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Protein unfolding by the mitochondrial membrane potential

Nature Structural Biology volume 9, pages 301–307 (2002)Cite this article

Abstract

Mitochondria can unfold importing precursor proteins by unraveling them from their N-termini. However, how this unraveling is induced is not known. Two candidates for the unfolding activity are the electrical potential across the inner mitochondrial membrane and mitochondrial Hsp70 in the matrix. Here, we propose that many precursors are unfolded by the electrical potential acting directly on positively charged amino acid side chains in the targeting sequences. Only precursor proteins with targeting sequences that are long enough to reach the matrix at the initial interaction with the import machinery are unfolded by mitochondrial Hsp70, and this unfolding occurs even in the absence of a membrane potential.

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Figure 1: Dependence of import on the membrane potential.
Figure 2: Membrane potential and precursor unfolding.
Figure 3: Membrane potential and targeting sequence charge.
Figure 4: Unfolding of precursors with long targeting sequences.
Figure 5: Unfolding by mtHsp70.

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References

  1. Horwich, A.L., Weber-Ban, E.U. & Finley, D. Chaperone rings in protein folding and degradation. Proc. Natl. Acad. Sci. USA 96, 11033–11040 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Huang, S., Ratliff, K.S., Schwartz, M.P., Spenner, J.M. & Matouschek, A. Mitochondria unfold precursor proteins by unraveling them from their N-termini. Nature Struct. Biol. 6, 1132–1138 (1999).

    Article  CAS  Google Scholar 

  4. Lee, C., Schwartz, M.P., Prakash, S., Iwakura, M. & Matouschek, A. ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol. Cell 7, 627–637 (2001).

    Article  CAS  Google Scholar 

  5. Eilers, M. & Schatz, G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature 322, 228–232 (1986).

    Article  CAS  Google Scholar 

  6. Schwartz, M.P., Huang, S. & Matouschek, A. The structure of precursor proteins during import into mitochondria. J. Biol. Chem. 274, 12759–12764 (1999).

    Article  CAS  Google Scholar 

  7. 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 

  8. Matouschek, A. et al. Active unfolding of precursor proteins during mitochondrial protein import. EMBO J. 16, 6727–6736 (1997).

    Article  CAS  Google Scholar 

  9. Huang, S., Murphy, S. & Matouschek, A. Effect of the protein import machinery at the mitochondrial surface on precursor stability. Proc. Natl. Acad. Sci. USA 97, 12991–12996 (2000).

    Article  CAS  Google Scholar 

  10. Kang, P.-L. 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 

  11. Manning-Krieg, U., Scherer, P.E. & Schatz, G. Sequential action of mitochondrial chaperones in protein import into the matrix. EMBO J. 10, 3273–3280 (1991).

    Article  CAS  Google Scholar 

  12. Wachter, C., Schatz, G. & Glick, B.S. Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix. Mol. Biol. Cell 5, 465–474 (1994).

    Article  CAS  Google Scholar 

  13. Gaume, B. et al. Unfolding of preproteins upon import into mitochondria. EMBO J. 17, 6497–6507 (1998).

    Article  CAS  Google Scholar 

  14. Schleyer, M., Schmidt, B. & Neupert, W. Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria. Eur. J. Biochem. 125, 109–116 (1982).

    Article  CAS  Google Scholar 

  15. Gasser, S.M., Daum, G. & Schatz, G. Import of proteins into mitochondria. Energy-dependent uptake of precursors by isolated mitochondria. J. Biol. Chem. 257, 13034–13041 (1982).

    CAS  PubMed  Google Scholar 

  16. Pfanner, N. & Neupert, W. Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier. EMBO J. 4, 2819–2825 (1985).

    Article  CAS  Google Scholar 

  17. Voos, W., Gambill, B.D., Guiard, B., Pfanner, N. & Craig, E.A. Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix. J. Cell Biol. 123, 119–126 (1993).

    Article  CAS  Google Scholar 

  18. 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 

  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. 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 

  21. 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 

  22. Ungermann, C., Neupert, W. & Cyr, D.M. The role of hsp70 in conferring unidirectionality on protein translocation into mitochondria. Science 266, 1250–1253 (1994).

    Article  CAS  Google Scholar 

  23. Costanzo, M.C. et al. YPD, PombePD and WormPD: model organism volumes of the BioKnowledge library, an integrated resource for protein information. Nucleic Acids Res. 29, 75–79 (2001).

    Article  CAS  Google Scholar 

  24. Costanzo, M.C. et al. The yeast proteome database (YPD) and Caenorhabditis elegans proteome database (WormPD): comprehensive resources for the organization and comparison of model organism protein information. Nucleic Acids Res. 28, 73–76 (2000).

