Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Oct 1;109(4):1421–1428. doi: 10.1083/jcb.109.4.1421

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites

PMCID: PMC2115798  PMID: 2529262

Abstract

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BAHR G. F., ZEITLER E. Study of mitochondria in rat liver. Quantitative electron microscopy. J Cell Biol. 1962 Dec;15:489–501. doi: 10.1083/jcb.15.3.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bonner W. M. Use of fluorography for sensitive isotope detection in polyacrylamide gel electrophoresis and related techniques. Methods Enzymol. 1983;96:215–222. doi: 10.1016/s0076-6879(83)96019-6. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  5. Chen W. J., Douglas M. G. 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. doi: 10.1016/0092-8674(87)90541-1. [DOI] [PubMed] [Google Scholar]
  6. Desel H., Zimmermann R., Janes M., Miller F., Neupert W. Biosynthesis of glyoxysomal enzymes in Neurospora crassa. Ann N Y Acad Sci. 1982;386:377–393. doi: 10.1111/j.1749-6632.1982.tb21429.x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Guiard B., Lederer F., Jacq C. More similarity between bakers'yeast L-(+)-lactate dehydrogenase and liver microsomal cytochrome b5. Nature. 1975 May 29;255(5507):422–423. doi: 10.1038/255422a0. [DOI] [PubMed] [Google Scholar]
  10. 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. doi: 10.1002/j.1460-2075.1985.tb04076.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Harnisch U., Weiss H., Sebald W. The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing. Eur J Biochem. 1985 May 15;149(1):95–99. doi: 10.1111/j.1432-1033.1985.tb08898.x. [DOI] [PubMed] [Google Scholar]
  13. Hartl F. U., Ostermann J., Guiard B., Neupert W. Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide. Cell. 1987 Dec 24;51(6):1027–1037. doi: 10.1016/0092-8674(87)90589-7. [DOI] [PubMed] [Google Scholar]
  14. Hartl F. U., Pfanner N., Nicholson D. W., Neupert W. Mitochondrial protein import. Biochim Biophys Acta. 1989 Jan 18;988(1):1–45. doi: 10.1016/0304-4157(89)90002-6. [DOI] [PubMed] [Google Scholar]
  15. Hartl F. U., Schmidt B., Wachter E., Weiss H., Neupert W. Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase. Cell. 1986 Dec 26;47(6):939–951. doi: 10.1016/0092-8674(86)90809-3. [DOI] [PubMed] [Google Scholar]
  16. Hawlitschek G., Schneider H., Schmidt B., Tropschug M., Hartl F. U., Neupert W. Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein. Cell. 1988 Jun 3;53(5):795–806. doi: 10.1016/0092-8674(88)90096-7. [DOI] [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. Kleene R., Pfanner N., Pfaller R., Link T. A., Sebald W., Neupert W., Tropschug M. Mitochondrial porin of Neurospora crassa: cDNA cloning, in vitro expression and import into mitochondria. EMBO J. 1987 Sep;6(9):2627–2633. doi: 10.1002/j.1460-2075.1987.tb02553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Knoll G., Brdiczka D. Changes in freeze-fractured mitochondrial membranes correlated to their energetic state. Dynamic interactions of the boundary membranes. Biochim Biophys Acta. 1983 Aug 24;733(1):102–110. doi: 10.1016/0005-2736(83)90095-0. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Matthews D. A., Bolin J. T., Burridge J. M., Filman D. J., Volz K. W., Kaufman B. T., Beddell C. R., Champness J. N., Stammers D. K., Kraut J. Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim. J Biol Chem. 1985 Jan 10;260(1):381–391. [PubMed] [Google Scholar]
  22. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pain D., Kanwar Y. S., Blobel G. Identification of a receptor for protein import into chloroplasts and its localization to envelope contact zones. Nature. 1988 Jan 21;331(6153):232–237. doi: 10.1038/331232a0. [DOI] [PubMed] [Google Scholar]
  24. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  25. Pfaller R., Steger H. F., Rassow J., Pfanner N., Neupert W. Import pathways of precursor proteins into mitochondria: multiple receptor sites are followed by a common membrane insertion site. J Cell Biol. 1988 Dec;107(6 Pt 2):2483–2490. doi: 10.1083/jcb.107.6.2483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pfanner N., Hartl F. U., Guiard B., Neupert W. Mitochondrial precursor proteins are imported through a hydrophilic membrane environment. Eur J Biochem. 1987 Dec 1;169(2):289–293. doi: 10.1111/j.1432-1033.1987.tb13610.x. [DOI] [PubMed] [Google Scholar]
  27. Pfanner N., Hartl F. U., Neupert W. Import of proteins into mitochondria: a multi-step process. Eur J Biochem. 1988 Aug 1;175(2):205–212. doi: 10.1111/j.1432-1033.1988.tb14185.x. [DOI] [PubMed] [Google Scholar]
  28. Pfanner N., Neupert W. Transport of F1-ATPase subunit beta into mitochondria depends on both a membrane potential and nucleoside triphosphates. FEBS Lett. 1986 Dec 15;209(2):152–156. doi: 10.1016/0014-5793(86)81101-2. [DOI] [PubMed] [Google Scholar]
  29. 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. 1985 Nov;4(11):2819–2825. doi: 10.1002/j.1460-2075.1985.tb04009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pfanner N., Pfaller R., Kleene R., Ito M., Tropschug M., Neupert W. Role of ATP in mitochondrial protein import. Conformational alteration of a precursor protein can substitute for ATP requirement. J Biol Chem. 1988 Mar 25;263(9):4049–4051. [PubMed] [Google Scholar]
  31. 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]
  32. Schauder B., Blöcker H., Frank R., McCarthy J. E. Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. Gene. 1987;52(2-3):279–283. doi: 10.1016/0378-1119(87)90054-0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Schmidt B., Wachter E., Sebald W., Neupert W. Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9. Eur J Biochem. 1984 Nov 2;144(3):581–588. doi: 10.1111/j.1432-1033.1984.tb08505.x. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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. doi: 10.1002/j.1460-2075.1984.tb02271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Van Venetië R., Verkleij A. J. Possible role of non-bilayer lipids in the structure of mitochondria. A freeze-fracture electron microscopy study. Biochim Biophys Acta. 1982 Nov 22;692(3):397–405. doi: 10.1016/0005-2736(82)90390-x. [DOI] [PubMed] [Google Scholar]
  38. Vestweber D., Schatz G. A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites. J Cell Biol. 1988 Dec;107(6 Pt 1):2037–2043. doi: 10.1083/jcb.107.6.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Volz K. W., Matthews D. A., Alden R. A., Freer S. T., Hansch C., Kaufman B. T., Kraut J. Crystal structure of avian dihydrofolate reductase containing phenyltriazine and NADPH. J Biol Chem. 1982 Mar 10;257(5):2528–2536. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES