Evolutionarily evolved discriminators in the 3-TPR domain of the Toc64 family involved in protein translocation at the outer membrane of chloroplasts and mitochondria

  • Oliver Mirus
  • Tihana Bionda
  • Arndt von Haeseler
  • Enrico Schleiff
Original Paper


Transport of polypeptides across membranes is a general and essential cellular process utilised by molecular machines. At least one component of these complexes contains a domain composed of three tetratricopeptide repeat (3-TPR) motifs. We have focussed on the receptor Toc64 to elucidate the evolved functional specifications of its 3-TPR domain. Toc64 is a component of the Toc core complex and functionally replaces Tom70 at the outer membrane of mitochondria in plants. Its 3-TPR domain recognises the conserved C-terminus of precursor-bound chaperones. We built homology models of the 3-TPR domain of chloroplastic Toc64 from different species and of the mitochondrial isoform from Arabidopsis. Guided by modelling, we identified residues essential for functional discrimination of the differently located isoforms to be located almost exclusively on the convex surface of the 3-TPR domain. The only exception is at568Ser/ps557Met, which is positioned in the ligand-binding groove. The functional implications of the homology models are discussed.


Motion contained within the 2nd eigenvector of the Calpha covariance matrix of the 3-TPR domain of atToc64-V indicated by a porcupine plot


Molecular dynamics simulation Homology modeling Toc64 Tetratricopeptide repeat Mitochondria Chloroplasts Chaperones 



We would like to thank Lutz Voigt for technical help, and Thomas Schlegel and Nicole Scherer for their bioinformatic support. Special thanks to Thomas Becker, Joanna Tripp and Jason Young for helpful discussions regarding the project. The work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB-TR01) and from the Volkswagenstiftung to E.S. and Wiener Wissenschafts-, Forschungs- und Technologiefonds (WWTF) to A.v.H.

Supplementary material

894_2008_449_MOESM1_ESM.pdf (5.6 mb)
ESM 1 (PDF 5.61 MB)


