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. 1995 May 15;14(10):2293–2297. doi: 10.1002/j.1460-2075.1995.tb07223.x

Dynamic interaction of the protein translocation systems in the inner and outer membranes of yeast mitochondria.

M Horst 1, S Hilfiker-Rothenfluh 1, W Oppliger 1, G Schatz 1
PMCID: PMC398336  PMID: 7774587

Abstract

Mitochondria contain two distinct protein import systems, one in the outer and the other in the inner membrane. These systems can act independently of one another in submitochondrial fractions of if a protein is transported to the outer membrane or to the intermembrane space. It has been proposed that the two systems associate reversibly when a protein is transported across both membranes, but this hypothesis has remained unproven. In order to address this question, we have checked whether antibodies against a subunit of one system can co-immunoprecipitate subunits of the other system. We find that the two systems associate stably if a matrix-targeted precursor is arrested during import; no association is seen in the absence of a stuck precursor. These experiments provide direct evidence that protein import into the mitochondrial matrix is mediated by the reversible interaction of the two translocation systems.

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Selected References

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  1. 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]
  2. Ey P. L., Prowse S. J., Jenkin C. R. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry. 1978 Jul;15(7):429–436. doi: 10.1016/0161-5890(78)90070-6. [DOI] [PubMed] [Google Scholar]
  3. Gasser S. M., Schatz G. Import of proteins into mitochondria. In vitro studies on the biogenesis of the outer membrane. J Biol Chem. 1983 Mar 25;258(6):3427–3430. [PubMed] [Google Scholar]
  4. Glick B., Wachter C., Schatz G. Protein import into mitochondria: two systems acting in tandem? Trends Cell Biol. 1991 Oct;1(4):99–103. doi: 10.1016/0962-8924(91)90037-a. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Horst M., Kronidou N. G., Schatz G. Protein translocation: through the mitochondrial inner membrane. Curr Biol. 1993 Mar;3(3):175–177. doi: 10.1016/0960-9822(93)90265-p. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Hwang S. T., 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] [Google Scholar]
  10. 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. doi: 10.1083/jcb.109.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jascur T., Goldenberg D. P., Vestweber D., Schatz G. Sequential translocation of an artificial precursor protein across the two mitochondrial membranes. J Biol Chem. 1992 Jul 5;267(19):13636–13641. [PubMed] [Google Scholar]
  12. Kellems R. E., Allison V. F., Butow R. A. Cytoplasmic type 80 S ribosomes associated with yeast mitochondria. II. Evidence for the association of cytoplasmic ribosomes with the outer mitochondrial membrane in situ. J Biol Chem. 1974 May 25;249(10):3297–3303. [PubMed] [Google Scholar]
  13. 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]
  14. Lill R., Stuart R. A., Drygas M. E., Nargang F. E., Neupert W. Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space. EMBO J. 1992 Feb;11(2):449–456. doi: 10.1002/j.1460-2075.1992.tb05074.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Manning-Krieg U. C., Scherer P. E., Schatz G. Sequential action of mitochondrial chaperones in protein import into the matrix. EMBO J. 1991 Nov;10(11):3273–3280. doi: 10.1002/j.1460-2075.1991.tb04891.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mayer A., Lill R., Neupert W. Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria. J Cell Biol. 1993 Jun;121(6):1233–1243. doi: 10.1083/jcb.121.6.1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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. doi: 10.1016/0092-8674(95)90457-3. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Ohba M., Schatz G. Protein import into yeast mitochondria is inhibited by antibodies raised against 45-kd proteins of the outer membrane. EMBO J. 1987 Jul;6(7):2109–2115. doi: 10.1002/j.1460-2075.1987.tb02477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pfanner N., Rassow J., van der Klei I. J., Neupert W. A dynamic model of the mitochondrial protein import machinery. Cell. 1992 Mar 20;68(6):999–1002. doi: 10.1016/0092-8674(92)90069-o. [DOI] [PubMed] [Google Scholar]
  21. Pon L., Moll T., Vestweber D., Marshallsay B., Schatz G. Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins. J Cell Biol. 1989 Dec;109(6 Pt 1):2603–2616. doi: 10.1083/jcb.109.6.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. Segui-Real B., Kispal G., Lill R., Neupert W. Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: passage of preproteins through the intermembrane space. EMBO J. 1993 May;12(5):2211–2218. doi: 10.1002/j.1460-2075.1993.tb05869.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Smith M., Hicks S., Baker K., McCauley R. Rupture of the mitochondrial outer membrane impairs porin assembly. J Biol Chem. 1994 Nov 11;269(45):28460–28464. [PubMed] [Google Scholar]
  29. Vestweber D., Brunner J., Baker A., Schatz G. A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site. Nature. 1989 Sep 21;341(6239):205–209. doi: 10.1038/341205a0. [DOI] [PubMed] [Google Scholar]
  30. 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]

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