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. 1996 Jun 3;15(11):2668–2677.

Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins.

W Voos 1, O von Ahsen 1, H Müller 1, B Guiard 1, J Rassow 1, N Pfanner 1
PMCID: PMC450202  PMID: 8654364

Abstract

The mitochondrial heat shock protein Hsp70 is essential for import of nuclear-encoded proteins, involved in both unfolding and membrane translocation of preproteins. mtHsp70 interacts reversibly with Tim44 of the mitochondrial inner membrane, yet the role of this interaction is unknown. We analysed this role by using two yeast mutants of mtHsp70 that differentially influenced its interaction with Tim44. One mutant mtHsp70 (Ssc1-2p) efficiently bound preproteins, but did not show a detectable complex formation with Tim44; the mitochondria imported loosely folded preproteins with wild-type kinetics, yet were impaired in unfolding of preproteins. The other mutant Hsp70 (Ssc1-3p') bound both Tim44 and preproteins, but the mitochondria did not import folded polypeptides and were impaired in import of unfolded preproteins; Ssc1-3p' was defective in its ATPase domain and did not undergo a nucleotide-dependent conformational change, resulting in permanent binding to Tim44. The following conclusions are suggested. (i) The import of loosely folded polypeptides (translocase function of mtHsp70) does not depend on formation of a detectable Hsp70-Tim44 complex. Two explanations are possible: a trapping mechanism by soluble mtHsp70, or a weak/very transient interaction of Ssc1-2p with Tim44 that leads to a weak force generation sufficient for import of loosely folded, but not folded, polypeptides. (ii) Import of folded preproteins (unfoldase function of mtHsp70) involves a reversible nucleotide-dependent interaction of mtHsp70 with Tim44, including a conformational change in mtHsp70. This is consistent with a model that the dynamic interaction of mtHsp70 with Tim44 generates a pulling force on preproteins which supports unfolding during translocation.

