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Pam16 has an essential role in the mitochondrial protein import motor

Nature Structural & Molecular Biology volume 11, pages 226–233 (2004)Cite this article

Abstract

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70–Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.

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Figure 1: Pam16 is associated with the mitochondrial presequence translocase.
Figure 2: Pam16 is exposed to the matrix and present in a functional translocase–preprotein supercomplex.
Figure 3: pam16 mutant mitochondria are defective in preprotein import into the matrix.
Figure 4: pam16 mutant mitochondria can insert preproteins into the inner membrane.
Figure 5: Pam16 interacts with Pam18.
Figure 6: Pam16 is required for the interaction of Pam18 with the presequence translocase and for formation of a stable mtHsp70–Tim44 complex.

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References

  1. Koehler, C.M., Merchant, S. & Schatz, G. How membrane proteins travel across the mitochondrial intermembrane space. Trends Biochem. Sci. 24, 428–432 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Endo, T. & Kohda, D. Functions of outer membrane receptors in mitochondrial protein import. Biochim. Biophys. Acta 1592, 3–14 (2002).

    Article  CAS  Google Scholar 

  4. Lithgow, T. Targeting of proteins to mitochondria. FEBS Lett. 476, 22–26 (2000).

    Article  CAS  Google Scholar 

  5. Neupert, W. & Brunner, M. The protein import motor of mitochondria. Nat. Rev. Mol. Cell Biol. 3, 555–565 (2002).

    Article  CAS  Google Scholar 

  6. Truscott, K.N., Brandner, K. & Pfanner, N. Mechanisms of protein import into mitochondria. Curr. Biol. 13, R326–337 (2003).

    Article  CAS  Google Scholar 

  7. Sickmann, A. et al. The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. USA 100, 13207–13212 (2003).

    Article  CAS  Google Scholar 

  8. Truscott, K.N. 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 

  9. Geissler, A. et al. The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel. Cell 111, 507–518 (2002).

    Article  CAS  Google Scholar 

  10. Yamamoto, H. et al. Tim50 is a subunit of the TIM23 complex that links protein translocation across the outer and inner mitochondrial membranes. Cell 111, 519–528 (2002).

    Article  CAS  Google Scholar 

  11. Mokranjac, D. et al. Tim50, a novel component of the TIM23 preprotein translocase of mitochondria. EMBO J. 22, 816–825 (2003).

    Article  CAS  Google Scholar 

  12. Martin, J., Mahlke, K. & Pfanner, N. Role of an energized inner membrane in mitochondrial protein import: ΔΨ drives the movement of presequences. J. Biol. Chem. 266, 18051–18057 (1991).

    CAS  PubMed  Google Scholar 

  13. 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 

  14. Geissler, A. et al. Membrane potential-driven protein import into mitochondria: the sorting sequence of cytochrome b2 modulates the ΔΨ dependence of translocation of the matrix-targeting sequence. Mol. Biol. Cell 11, 3977–3991 (2000).

    Article  CAS  Google Scholar 

  15. 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 

  16. Glick, B.S. Can Hsp70 proteins act as force-generating motors? Cell 80, 11–14 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. 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 

  20. 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 

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

    Article  CAS  Google Scholar 

  22. Matlack, K.E., Misselwitz, B., Plath, K. & Rapoport, T.A. BiP acts as a molecular ratchet during posttranslational transport of prepro-α factor across the ER membrane. Cell 97, 553–564 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Mayer, M.P., Brehmer, D., Gässler, C.S. & Bukau, B. Hsp70 chaperone machines. Adv. Protein Chem. 59, 1–44 (2002).

    CAS  Google Scholar 

  25. 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 

  26. 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 

  27. 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 

  28. 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 

  29. Chacinska, A. et al. Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM–TIM-preprotein supercomplex. EMBO J. 22, 5370–5381 (2003).

    Article  CAS  Google Scholar 

  30. Meisinger, C. et al. Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small Tom proteins, and import receptors. Mol. Cell. Biol. 21, 2337–2348 (2001).

    Article  CAS  Google Scholar 

  31. Jensen, R.E. & Dunn, C.D. Protein import into and across the mitochondrial inner membrane: role of the TIM23 and TIM22 translocons. Biochim. Biophys. Acta 1592, 25–34 (2002).

    Article  CAS  Google Scholar 

  32. 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 

  33. Rehling, P., Pfanner, N. & Meisinger, C. Insertion of hydrophobic membrane proteins into the inner mitochondrial membrane—a guided tour. J. Mol. Biol. 326, 639–657 (2003).

    Article  CAS  Google Scholar 

  34. Rehling, P. et al. Protein insertion into the mitochondrial inner membrane by a twin-pore translocase. Science 299, 1747–1751 (2003).

    Article  CAS  Google Scholar 

  35. Wachter, C., Schatz, G. & Glick, B.S. Role of ATP in the intramitochondrial sorting of cytochrome c1 and the adenine nucleotide translocator. EMBO J. 11, 4787–4794 (1992).

    Article  CAS  Google Scholar 

  36. Glick, B.S., Brandt, A., Cunnigham, K., Müller, S. & Hallberg, L.R. Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism. Cell 69, 809–822 (1992).

    Article  CAS  Google Scholar 

  37. Arnold, I., Fölsch, H., Neupert, W. & Stuart, R.A. Two distinct and independent mitochondrial targeting signals function in the sorting of an inner membrane protein, cytochrome c1 . J. Biol. Chem. 273, 1469–1476 (1998).

    Article  CAS  Google Scholar 

  38. Gärtner, F. et al. Mitochondrial import of subunit Va of cytochrome c oxidase characterized with yeast mutants: independence from receptors, but requirement for matrix hsp70 translocase function. J. Biol. Chem. 270, 3788–3795 (1995).

    Article  Google Scholar 

  39. Nunnari, J., Fox, T.D. & Walter, P. A mitochondrial protease with two catalytic subunits of nonoverlapping specificities. Science 262, 1997–2004 (1993).

    Article  CAS  Google Scholar 

  40. Gakh, O., Cavadini, P. & Isaya, G. Mitochondrial processing peptidases. Biochim. Biophys. Acta 1592, 63–77 (2002).

    Article  CAS  Google Scholar 

  41. 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 

  42. 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 

  43. Schneider, H.C., Westermann, B., Neupert, W. & Brunner, M. The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import. EMBO J. 15, 5796–5803 (1996).

    Article  CAS  Google Scholar 

  44. Schwarz, E., Seytter, T., Guiard, B. & Neupert, W. Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal. EMBO J. 12, 2295–2302 (1993).

    Article  CAS  Google Scholar 

  45. Bömer, U. et al. Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins. EMBO J. 17, 4226–4237 (1998).

    Article  Google Scholar 

  46. Becker, S., Gehrsitz, A., Bork, P., Buchner, S. & Buchner, E. The black-pearl gene of Drosophila defines a novel conserved protein family and is required for larval growth and survival. Gene 262, 15–22 (2001).

    Article  CAS  Google Scholar 

  47. Jubinsky, P.T. et al. Identification and characterization of Magmas, a novel mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction. Exp. Hematol. 29, 1392–1402 (2001).

    Article  CAS  Google Scholar 

  48. 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 

  49. 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 

  50. Sikorski, R.S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Ryan, M.T., Voos, W. & Pfanner, N. Assaying protein import into mitochondria. Methods Cell Biol. 65, 189–215 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank R. Jensen for antiserum against Tim17, S. Rospert for antiserum against Pam18 and I. Perschil, N. Zufall and H. Müller for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 388, Max Planck Research Award, Bundesministerium für Bildung und Forschung, Nationales Genomforschungsnetz, and the Fonds der Chemischen Industrie. M.L. is a recipient of a postdoctoral fellowship from the Wenner-Gren foundations. Work in the laboratory of M.G.C. was supported by grant GM 57017 from the US National Institutes of Health.

Author information

Author notes

  1. Maria Lind

    Present address: Department of Comparative Physiology, Evolutionary Biology Centre, Uppsala University, SE-75236, Uppsala, Sweden

  2. Andreas Geissler

    Present address: Curacyte AG, D-80339, München, Germany

  3. Ann E Frazier and Jan Dudek: 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

    Ann E Frazier, Jan Dudek, Wolfgang Voos, Yanfeng Li, Maria Lind, Chris Meisinger, Andreas Geissler, Kaye N Truscott, Nikolaus Pfanner & Peter Rehling

  2. Fakultät für Biologie, Universität Freiburg, Freiburg, D-79104, Germany

    Ann E Frazier, Jan Dudek & Yanfeng Li

  3. Laboratoire propre du CNRS Université Pierre et Marie Curie, Gif-sur-Yvette, F-91190, France

    Bernard Guiard

  4. Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, D-44780, Germany

    Albert Sickmann & Helmut E Meyer

  5. Rudolf-Virchow-Center for Experimental Biomedicine, Universität Würzburg, Würzburg, D-97078, Germany

    Albert Sickmann

  6. Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, 92697-3900, California, USA

    Virginia Bilanchone & Michael G Cumsky

  7. Department of Comparative Physiology, Evolutionary Biology Centre, Uppsala University, SE-75236, Uppsala, Sweden

    Andreas Geissler

  8. Curacyte AG, D-80339, München, Germany

    Maria Lind

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Frazier, A., Dudek, J., Guiard, B. et al. Pam16 has an essential role in the mitochondrial protein import motor. Nat Struct Mol Biol 11, 226–233 (2004). https://doi.org/10.1038/nsmb735

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