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EMBO J. Nov 17, 1997; 16(22): 6727–6736.
PMCID: PMC1170277

Active unfolding of precursor proteins during mitochondrial protein import.

Abstract

Precursor proteins made in the cytoplasm must be in an unfolded conformation during import into mitochondria. Some precursor proteins have tightly folded domains but are imported faster than they unfold spontaneously, implying that mitochondria can unfold proteins. We measured the import rates of artificial precursors containing presequences of varying length fused to either mouse dihydrofolate reductase or bacterial barnase, and found that unfolding of a precursor at the mitochondrial surface is dramatically accelerated when its presequence is long enough to span both membranes and to interact with mhsp70 in the mitochondrial matrix. If the presequence is too short, import is slow but can be strongly accelerated by urea-induced unfolding, suggesting that import of these 'short' precursors is limited by spontaneous unfolding at the mitochondrial surface. With precursors that have sufficiently long presequences, unfolding by the inner membrane import machinery can be orders of magnitude faster than spontaneous unfolding, suggesting that mhsp70 can act as an ATP-driven force-generating motor during protein import.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Arretz M, Schneider H, Guiard B, Brunner M, Neupert W. Characterization of the mitochondrial processing peptidase of Neurospora crassa. J Biol Chem. 1994 Feb 18;269(7):4959–4967. [PubMed]
  • Bai Y, Sosnick TR, Mayne L, Englander SW. Protein folding intermediates: native-state hydrogen exchange. Science. 1995 Jul 14;269(5221):192–197. [PMC free article] [PubMed]
  • Beasley EM, Müller S, Schatz G. The signal that sorts yeast cytochrome b2 to the mitochondrial intermembrane space contains three distinct functional regions. EMBO J. 1993 Jun;12(6):2303–2311. [PMC free article] [PubMed]
  • Bolliger L, Deloche O, Glick BS, Georgopoulos C, Jenö P, Kronidou N, Horst M, Morishima N, Schatz G. A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability. EMBO J. 1994 Apr 15;13(8):1998–2006. [PMC free article] [PubMed]
  • Bujard H, Gentz R, Lanzer M, Stueber D, Mueller M, Ibrahimi I, Haeuptle MT, Dobberstein B. A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo. Methods Enzymol. 1987;155:416–433. [PubMed]
  • Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. [PMC free article] [PubMed]
  • Chen WJ, Douglas MG. Phosphodiester bond cleavage outside mitochondria is required for the completion of protein import into the mitochondrial matrix. Cell. 1987 Jun 5;49(5):651–658. [PubMed]
  • Cyr DM, Stuart RA, 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]
  • Deshaies RJ, Koch BD, Werner-Washburne M, Craig EA, Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. [PubMed]
  • Eilers M, Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature. 1986 Jul 17;322(6076):228–232. [PubMed]
  • Eilers M, Hwang S, Schatz G. Unfolding and refolding of a purified precursor protein during import into isolated mitochondria. EMBO J. 1988 Apr;7(4):1139–1145. [PMC free article] [PubMed]
  • Endo T, Schatz G. Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding. EMBO J. 1988 Apr;7(4):1153–1158. [PMC free article] [PubMed]
  • Gärtner F, Bömer U, Guiard B, Pfanner N. The sorting signal of cytochrome b2 promotes early divergence from the general mitochondrial import pathway and restricts the unfoldase activity of matrix Hsp70. EMBO J. 1995 Dec 1;14(23):6043–6057. [PMC free article] [PubMed]
  • Gasser SM, Ohashi A, Daum G, Böhni PC, Gibson J, Reid GA, Yonetani T, Schatz G. Imported mitochondrial proteins cytochrome b2 and cytochrome c1 are processed in two steps. Proc Natl Acad Sci U S A. 1982 Jan;79(2):267–271. [PMC free article] [PubMed]
  • Glick BS. Can Hsp70 proteins act as force-generating motors? Cell. 1995 Jan 13;80(1):11–14. [PubMed]
  • Glick BS, Pon LA. Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol. 1995;260:213–223. [PubMed]
  • Glick BS, Wachter C, Reid GA, 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. [PMC free article] [PubMed]
  • Guiard B. Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2). EMBO J. 1985 Dec 1;4(12):3265–3272. [PMC free article] [PubMed]
  • Gray TE, Fersht AR. Refolding of barnase in the presence of GroE. J Mol Biol. 1993 Aug 20;232(4):1197–1207. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Hartley RW. Barnase and barstar: two small proteins to fold and fit together. Trends Biochem Sci. 1989 Nov;14(11):450–454. [PubMed]
  • Hase T, Müller U, Riezman H, Schatz G. A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus. EMBO J. 1984 Dec 20;3(13):3157–3164. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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]
  • Hurt EC, Pesold-Hurt B, Schatz G. The amino-terminal region of an imported mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix. EMBO J. 1984 Dec 20;3(13):3149–3156. [PMC free article] [PubMed]
  • Hwang S, Jascur T, Vestweber D, Pon L, Schatz G. Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane. J Cell Biol. 1989 Aug;109(2):487–493. [PMC free article] [PubMed]
  • Hwang ST, Wachter C, Schatz G. Protein import into the yeast mitochondrial matrix. A new translocation intermediate between the two mitochondrial membranes. J Biol Chem. 1991 Nov 5;266(31):21083–21089. [PubMed]