  1. 1.
    Gray MW, Burger G, Lang BF (1999) Mitochondrial evolution. Science 283:1476–1481CrossRefGoogle Scholar
  2. 2.
    McFadden GI (1999) Endosymbiosis and evolution of the plant cell. Curr Opin Plant Biol 2:513–519CrossRefGoogle Scholar
  3. 3.
    Wickner W, Schekman R (2005) Protein translocation across biological membranes. Science 310:1452–1456CrossRefGoogle Scholar
  4. 4.
    Dolezal P, Likic V, Tachezy J et al (2006) Evolution of the molecular machines for protein import into mitochondria. Science 313:314–318CrossRefGoogle Scholar
  5. 5.
    Löffelhardt W, von Haeseler A, Schleiff E (2007) The β-barrel shaped polypeptide transporter, an old concept for precursor protein transfer across membranes. Symbiosis 44:33–42Google Scholar
  6. 6.
    Kalanon M, McFadden GI (2008) The chloroplast protein translocation complexes of Chlamydomonas reinhardtii: a bioinformatic comparison of Toc and Tic components in plants, green algae and red algae. Genetics 179:95–112CrossRefGoogle Scholar
  7. 7.
    Hulett JM, Lueder F, Chan NC et al (2008) The transmembrane segment of Tom20 is recognized by Mim1 for docking to the mitochondrial TOM complex. J Mol Biol 376:694–704CrossRefGoogle Scholar
  8. 8.
    Chew O, Lister R, Qbadou S et al (2004) A plant outer mitochondrial membrane protein with high amino acid sequence identity to a chloroplast protein import receptor. FEBS Lett 557:109–114CrossRefGoogle Scholar
  9. 9.
    Sohrt K, Soll J (2000) Toc64, a new component of the protein translocon of chloroplasts. J Cell Biol 148:1213–1221CrossRefGoogle Scholar
  10. 10.
    Qbadou S, Becker T, Bionda T (2007) Toc64—a preprotein receptor at the outer membrane with bipartide function. J Mol Biol 367:1330–1346CrossRefGoogle Scholar
  11. 11.
    Schultz J, Marshall-Carlson L, Carlson M (1990) The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae. Mol Cell Biol 10:4744–4756Google Scholar
  12. 12.
    Sikorski RS, Boguski MS, Goebl M et al (1990) A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell 60:307–317CrossRefGoogle Scholar
  13. 13.
    Hirano T, Kinoshita N, Morikawa K et al (1990) Snap helix with knob and hole: essential repeats in S. pombe nuclear protein nuc2+. Cell 60:319–328CrossRefGoogle Scholar
  14. 14.
    Goebl M, Yanagida M (1991) The TPR snap helix: a novel protein repeat motif from mitosis to transcription. Trends Biochem Sci 16:173–177CrossRefGoogle Scholar
  15. 15.
    Lamb JR, Tugendreich S, Hieter P (1995) Tetratrico peptide repeat interactions: to TPR or not to TPR? Trends Biochem Sci 20:257–259CrossRefGoogle Scholar
  16. 16.
    D’Andrea LD, Regan L (2003) TPR proteins: the versatile helix. Trends Biochem Sci 28:655–662CrossRefGoogle Scholar
  17. 17.
    Letunic I, Copley RR, Schmidt S, Ciccarelli FD, Doerks T, Schultz J, Ponting CP, Bork P (2004) SMART 4.0: towards genomic data integration. Nucleic Acids Res 32:D142–D144CrossRefGoogle Scholar
  18. 18.
    Finn RD, Tate J, Mistry J, Coggill PC, Sammut JS, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer EL, Bateman A (2008) The Pfam protein families database. Nucleic Acids Res 36:D281–D288CrossRefGoogle Scholar
  19. 19.
    Haucke V, Horst M, Schatz G et al (1996) The Mas20p and Mas70p subunits of the protein import receptor of yeast mitochondria interact via the tetratricopeptide repeat motif in Mas20p: evidence for a single hetero-oligomeric receptor. EMBO J 15:1231–1237Google Scholar
  20. 20.
    Young JC, Hoogenraad NJ, Hartl FU (2003) Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70. Cell 112:41–50CrossRefGoogle Scholar
  21. 21.
    Nuttall SD, Hanson BJ, Mori M et al (1997) hTom34: a novel translocase for the import of proteins into human mitochondria. DNA Cell Biol 16:1067–1074CrossRefGoogle Scholar
  22. 22.
    Young JC, Obermann WM, Hartl FU (1998) Specific binding of tetratricopeptide repeat proteins to the C-terminal 12-kDa domain of hsp90. J Biol Chem 273:18007–18010CrossRefGoogle Scholar
  23. 23.
    Chewawiwat N, Yano M, Terada K et al (1999) Characterization of the novel mitochondrial protein import component, Tom34, in mammalian cells. J Biochem (Tokyo) 125:721–727Google Scholar
  24. 24.
    Yang CS, Weiner H (2002) Yeast two-hybrid screening identifies binding partners of human Tom34 that have ATPase activity and form a complex with Tom34 in the cytosol. Arch Biochem Biophys 400:105–110CrossRefGoogle Scholar
  25. 25.
    Gatto GJ Jr, Geisbrecht BV, Gould SJ et al (2000) Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat Struct Biol 7:1091–1095CrossRefGoogle Scholar
  26. 26.
    Kumar A, Roach C, Hirsh IS et al (2001) An unexpected extended conformation for the third TPR motif of the peroxin PEX5 from Trypanosoma brucei. J Mol Biol 307:271–282CrossRefGoogle Scholar
  27. 27.
    Feldheim D, Schekman R (1994) Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex. J Cell Biol 126:935–943CrossRefGoogle Scholar
  28. 28.
    Ponting CP (2000) Proteins of the endoplasmic-reticulum-associated degradation pathway: domain detection and function prediction. Biochem J 351:527–535CrossRefGoogle Scholar
  29. 29.
    Scheufler C, Brinker A, Bourenkov G et al (2000) Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101:199–210CrossRefGoogle Scholar
  30. 30.
    Jabet C, Sprague ER, VanDemark AP et al (2000) Characterization of the N-terminal domain of the yeast transcriptional repressor Tup1. Proposal for an association model of the repressor complex Tup1 x Ssn6. J Biol Chem 275:9011–9018CrossRefGoogle Scholar
  31. 31.
    Cheng CY, Jarymowycz VA, Cortajarena AL et al (2006) Repeat motions and backbone flexibility in designed proteins with different numbers of identical consensus tetratricopeptide repeats. Biochem 45:12175–12183CrossRefGoogle Scholar
  32. 32.
    Qbadou S, Becker T, Mirus O et al (2006) The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64. EMBO J 25:1836–1847CrossRefGoogle Scholar
  33. 33.
    Jackson-Constan D, Keegstra K (2001) Arabidopsis genes encoding components of the chloroplastic protein import apparatus. Plant Physiol 125:1667–1676CrossRefGoogle Scholar
  34. 34.
    Oreb M, Reger K, Schleiff E (2006) Chloroplast protein import: reverse genetic approaches. Curr Genom 7:235–244CrossRefGoogle Scholar
  35. 35.
    Pollmann S, Neu D, Weiler EW (2003) Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid. Phytochemistry 62:293–300CrossRefGoogle Scholar
  36. 36.
    Pollmann S, Neu D, Lehmann T et al (2006) Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana. Planta 224:1241–1253CrossRefGoogle Scholar
  37. 37.
    Chan NC, Likic VA, Waller RF et al (2006) The C-terminal TPR domain of Tom70 defines a family of mitochondrial protein import receptors found only in animals and fungi. J Mol Biol 358:1010–1022CrossRefGoogle Scholar
  38. 38.
    Lister R, Carrie C, Duncan O et al (2007) Functional definition of outer membrane proteins involved in preprotein import into mitochondria. Plant Cell 19:3739–3759CrossRefGoogle Scholar
  39. 39.
    Rosenbaum Hofmann N, Theg SM (2005) Toc64 is not required for import of proteins into chloroplasts in the moss Physcomitrella patens. Plant J 43:675–687CrossRefGoogle Scholar
  40. 40.
    Aronsson H, Boij P, Patel R et al (2007) Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis thaliana. Plant J 52:53–68CrossRefGoogle Scholar
  41. 41.
    Rost B (1999) Twilight zone of protein sequence alignments. Protein Eng 12:85–94CrossRefGoogle Scholar
  42. 42.
    Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815CrossRefGoogle Scholar
  43. 43.
    Vriend G (1990) WHAT IF: a molecular modeling and drug design program. J Mol Graph 8:52–56CrossRefGoogle Scholar
  44. 44.
    Hess B (2002) Convergence of sampling in protein simulations. Phys Rev E 65:031910CrossRefGoogle Scholar
  45. 45.
    Krieger E, Darden T, Nabuurs S et al (2004) Making optimal use of empirical energy functions: force field parameterization in crystal space. Proteins 57:678–683CrossRefGoogle Scholar
  46. 46.
    Otto S (2006) Reinforced molecular recognition as an alternative to rigid receptors. Dalton Trans 23:2861–2864CrossRefGoogle Scholar
  47. 47.
    Main ER, Xiong Y, Cocco MJ, D’Andrea L, Regan L (2003) Design of stable alpha-helical arrays from an idealized TPR motif. Structure 11:497–50848CrossRefGoogle Scholar
  48. 48.
    Cliff MJ, Harris R, Barford D et al (2006) Conformational diversity in the TPR domain-mediated interaction of protein phosphatase 5 with Hsp90. Structure 14:415–426CrossRefGoogle Scholar
  49. 49.
    Cortajarena AL, Regan L (2006) Ligand binding by TPR domains. Protein Sci 15:1193–1198CrossRefGoogle Scholar
  50. 50.
    Cortajarena AL, Kajander T, Pan W, Cocco MJ, Regan L (2004) Protein design to understand peptide ligand recognition by tetratricopeptide repeat proteins. Protein Eng Des Sel 17:399–409CrossRefGoogle Scholar
  51. 51.
    Schlegel T, Mirus O, von Haeseler A et al (2007) The tetratricopeptide repeats of receptors involved in protein translocation across membranes. Mol Biol Evol 24:2763–2774CrossRefGoogle Scholar
  52. 52.
    Fan AC, Bhangoo MK, Young JC (2006) Hsp90 functions in the targeting and outer membrane translocation steps of Tom70-mediated mitochondrial import. J Biol Chem 281:33313–33324CrossRefGoogle Scholar
  53. 53.
    Yang J, Roe SM, Cliff MJ et al (2005) Molecular basis for TPR domain-mediated regulation of protein phosphatase 5. EMBO J 24:1–10CrossRefGoogle Scholar
  54. 54.
    Schleiff E, Turnbull JL (1998) Functional and structural properties of the mitochondrial outer membrane receptor Tom20. Biochemistry 37:13043–13051CrossRefGoogle Scholar
  55. 55.
    Schleiff E, Soll J, Sveshnikova N et al (2002) Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34. Biochemistry 41:1934–1946CrossRefGoogle Scholar
  56. 56.
    Bennett-Lovsey RM, Herbert AD, Sternberg MJE et al (2008) Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre. Proteins 70:3CrossRefGoogle Scholar
  57. 57.
    Das AK, Cohen PW, Barford D (1998) The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions. EMBO J 17:1192–1199CrossRefGoogle Scholar
  58. 58.
    Fiser A, Do RK, Sali A (2000) Modeling of loops in protein structures. Protein Sci 9:1753–1773CrossRefGoogle Scholar
  59. 59.
    Schwede T, Kopp J, Guex N et al (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385CrossRefGoogle Scholar
  60. 60.
    Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC (2003) Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50:437–450CrossRefGoogle Scholar
  61. 61.
    Bowie JU, Lüthy R, Eisenberg D (1991) A method to identify protein sequences that fold into a known three-dimensional structure. Science 253:164–170CrossRefGoogle Scholar
  62. 62.
    Lüthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with three-dimensional profiles. Nature 356:83–85CrossRefGoogle Scholar
  63. 63.
    Berendsen HJC, van der Spoel D, van Drunen R (1995) GROMACS: a message-passing parallel molecular dynamics implementation. Comp Phys Commun 91:43–56CrossRefGoogle Scholar
  64. 64.
    Lindahl E, Hess B, van der Spoel D (2001) Gromacs 3.0: a package for molecular simulation and trajectory analysis. J Mol Model 7:306–317Google Scholar
  65. 65.
    van der Spoel D, Lindahl E, Hess B et al (2005) GROMACS: fast, flexible, and free. J Comput Chem 26:1701–1718CrossRefGoogle Scholar
  66. 66.
    van Gunsteren WF, Billeter SR, Eising AA et al (1996) Biomolecular asimulation: the GROMOS96 manual and user guide. ETH, ZürichGoogle Scholar
  67. 67.
    Berendsen HJC, Postma JPM, van Gunsteren WF et al (1981) Interaction models for water in relation to protein hydration. In: Pullman B (ed) Intermolecular forces. Reidel, Dordrecht, pp 331–342Google Scholar
  68. 68.
    Berendsen HJC, Postma JPM, van Gunsteren WF et al (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684–3690CrossRefGoogle Scholar
  69. 69.
    Essmann U, Perera L, Berkowitz ML et al (1995) A smooth particle mesh Ewald method. J Chem Phys 103:8577–8593CrossRefGoogle Scholar
  70. 70.
    Miyamoto S, Kollman P (1992) Settle: an analytical version of the SHAKE and RATTLE algorithm for rigid water models. J Comp Chem 13:952–962CrossRefGoogle Scholar
  71. 71.
    Hess B, Bekker H, Berendsen HJC et al (1997) LINCS: a linear constraint solver for molecular simulations. J Comp Chem 18:1463–1472CrossRefGoogle Scholar
  72. 72.
    Hess B (2000) Similarities between principal components of protein dynamics and random diffusion. Phys Rev E 62:8438–8448CrossRefGoogle Scholar
  73. 73.
    Humphrey W, Dalke A, Schulten K (1996) VMD - Visual Molecular Dynamics. J Mol Graph 14:33–38CrossRefGoogle Scholar
  74. 74.
    Tai K, Shen T, Börjesson U et al (2001) Analysis of a 10-ns molecular dynamics simulation of mouse acetylcholinesterase. Biophys J 81:715–724CrossRefGoogle Scholar
  75. 75.
    Barrett CP, Hall BA, Noble ME (2004) Dynamite: a simple way to gain insight into protein motions. Acta Crystallogr D Biol Crystallogr 60:2280–2287CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Oliver Mirus
    • 1
  • Tihana Bionda
    • 1
  • Arndt von Haeseler
    • 2
  • Enrico Schleiff
    • 1
  1. 1.Cluster of Excellence and Center of Membrane Proteomics, Institute for Molecular BiosciencesJohann Wolfgang Goethe UniversityFrankfurt am MainGermany
  2. 2.Center for Integrative Bioinformatics Vienna, Max F. Perutz LaboratoriesUniversity of ViennaViennaAustria

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