Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23

Nature Structural Biology volume 8, pages 1074–1082 (2001)Cite this article

Abstract

Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes. The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel. Here we report that purified Tim23 forms a hydrophilic, ∼13–24 Å wide channel characteristic of the mitochondrial presequence translocase. The Tim23 channel is cation selective and activated by a membrane potential and presequences. The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction. Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Reconstituted Tim23 forms a cation-selective, voltage-sensitive channel.
Figure 2: The Tim23 channel is specifically influenced by a mitochondrial presequence.
Figure 3: Reduction of the presequence sensitivity in the mutant protein Tim23-1.
Figure 4: Tim23-specific characteristics of the inner membrane translocase channel.
Figure 5: The translocase channel is present in tim23-2 mutant mitochondria despite a destabilization of the Tim23–Tim17 interaction.
Figure 6: The C-terminal domain of Tim23 forms the channel.
Figure 7: Estimation of the Tim23 pore size by the polymer exclusion method.

Similar content being viewed by others

References

  1. Schatz, G. & Dobberstein, B. Common principles of protein translocation across membranes. Science 271, 1519–1526 (1996).

    Article  CAS  Google Scholar 

  2. Jensen, R.E. & Johnson, A.E. Protein translocation: is Hsp70 pulling my chain? Curr. Biol. 9, R779–R782 (1999).

    Article  CAS  Google Scholar 

  3. Bauer, M.F., Hofmann, S., Neupert, W. & Brunner, M. Protein translocation into mitochondria: the role of TIM complexes. Trends Cell Biol. 10, 25–31 (2000).

    Article  CAS  Google Scholar 

  4. Matouschek, A., Pfanner, N. & Voos, W. Protein unfolding by mitochondria: the Hsp70 import motor. EMBO Rep. 1, 404–410 (2000).

    Article  CAS  Google Scholar 

  5. Hill, K. et al. Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins. Nature 395, 516–521 (1998).

    Article  CAS  Google Scholar 

  6. Künkele, K.-P. et al. The preprotein translocation channel of the outer membrane of mitochondria. Cell 93, 1009–1019 (1998).

    Article  Google Scholar 

  7. Ryan, K.R. & Jensen, R.E. Mas6p can be cross-linked to an arrested precursor and interacts with other proteins during mitochondrial protein import. J. Biol. Chem. 268, 23743–23746 (1993).

    CAS  PubMed  Google Scholar 

  8. Lohret, T.A. & Kinnally, K.W. Targeting peptides transiently block a mitochondrial channel. J. Biol. Chem. 270, 15950–15953 (1995).

    Article  CAS  Google Scholar 

  9. Berthold, J. et al. The MIM complex mediates preprotein translocation across the mitochondrial inner membrane and couples it to the mt-Hsp70/ATP driving system. Cell 81, 1085–1093 (1995).

    Article  CAS  Google Scholar 

  10. Blom, J., Dekker, P.J.T. & Meijer, M. Functional and physical interactions of components of the yeast mitochondrial inner-membrane import machinery (MIM). Eur. J. Biochem. 232, 309–314 (1995).

    Article  CAS  Google Scholar 

  11. Lohret, T.A., Jensen, R.E. & Kinnally, K.W. Tim23, a protein import component of the mitochondrial inner membrane, is required for normal activity of the multiple conductance channel, MCC. J. Cell Biol. 137, 377–386 (1997).

    Article  CAS  Google Scholar 

  12. Moro, F., Sirrenberg, C., Schneider, H.-C., Neupert, W. & Brunner, M. The TIM17·23 preprotein translocase of mitochondria: composition and function in protein transport into the matrix. EMBO J. 18, 3667–3675 (1999).

    Article  CAS  Google Scholar 

  13. Emtage, J.L.T. & Jensen, R.E. MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway. J. Cell Biol. 122, 1003–1012 (1993).

    Article  CAS  Google Scholar 

  14. Dekker, P.J.T. et al. Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane. FEBS Lett. 330, 66–70 (1993).

    Article  CAS  Google Scholar 

  15. Allison, D.S. & Schatz, G. Artificial mitochondrial presequences. Proc. Natl Acad. Sci. USA 83, 9011–9015 (1986).

    Article  CAS  Google Scholar 

  16. Bölter, B., Soll, J., Hill, K., Hemmler, R. & Wagner, R. A rectifying ATP-regulated solute channel in the chloroplastic outer envelope from pea. EMBO J. 18, 5505–5516 (1999).

    Article  Google Scholar 

  17. Kübrich, M. et al. The polytopic mitochondrial inner membrane proteins MIM17 and MIM23 operate at the same preprotein import site. FEBS Lett. 349, 222–228 (1994).

    Article  Google Scholar 

  18. Maarse, A.C., Blom, J., Keil, P., Pfanner, N. & Meijer, M. Identification of the essential yeast protein MIM17, an integral mitochondrial inner membrane protein involved in protein import. FEBS Lett. 349, 215–221 (1994).

    Article  CAS  Google Scholar 

  19. Ryan, K.R., Menold, M.M., Garrett, S. & Jensen, R.E. SMS1, a high-copy suppressor of the yeast mas6 mutant, encodes an essential inner membrane protein required for mitochondrial protein import. Mol. Biol. Cell 5, 529–538 (1994).