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Selected References

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  1. 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]
  2. Berthold J., Bauer M. F., Schneider H. C., Klaus C., Dietmeier K., Neupert W., Brunner M. The MIM complex mediates preprotein translocation across the mitochondrial inner membrane and couples it to the mt-Hsp70/ATP driving system. Cell. 1995 Jun 30;81(7):1085–1093. doi: 10.1016/s0092-8674(05)80013-3. [DOI] [PubMed] [Google Scholar]
  3. Blom J., Kübrich M., Rassow J., Voos W., Dekker P. J., Maarse A. C., Meijer M., Pfanner N. The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane. Mol Cell Biol. 1993 Dec;13(12):7364–7371. doi: 10.1128/mcb.13.12.7364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brodsky J. L., Goeckeler J., Schekman R. BiP and Sec63p are required for both co- and posttranslational protein translocation into the yeast endoplasmic reticulum. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9643–9646. doi: 10.1073/pnas.92.21.9643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brodsky J. L., Schekman R. A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome. J Cell Biol. 1993 Dec;123(6 Pt 1):1355–1363. doi: 10.1083/jcb.123.6.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buchberger A., Theyssen H., Schröder H., McCarty J. S., Virgallita G., Milkereit P., Reinstein J., Bukau B. Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication. J Biol Chem. 1995 Jul 14;270(28):16903–16910. doi: 10.1074/jbc.270.28.16903. [DOI] [PubMed] [Google Scholar]
  7. Chappell T. G., Konforti B. B., Schmid S. L., Rothman J. E. The ATPase core of a clathrin uncoating protein. J Biol Chem. 1987 Jan 15;262(2):746–751. [PubMed] [Google Scholar]
  8. Craig E. A., Kramer J., Kosic-Smithers J. SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4156–4160. doi: 10.1073/pnas.84.12.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cyr D. M., Stuart R. A., Neupert W. A matrix ATP requirement for presequence translocation across the inner membrane of mitochondria. J Biol Chem. 1993 Nov 15;268(32):23751–23754. [PubMed] [Google Scholar]
  10. Daum G., Böhni P. C., Schatz G. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13028–13033. [PubMed] [Google Scholar]
  11. Eilers M., Oppliger W., Schatz G. Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria. EMBO J. 1987 Apr;6(4):1073–1077. doi: 10.1002/j.1460-2075.1987.tb04860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ellis R. J., Hemmingsen S. M. Molecular chaperones: proteins essential for the biogenesis of some macromolecular structures. Trends Biochem Sci. 1989 Aug;14(8):339–342. doi: 10.1016/0968-0004(89)90168-0. [DOI] [PubMed] [Google Scholar]
  13. Freeman B. C., Myers M. P., Schumacher R., Morimoto R. I. Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1. EMBO J. 1995 May 15;14(10):2281–2292. doi: 10.1002/j.1460-2075.1995.tb07222.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gambill B. D., Voos W., Kang P. J., Miao B., Langer T., Craig E. A., Pfanner N. A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins. J Cell Biol. 1993 Oct;123(1):109–117. doi: 10.1083/jcb.123.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Glick B. S., Wachter C., Reid G. A., Schatz G. Import of cytochrome b2 to the mitochondrial intermembrane space: the tightly folded heme-binding domain makes import dependent upon matrix ATP. Protein Sci. 1993 Nov;2(11):1901–1917. doi: 10.1002/pro.5560021112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gärtner F., Voos W., Querol A., Miller B. R., Craig E. A., Cumsky M. G., Pfanner N. Mitochondrial import of subunit Va of cytochrome c oxidase characterized with yeast mutants. J Biol Chem. 1995 Feb 24;270(8):3788–3795. doi: 10.1074/jbc.270.8.3788. [DOI] [PubMed] [Google Scholar]
  18. Horwich A. L., Kalousek F., Mellman I., Rosenberg L. E. A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. EMBO J. 1985 May;4(5):1129–1135. doi: 10.1002/j.1460-2075.1985.tb03750.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hurt E. C., Pesold-Hurt B., Schatz G. The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix. FEBS Lett. 1984 Dec 10;178(2):306–310. doi: 10.1016/0014-5793(84)80622-5. [DOI] [PubMed] [Google Scholar]
  20. Kang P. J., Ostermann J., Shilling J., Neupert W., Craig E. A., Pfanner N. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature. 1990 Nov 8;348(6297):137–143. doi: 10.1038/348137a0. [DOI] [PubMed] [Google Scholar]
  21. Kassenbrock C. K., Kelly R. B. Interaction of heavy chain binding protein (BiP/GRP78) with adenine nucleotides. EMBO J. 1989 May;8(5):1461–1467. doi: 10.1002/j.1460-2075.1989.tb03529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koll H., Guiard B., Rassow J., Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space. Cell. 1992 Mar 20;68(6):1163–1175. doi: 10.1016/0092-8674(92)90086-r. [DOI] [PubMed] [Google Scholar]
  23. Kronidou N. G., Oppliger W., Bolliger L., Hannavy K., Glick B. S., Schatz G., Horst M. Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12818–12822. doi: 10.1073/pnas.91.26.12818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kübrich M., Dietmeier K., Pfanner N. Genetic and biochemical dissection of the mitochondrial protein-import machinery. Curr Genet. 1995 Apr;27(5):393–403. doi: 10.1007/BF00311207. [DOI] [PubMed] [Google Scholar]
  25. Laloraya S., Dekker P. J., Voos W., Craig E. A., Pfanner N. Mitochondrial GrpE modulates the function of matrix Hsp70 in translocation and maturation of preproteins. Mol Cell Biol. 1995 Dec;15(12):7098–7105. doi: 10.1128/mcb.15.12.7098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Liberek K., Skowyra D., Zylicz M., Johnson C., Georgopoulos C. The Escherichia coli DnaK chaperone, the 70-kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein. J Biol Chem. 1991 Aug 5;266(22):14491–14496. [PubMed] [Google Scholar]
  27. Maarse A. C., Blom J., Grivell L. A., Meijer M. MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria. EMBO J. 1992 Oct;11(10):3619–3628. doi: 10.1002/j.1460-2075.1992.tb05446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martin J., Mahlke K., Pfanner N. Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences. J Biol Chem. 1991 Sep 25;266(27):18051–18057. [PubMed] [Google Scholar]
  29. Nicchitta C. V., Blobel G. Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation. Cell. 1993 Jun 4;73(5):989–998. doi: 10.1016/0092-8674(93)90276-v. [DOI] [PubMed] [Google Scholar]
  30. Pace C. N. Conformational stability of globular proteins. Trends Biochem Sci. 1990 Jan;15(1):14–17. doi: 10.1016/0968-0004(90)90124-t. [DOI] [PubMed] [Google Scholar]
  31. Panzner S., Dreier L., Hartmann E., Kostka S., Rapoport T. A. Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p. Cell. 1995 May 19;81(4):561–570. doi: 10.1016/0092-8674(95)90077-2. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Pfanner N., Douglas M. G., Endo T., Hoogenraad N. J., Jensen R. E., Meijer M., Neupert W., Schatz G., Schmitz U. K., Shore G. C. Uniform nomenclature for the protein transport machinery of the mitochondrial membranes. Trends Biochem Sci. 1996 Feb;21(2):51–52. [PubMed] [Google Scholar]
  34. Pfanner N., Meijer M. Protein sorting. Pulling in the proteins. Curr Biol. 1995 Feb 1;5(2):132–135. doi: 10.1016/s0960-9822(95)00033-9. [DOI] [PubMed] [Google Scholar]
  35. Pfanner N., Tropschug M., Neupert W. Mitochondrial protein import: nucleoside triphosphates are involved in conferring import-competence to precursors. Cell. 1987 Jun 19;49(6):815–823. doi: 10.1016/0092-8674(87)90619-2. [DOI] [PubMed] [Google Scholar]
  36. Rassow J., Maarse A. C., Krainer E., Kübrich M., Müller H., Meijer M., Craig E. A., Pfanner N. Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44. J Cell Biol. 1994 Dec;127(6 Pt 1):1547–1556. doi: 10.1083/jcb.127.6.1547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rassow J., Voos W., Pfanner N. Partner proteins determine multiple functions of Hsp70. Trends Cell Biol. 1995 May;5(5):207–212. doi: 10.1016/s0962-8924(00)89001-7. [DOI] [PubMed] [Google Scholar]
  38. Rothman J. E. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell. 1989 Nov 17;59(4):591–601. doi: 10.1016/0092-8674(89)90005-6. [DOI] [PubMed] [Google Scholar]
  39. Scherer P. E., Krieg U. C., Hwang S. T., Vestweber D., Schatz G. A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J. 1990 Dec;9(13):4315–4322. doi: 10.1002/j.1460-2075.1990.tb07880.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Scherer P. E., Manning-Krieg U. C., Jenö P., Schatz G., Horst M. Identification of a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11930–11934. doi: 10.1073/pnas.89.24.11930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schleyer M., Neupert W. Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes. Cell. 1985 Nov;43(1):339–350. doi: 10.1016/0092-8674(85)90039-x. [DOI] [PubMed] [Google Scholar]
  42. Schmitt M., Neupert W., Langer T. Hsp78, a Clp homologue within mitochondria, can substitute for chaperone functions of mt-hsp70. EMBO J. 1995 Jul 17;14(14):3434–3444. doi: 10.1002/j.1460-2075.1995.tb07349.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schneider H. C., Berthold J., Bauer M. F., Dietmeier K., Guiard B., Brunner M., Neupert W. Mitochondrial Hsp70/MIM44 complex facilitates protein import. Nature. 1994 Oct 27;371(6500):768–774. doi: 10.1038/371768a0. [DOI] [PubMed] [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. 1993 Jun;12(6):2295–2302. doi: 10.1002/j.1460-2075.1993.tb05883.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Simon S. M., Peskin C. S., Oster G. F. What drives the translocation of proteins? Proc Natl Acad Sci U S A. 1992 May 1;89(9):3770–3774. doi: 10.1073/pnas.89.9.3770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sims P. J., Waggoner A. S., Wang C. H., Hoffman J. F. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry. 1974 Jul 30;13(16):3315–3330. doi: 10.1021/bi00713a022. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Stuart R. A., Gruhler A., van der Klei I., Guiard B., Koll H., Neupert W. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space. Eur J Biochem. 1994 Feb 15;220(1):9–18. doi: 10.1111/j.1432-1033.1994.tb18593.x. [DOI] [PubMed] [Google Scholar]
  49. Söllner T., Rassow J., Pfanner N. Analysis of mitochondrial protein import using translocation intermediates and specific antibodies. Methods Cell Biol. 1991;34:345–358. doi: 10.1016/s0091-679x(08)61689-1. [DOI] [PubMed] [Google Scholar]
  50. Ungermann C., Neupert W., Cyr D. M. The role of Hsp70 in conferring unidirectionality on protein translocation into mitochondria. Science. 1994 Nov 18;266(5188):1250–1253. doi: 10.1126/science.7973708. [DOI] [PubMed] [Google Scholar]
  51. 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. 1993 Oct;123(1):119–126. doi: 10.1083/jcb.123.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Voos W., Gambill B. D., Laloraya S., Ang D., Craig E. A., Pfanner N. Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes. Mol Cell Biol. 1994 Oct;14(10):6627–6634. doi: 10.1128/mcb.14.10.6627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wagner I., Arlt H., van Dyck L., Langer T., Neupert W. Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria. EMBO J. 1994 Nov 1;13(21):5135–5145. doi: 10.1002/j.1460-2075.1994.tb06843.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wickner W. T. How ATP drives proteins across membranes. Science. 1994 Nov 18;266(5188):1197–1198. doi: 10.1126/science.7973701. [DOI] [PubMed] [Google Scholar]
  55. Xia Z. X., Mathews F. S. Molecular structure of flavocytochrome b2 at 2.4 A resolution. J Mol Biol. 1990 Apr 20;212(4):837–863. doi: 10.1016/0022-2836(90)90240-M. [DOI] [PubMed] [Google Scholar]
  56. von Ahsen O., Voos W., Henninger H., Pfanner N. The mitochondrial protein import machinery. Role of ATP in dissociation of the Hsp70.Mim44 complex. J Biol Chem. 1995 Dec 15;270(50):29848–29853. doi: 10.1074/jbc.270.50.29848. [DOI] [PubMed] [Google Scholar]

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