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. 1996 Jan 15;15(2):399–407.

Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria.

T Komiya 1, M Sakaguchi 1, K Mihara 1
PMCID: PMC449955  PMID: 8617215

Abstract

Two ATP-dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria. Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM-sensitive manner. Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70. Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane. The MSF-dependent pathway is NEM-sensitive and requires the hydrolysis of extra-mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70-dependent pathway is NEM-sensitive and does not require extra-mitochondrial ATP. The NEM-insensitive, hsp70-dependent import became NEM-sensitive depending on the amount of MSF added. The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins.

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  1. Alam R., Hachiya N., Sakaguchi M., Kawabata S., Iwanaga S., Kitajima M., Mihara K., Omura T. cDNA cloning and characterization of mitochondrial import stimulation factor (MSF) purified from rat liver cytosol. J Biochem. 1994 Aug;116(2):416–425. doi: 10.1093/oxfordjournals.jbchem.a124541. [DOI] [PubMed] [Google Scholar]
  2. Attardi G., Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol. 1988;4:289–333. doi: 10.1146/annurev.cb.04.110188.001445. [DOI] [PubMed] [Google Scholar]
  3. Baker K. P., Schaniel A., Vestweber D., Schatz G. A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature. 1990 Dec 13;348(6302):605–609. doi: 10.1038/348605a0. [DOI] [PubMed] [Google Scholar]
  4. Baker K. P., Schatz G. Mitochondrial proteins essential for viability mediate protein import into yeast mitochondria. Nature. 1991 Jan 17;349(6306):205–208. doi: 10.1038/349205a0. [DOI] [PubMed] [Google Scholar]
  5. Becker K., Guiard B., Rassow J., Söllner T., Pfanner N. Targeting of a chemically pure preprotein to mitochondria does not require the addition of a cytosolic signal recognition factor. J Biol Chem. 1992 Mar 15;267(8):5637–5643. [PubMed] [Google Scholar]
  6. Blond-Elguindi S., Cwirla S. E., Dower W. J., Lipshutz R. J., Sprang S. R., Sambrook J. F., Gething M. J. Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell. 1993 Nov 19;75(4):717–728. doi: 10.1016/0092-8674(93)90492-9. [DOI] [PubMed] [Google Scholar]
  7. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  8. Craig E. A. Chaperones: helpers along the pathways to protein folding. Science. 1993 Jun 25;260(5116):1902–1903. doi: 10.1126/science.8100364. [DOI] [PubMed] [Google Scholar]
  9. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
  10. Flynn G. C., Chappell T. G., Rothman J. E. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 1989 Jul 28;245(4916):385–390. doi: 10.1126/science.2756425. [DOI] [PubMed] [Google Scholar]
  11. Flynn G. C., Pohl J., Flocco M. T., Rothman J. E. Peptide-binding specificity of the molecular chaperone BiP. Nature. 1991 Oct 24;353(6346):726–730. doi: 10.1038/353726a0. [DOI] [PubMed] [Google Scholar]
  12. Fourie A. M., Sambrook J. F., Gething M. J. Common and divergent peptide binding specificities of hsp70 molecular chaperones. J Biol Chem. 1994 Dec 2;269(48):30470–30478. [PubMed] [Google Scholar]
  13. Glick B. S., Beasley E. M., Schatz G. Protein sorting in mitochondria. Trends Biochem Sci. 1992 Nov;17(11):453–459. doi: 10.1016/0968-0004(92)90487-t. [DOI] [PubMed] [Google Scholar]
  14. Glick B. S. Can Hsp70 proteins act as force-generating motors? Cell. 1995 Jan 13;80(1):11–14. doi: 10.1016/0092-8674(95)90444-1. [DOI] [PubMed] [Google Scholar]
