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. 1998 Jul 1;333(Pt 1):151–158. doi: 10.1042/bj3330151

Targeting and assembly of the oxoglutarate carrier: general principles for biogenesis of carrier proteins of the mitochondrial inner membrane.

A Palmisano 1, V Zara 1, A Hönlinger 1, A Vozza 1, P J Dekker 1, N Pfanner 1, F Palmieri 1
PMCID: PMC1219567  PMID: 9639574

Abstract

We have studied the targeting and assembly of the 2-oxoglutarate carrier (OGC), an integral inner-membrane protein of mitochondria. The precursor of OGC, synthesized without a cleavable presequence, is transported into mitochondria in an ATP- and membrane potential-dependent manner. Import of the mammalian OGC occurs efficiently into both mammalian and yeast mitochondria. Targeting of OGC reveals a clear dependence on the mitochondrial surface receptor Tom70 (the 70 kDa subunit of the translocase of the outer mitochondrial membrane), whereas a cleavable preprotein depends on Tom20 (the 20 kDa subunit), supporting a model of specificity differences of the receptors and the existence of distinct targeting pathways to mitochondria. The assembly of minute amounts of OGC imported in vitro to the dimeric form can be monitored by blue native electrophoresis of digitonin-lysed mitochondria. The assembly of mammalian OGC and fungal ADP/ATP carrier occurs with high efficiency in both mammalian and yeast mitochondria. These findings indicate a dynamic behaviour of the carrier dimers in the mitochondrial inner membrane and suggest a high conservation of the assembly reactions from mammals to fungi.

