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:

Regulated interactions of mtHsp70 with Tim44 at the translocon in the mitochondrial inner membrane

Nature Structural & Molecular Biology volume 11, pages 1084–1091 (2004)Cite this article

Abstract

Preproteins synthesized on cytosolic ribosomes, but destined for the mitochondrial matrix, pass through the presequence translocase of the inner membrane. Translocation is driven by the import motor, having at its core the essential chaperone mtHsp70 (Ssc1 in yeast). MtHsp70 is tethered to the translocon channel at the matrix side of the inner membrane by the peripheral membrane protein Tim44. A key question in mitochondrial import is how the mtHsp70-Tim44 interaction is regulated. Here we report that Tim44 interacts with both the ATPase and peptide-binding domains of mtHsp70. Disruption of these interactions upon binding of polypeptide substrates requires concerted conformational changes involving both domains of mtHsp70. Our results fit a model in which regulated interactions between Tim44 and mtHsp70, controlled by polypeptide binding, are required for efficient translocation across the mitochondrial inner membrane in vivo.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Interaction of Tim44 with the ATPase domain and PBD of Ssc1.
Figure 2: Effect of peptide on the Ssc1-Tim44 interaction in vitro.
Figure 3: Analysis of SSC1 mutants defective in interaction with Tim44.
Figure 4: Stimulation of ATPase activity of Ssc1 by a J protein and peptide.
Figure 5: Effect of peptide on mutant Ssc1-Tim44 interactions in vitro.
Figure 6: In vitro interaction of Ssc1-2 and Ssc1-201 with Tim44.

Similar content being viewed by others

References

  1. Neupert, W. & Brunner, M. The protein import motor of mitochondria. Nat. Rev. Mol. Cell Biol. 8, 555–565 (2002).

    Article  Google Scholar 

  2. Wiedemann, N., Frazier, A.E. & Pfanner, N. The protein import machinery of mitochondria. J. Biol. Chem. 279, 14473–14476 (2004).

    Article  CAS  Google Scholar 

  3. Jensen, R.E. & Johnson, A.E. Opening the door to mitochondrial protein import. Nat. Struct. Biol. 8, 1008–1010 (2001).

    Article  CAS  Google Scholar 

  4. 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 

  5. Truscott, K. et al. A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23. Nat. Struct. Biol. 8, 1074–1082 (2001).

    Article  CAS  Google Scholar 

  6. Huang, S., Ratliff, K.S. & Matouschek, A. Protein unfolding by the mitochondrial membrane potential. Nat. Struct. Biol. 9, 301–307 (2002).

    Article  CAS  Google Scholar 

  7. Kang, P.J. et al. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature 348, 137–143 (1990).

    Article  CAS  Google Scholar 

  8. Scherer, P., Krieg, U., Hwang, S., Vestweber, D. & Schatz, G. A precursor protein partially translocated into yeast mitochondria is bound to a 70kd mitochondrial stress protein. EMBO J. 9, 4315–4322 (1990).

    Article  CAS  Google Scholar 

  9. Gambill, B.D. et al. A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins. J. Cell Biol. 123, 109–117 (1993).

    Article  CAS  Google Scholar 

  10. Pilon, M. & Schekman, R. Protein translocation: how Hsp70 pulls it off. Cell 97, 679–682 (1999).

    Article  CAS  Google Scholar 

  11. Schneider, H.-C. et al. Mitochondrial Hsp70/MIM44 complex facilitates protein import. Nature 371, 768–774 (1994).

    Article  CAS  Google Scholar 

  12. Voisine, C. et al. The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70. Cell 97, 565–574 (1999).

    Article  CAS  Google Scholar 

  13. Liu, Q., D'Silva, P., Walter, W., Marszalek, J. & Craig, E.A. Regulated cycling of mitochondrial Hsp70 at the protein import channel. Science 300, 139–141 (2003).

    Article  CAS  Google Scholar 

  14. Maarse, A.C., Blom, J., Grivell, L.A. & Meijer, M. MPI, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria. EMBO J. 11, 3619–3628 (1992).

    Article  CAS  Google Scholar 

  15. Scherer, P.E., Manning-Krieg, U.C., Jeno, P., Schatz, G. & Horst, M. Identification of a 45-kDa protein at the import site of the yeast mitochondrial inner membrane. Proc. Natl. Acad. Sci. USA 89, 11930–11934 (1992).

    Article  CAS  Google Scholar 

  16. Mokranjac, D., Sichting, M., Neupert, W. & Hell, K. Tim14, a novel key component of the import motor of the Tim23 protein translocase of mitochondria. EMBO J. 22, 4945–4956 (2003).

    Article  CAS  Google Scholar 

  17. Truscott, K.N. et al. A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria. J. Cell Biol. 163, 707–713 (2003).

    Article  CAS  Google Scholar 

  18. D'Silva, P.D., Schilke, B., Walter, W., Andrew, A. & Craig, E.A. J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix. Proc. Natl. Acad. Sci USA 100, 13839–13844 (2003).

    Article  CAS  Google Scholar 

  19. Kozany, C., Mokranjac, D., Sichting, M., Neupert, W. & Hell, K. The J domain-related cochaperone Tim16 is a constituent of the mitochondrial TIM23 preprotein translocase. Nat. Struct. Mol. Biol. 11, 234–241 (2004).

    Article  CAS  Google Scholar 

  20. Frazier, A.E. et al. Pam16 has an essential role in the mitochondrial protein import motor. Nat. Struct. Mol. Biol. 11, 226–233 (2004).

    Article  CAS  Google Scholar 

  21. Liu, Q., Krzewska, J., Liberek, K. & Craig, E.A. Mitochondrial Hsp70 Ssc1:role in protein folding. J. Biol. Chem. 276, 6112–6118 (2001).

    Article  CAS  Google Scholar 

  22. Bukau, B. & Horwich, A.L. The Hsp70 and Hsp60 chaperone machines. Cell 92, 351–366 (1998).

    Article  CAS  Google Scholar 

  23. Hartl, F. & Hayer-Hartl, M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852–1858 (2002).

    Article  CAS  Google Scholar 

  24. Rudiger, S., Buchberger, A. & Bukau, B. Interaction of Hsp70 chaperones with substrates. Nat. Struct. Biol. 4, 342–349 (1997).

    Article  CAS  Google Scholar 

  25. Mayer, M.P. et al. Multistep mechanism of substrate binding determines chaperone activity of Hsp70. Nat. Struct. Biol. 7, 586–593 (2000).

    Article  CAS  Google Scholar 

  26. Buchberger, A., Reinstein, J. & Bukau, B. The DnaK chaperone system: mechanism and comparison with other Hsp70 systems. In Molecular Chaperones and Folding Catalysts: Regulation, Cellular Function and Mechanisms (ed. Bukau, B.) 609–635 (Harwood Academic, 1999).

    Google Scholar 

  27. Laufen, T. et al. Mechanism of regulation of Hsp70 chaperones by DnaJ co-chaperones. Proc. Natl. Acad. Sci. USA 96, 5452–5457 (1999).

    Article  CAS  Google Scholar 

  28. Krimmer, T., Rassow, J., Kunau, W.H., Voos, W. & Pfanner, N. Mitochondrial protein import motor: the ATPase domain of matrix Hsp70 is crucial for binding to Tim44, while the peptide binding domain and the carboxy-terminal segment play a stimulatory role. Mol. Cell. Biol. 20, 5879–5887 (2000).

    Article  CAS  Google Scholar 

  29. Moro, F., Okamoto, K., Donzeau, M., Neupert, W. & Brunner, M. Mitochondrial protein import: molecular basis of the ATP-dependent interaction of MtHsp70 with Tim44. J. Biol. Chem. 277, 6874–6880 (2002).

    Article  CAS  Google Scholar 

  30. Strub, A., Rottgers, K. & Voos, W. The Hsp70 peptide-binding domain determines the interaction of the ATPase domain with Tim44 in mitochondria. EMBO J. 21, 2626–2635 (2002).

    Article  CAS  Google Scholar 

  31. Craig, E.A. & Marszalek, J. A specialized mitochondrial molecular chaperone system: a role in formation of Fe/S centers. Cell Mol. Life Sci. 59, 1658–1665 (2002).

    Article  CAS  Google Scholar 

  32. Zhu, X. et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606–1614 (1996).

    Article  CAS  Google Scholar 

  33. Voos, W. et al. Differential requirement for the mitochondrial Hsp70–Tim44 complex in unfolding and translocation of preproteins. EMBO J. 15, 2668–2677 (1996).