    Article  CAS  Google Scholar 

  20. Dekker, P.J.T. et al. The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44. EMBO J. 16, 5408–5419 (1997).

    Article  CAS  Google Scholar 

  21. Bauer, M.F., Sirrenberg, C., Neupert, W. & Brunner, M. Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria. Cell 87, 33–41 (1996).

    Article  CAS  Google Scholar 

  22. Komiya, T. et al. Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the 'acid chain' hypothesis. EMBO J. 17, 3886–3898 (1998).

    Article  CAS  Google Scholar 

  23. Zoratti, M. & Szabo, I. Electrophysiology of the inner mitochondrial membrane. J. Bioenerg. Biomembr. 26, 543–553 (1994).

    Article  CAS  Google Scholar 

  24. Ryan, K.R., Leung, R.S. & Jensen, R.E. Characterization of the mitochondrial inner membrane translocase complex: the Tim23p hydrophobic domain interacts with Tim17p but not with other Tim23p molecules. Mol. Cell. Biol. 18, 178–187 (1998).

    Article  CAS  Google Scholar 

  25. Donzeau, M. et al. Tim23 links the inner and outer mitochondrial membranes. Cell 101, 401–412 (2000).

    Article  CAS  Google Scholar 

  26. Schwartz, M.P. & Matouschek, A. The dimensions of the protein import channels in the outer and inner mitochondrial membranes. Proc. Natl Acad. Sci. USA 96, 13086–13090 (1999).

    Article  CAS  Google Scholar 

  27. Rassow, J., Hartl, F.-U., Guiard, B., Pfanner, N. & Neupert, W. Polypeptides traverse the mitochondrial envelope in an extended state. FEBS Lett. 275, 190–194 (1990).

    Article  CAS  Google Scholar 

  28. Smart, O.S., Breed, J., Smith, G.R. & Sansom, M.S. A novel method for structure-based prediction of ion channel conductance properties. Biophys. J. 72, 1109–1126 (1997).

    Article  CAS  Google Scholar 

  29. Krasilnikow, O.V., Sabirov, R.Z., Ternovsky, V.L., Merzliak, P.G. & Muratkhodjaev, J.N. A simple method for the determination of the pore radius of ion channels in planar lipid bilayer membranes. FEMS Microbiol. Immunol. 5, 93–100 (1992).

    Article  Google Scholar 

  30. Hamman, B.D., Hendershot, L.M. & Johnson, A.E. BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation. Cell 92, 747–758 (1998).

    Article  CAS  Google Scholar 

  31. Beckmann, R. et al. Alignment of conduits for the nascent polypeptide chain in the ribosome–Sec61 complex. Science 278, 2123–2126 (1997).

    Article  CAS  Google Scholar 

  32. Ménétret, J.-F. et al. The structure of ribosome-channel complexes engaged in protein translocation. Mol. Cell 6, 1219–1232 (2000).

    Article  Google Scholar 

  33. Nissen, P., Hansen, J., Ban, N., Moore, P.B. & Steitz, T.A. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930 (2000).

    Article  CAS  Google Scholar 

  34. Miroux, B. & Walker, J.E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996).

    Article  CAS  Google Scholar 

  35. Bezrukov, S.M. & Kasianowicz J.J. The charge state of an ion channel controls neutral polymer entry into its pore. Eur. Biophys. J. 26, 471–476 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to J. Walker for the E. coli strain C43 (DE3), R. Jensen for antibody against Tim17, B. Guiard, J. H. Lim, A. Chacinska and W. Voos for b2Δ-DHFR, K. Model and C. Meisinger for technical advice, and H. Müller for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich 388, the Fonds der Chemischen Industrie/BMBF (N.P.), the Sonderforschungsbereich 431 (R.W.) and a long-term fellowship from the Alexander von Humboldt Foundation (K.N.T.).

Author information

Author notes

  1. Alessio Merlin

    Present address: Schering AG, D-13342, Berlin, Germany

  2. Kaye N. Truscott, Peter Kovermann and Arnold J.M. Driessen: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Strasse 7, Freiburg, D-79104, Germany

    Kaye N. Truscott, Peter Kovermann, Andreas Geissler, Alessio Merlin & Nikolaus Pfanner

  2. Biophysik, Universität Osnabrück, FB Biologie/Chemie, Osnabrück, D-49034, Germany

    Peter Kovermann & Richard Wagner

  3. Section for Plant Pathology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, 1098 SM, The Netherlands

    Michiel Meijer

  4. Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, 9751 NN, The Netherlands

    Arnold J.M. Driessen

  5. Universität Hohenheim, Institut für Mikrobiologie, Stuttgart, D-70593, Germany

    Joachim Rassow

Authors
  1. Kaye N. Truscott
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter Kovermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andreas Geissler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alessio Merlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michiel Meijer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Arnold J.M. Driessen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joachim Rassow
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Nikolaus Pfanner
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Richard Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Nikolaus Pfanner or Richard Wagner.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Truscott, K., Kovermann, P., Geissler, A. et al. A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23. Nat Struct Mol Biol 8, 1074–1082 (2001). https://doi.org/10.1038/nsb726

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb726

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing