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. 1988 Dec 1;107(6):2037–2043. doi: 10.1083/jcb.107.6.2037

A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites

PMCID: PMC2115651  PMID: 2904445

Abstract

Bovine pancreatic trypsin inhibitor (which contains three intramolecular disulfide bridges) was chemically coupled to the COOH terminus of a purified artificial mitochondrial precursor protein. When the resulting chimeric precursor was presented to energized isolated yeast mitochondria, its trypsin inhibitor moiety prevented the protein from completely entering the organelle; the protein remained stuck across both mitochondrial membranes, with its NH2 terminus in the matrix and its trypsin inhibitor moiety still exposed on the mitochondrial surface. The incompletely imported protein appeared to "jam" mitochondrial protein import sites since it blocked import of three authentic mitochondrial precursor proteins; it did not collapse the potential across the mitochondrial inner membrane. Quantification of the inhibition indicated that each isolated mitochondrial particle contains between 10(2) and 10(3) protein import sites.

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Selected References

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  1. Ades I. Z., Butow R. A. The products of mitochondria-bound cytoplasmic polysomes in yeast. J Biol Chem. 1980 Oct 25;255(20):9918–9924. [PubMed] [Google Scholar]
  2. Chen W. J., Douglas M. G. The role of protein structure in the mitochondrial import pathway. Unfolding of mitochondrially bound precursors is required for membrane translocation. J Biol Chem. 1987 Nov 15;262(32):15605–15609. [PubMed] [Google Scholar]
  3. 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]
  4. Della-Cioppa G., Kishore G. M. Import of a precursor protein into chloroplasts is inhibited by the herbicide glyphosate. EMBO J. 1988 May;7(5):1299–1305. doi: 10.1002/j.1460-2075.1988.tb02944.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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. doi: 10.1002/j.1460-2075.1988.tb02923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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. doi: 10.1038/322228a0. [DOI] [PubMed] [Google Scholar]
  7. Eilers M., Schatz G. Protein unfolding and the energetics of protein translocation across biological membranes. Cell. 1988 Feb 26;52(4):481–483. doi: 10.1016/0092-8674(88)90458-8. [DOI] [PubMed] [Google Scholar]
  8. 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. doi: 10.1002/j.1460-2075.1988.tb02925.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eytan G. D., Schatz G. Cytochrome c oxidase from bakers' yeast. V. Arrangement of the subunits in the isolated and membrane-bound enzyme. J Biol Chem. 1975 Jan 25;250(2):767–774. [PubMed] [Google Scholar]
  10. Gasser S. M., Daum G., Schatz G. Import of proteins into mitochondria. Energy-dependent uptake of precursors by isolated mitochondria. J Biol Chem. 1982 Nov 10;257(21):13034–13041. [PubMed] [Google Scholar]
  11. Gillespie L. L., Argan C., Taneja A. T., Hodges R. S., Freeman K. B., Shore G. C. A synthetic signal peptide blocks import of precursor proteins destined for the mitochondrial inner membrane or matrix. J Biol Chem. 1985 Dec 25;260(30):16045–16048. [PubMed] [Google Scholar]
  12. Hackenbrock C. R. Chemical and physical fixation of isolated mitochondria in low-energy and high-energy states. Proc Natl Acad Sci U S A. 1968 Oct;61(2):598–605. doi: 10.1073/pnas.61.2.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haid A., Suissa M. Immunochemical identification of membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methods Enzymol. 1983;96:192–205. doi: 10.1016/s0076-6879(83)96017-2. [DOI] [PubMed] [Google Scholar]
  14. Hurt E. C., 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. doi: 10.1002/j.1460-2075.1984.tb02272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hurt E. C., Pesold-Hurt B., Suda K., Oppliger W., Schatz G. The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. EMBO J. 1985 Aug;4(8):2061–2068. doi: 10.1002/j.1460-2075.1985.tb03892.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kellems R. E., Allison V. F., Butow R. A. Cytoplasmic type 80S ribosomes associated with yeast mitochondria. IV. Attachment of ribosomes to the outer membrane of isolated mitochondria. J Cell Biol. 1975 Apr;65(1):1–14. doi: 10.1083/jcb.65.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mori M., Matsue H., Miura S., Tatibana M., Hashimoto T. Transport of proteins into mitochondrial matrix. Evidence suggesting a common pathway for 3-ketoacyl-CoA thiolase and enzymes having presequences. Eur J Biochem. 1985 May 15;149(1):181–186. doi: 10.1111/j.1432-1033.1985.tb08909.x. [DOI] [PubMed] [Google Scholar]
  19. Müller G., Zimmermann R. Import of honeybee prepromelittin into the endoplasmic reticulum: energy requirements for membrane insertion. EMBO J. 1988 Mar;7(3):639–648. doi: 10.1002/j.1460-2075.1988.tb02858.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ohba M., Schatz G. Disruption of the outer membrane restores protein import to trypsin-treated yeast mitochondria. EMBO J. 1987 Jul;6(7):2117–2122. doi: 10.1002/j.1460-2075.1987.tb02478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ono H., Tuboi S. The cytosolic factor required for import of precursors of mitochondrial proteins into mitochondria. J Biol Chem. 1988 Mar 5;263(7):3188–3193. [PubMed] [Google Scholar]
  22. Pfaller R., Neupert W. High-affinity binding sites involved in the import of porin into mitochondria. EMBO J. 1987 Sep;6(9):2635–2642. doi: 10.1002/j.1460-2075.1987.tb02554.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Poyton R. O., Schatz G. Cytochrome c oxidase from bakers' yeast. IV. Immunological evidence for the participation of a mitochondrially synthesized subunit in enzymatic activity. J Biol Chem. 1975 Jan 25;250(2):762–766. [PubMed] [Google Scholar]
  24. Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
  25. Roise D., Schatz G. Mitochondrial presequences. J Biol Chem. 1988 Apr 5;263(10):4509–4511. [PubMed] [Google Scholar]
  26. 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]
  27. Schwaiger M., Herzog V., Neupert W. Characterization of translocation contact sites involved in the import of mitochondrial proteins. J Cell Biol. 1987 Jul;105(1):235–246. doi: 10.1083/jcb.105.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Suissa M., Schatz G. Import of proteins into mitochondria. Translatable mRNAs for imported mitochondrial proteins are present in free as well as mitochondria-bound cytoplasmic polysomes. J Biol Chem. 1982 Nov 10;257(21):13048–13055. [PubMed] [Google Scholar]
  30. Vestweber D., Schatz G. Mitochondria can import artificial precursor proteins containing a branched polypeptide chain or a carboxy-terminal stilbene disulfonate. J Cell Biol. 1988 Dec;107(6 Pt 1):2045–2049. doi: 10.1083/jcb.107.6.2045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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. doi: 10.1002/j.1460-2075.1988.tb02924.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wolfe P. B., Wickner W. Bacterial leader peptidase, a membrane protein without a leader peptide, uses the same export pathway as pre-secretory proteins. Cell. 1984 Apr;36(4):1067–1072. doi: 10.1016/0092-8674(84)90056-4. [DOI] [PubMed] [Google Scholar]

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