  15. Gragerov A., Zeng L., Zhao X., Burkholder W., Gottesman M. E. Specificity of DnaK-peptide binding. J Mol Biol. 1994 Jan 21;235(3):848–854. doi: 10.1006/jmbi.1994.1043. [DOI] [PubMed] [Google Scholar]
  16. Gratzer S., Lithgow T., Bauer R. E., Lamping E., Paltauf F., Kohlwein S. D., Haucke V., Junne T., Schatz G., Horst M. Mas37p, a novel receptor subunit for protein import into mitochondria. J Cell Biol. 1995 Apr;129(1):25–34. doi: 10.1083/jcb.129.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hachiya N., Alam R., Sakasegawa Y., Sakaguchi M., Mihara K., Omura T. A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins. EMBO J. 1993 Apr;12(4):1579–1586. doi: 10.1002/j.1460-2075.1993.tb05802.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hachiya N., Komiya T., Alam R., Iwahashi J., Sakaguchi M., Omura T., Mihara K. MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria. EMBO J. 1994 Nov 1;13(21):5146–5154. doi: 10.1002/j.1460-2075.1994.tb06844.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hachiya N., Mihara K., Suda K., Horst M., Schatz G., Lithgow T. Reconstitution of the initial steps of mitochondrial protein import. Nature. 1995 Aug 24;376(6542):705–709. doi: 10.1038/376705a0. [DOI] [PubMed] [Google Scholar]
  20. Hartl F. U., Hlodan R., Langer T. Molecular chaperones in protein folding: the art of avoiding sticky situations. Trends Biochem Sci. 1994 Jan;19(1):20–25. doi: 10.1016/0968-0004(94)90169-4. [DOI] [PubMed] [Google Scholar]
  21. Haucke V., Lithgow T., Rospert S., Hahne K., Schatz G. The yeast mitochondrial protein import receptor Mas20p binds precursor proteins through electrostatic interaction with the positively charged presequence. J Biol Chem. 1995 Mar 10;270(10):5565–5570. doi: 10.1074/jbc.270.10.5565. [DOI] [PubMed] [Google Scholar]
  22. Hines V., Brandt A., Griffiths G., Horstmann H., Brütsch H., Schatz G. Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70. EMBO J. 1990 Oct;9(10):3191–3200. doi: 10.1002/j.1460-2075.1990.tb07517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hines V., Schatz G. Precursor binding to yeast mitochondria. A general role for the outer membrane protein Mas70p. J Biol Chem. 1993 Jan 5;268(1):449–454. [PubMed] [Google Scholar]
  24. Hönlinger A., Kübrich M., Moczko M., Gärtner F., Mallet L., Bussereau F., Eckerskorn C., Lottspeich F., Dietmeier K., Jacquet M. The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins. Mol Cell Biol. 1995 Jun;15(6):3382–3389. doi: 10.1128/mcb.15.6.3382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Iwahashi J., Furuya S., Mihara K., Omura T. Characterization of adrenodoxin precursor expressed in Escherichia coli. J Biochem. 1992 Apr;111(4):451–455. doi: 10.1093/oxfordjournals.jbchem.a123778. [DOI] [PubMed] [Google Scholar]
  26. Kiebler M., Keil P., Schneider H., van der Klei I. J., Pfanner N., Neupert W. The mitochondrial receptor complex: a central role of MOM22 in mediating preprotein transfer from receptors to the general insertion pore. Cell. 1993 Aug 13;74(3):483–492. doi: 10.1016/0092-8674(93)80050-o. [DOI] [PubMed] [Google Scholar]
  27. Kiebler M., Pfaller R., Söllner T., Griffiths G., Horstmann H., Pfanner N., Neupert W. Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature. 1990 Dec 13;348(6302):610–616. doi: 10.1038/348610a0. [DOI] [PubMed] [Google Scholar]
  28. Komiya T., Hachiya N., Sakaguchi M., Omura T., Mihara K. Recognition of mitochondria-targeting signals by a cytosolic import stimulation factor, MSF. J Biol Chem. 1994 Dec 9;269(49):30893–30897. [PubMed] [Google Scholar]