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Selected References

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  1. Alconada A., Gärtner F., Hönlinger A., Kübrich M., Pfanner N. Mitochondrial receptor complex from Neurospora crassa and Saccharomyces cerevisiae. Methods Enzymol. 1995;260:263–286. doi: 10.1016/0076-6879(95)60144-9. [DOI] [PubMed] [Google Scholar]
  2. Aquila H., Link T. A., Klingenberg M. The uncoupling protein from brown fat mitochondria is related to the mitochondrial ADP/ATP carrier. Analysis of sequence homologies and of folding of the protein in the membrane. EMBO J. 1985 Sep;4(9):2369–2376. doi: 10.1002/j.1460-2075.1985.tb03941.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bisaccia F., Indiveri C., Palmieri F. Purification of reconstitutively active alpha-oxoglutarate carrier from pig heart mitochondria. Biochim Biophys Acta. 1985 Dec 16;810(3):362–369. doi: 10.1016/0005-2728(85)90222-1. [DOI] [PubMed] [Google Scholar]
  4. Bisaccia F., Zara V., Capobianco L., Iacobazzi V., Mazzeo M., Palmieri F. The formation of a disulfide cross-link between the two subunits demonstrates the dimeric structure of the mitochondrial oxoglutarate carrier. Biochim Biophys Acta. 1996 Feb 8;1292(2):281–288. doi: 10.1016/0167-4838(95)00215-4. [DOI] [PubMed] [Google Scholar]
  5. Brandolin G., Doussiere J., Gulik A., Gulik-Krzywicki T., Lauquin G. J., Vignais P. V. Kinetic, binding and ultrastructural properties of the beef heart adenine nucleotide carrier protein after incorporation into phospholipid vesicles. Biochim Biophys Acta. 1980 Oct 3;592(3):592–614. doi: 10.1016/0005-2728(80)90103-6. [DOI] [PubMed] [Google Scholar]
  6. Brandolin G., Le Saux A., Trezeguet V., Lauquin G. J., Vignais P. V. Chemical, immunological, enzymatic, and genetic approaches to studying the arrangement of the peptide chain of the ADP/ATP carrier in the mitochondrial membrane. J Bioenerg Biomembr. 1993 Oct;25(5):459–472. doi: 10.1007/BF01108403. [DOI] [PubMed] [Google Scholar]
  7. Brix J., Dietmeier K., Pfanner N. Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70. J Biol Chem. 1997 Aug 15;272(33):20730–20735. doi: 10.1074/jbc.272.33.20730. [DOI] [PubMed] [Google Scholar]
  8. Bömer U., Rassow J., Zufall N., Pfanner N., Meijer M., Maarse A. C. The preprotein translocase of the inner mitochondrial membrane: evolutionary conservation of targeting and assembly of Tim17. J Mol Biol. 1996 Oct 4;262(4):389–395. doi: 10.1006/jmbi.1996.0522. [DOI] [PubMed] [Google Scholar]
  9. Dekker P. J., Martin F., Maarse A. C., Bömer U., Müller H., Guiard B., Meijer M., Rassow J., Pfanner N. The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44. EMBO J. 1997 Sep 1;16(17):5408–5419. doi: 10.1093/emboj/16.17.5408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dekker P. J., Müller H., Rassow J., Pfanner N. Characterization of the preprotein translocase of the outer mitochondrial membrane by blue native electrophoresis. Biol Chem. 1996 Jul-Aug;377(7-8):535–538. [PubMed] [Google Scholar]
  11. Dietmeier K., Zara V., Palmisano A., Palmieri F., Voos W., Schlossmann J., Moczko M., Kispal G., Pfanner N. Targeting and translocation of the phosphate carrier/p32 to the inner membrane of yeast mitochondria. J Biol Chem. 1993 Dec 5;268(34):25958–25964. [PubMed] [Google Scholar]
  12. Dolce V., Iacobazzi V., Palmieri F., Walker J. E. The sequences of human and bovine genes of the phosphate carrier from mitochondria contain evidence of alternatively spliced forms. J Biol Chem. 1994 Apr 8;269(14):10451–10460. [PubMed] [Google Scholar]
  13. Emmermann M., Braun H. P., Schmitz U. K. The ADP/ATP translocator from potato has a long amino-terminal extension. Curr Genet. 1991 Nov;20(5):405–410. doi: 10.1007/BF00317069. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Hackenberg H., Klingenberg M. Molecular weight and hydrodynamic parameters of the adenosine 5'-diphosphate--adenosine 5'-triphosphate carrier in Triton X-100. Biochemistry. 1980 Feb 5;19(3):548–555. doi: 10.1021/bi00544a024. [DOI] [PubMed] [Google Scholar]
  16. Hines V., Brandt A., Griffiths G., Horstmann H., Brütsch H., Schatz G. Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70. EMBO J. 1990 Oct;9(10):3191–3200. doi: 10.1002/j.1460-2075.1990.tb07517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Iacobazzi V., Lauria G., Palmieri F. Organization and sequence of the human gene for the mitochondrial citrate transport protein. DNA Seq. 1997;7(3-4):127–139. doi: 10.3109/10425179709034029. [DOI] [PubMed] [Google Scholar]
  18. Indiveri C., Palmieri F., Bisaccia F., Krämer R. Kinetics of the reconstituted 2-oxoglutarate carrier from bovine heart mitochondria. Biochim Biophys Acta. 1987 Mar 4;890(3):310–318. doi: 10.1016/0005-2728(87)90158-7. [DOI] [PubMed] [Google Scholar]
  19. Klingenberg M., Appel M. The uncoupling protein dimer can form a disulfide cross-link between the mobile C-terminal SH groups. Eur J Biochem. 1989 Mar 1;180(1):123–131. doi: 10.1111/j.1432-1033.1989.tb14622.x. [DOI] [PubMed] [Google Scholar]
  20. Koehler C. M., Jarosch E., Tokatlidis K., Schmid K., Schweyen R. J., Schatz G. Import of mitochondrial carriers mediated by essential proteins of the intermembrane space. Science. 1998 Jan 16;279(5349):369–373. doi: 10.1126/science.279.5349.369. [DOI] [PubMed] [Google Scholar]
  21. Komiya T., Rospert S., Schatz G., Mihara K. Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones. EMBO J. 1997 Jul 16;16(14):4267–4275. doi: 10.1093/emboj/16.14.4267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Komiya T., Sakaguchi M., Mihara K. Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria. EMBO J. 1996 Jan 15;15(2):399–407. [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. LaNoue K. F., Schoolwerth A. C. Metabolite transport in mitochondria. Annu Rev Biochem. 1979;48:871–922. doi: 10.1146/annurev.bi.48.070179.004255. [DOI] [PubMed] [Google Scholar]
  25. Moczko M., Ehmann B., Gärtner F., Hönlinger A., Schäfer E., Pfanner N. Deletion of the receptor MOM19 strongly impairs import of cleavable preproteins into Saccharomyces cerevisiae mitochondria. J Biol Chem. 1994 Mar 25;269(12):9045–9051. [PubMed] [Google Scholar]
  26. Neupert W. Protein import into mitochondria. Annu Rev Biochem. 1997;66:863–917. doi: 10.1146/annurev.biochem.66.1.863. [DOI] [PubMed] [Google Scholar]
  27. Palmieri F. Mitochondrial carrier proteins. FEBS Lett. 1994 Jun 6;346(1):48–54. doi: 10.1016/0014-5793(94)00329-7. [DOI] [PubMed] [Google Scholar]
  28. Palmieri L., De Marco V., Iacobazzi V., Palmieri F., Runswick M. J., Walker J. E. Identification of the yeast ARG-11 gene as a mitochondrial ornithine carrier involved in arginine biosynthesis. FEBS Lett. 1997 Jun 30;410(2-3):447–451. doi: 10.1016/s0014-5793(97)00630-3. [DOI] [PubMed] [Google Scholar]
  29. Palmieri L., Palmieri F., Runswick M. J., Walker J. E. Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein. FEBS Lett. 1996 Dec 16;399(3):299–302. doi: 10.1016/s0014-5793(96)01350-6. [DOI] [PubMed] [Google Scholar]
  30. Perkins M., Haslam J. M., Linnane A. W. Biogenesis of mitochondria. The effects of physiological and genetic manipulation of Saccharomyces cerevisiae on the mitochondrial transport systems for tricarboxylate-cycle anions. Biochem J. 1973 Aug;134(4):923–934. doi: 10.1042/bj1340923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pfanner N., Meijer M. The Tom and Tim machine. Curr Biol. 1997 Feb 1;7(2):R100–R103. doi: 10.1016/s0960-9822(06)00048-0. [DOI] [PubMed] [Google Scholar]
  32. Ramage L., Junne T., Hahne K., Lithgow T., Schatz G. Functional cooperation of mitochondrial protein import receptors in yeast. EMBO J. 1993 Nov;12(11):4115–4123. doi: 10.1002/j.1460-2075.1993.tb06095.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rassow J., Guiard B., Wienhues U., Herzog V., Hartl F. U., Neupert W. Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites. J Cell Biol. 1989 Oct;109(4 Pt 1):1421–1428. doi: 10.1083/jcb.109.4.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Runswick M. J., Powell S. J., Nyren P., Walker J. E. Sequence of the bovine mitochondrial phosphate carrier protein: structural relationship to ADP/ATP translocase and the brown fat mitochondria uncoupling protein. EMBO J. 1987 May;6(5):1367–1373. doi: 10.1002/j.1460-2075.1987.tb02377.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Runswick M. J., Walker J. E., Bisaccia F., Iacobazzi V., Palmieri F. Sequence of the bovine 2-oxoglutarate/malate carrier protein: structural relationship to other mitochondrial transport proteins. Biochemistry. 1990 Dec 18;29(50):11033–11040. doi: 10.1021/bi00502a004. [DOI] [PubMed] [Google Scholar]
  36. Saraste M., Walker J. E. Internal sequence repeats and the path of polypeptide in mitochondrial ADP/ATP translocase. FEBS Lett. 1982 Aug 2;144(2):250–254. doi: 10.1016/0014-5793(82)80648-0. [DOI] [PubMed] [Google Scholar]
  37. Schatz G., Dobberstein B. Common principles of protein translocation across membranes. Science. 1996 Mar 15;271(5255):1519–1526. doi: 10.1126/science.271.5255.1519. [DOI] [PubMed] [Google Scholar]
  38. Schleiff E., Shore G. C., Goping I. S. Interactions of the human mitochondrial protein import receptor, hTom20, with precursor proteins in vitro reveal pleiotropic specificities and different receptor domain requirements. J Biol Chem. 1997 Jul 11;272(28):17784–17789. doi: 10.1074/jbc.272.28.17784. [DOI] [PubMed] [Google Scholar]
  39. Schleyer M., Neupert W. Transport of ADP/ATP carrier into mitochondria. Precursor imported in vitro acquires functional properties of the mature protein. J Biol Chem. 1984 Mar 25;259(6):3487–3491. [PubMed] [Google Scholar]
  40. Schneider H., Söllner T., Dietmeier K., Eckerskorn C., Lottspeich F., Trülzsch B., Neupert W., Pfanner N. Targeting of the master receptor MOM19 to mitochondria. Science. 1991 Dec 13;254(5038):1659–1662. doi: 10.1126/science.1661031. [DOI] [PubMed] [Google Scholar]
  41. Schägger H., Cramer W. A., von Jagow G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem. 1994 Mar;217(2):220–230. doi: 10.1006/abio.1994.1112. [DOI] [PubMed] [Google Scholar]
  42. Sirrenberg C., Bauer M. F., Guiard B., Neupert W., Brunner M. Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22. Nature. 1996 Dec 12;384(6609):582–585. doi: 10.1038/384582a0. [DOI] [PubMed] [Google Scholar]
  43. Steger H. F., Söllner T., Kiebler M., Dietmeier K. A., Pfaller R., Trülzsch K. S., Tropschug M., Neupert W., Pfanner N. Import of ADP/ATP carrier into mitochondria: two receptors act in parallel. J Cell Biol. 1990 Dec;111(6 Pt 1):2353–2363. doi: 10.1083/jcb.111.6.2353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wills C., Benhaim P., Martin T. Effect of mutants and inhibitors on mitochondrial transport systems in vivo in yeast. Biochim Biophys Acta. 1984 Nov 21;778(1):57–66. doi: 10.1016/0005-2736(84)90447-4. [DOI] [PubMed] [Google Scholar]
  45. Zara V., Palmieri F., Mahlke K., Pfanner N. The cleavable presequence is not essential for import and assembly of the phosphate carrier of mammalian mitochondria but enhances the specificity and efficiency of import. J Biol Chem. 1992 Jun 15;267(17):12077–12081. [PubMed] [Google Scholar]
  46. Zara V., Rassow J., Wachter E., Tropschug M., Palmieri F., Neupert W., Pfanner N. Biogenesis of the mitochondrial phosphate carrier. Eur J Biochem. 1991 Jun 1;198(2):405–410. doi: 10.1111/j.1432-1033.1991.tb16029.x. [DOI] [PubMed] [Google Scholar]

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