The Protein Journal

, Volume 38, Issue 3, pp 306–316 | Cite as

Membrane Protein Integration and Topogenesis at the ER

  • Martin SpiessEmail author
  • Tina Junne
  • Marco Janoschke


Most membrane proteins are composed of hydrophobic α-helical transmembrane segments and are integrated into the lipid bilayer of the endoplasmic reticulum by the highly conserved Sec61 translocon. With respect to the integration mechanism, three types of transmembrane segments can be distinguished—the signal, the stop-transfer sequence, and the re-integration sequence—which in linear succession can account for all kinds of membrane protein topologies. The transmembrane orientation of the initial signal and to a weaker extent also of downstream transmembrane segments is affected by charged flanking residues according to the so-called positive-inside rule. The main driving force for transmembrane integration is hydrophobicity. Systematic analysis suggested thermodynamic equilibration of each peptide segment in the translocon with the membrane as the underlying mechanism. However, there is evidence that integration is not entirely sequence-autonomous, but depends also on the sequence context, from very closely spaced transmembrane segments to the folding state and properties of neighboring sequences. Topogenesis is even influenced by accessory proteins that appear to act as intramembrane chaperones.


Sec61 Signal sequence Translocon Transmembrane helix 



Endoplasmic reticulum


Cytoplasmic N- and exoplasmic C-terminus


Exoplasmic N- and cytoplasmic C-terminus




Translocating chain-associated membrane protein



Our work was supported by Grant No. 31003A-182519 from the Swiss National Science Foundation.


  1. 1.
    Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132. CrossRefGoogle Scholar
  2. 2.
    Wimley WC (2002) Toward genomic identification of beta-barrel membrane proteins: composition and architecture of known structures. Protein Sci 11:301–312. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Schiffrin B, Brockwell DJ, Radford SE (2017) Outer membrane protein folding from an energy landscape perspective. BMC Biol 15:123. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Gruss F, Zähringer F, Jakob RP et al (2013) The structural basis of autotransporter translocation by TamA. Nat Struct Mol Biol 20:1318–1320. CrossRefPubMedGoogle Scholar
  5. 5.
    Höhr AIC, Lindau C, Wirth C et al (2018) Membrane protein insertion through a mitochondrial β-barrel gate. Science 359:eaah6834. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Park E, Ménétret J-F, Gumbart JC et al (2014) Structure of the SecY channel during initiation of protein translocation. Nature 506:102–106. CrossRefPubMedGoogle Scholar
  7. 7.
    Gogala M, Becker T, Beatrix B et al (2014) Structures of the Sec61 complex engaged in nascent peptide translocation or membrane insertion. Nature 506:107–110. CrossRefPubMedGoogle Scholar
  8. 8.
    Voorhees RM, Hegde RS (2016) Structure of the Sec61 channel opened by a signal sequence. Science 351:88–91. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    MacKinnon AL, Paavilainen VO, Sharma A et al (2014) An allosteric Sec61 inhibitor traps nascent transmembrane helices at the lateral gate. Elife 3:e01483. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Junne T, Wong J, Studer C et al (2015) Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon. J Cell Sci 128:1217–1229. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Paatero AO, Kellosalo J, Dunyak BM et al (2016) Apratoxin kills cells by direct blockade of the Sec61 protein translocation channel. Cell Chem Biol 23:561–566. CrossRefPubMedGoogle Scholar
  12. 12.
    Blobel G, Dobberstein B (1975) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 67:835–851. CrossRefPubMedGoogle Scholar
  13. 13.
    Blobel G (1980) Intracellular protein topogenesis. Proc Natl Acad Sci USA 77:1496–1500CrossRefPubMedGoogle Scholar
  14. 14.
    von Heijne G (1990) The signal peptide. J Membr Biol 115:195–201CrossRefGoogle Scholar
  15. 15.
    Heijne G (1986) The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBO J 5:3021–3027CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Nilsson J, Persson B, von Heijne G (2005) Comparative analysis of amino acid distributions in integral membrane proteins from 107 genomes. Proteins 60:606–616. CrossRefPubMedGoogle Scholar
  17. 17.
    Hartmann E, Rapoport TA, Lodish HF (1989) Predicting the orientation of eukaryotic membrane-spanning proteins. Proc Natl Acad Sci USA 86:5786–5790CrossRefPubMedGoogle Scholar
  18. 18.
    Szczesna-Skorupa E, Kemper B (1989) NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2. J Cell Biol 108:1237–1243CrossRefPubMedGoogle Scholar
  19. 19.
    Beltzer JP, Fiedler K, Fuhrer C et al (1991) Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence. J Biol Chem 266:973–978PubMedGoogle Scholar
  20. 20.
    Parks GD, Lamb RA (1991) Topology of eukaryotic type II membrane proteins: importance of N-terminal positively charged residues flanking the hydrophobic domain. Cell 64:777–787. CrossRefPubMedGoogle Scholar
  21. 21.
    Denzer AJ, Nabholz CE, Spiess M (1995) Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain. EMBO J 14:6311–6317CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Sakaguchi M, Tomiyoshi R, Kuroiwa T et al (1992) Functions of signal and signal-anchor sequences are determined by the balance between the hydrophobic segment and the N-terminal charge. Proc Natl Acad Sci USA 89:16–19. CrossRefPubMedGoogle Scholar
  23. 23.
    Wahlberg JM, Spiess M (1997) Multiple determinants direct the orientation of signal-anchor proteins: the topogenic role of the hydrophobic signal domain. 137:555–562Google Scholar
  24. 24.
    Rösch K, Naeher D, Laird V et al (2000) The topogenic contribution of uncharged amino acids on signal sequence orientation in the endoplasmic reticulum. J Biol Chem 275:14916–14922. CrossRefPubMedGoogle Scholar
  25. 25.
    Goder V, Spiess M (2003) Molecular mechanism of signal sequence orientation in the endoplasmic reticulum. EMBO J 22:3645–3653. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Devaraneni PK, Conti B, Matsumura Y et al (2011) Stepwise insertion and inversion of a type II signal anchor sequence in the ribosome-Sec61 translocon complex. Cell 146:134–147. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Kocik L, Junne T, Spiess M (2012) Orientation of internal signal-anchor sequences at the Sec61 translocon. J Mol Biol 424:368–378. CrossRefPubMedGoogle Scholar
  28. 28.
    McKenna M, Simmonds RE, High S (2017) Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis. J Cell Sci 130:1307–1320. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Goder V, Junne T, Spiess M (2004) Sec61p contributes to signal sequence orientation according to the positive-inside rule. Mol Biol Cell 15:1470–1478. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Junne T, Schwede T, Goder V, Spiess M (2007) Mutations in the Sec61p channel affecting signal sequence recognition and membrane protein topology. J Biol Chem 282:33201–33209. CrossRefPubMedGoogle Scholar
  31. 31.
    Emr SD, Hanley-Way S, Silhavy TJ (1981) Suppressor mutations that restore export of a protein with a defective signal sequence. Cell 23:79–88CrossRefPubMedGoogle Scholar
  32. 32.
    Tam PCK, Maillard AP, Chan KKY, Duong F (2005) Investigating the SecY plug movement at the SecYEG translocation channel. EMBO J 24:3380–3388. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Saparov SM, Erlandson K, Cannon K et al (2007) Determining the conductance of the SecY protein translocation channel for small molecules. Mol Cell 26:501–509. CrossRefPubMedGoogle Scholar
  34. 34.
    Flower AM (2007) The SecY translocation complex: convergence of genetics and structure. Trends Microbiol. CrossRefPubMedGoogle Scholar
  35. 35.
    Audigier Y, Friedlander M, Blobel G (1987) Multiple topogenic sequences in bovine opsin. Proc Natl Acad Sci USA 84:5783–5787. CrossRefPubMedGoogle Scholar
  36. 36.
    Wessels HP, Spiess M (1988) Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence. Cell 55:61–70CrossRefPubMedGoogle Scholar
  37. 37.
    Lipp J, Flint N, Haeuptle MT, Dobberstein B (1989) Structural requirements for membrane assembly of proteins spanning the membrane several times. J Cell Biol 109:2013–2022. CrossRefPubMedGoogle Scholar
  38. 38.
    Sato M, Hresko R, Mueckler M (1998) Testing the charge difference hypothesis for the assembly of a eucaryotic multispanning membrane protein. J Biol Chem 273:25203–25208. CrossRefPubMedGoogle Scholar
  39. 39.
    Sato M, Mueckler M (1999) A conserved amino acid motif (R-X-G-R-R) in the Glut1 glucose transporter is an important determinant of membrane topology. J Biol Chem 274:24721–24725. CrossRefPubMedGoogle Scholar
  40. 40.
    Gafvelin G, von Heijne G (1994) Topological “frustration” in multispanning E. coli inner membrane proteins. Cell 77:401–412. CrossRefPubMedGoogle Scholar
  41. 41.
    Goder V, Bieri C, Spiess M (1999) Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon. J Cell Biol 147:257–266CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Tu L, Khanna P, Deutsch C (2014) Transmembrane segments form tertiary hairpins in the folding vestibule of the ribosome. J Mol Biol 426:185–198. CrossRefPubMedGoogle Scholar
  43. 43.
    Hessa T, Kim H, Bihlmaier K et al (2005) Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433:377–381. CrossRefPubMedGoogle Scholar
  44. 44.
    Hessa T, Meindl-Beinker NM, Bernsel A et al (2007) Molecular code for transmembrane-helix recognition by the Sec61 translocon. Nature 450:1026–1030. CrossRefPubMedGoogle Scholar
  45. 45.
    Hedin LE, Ojemalm K, Bernsel A et al (2010) Membrane insertion of marginally hydrophobic transmembrane helices depends on sequence context. J Mol Biol 396:221–229. CrossRefPubMedGoogle Scholar
  46. 46.
    MacCallum JL, Tieleman DP (2011) Hydrophobicity scales: a thermodynamic looking glass into lipid-protein interactions. Trends Biochem Sci 36:653–662. CrossRefPubMedGoogle Scholar
  47. 47.
    Schow EV, Freites JA, Cheng P et al (2011) Arginine in membranes: the connection between molecular dynamics simulations and translocon-mediated insertion experiments. J Membr Biol 239:35–48. CrossRefPubMedGoogle Scholar
  48. 48.
    Gumbart JC, Teo I, Roux B, Schulten K (2013) Reconciling the roles of kinetic and thermodynamic factors in membrane-protein insertion. J Am Chem Soc 135:2291–2297. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Junne T, Kocik L, Spiess M (2010) The hydrophobic core of the Sec61 translocon defines the hydrophobicity threshold for membrane integration. Mol Biol Cell 21:1662–1670. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Demirci E, Junne T, Baday S et al (2013) Functional asymmetry within the Sec61p translocon. Proc Natl Acad Sci USA 110:18856–18861. CrossRefPubMedGoogle Scholar
  51. 51.
    Ismail N, Hedman R, Schiller N, von Heijne G (2012) A biphasic pulling force acts on transmembrane helices during translocon-mediated membrane integration. Nat Struct Mol Biol 19:1018–1022. CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Junne T, Spiess M (2017) Integration of transmembrane domains is regulated by their downstream sequences. J Cell Sci 130:372–381. CrossRefPubMedGoogle Scholar
  53. 53.
    Lundin C, Kim H, Nilsson I et al (2008) Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N(in)-C(out) and N(out)-C(in) transmembrane helices. Proc Natl Acad Sci USA 105:15702–15707. CrossRefPubMedGoogle Scholar
  54. 54.
    Cymer F, Ismail N, von Heijne G (2014) Weak pulling forces exerted on Nin-orientated transmembrane segments during co-translational insertion into the inner membrane of Escherichia coli. FEBS Lett 588:1930–1934. CrossRefPubMedGoogle Scholar
  55. 55.
    Deshaies RJ, Sanders SL, Feldheim DA, Schekman R (1991) Assembly of yeast Sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex. Nature 349:806–808. CrossRefPubMedGoogle Scholar
  56. 56.
    Panzner S, Dreier L, Hartmann E et al (1995) Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p. Cell 81:561–570CrossRefPubMedGoogle Scholar
  57. 57.
    Itskanov S, Park E (2019) Structure of the posttranslational Sec protein-translocation channel complex from yeast. Science 363:84–87. CrossRefPubMedGoogle Scholar
  58. 58.
    Wu X, Cabanos C, Rapoport TA (2019) Structure of the post-translational protein translocation machinery of the ER membrane. Nature 566:136–139. CrossRefPubMedGoogle Scholar
  59. 59.
    Brodsky JL, Goeckeler J, Schekman R (1995) BiP and Sec63p are required for both co- and posttranslational protein translocation into the yeast endoplasmic reticulum. Proc Natl Acad Sci USA 92:9643–9646CrossRefPubMedGoogle Scholar
  60. 60.
    Young BP, Craven RA, Reid PJ et al (2001) Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo. EMBO J 20:262–271. CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Lang S, Benedix J, Fedeles SV et al (2012) Different effects of Sec61α, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells. J Cell Sci 125:1958–1969. CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Voigt S, Jungnickel B, Hartmann E, Rapoport TA (1996) Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane. J Cell Biol 134:25–35CrossRefPubMedGoogle Scholar
  63. 63.
    Chen Q, Denard B, Lee C-E et al (2016) Inverting the topology of a transmembrane protein by regulating the translocation of the first transmembrane helix. Mol Cell 63:567–578. CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Jonikas MC, Collins SR, Denic V et al (2009) Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 323:1693–1697. CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Chitwood PJ, Juszkiewicz S, Guna A et al (2018) EMC is required to initiate accurate membrane protein topogenesis. Cell 175:1507–1519.e16. CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Shurtleff MJ, Itzhak DN, Hussmann JA et al (2018) The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins. Elife 7:382. CrossRefGoogle Scholar
  67. 67.
    van den Berg B, Clemons WM, Collinson I et al (2004) X-ray structure of a protein-conducting channel. Nature 427:36–44. CrossRefPubMedGoogle Scholar
  68. 68.
    Sommer N, Junne T, Kalies K-U et al (2013) TRAP assists membrane protein topogenesis at the mammalian ER membrane. Biochim Biophys Acta 1833:3104–3111. CrossRefPubMedGoogle Scholar
  69. 69.
    Hessa T, Reithinger JH, von Heijne G, Kim H (2009) Analysis of transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae. J Mol Biol 386:1222–1228CrossRefPubMedGoogle Scholar
  70. 70.
    Xie K, Hessa T, Seppälä S et al (2007) Features of transmembrane segments that promote the lateral release from the translocase into the lipid phase. Biochemistry 46:15153–15161. CrossRefPubMedGoogle Scholar
  71. 71.
    Ojemalm K, Botelho SC, Stüdle C, von Heijne G (2013) Quantitative analysis of SecYEG-mediated insertion of transmembrane α-helices into the bacterial inner membrane. J Mol Biol 425:2813–2822. CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.BiozentrumUniversity of BaselBaselSwitzerland

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