Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a ‘biological’ hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein–lipid interactions are critical during translocon-mediated membrane insertion.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. Predicting transmembrane protein topology with a hidden Markov model. Application to complete genomes. J. Mol. Biol. 305, 567–580 (2001)
von Heijne, G. Recent advances in the understanding of membrane protein assembly and structure. Q. Rev. Biophys. 32, 285–307 (2000)
von Heijne, G. Membrane protein assembly in vivo. Adv. Protein Chem. 63, 1–18 (2003)
Snapp, E., Reinhart, G., Bogert, B., Lippincott-Schwartz, J. & Hegde, R. The organization of engaged and quiescent translocons in the endoplasmic reticulum of mammalian cells. J. Cell Biol. 164, 997–1007 (2004)
Rapoport, T. A., Goder, V., Heinrich, S. U. & Matlack, K. E. Membrane-protein integration and the role of the translocation channel. Trends Cell Biol. 14, 568–575 (2004)
Alder, N. N. & Johnson, A. E. Cotranslational membrane protein biogenesis at the endoplasmic reticulum. J. Biol. Chem. 279, 22787–22790 (2004)
van den Berg, B. et al. X-ray structure of a protein-conducting channel. Nature 427, 36–44 (2004)
Woolhead, C. A., McCormick, P. J. & Johnson, A. E. Nascent membrane and secretory proteins differ in FRET-detected folding. Cell 116, 725–736 (2004)
de Planque, M. R. R. & Killian, J. A. Protein-lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring. Mol. Membr. Biol. 20, 271–284 (2003)
White, S. H. & Wimley, W. C. Membrane protein folding and stability: Physical principles. Annu. Rev. Biophys. Biomol. Struct. 28, 319–365 (1999)
Ulmschneider, M. B. & Sansom, M. S. P. Amino acid distributions in integral membrane protein structures. Biochim. Biophys. Acta 1512, 1–14 (2001)
Beuming, T. & Weinstein, H. A knowledge-based scale for the analysis and prediction of buried and exposed faces of transmembrane domain proteins. Bioinformatics 20, 1822–1835 (2004)
Sääf, A., Wallin, E. & von Heijne, G. Stop-transfer function of pseudo-random amino acid segments during translocation across prokaryotic and eukaryotic membranes. Eur. J. Biochem. 251, 821–829 (1998)
Wimley, W. C., Creamer, T. P. & White, S. H. Solvation energies of amino acid sidechains and backbone in a family of host-guest pentapeptides. Biochemistry 35, 5109–5124 (1996)
Jayasinghe, S., Hristova, K. & White, S. H. Energetics, stability, and prediction of transmembrane helices. J. Mol. Biol. 312, 927–934 (2001)
Cornette, J. L. et al. Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins. J. Mol. Biol. 195, 659–685 (1987)
Degli Esposti, M., Crimi, M. & Venturoli, G. A critical evaluation of the hydropathy profile of membrane proteins. Eur. J. Biochem. 190, 207–219 (1990)
Heinrich, S., Mothes, W., Brunner, J. & Rapoport, T. The Sec61p complex mediates the integration of a membrane protein by allowing lipid partitioning of the transmembrane domain. Cell 102, 233–244 (2000)
Lu, L. P. & Deber, C. M. Guidelines for membrane protein engineering derived from de novo designed model peptides. Biopolymers 47, 41–62 (1998)
Bechinger, B. Membrane insertion and orientation of polyalanine peptides: A N-15 solid-state NMR spectroscopy investigation. Biophys. J. 81, 2251–2256 (2001)
Lewis, R. N. et al. A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers. Biochemistry 40, 12103–12111 (2001)
Wallin, E., Tsukihara, T., Yoshikawa, S., von Heijne, G. & Elofsson, A. Architecture of helix bundle membrane proteins: An analysis of cytochrome c oxidase from bovine mitochondria. Protein Sci. 6, 808–815 (1997)
Killian, J. A. & von Heijne, G. How proteins adapt to a membrane-water interface. Trends Biochem. Sci. 25, 429–434 (2000)
Yau, W. M., Wimley, W. C., Gawrisch, K. & White, S. H. The preference of tryptophan for membrane interfaces. Biochemistry 37, 14713–14718 (1998)
Wimley, W. C. & White, S. H. Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nature Struct. Biol. 3, 842–848 (1996)
Eisenberg, D., Schwarz, E., Komaromy, M. & Wall, R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J. Mol. Biol. 179, 125–142 (1984)
Plath, K., Mothes, W., Wilkinson, B. M., Stirling, C. J. & Rapoport, T. A. Signal sequence recognition in posttranslational protein transport across the yeast ER membrane. Cell 94, 795–807 (1998)
McCormick, P. J., Miao, Y., Shao, Y., Lin, J. & Johnson, A. E. Cotranslational protein integration into the ER membrane is mediated by the binding of nascent chains to translocon proteins. Mol. Cell 12, 329–341 (2003)
Presta, L. G. & Rose, G. D. Helix signals in proteins. Science 240, 1632–1641 (1988)
Richardson, J. S. & Richardson, D. C. Amino acid preferences for specific locations at the ends of α-helices. Science 240, 1648–1652 (1988)
Yohannan, S. et al. Proline substitutions are not easily accommodated in a membrane protein. J. Mol. Biol. 341, 1–6 (2004)
Kuroiwa, T., Sakaguchi, M., Mihara, K. & Omura, T. Systematic analysis of stop-transfer sequence for microsomal membrane. J. Biol. Chem. 266, 9251–9255 (1991)
Anthony, V. & Skach, W. R. Molecular mechanism of P-glycoprotein assembly into cellular membranes. Curr. Protein Pept. Sci. 3, 485–501 (2002)
Kozak, M. Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187–208 (1999)
Liljeström, P. & Garoff, H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology 9, 1356–1361 (1991)
Liljeström, P., Lusa, S., Huylebroeck, D. & Garoff, H. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J. Virol. 65, 4107–4113 (1991)
We wish to thank E. Missioux for technical assistance and R. MacKinnon, D. Rees, and T. Rapoport for comments. This work was supported by grants from the Swedish Cancer Foundation to G.v.H. and I.M.N., the Marianne and Marcus Wallenberg Foundation and the Swedish Research Council to G.v.H., the Magnus Bergvall Foundation to I.M.N., and the National Institute of General Medical Sciences to S.H.W.
The authors declare that they have no competing financial interests.
About this article
Cite this article
Hessa, T., Kim, H., Bihlmaier, K. et al. Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433, 377–381 (2005). https://doi.org/10.1038/nature03216
Proceedings of the National Academy of Sciences (2020)
Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite
The FEBS Journal (2020)
Science Advances (2020)
Single‐Molecule Study of Peptides with the Same Amino Acid Composition but Different Sequences by Using an Aerolysin Nanopore