  29. Lithgow T., Glick B. S., Schatz G. The protein import receptor of mitochondria. Trends Biochem Sci. 1995 Mar;20(3):98–101. doi: 10.1016/s0968-0004(00)88972-0. [DOI] [PubMed] [Google Scholar]
  30. Lithgow T., Høj P. B., Hoogenraad N. J. Do cytosolic factors prevent promiscuity at the membrane surface? FEBS Lett. 1993 Aug 23;329(1-2):1–4. doi: 10.1016/0014-5793(93)80179-x. [DOI] [PubMed] [Google Scholar]
  31. Lithgow T., Junne T., Suda K., Gratzer S., Schatz G. The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11973–11977. doi: 10.1073/pnas.91.25.11973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mihara K., Blobel G., Sato R. In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7102–7106. doi: 10.1073/pnas.79.23.7102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Murakami H., Pain D., Blobel G. 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria. J Cell Biol. 1988 Dec;107(6 Pt 1):2051–2057. doi: 10.1083/jcb.107.6.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nakai M., Endo T., Hase T., Matsubara H. Intramitochondrial protein sorting. Isolation and characterization of the yeast MSP1 gene which belongs to a novel family of putative ATPases. J Biol Chem. 1993 Nov 15;268(32):24262–24269. [PubMed] [Google Scholar]
  35. Nakai M., Endo T. Identification of yeast MAS17 encoding the functional counterpart of the mitochondrial receptor complex protein MOM22 of Neurospora crassa. FEBS Lett. 1995 Jan 3;357(2):202–206. doi: 10.1016/0014-5793(94)01362-5. [DOI] [PubMed] [Google Scholar]
  36. Neupert W., Hartl F. U., Craig E. A., Pfanner N. How do polypeptides cross the mitochondrial membranes? Cell. 1990 Nov 2;63(3):447–450. doi: 10.1016/0092-8674(90)90437-j. [DOI] [PubMed] [Google Scholar]
  37. Ono H., Tuboi S. Purification of the putative import-receptor for the precursor of the mitochondrial protein. J Biochem. 1990 Jun;107(6):840–845. doi: 10.1093/oxfordjournals.jbchem.a123135. [DOI] [PubMed] [Google Scholar]
  38. Pfanner N., Craig E. A., Meijer M. The protein import machinery of the mitochondrial inner membrane. Trends Biochem Sci. 1994 Sep;19(9):368–372. doi: 10.1016/0968-0004(94)90113-9. [DOI] [PubMed] [Google Scholar]
  39. Pfanner N., Neupert W. The mitochondrial protein import apparatus. Annu Rev Biochem. 1990;59:331–353. doi: 10.1146/annurev.bi.59.070190.001555. [DOI] [PubMed] [Google Scholar]
  40. Pfanner N., Söllner T., Neupert W. Mitochondrial import receptors for precursor proteins. Trends Biochem Sci. 1991 Feb;16(2):63–67. doi: 10.1016/0968-0004(91)90026-r. [DOI] [PubMed] [Google Scholar]
  41. Sheffield W. P., Shore G. C., Randall S. K. Mitochondrial precursor protein. Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence. J Biol Chem. 1990 Jul 5;265(19):11069–11076. [PubMed] [Google Scholar]
  42. Stuart R. A., Cyr D. M., Craig E. A., Neupert W. Mitochondrial molecular chaperones: their role in protein translocation. Trends Biochem Sci. 1994 Feb;19(2):87–92. doi: 10.1016/0968-0004(94)90041-8. [DOI] [PubMed] [Google Scholar]
  43. Söllner T., Griffiths G., Pfaller R., Pfanner N., Neupert W. MOM19, an import receptor for mitochondrial precursor proteins. Cell. 1989 Dec 22;59(6):1061–1070. doi: 10.1016/0092-8674(89)90762-9. [DOI] [PubMed] [Google Scholar]
  44. Söllner T., Pfaller R., Griffiths G., Pfanner N., Neupert W. A mitochondrial import receptor for the ADP/ATP carrier. Cell. 1990 Jul 13;62(1):107–115. doi: 10.1016/0092-8674(90)90244-9. [DOI] [PubMed] [Google Scholar]
  45. 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. 1994 Apr;5(4):465–474. doi: 10.1091/mbc.5.4.465. [DOI] [PMC free article] [PubMed] [Google Scholar]

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