    Article  CAS  Google Scholar 

  34. Merlin, A. et al. The J-related segment of Tim44 is essential for cell Viability: A mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHSP70 and impairs protein translocation. J. Cell Biol. 145, 961–972 (1999).

    Article  CAS  Google Scholar 

  35. Lim, J.H., Martin, F., Guiard, B., Pfanner, N. & Voos, W. The mitochondrial Hsp70-dependent import system actively unfolds preproteins and shortens the lag phase of translocation. EMBO J. 20, 941–950 (2001).

    Article  CAS  Google Scholar 

  36. Geissler, A., Rassow, J., Pfanner, N. & Voos, W. Mitochondrial import driving forces: enhanced trapping by matrix Hsp70 stimulates the translocation and reduces the membrane potential dependence of loosely folded preproteins. Mol. Cell. Biol. 21, 7097–7104 (2001).

    Article  CAS  Google Scholar 

  37. Rassow, J. et al. Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix Hsp70 and the inner membrane protein Mim44. J. Cell Biol. 127, 1547–1556 (1994).

    Article  CAS  Google Scholar 

  38. Strub, A., Zufall, N. & Voos, W. The putative helical lid of the Hsp70 peptide-binding domain is required for efficient preprotein translocation into mitochondria. J. Mol. Biol. 334, 1087–1099 (2003).

    Article  CAS  Google Scholar 

  39. Jordan, R. & McMacken, R. Modulation of the ATPase activity of the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat shock proteins. J. Biol. Chem. 270, 4563–4569 (1995).

    Article  CAS  Google Scholar 

  40. O'Brien, M. & McKay, D. Threonine 204 of the chaperone protein hsc70 influences the structure of the active site but is not essential for ATP hydrolysis. J. Biol. Chem. 268, 24323–24329 (1993).

    CAS  PubMed  Google Scholar 

  41. Lopez-Buesca, P., Pfund, C. & Craig, E.A. The biochemical properties of the ATPase activity of a 70-kDa heat shock protein (Hsp70) are governed by the C-terminal domains. Proc. Natl. Acad. Sci. USA 95, 15253–15258 (1998).

    Article  Google Scholar 

  42. Slepenkov, S.V. & Witt, S.N. Peptide-induced conformational changes in the molecular chaperone DnaK. Biochemistry 37, 16749–16756 (1998).

    Article  CAS  Google Scholar 

  43. Slepenkov, S. & Witt, S. Detection of a concerted conformational change in the ATPase domain of DnaK triggered by peptide binding. FEBS Lett. 539, 100–104 (2003).

    Article  CAS  Google Scholar 

  44. Miao, B., Davis, J.E. & Craig, E.A. Mge1 functions as a nucleotide release factor for Ssc1, a mitochondrial Hsp70 of Saccharomyces cerevisiae. J. Mol. Biol. 265, 541–552 (1997).

    Article  CAS  Google Scholar 

  45. 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 

  46. Horst, M. et al. Sequential action of two hsp70 complexes during protein import into mitochondria. EMBO J. 16, 1842–1849 (1997).

    Article  CAS  Google Scholar 

  47. Dutkiewicz, R. et al. Ssq1, a mitochondrial Hsp70 involved in iron-sulfur (Fe/S) center biogenesis: Similarities to and differences from its bacterial counterparts. J. Biol. Chem. 278, 29719–29727 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Marszalek for helpful comments on the manuscript. This work was supported by National Institutes of Health Grant GM278709 (to E.A.C.) and American Heart Association Fellowship 0420049Z (P.D.).

Author information

Author notes

  1. Qinglian Liu

    Present address: Department of Biochemistry and Biophysics, Columbia University, 650 West 168th Street, New York, New York, 10032, USA

Authors and Affiliations

  1. Department of Biochemistry, 433 Babcock Drive, University of Wisconsin–Madison, Madison, 53706, Wisconsin, USA

    Patrick D'Silva, Qinglian Liu, William Walter & Elizabeth A Craig

Authors
  1. Patrick D'Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qinglian Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William Walter
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elizabeth A Craig
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elizabeth A Craig.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Effect of CoxIV peptide on Ssc1-Tim44 interactions in vitro. (PDF 111 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

D'Silva, P., Liu, Q., Walter, W. et al. Regulated interactions of mtHsp70 with Tim44 at the translocon in the mitochondrial inner membrane. Nat Struct Mol Biol 11, 1084–1091 (2004). https://doi.org/10.1038/nsmb846

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb846

This article is cited by

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