  • Jascur T. Import of precursor proteins into yeast submitochondrial particles. Methods Cell Biol. 1991;34:359–368. [PubMed]
  • Kang PJ, Ostermann J, Shilling J, Neupert W, Craig EA, Pfanner N. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature. 1990 Nov 8;348(6297):137–143. [PubMed]
  • Kronidou NG, Oppliger W, Bolliger L, Hannavy K, Glick BS, 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. [PMC free article] [PubMed]
  • Laloraya S, Gambill BD, Craig EA. A role for a eukaryotic GrpE-related protein, Mge1p, in protein translocation. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6481–6485. [PMC free article] [PubMed]
  • 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]
  • Maddox J. More models of muscle movement. Nature. 1994 Mar 24;368(6469):287–287. [PubMed]
  • Manning-Krieg UC, Scherer PE, Schatz G. Sequential action of mitochondrial chaperones in protein import into the matrix. EMBO J. 1991 Nov;10(11):3273–3280. [PMC free article] [PubMed]
  • Matouschek A, Matthews JM, Johnson CM, Fersht AR. Extrapolation to water of kinetic and equilibrium data for the unfolding of barnase in urea solutions. Protein Eng. 1994 Sep;7(9):1089–1095. [PubMed]
  • Matouschek A, Otzen DE, Itzhaki LS, Jackson SE, Fersht AR. Movement of the position of the transition state in protein folding. Biochemistry. 1995 Oct 17;34(41):13656–13662. [PubMed]
  • Mayer A, Neupert W, Lill R. Mitochondrial protein import: reversible binding of the presequence at the trans side of the outer membrane drives partial translocation and unfolding. Cell. 1995 Jan 13;80(1):127–137. [PubMed]
  • Mayo SL, Baldwin RL. Guanidinium chloride induction of partial unfolding in amide proton exchange in RNase A. Science. 1993 Nov 5;262(5135):873–876. [PubMed]
  • Ostermann J, Voos W, Kang PJ, Craig EA, Neupert W, Pfanner N. Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix. FEBS Lett. 1990 Dec 17;277(1-2):281–284. [PubMed]
  • Paddon CJ, Hartley RW. Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation. Gene. 1987;53(1):11–19. [PubMed]
  • Pelham HR. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell. 1986 Sep 26;46(7):959–961. [PubMed]
  • Pfanner N, Meijer M. Protein sorting. Pulling in the proteins. Curr Biol. 1995 Feb 1;5(2):132–135. [PubMed]
  • Rassow J, Hartl FU, Guiard B, Pfanner N, Neupert W. Polypeptides traverse the mitochondrial envelope in an extended state. FEBS Lett. 1990 Nov 26;275(1-2):190–194. [PubMed]
  • Rassow J, Maarse AC, Krainer E, Kübrich M, Müller H, Meijer M, Craig EA, 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. [PMC free article] [PubMed]
  • Rospert S, Looser R, Dubaquie Y, Matouschek A, Glick BS, Schatz G. Hsp60-independent protein folding in the matrix of yeast mitochondria. EMBO J. 1996 Feb 15;15(4):764–774. [PMC free article] [PubMed]
  • Scherer PE, Krieg UC, Hwang ST, 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. [PMC free article] [PubMed]
  • Schmid D, Jaussi R, Christen P. Precursor of mitochondrial aspartate aminotransferase synthesized in Escherichia coli is complexed with heat-shock protein DnaK. Eur J Biochem. 1992 Sep 15;208(3):699–704. [PubMed]
  • Schneider HC, Berthold J, Bauer MF, Dietmeier K, Guiard B, Brunner M, Neupert W. Mitochondrial Hsp70/MIM44 complex facilitates protein import. Nature. 1994 Oct 27;371(6500):768–774. [PubMed]
  • Shi L, Kataoka M, Fink AL. Conformational characterization of DnaK and its complexes by small-angle X-ray scattering. Biochemistry. 1996 Mar 12;35(10):3297–3308. [PubMed]
  • Simon SM, Peskin CS, Oster GF. What drives the translocation of proteins? Proc Natl Acad Sci U S A. 1992 May 1;89(9):3770–3774. [PMC free article] [PubMed]
  • Smith BJ, Yaffe MP. A mutation in the yeast heat-shock factor gene causes temperature-sensitive defects in both mitochondrial protein import and the cell cycle. Mol Cell Biol. 1991 May;11(5):2647–2655. [PMC free article] [PubMed]
  • Stuart RA, Cyr DM, Craig EA, Neupert W. Mitochondrial molecular chaperones: their role in protein translocation. Trends Biochem Sci. 1994 Feb;19(2):87–92. [PubMed]
  • Stueber D, Ibrahimi I, Cutler D, Dobberstein B, Bujard H. A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences. EMBO J. 1984 Dec 20;3(13):3143–3148. [PMC free article] [PubMed]
  • Ungermann C, Neupert W, Cyr DM. The role of Hsp70 in conferring unidirectionality on protein translocation into mitochondria. Science. 1994 Nov 18;266(5188):1250–1253. [PubMed]
  • Vestweber D, Schatz G. Point mutations destabilizing a precursor protein enhance its post-translational import into mitochondria. EMBO J. 1988 Apr;7(4):1147–1151. [PMC free article] [PubMed]
  • Viitanen PV, Donaldson GK, Lorimer GH, Lubben TH, Gatenby AA. Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase. Biochemistry. 1991 Oct 8;30(40):9716–9723. [PubMed]
  • Voos W, Gambill BD, Guiard B, Pfanner N, Craig EA. 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. [PMC free article] [PubMed]
  • Voos W, Gambill BD, Laloraya S, Ang D, Craig EA, 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. [PMC free article] [PubMed]
  • Wilbanks SM, Chen L, Tsuruta H, Hodgson KO, McKay DB. Solution small-angle X-ray scattering study of the molecular chaperone Hsc70 and its subfragments. Biochemistry. 1995 Sep 26;34(38):12095–12106. [PubMed]

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