    Article  CAS  Google Scholar 

  25. Hwang, S.T., Wachter, C. & Schatz, G. Protein import into the yeast mitochondrial matrix. A new translocation intermediate between the two mitochondrial membranes. J. Biol. Chem. 266, 21083–21089 (1991).

    CAS  PubMed  Google Scholar 

  26. 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 

  27. 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 

  28. Reers, M., Smith, T.W. & Chen, L.B. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480–4486 (1991).

    Article  CAS  Google Scholar 

  29. Reers, M. et al. Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol. 260, 406–417 (1995).

    Article  CAS  Google Scholar 

  30. Smiley, S.T. et al. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA 88, 3671–3675 (1991).

    Article  CAS  Google Scholar 

  31. Pfanner, N. & Geissler, A. Versatility of the mitochondrial protein import machinery. Nature Rev. Mol. Cell Biol. 2, 339–349 (2001).

    Article  CAS  Google Scholar 

  32. Brix, J., Dietmeier, K. & Pfanner, N. Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70. J. Biol. Chem. 272, 20730–20735 (1997).

    Article  CAS  Google Scholar 

  33. Abe, Y. et al. Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20. Cell 100, 551–560 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Ungermann, C., Guiard, B., Neupert, W. & Cyr, D.M. The ΔΨ and Hsp70/MIM44-dependent reaction cycle driving early steps of protein import into mitochondria. EMBO J. 15, 735–744 (1996).

    Article  CAS  Google Scholar 

  36. Rudiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 16, 1501–1517 (1997).

    Article  CAS  Google Scholar 

  37. Blond-Elguindi, S. et al. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell 75, 717–728 (1993).

    Article  CAS  Google Scholar 

  38. Chauwin, J.F., Oster, G. & Glick, B.S. Strong precursor–pore interactions constrain models for mitochondrial protein import. Biophys. J. 74, 1732–1743 (1998).

    Article  CAS  Google Scholar 

  39. Dekker, P.J. 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 

  40. Hartley, R.W. A two state conformational transition of the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) induced by sodium dodecyl sulfate. Biochemistry 14, 2367–2370 (1975).

    Article  CAS  Google Scholar 

  41. Arretz, M., Schneider, H., Guiard, B., Brunner, M. & Neupert, W. Characterization of the mitochondrial processing peptidase of Neurospora crassa. J. Biol. Chem. 269, 4959–4967 (1994).

    CAS  PubMed  Google Scholar 

  42. Beasley, E.M., Müller, S. & Schatz, G. The signal that sorts yeast cytochrome b 2 to the mitochondrial intermembrane space contains three distinct functional regions. EMBO J. 12, 2303–2311 (1993).

    Article  CAS  Google Scholar 

  43. Mossakowska, D.E., Nyberg, K. & Fersht, A.R. Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis. Biochemistry 28, 3843–3850 (1989).

    Article  CAS  Google Scholar 

  44. Serrano, L., Kellis, J.T. Jr, Cann, P., Matouschek, A. & Fersht, A.R. The folding of an enzyme II. Substructure of barnase and the contribution of different interactions to protein stability. J. Mol. Biol. 224, 783–804 (1992).

    Article  CAS  Google Scholar 

  45. Hartley, R.W. Directed mutagenesis and barnase–barstar recognition. Biochemistry 32, 5978–5984 (1993).

    Article  CAS  Google Scholar 

  46. Glick, B.S. & Pon, L.A. Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol. 260, 213–223 (1995).

    Article  CAS  Google Scholar 

  47. Heytler, P.G. & Prichard, W.W. A new class of uncoupling agents – carbonyl cyanide phenylhydrazones. Biochem. Biophys. Res. Commun. 7, 272–275 (1962).

    Article  CAS  Google Scholar 

  48. Geissler, A. et al. Membrane potential-driven protein import into mitochondria: the sorting sequence of cytochrome b 2 modulates the ΔΨ-dependence of translocation of the matrix-targeting sequence. Mol. Biol. Cell 11, 3977–3991 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank C. Voisine and E. Craig, University of Wisconsin, for generously sending us the ssc1-2 and congenic wild type yeast strains; acknowledge the use of the Keck Biophysics Facility at Northwestern University; and are grateful to L. Feferman for his help with experiments. This work was funded by the National Institutes of Health. S.H. gratefully acknowledges the support by a Gramm Travel Fellowship Award from the Robert H. Lurie Comprehensive Cancer Center of Northwestern University.

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  1. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, 60208-3500, Illinois, USA

    Shihai Huang, Kevin S. Ratliff & Andreas Matouschek

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Correspondence to Andreas Matouschek.

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Huang, S., Ratliff, K. & Matouschek, A. Protein unfolding by the mitochondrial membrane potential. Nat Struct Mol Biol 9, 301–307 (2002). https://doi.org/10.1038/nsb772

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