. A. Ovalbumin: a secreted protein without a transient hydrophobic leader sequence. Proc Natl Acad Sci U S A. 1978 Jan;75(1):94–98. doi: 10.1073/pnas.75.1.94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pelham H. R. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell. 1986 Sep 26;46(7):959–961. doi: 10.1016/0092-8674(86)90693-8. [DOI] [PubMed] [Google Scholar]
  26. Rose J. K., Welch W. J., Sefton B. M., Esch F. S., Ling N. C. Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3884–3888. doi: 10.1073/pnas.77.7.3884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rothman R. E., Andrews D. W., Calayag M. C., Lingappa V. R. Construction of defined polytopic integral transmembrane proteins. The role of signal and stop transfer sequence permutations. J Biol Chem. 1988 Jul 25;263(21):10470–10480. [PubMed] [Google Scholar]
  28. 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]
  29. Szczesna-Skorupa E., Browne N., Mead D., Kemper B. Positive charges at the NH2 terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide. Proc Natl Acad Sci U S A. 1988 Feb;85(3):738–742. doi: 10.1073/pnas.85.3.738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Toyn J., Hibbs A. R., Sanz P., Crowe J., Meyer D. I. In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation. EMBO J. 1988 Dec 20;7(13):4347–4353. doi: 10.1002/j.1460-2075.1988.tb03333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Trun N. J., Silhavy T. J. PrlC, a suppressor of signal sequence mutations in Escherichia coli, can direct the insertion of the signal sequence into the membrane. J Mol Biol. 1989 Feb 20;205(4):665–676. doi: 10.1016/0022-2836(89)90312-4. [DOI] [PubMed] [Google Scholar]
  32. Wessels H. P., Spiess M. Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence. Cell. 1988 Oct 7;55(1):61–70. doi: 10.1016/0092-8674(88)90009-8. [DOI] [PubMed] [Google Scholar]
  33. Wiedmann M., Kurzchalia T. V., Hartmann E., Rapoport T. A. A signal sequence receptor in the endoplasmic reticulum membrane. 1987 Aug 27-Sep 2Nature. 328(6133):830–833. doi: 10.1038/328830a0. [DOI] [PubMed] [Google Scholar]
  34. Yost C. S., Hedgpeth J., Lingappa V. R. A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems. Cell. 1983 Oct;34(3):759–766. doi: 10.1016/0092-8674(83)90532-9. [DOI] [PubMed] [Google Scholar]
  35. Zerial M., Huylebroeck D., Garoff H. Foreign transmembrane peptides replacing the internal signal sequence of transferrin receptor allow its translocation and membrane binding. Cell. 1987 Jan 16;48(1):147–155. doi: 10.1016/0092-8674(87)90365-5. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES