Signal Peptide Interactions during ER Translocation: Comparison
Please note this is a comparison between Version 2 by Lindsay Dong and Version 1 by Friedrich Förster.

Cleavable endoplasmic reticulum (ER) signal peptides (SPs) and other non-cleavable signal sequences target roughly a quarter of the human proteome to the ER. These short peptides, mostly located at the N-termini of proteins, are highly diverse. For most proteins targeted to the ER, it is the interactions between the signal sequences and the various ER targeting and translocation machineries such as the signal recognition particle (SRP), the protein-conducting channel Sec61, and the signal peptidase complex (SPC) that determine the proteins’ target location and provide translocation fidelity. 

  • signal peptide
  • signal peptidase
  • ER translocon
  • endoplasmic reticulum

1. Introduction

The secretory pathway is a protein trafficking highway utilized by more than a quarter of the human proteome [1,2][1][2]. Soluble secreted proteins such as antibodies and protein hormones rely on this pathway. The pathway also delivers transmembrane proteins (TMPs) to the endoplasmic reticulum (ER), its downstream organelles such as the Golgi apparatus, and the plasma membrane.
All secretory proteins are translated by cytosolic ribosomes and must be first targeted to and then transported across (or inserted into) the ER membrane at the early stage of their life, either co- or post-translationally [3,4][3][4]. A complex network of cytosolic and ER membrane-resident macromolecules facilitate and assist the ER targeting and translocation.
Both ER targeting and translocation/insertion critically depend on so-called signal sequences (SSs), short hydrophobic peptide stretches in the amino acid sequence of the newly synthesized proteins that are recognized by the secretory machinery as trafficking signals. While SSs may appear exceedingly simple, they possess a remarkably versatile and complex physiology. Besides the choice of trafficking routes, SSs carry information about translocation efficiency, occurrence and timing of cleavage, and post-targeting functions.
There are four main classes of SSs: (i) cleavable signal peptides (SPs), found on secreted proteins such as insulin and type I membrane proteins such as HLA molecules; (ii) type II signal anchor sequences (SASs), found on single- and multi-pass transmembrane proteins (TMPs) such as the membrane-bound form of tumor necrosis factor (TNF); (iii) type III SASs found, e.g., on Sec61β; and (iv) tail anchors (TAs) found on proteins such as Sec61γ (Figure 1a). Signal peptides (SPs) are by far the most populous class of SSs. In humans alone, there are an estimated >3000 different SP-containing proteins, constituting >10% of the whole proteome. SPs are usually localized within the first 30 amino acids of the coding sequence but can in some cases also be found more internally. The defining trait of SPs is the capacity to be cleaved by the aptly named signal peptidase complex (SPC).
The primary sequence of SPs is only loosely defined. In fact, approximately 20% of randomized sequences can promote protein secretion in yeast [6][5]. SPs are characterized by a tripartite structure (Figure 1b–d): (i) an often positively charged, N-terminal ‘n-region’ that faces the cytosol; (ii) a short hydrophobic core—most commonly between 7 and 15, but not longer than 18–20 amino acids called ‘h-region’; and (iii) a polar luminal C-terminal ‘c-region’ that contains the scissile bond and must be occupied by short, hydrophobic residues at positions −1 and −3 relative to the cleavage site [7,8][6][7]. Initially, SPs are inserted into the ER membrane with the N-terminus facing towards the cytosol (Nin) and the mature sequence facing the organellar lumen (Cout). In the case of type I TMPs, the removal of the SP leads to an ‘inverted’ topology of the mature sequence in which the N-terminus is facing ‘outside’ (Nout), while the C-terminus is facing the cytosol (Cin) (Figure 1a).
Figure 1. Types of signal sequences. (a) Depiction of the four types of SSs with their membrane topology indicated. Hydrophobic segments are depicted in magenta. (b) Signal peptides have a tripartite structure, consisting of an n- (cyan), h- (magenta), and c-region (yellow) and are cleaved in the ER lumen by the SPC (green flash). (c) Frequency of residue types relative to the cleavage site [8]. (d) Relative length of the respective regions (colored as in (b)) as a function of total SP length. The bulk of the length variation stems from the n-region. Panels c–d were adapted from [8].

2. SP Recognition in the Cytosol and ER Targeting

As the first step of their life cycle, secreted proteins and TMPs must be targeted to the ER membrane (Figure 2). The timing of translation, folding, and ER transport is of particular importance: on one hand, nascent chains (NCs) can only cross the ER membrane in an unfolded state, while on the other hand, this prerequisite exposes their hydrophobic segments and makes them prone to aggregation and proteolysis. Therefore, these proteins must be shielded from the hydrophilic cytosol, which is achieved by one of two separate strategies: (i) direct recognition of the nascent protein at the ribosomal exit tunnel by the SRP, leading to co-translational translocation/insertion through the recruitment of the ribosome–nascent chain complex (RNC) to the ER; or (ii) post-translational transport to the ER, which requires the involvement of chaperones to protect the clients from the aqueous environment.
Figure 2. ER delivery pathways for SP-containing proteins. The upper panel shows a schematic of each delivery pathway. The central component of each pathway is underscored. Left: SRP (blue/brown subunits) recognizes SPs emerging from the ribosomal exit tunnel and shields them through the SRP54 M-domain. SP binding triggers the heterodimerization of the SRP54 NG domain (blue) with that of SRα (green), guiding the RNC to the ER. A large conformational rearrangement partially exposes the SP for handover to Sec61 [9]. Middle: Cytosolic calmodulin or Hsp40-assisted Hsc70 recognize SP-containing proteins and guide them to the ER. Right: The recently discovered Snd pathway likely consists of a cytosolic component, Snd1, which might act as a chaperone, and two ER membrane-resident components, Snd2/3, which might facilitate handover to Sec61 in some unknown way [10][11]. Lower panels show specifics of each pathway.


  1. Palade, G.; Arch, A.; Locke, M.; Locke, E.; Palade, G. Intracellular aspects of the process of protein synthesis. Science 1975, 189, 347–358.
  2. Uhlén, M.; Fagerberg, L.; Hallström, B.M.; Lindskog, C.; Oksvold, P.; Mardinoglu, A.; Sivertsson, Å.; Kampf, C.; Sjöstedt, E.; Asplund, A.; et al. Tissue-based map of the human proteome. Science 2015, 347, 1260419.
  3. Aviram, N.; Schuldiner, M. Targeting and translocation of proteins to the endoplasmic reticulum at a glance. J. Cell Sci. 2017, 130, 4079–4085.
  4. Rapoport, T.A.; Li, L.; Park, E. Structural and mechanistic insights into protein translocation. Annu. Rev. Cell Dev. Biol. 2017, 33, 369–390.
  5. Kaiser, C.A.; Preuss, D.; Grisafi, P.; Botstein, D. Many random sequences functionally replace the secretion signal sequence of yeast invertase. Science 1987, 235, 312–317.
  6. Von Heijne, G. The signal peptide. J. Membr. Biol. 1990, 115, 195–201.
  7. Liaci, A.M.; Förster, F. Empirical Analysis of Eukaryotic ER Signal Peptides; Mendeley Data; Data Archiving and Networked Services (DANS): The Hague, The Netherlands, 2021.
  8. Liaci, A.M.; Steigenberger, B.; Tamara, S.; Telles de Souza, P.C.; Gröllers-Mulderij, M.; Ogrissek, P.; Marrink, S.J.; Scheltema, R.; Förster, F. Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage. Mol. Cell 2021, 81, 3934–3948.
  9. Jomaa, A.; Eitzinger, S.; Zhu, Z.; Chandrasekar, S.; Kobayashi, K.; Shan, S.; Ban, N. Molecular mechanism of cargo recognition and handover by the mammalian signal recognition particle. Cell Rep. 2021, 36, 109350.
  10. Aviram, N.; Ast, T.; Costa, E.A.; Arakel, E.C.; Chartzman, S.G.; Jan, C.H.; Haßdenteufel, S.; Dudek, J.; Jung, M.; Schorr, S.; et al. The SND proteins constitute an alternative targeting route to the endoplasmic reticulum. Nature 2016, 540, 134–138.
  11. Haßdenteufel, S.; Sicking, M.; Schorr, S.; Aviram, N.; Fecher-Trost, C.; Schuldiner, M.; Jung, M.; Zimmermann, R.; Lang, S. hSnd2 protein represents an alternative targeting factor to the endoplasmic reticulum in human cells. FEBS Lett. 2017, 591, 3211–3224.
  12. Gamerdinger, M.; Hanebuth, M.A.; Frickey, T.; Deuerling, E. The principle of antagonism ensures protein targeting specificity at the endoplasmic reticulum. Science 2015, 348, 201–207.
  13. Goder, V.; Crottet, P.; Spiess, M. In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site-specific phosphorylation. EMBO J. 2000, 19, 6704–6712.
  14. Chartron, J.W.; Hunt, K.C.L.; Frydman, J. Cotranslational signal-independent SRP preloading during membrane targeting. Nature 2016, 536, 224–228.
  15. Voorhees, R.M.; Hegde, R.S. Structures of the scanning and engaged states of the mammalian SRP-ribosome complex. eLife 2015, 4, e07975.
  16. Bornemann, T.; Jöckel, J.; Rodnina, M.V.; Wintermeyer, W. Signal sequence-independent membrane targeting of ribosomes containing short nascent peptides within the exit tunnel. Nat. Struct. Mol. Biol. 2008, 15, 494–499.
  17. Denks, K.; Sliwinski, N.; Erichsen, V.; Borodkina, B.; Origi, A.; Koch, H.G. The signal recognition particle contacts uL23 and scans substrate translation inside the ribosomal tunnel. Nat. Microbiol. 2017, 2, 16265.
  18. Jomaa, A.; Boehringer, D.; Leibundgut, M.; Ban, N. Structures of the E. coli translating ribosome with SRP and its receptor and with the translocon. Nat. Commun. 2016, 7, 10471.
  19. Berndt, U.; Oellerer, S.; Zhang, Y.; Johnson, A.E.; Rospert, S. A signal-anchor sequence stimulates signal recognition particle binding to ribosomes from inside the exit tunnel. Proc. Natl. Acad. Sci. USA 2009, 106, 1398–1403.
  20. Flanagan, J.J.; Chen, J.C.; Miao, Y.; Shao, Y.; Lin, J.; Bock, P.E.; Johnson, A.E. Signal recognition particle binds to ribosome-bound signal sequences with fluorescence-detected subnanomolar affinity that does not diminish as the nascent chain lengthens. J. Biol. Chem. 2003, 278, 18628–18637.
  21. Lee, J.H.; Chandrasekar, S.; Chung, S.Y.; Fu, Y.H.H.; Liu, D.; Weiss, S.; Shan, S.O. Sequential activation of human signal recognition particle by the ribosome and signal sequence drives efficient protein targeting. Proc. Natl. Acad. Sci. USA 2018, 115, E5487–E5496.
  22. Von Heijne, G. Signal sequences. The limits of variation. J. Mol. Biol. 1985, 184, 99–105.
  23. Nilsson, I.; Lara, P.; Hessa, T.; Johnson, A.E.; von Heijne, G.; Karamyshev, A.L. The code for directing proteins for translocation across ER membrane: SRP cotranslationally recognizes specific features of a signal sequence. J. Mol. 2015, 427, 1191–1201.
  24. Costa, E.A.; Subramanian, K.; Nunnari, J.; Weissman, J.S. Defining the physiological role of SRP in protein-targeting efficiency and specificity. Science 2018, 359, 689–692.
  25. Ng, D.T.W.; Brown, J.D.; Walter, P. Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J. Cell Biol. 1996, 134, 269–278.
  26. Schorr, S.; Nguyen, D.; Haßdenteufel, S.; Nagaraj, N.; Cavalié, A.; Greiner, M.; Weissgerber, P.; Loi, M.; Paton, A.W.; Paton, J.C.; et al. Identification of signal peptide features for substrate specificity in human Sec62/Sec63-dependent ER protein import. FEBS J. 2020, 287, 4612–4640.
  27. Nguyen, D.; Stutz, R.; Schorr, S.; Lang, S.; Pfeffer, S.; Freeze, H.H.; Förster, F.; Helms, V.; Dudek, J.; Zimmermann, R. Proteomics reveals signal peptide features determining the client specificity in human TRAP-dependent ER protein import. Nat. Commun. 2018, 9, 3765.
  28. Hegde, R.S.; Bernstein, H.D. The surprising complexity of signal sequences. Trends Biochem. Sci. 2006, 31, 563–571.
  29. Rüdiger, S.; Germeroth, L.; Schneider-Mergener, J.; Bukau, B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 1997, 16, 1501–1507.
  30. Tripathi, A.; Mandon, E.C.; Gilmore, R.; Rapoport, T.A. Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins. J. Biol. Chem. 2017, 292, 8007–8018.
  31. Ngosuwan, J.; Wang, N.M.; Fung, K.L.; Chirico, W.J. Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum. J. Biol. Chem. 2003, 278, 7034–7042.
  32. Ast, T.; Cohen, G.; Schuldiner, M. A network of cytosolic factors targets SRP-independent proteins to the endoplasmic reticulum. Cell 2013, 152, 1134–1145.
  33. Becker, T.; Song, J.; Pfanner, N. Versatility of preprotein transfer from the cytosol to mitochondria. Trends Cell Biol. 2019, 29, 534–548.
  34. Caplan, A.J.; Cyr, D.M.; Douglas, M.G. YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism. Cell 1992, 71, 1143–1155.
  35. Craig, E.A. Hsp70 at the membrane: Driving protein translocation. BMC Biol. 2018, 16, 11.
  36. Kityk, R.; Kopp, J.; Mayer, M.P. Molecular mechanism of J-domain-triggered ATP hydrolysis by Hsp70 chaperones. Mol. Cell 2018, 69, 227–237.
  37. Craig, E.A.; Marszalek, J. How do J-proteins get Hsp70 to do so many different things? Trends Biochem. Sci. 2017, 42, 355–368.
  38. Rabu, C.; Wipf, P.; Brodsky, J.L.; High, S. A precursor-specific role for Hsp40/Hsc70 during tail-anchored protein integration at the endoplasmic reticulum. J. Biol. Chem. 2008, 283, 27504–27513.
  39. Haßdenteufel, S.; Johnson, N.; Paton, A.W.; Paton, J.C.; High, S.; Zimmermann, R. Chaperone-mediated Sec61 channel gating during ER import of small precursor proteins overcomes Sec61 inhibitor-reinforced energy barrier. Cell Rep. 2018, 23, 1373–1386.
  40. Casson, J.; McKenna, M.; Haßdenteufel, S.; Aviram, N.; Zimmerman, R.; High, S. Multiple pathways facilitate the biogenesis of mammalian tail-anchored proteins. J. Cell Sci. 2017, 130, 3851–3861.
  41. Gemmer, M.; Förster, F. A clearer picture of the ER translocon complex. J. Cell Sci. 2020, 133, jcs231340.
  42. O’Keefe, S.; Pool, M.R.; High, S. Membrane protein biogenesis at the ER: The highways and byways. FEBS J. 2021, 1–28.
  43. Pfeffer, S.; Dudek, J.; Schaffer, M.; Ng, B.G.; Albert, S.; Plitzko, J.M.; Baumeister, W.; Zimmermann, R.; Freeze, H.H.; Engel, B.D.; et al. Dissecting the molecular organization of the translocon-associated protein complex. Nat. Commun. 2017, 8, 14516.
  44. McGilvray, P.T.; Anghel, S.A.; Sundaram, A.; Zhong, F.; Trnka, M.J.; Fuller, J.R.; Hu, H.; Burlingame, A.L.; Keenan, R.J. An ER translocon for multi-pass membrane protein biogenesis. eLife 2020, 9, e56889.
  45. Wu, X.; Siggel, M.; Ovchinnikov, S.; Mi, W.; Svetlov, V.; Nudler, E.; Liao, M.; Hummer, G.; Rapoport, T.A. Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex. Science 2020, 368, eaaz2449.
  46. Itskanov, S.; Park, E. Structure of the posttranslational Sec protein-translocation channel complex from yeast. Science 2019, 363, 84–87.
  47. Pleiner, T.; Tomaleri, G.P.; Januszyk, K.; Inglis, A.J.; Hazu, M.; Voorhees, R.M. Structural basis for membrane insertion by the human ER membrane protein complex. Science 2020, 369, 433–436.
  48. Bai, L.; You, Q.; Feng, X.; Kovach, A.; Li, H. Structure of the ER membrane complex, a transmembrane-domain insertase. Nature 2020, 584, 475–478.
  49. Miller-Vedam, L.E.; Bräuning, B.; Popova, K.D.; Oakdale, N.T.S.; Bonnar, J.L.; Prabu, J.R.; Boydston, E.A.; Sevillano, N.; Shurtleff, M.J.; Stroud, R.M.; et al. Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients. eLife 2020, 9, e62611.
  50. O’Donnell, J.P.; Phillips, B.P.; Yagita, Y.; Juszkiewicz, S.; Wagner, A.; Malinverni, D.; Keenan, R.J.; Miller, E.A.; Hegde, R.S. The architecture of EMC reveals a path for membrane protein insertion. eLife 2020, 9, e57887.
  51. Wild, R.; Kowal, J.; Eyring, J.; Ngwa, E.M.; Aebi, M.; Locher, K.P. Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation. Science 2018, 5140, 545–550.
  52. Braunger, K.; Pfeffer, S.; Shrimal, S.; Gilmore, R.; Berninghausen, O.; Mandon, E.C.; Becker, T.; Förster, F.; Beckmann, R. Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum. Science 2018, 360, 215–219.
  53. McDowell, M.A.; Heimes, M.; Sinning, I. Structural and molecular mechanisms for membrane protein biogenesis by the Oxa1 superfamily. Nat. Struct. Mol. Biol. 2021, 28, 234–239.
  54. Hegde, R.S.; Keenan, R.J. The mechanisms of integral membrane protein biogenesis. Nat. Rev. Mol. Cell Biol. 2021, 1–18.
  55. Guna, A.; Volkmar, N.; Christianson, J.C.; Hegde, R.S. The ER membrane protein complex is a transmembrane domain insertase. Science 2018, 359, 470–473.
  56. Chitwood, P.J.; Juszkiewicz, S.; Guna, A.; Shao, S.; Hegde, R.S. EMC is required to initiate accurate membrane protein topogenesis. Cell 2018, 175, 1507–1519.
  57. Voorhees, R.M.; Hegde, R.S. Structure of the Sec61 channel opened by a signal sequence. Science 2016, 351, 88–91.
  58. Weng, T.; Steinchen, W.; Beatrix, B.; Berninghausen, O.; Becker, T.; Bange, G.; Cheng, J.; Beckmann, R. Architecture of the active post-translational Sec translocon. EMBO J. 2021, 40, e105643.
  59. Van Den Berg, B.; Clemons, W.M.; Collinson, I.; Modis, Y.; Hartmann, E.; Harrison, S.C.; Rapoport, T.A. X-ray structure of a protein-conducting channel. Nature 2004, 427, 36–44.
  60. Voorhees, R.M.; Fernández, I.S.; Scheres, S.H.W.; Hegde, R.S. Structure of the mammalian ribosome-Sec61 complex to 3.4 Å resolution. Cell 2014, 157, 1632–1643.
  61. Gogala, M.; Becker, T.; Beatrix, B.; Armache, J.P.; Barrio-Garcia, C.; Berninghausen, O.; Beckmann, R. Structures of the Sec61 complex engaged in nascent peptide translocation or membrane insertion. Nature 2014, 506, 107–110.
  62. Park, E.; Rapoport, T.A. Mechanisms of Sec61SecY-mediated protein translocation across membranes. Annu. Rev. Biophys. 2012, 41, 21–40.
  63. Li, L.; Park, E.; Ling, J.J.; Ingram, J.; Ploegh, H.; Rapoport, T.A. Crystal structure of a substrate-engaged SecY protein-translocation channel. Nature 2016, 531, 395–399.
  64. Gamayun, I.; O’Keefe, S.; Pick, T.; Klein, M.C.; Nguyen, D.; McKibbin, C.; Piacenti, M.; Williams, H.M.; Flitsch, S.L.; Whitehead, R.C.; et al. Eeyarestatin compounds selectively enhance Sec61-mediated Ca2+ leakage from the endoplasmic reticulum. Cell Chem. Biol. 2019, 26, 571–583.
  65. Kriegler, T.; Magoulopoulou, A.; Amate Marchal, R.; Hessa, T. Measuring endoplasmic reticulum signal sequences translocation efficiency using the Xbp1 arrest peptide. Cell Chem. Biol. 2018, 25, 880–890.
  66. Niesen, M.J.M.; Müller-Lucks, A.; Hedman, R.; von Heijne, G.; Miller, T.F. Forces on nascent polypeptides during membrane insertion and translocation via the Sec translocon. Biophys. J. 2018, 115, 1885–1894.
  67. 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, 13, 3973–3982.
  68. Nilsson, I.M.; Johnson, A.E.; Von Heijne, G. Cleavage of a tail-anchored protein by signal peptidase. FEBS Lett. 2002, 516, 106–108.
  69. Nilsson, I.M.; 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, 126, 1127–1132.
  70. Alzahrani, N.; Wu, M.J.; Shanmugam, S.; Yi, M.K. Delayed by design: Role of suboptimal signal peptidase processing of viral structural protein precursors in flaviviridae virus assembly. Viruses 2020, 12, 1090.
  71. Pfeffer, S.; Burbaum, L.; Unverdorben, P.; Pech, M.; Chen, Y.; Zimmermann, R.; Beckmann, R.; Forster, F. Structure of the native Sec61 protein-conducting channel. Nat. Commun. 2015, 6, 8403.
  72. Pfeffer, S.; Dudek, J.; Gogala, M.; Schorr, S.; Linxweiler, J.; Lang, S.; Becker, T.; Beckmann, R.; Zimmermann, R.; Förster, F. Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon. Nat. Commun. 2014, 5, 3072.
  73. Kapp, K.; Schrempf, S.; Lemberg, M.K.; Dobberstein, B. Protein Transport into the Endoplasmic Reticulum. In Protein Transport into the Endoplasmic Reticulum; Landes Bioscience: Austin, TX, USA, 2009; Volume 1, pp. 1–16.
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