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Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus

Nature Genetics volume 30, pages 394–399 (2002)Cite this article

Abstract

A T→G transversion at nt 8993 in mitochondrial DNA of MTATP6 (encoding ATPase 6 of complex V of the respiratory chain) causes impaired mitochondrial ATP synthesis in two related mitochondrial disorders: neuropathy, ataxia and retinitis pigmentosa1 and maternally inherited Leigh syndrome2. To overcome the biochemical defect, we expressed wildtype ATPase 6 protein allotopically3 from nucleus-transfected constructs encoding an amino-terminal mitochondrial targeting signal appended to a recoded ATPase 6 gene (made compatible with the universal genetic code) that also contained a carboxy-terminal FLAG epitope tag. After transfection of human cells, the precursor polypeptide was expressed, imported into and processed within mitochondria, and incorporated into complex V. Allotopic expression of stably transfected constructs in cytoplasmic hybrids (cybrids) homoplasmic with respect to the 8993T→G mutation showed a significantly improved recovery after growth in selective medium as well as a significant increase in ATP synthesis. This is the first successful demonstration of allotopic expression of an mtDNA-encoded polypeptide in mammalian cells and could form the basis of a genetic approach to treat a number of human mitochondrial disorders.

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Figure 1: Constructs.
Figure 2: Subcellular localization of rA6F in human 293T cells.
Figure 3: Importation of rA6F into mitochondria.
Figure 4: RT–PCR of stably transfected cybrids.
Figure 5: Phenotypes of homoplasmic mutated (8993T→G) cybrids transfected with P1A6F constructs.

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References

  1. Holt, I.J., Harding, A.E., Petty, R.K.H. & Morgan-Hughes, J.A. A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am. J. Hum. Genet. 46, 428–433 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Tatuch, Y. et al. Heteroplasmic mtDNA mutation (T→G) at 8993 can cause Leigh's disease when the percentage of abnormal mtDNA is high. Am. J. Hum. Genet. 50, 852–858 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Law, R.H., Farrell, L.B., Nero, D., Devenish, R.J. & Nagley, P. Studies on the import into mitochondria of yeast ATP synthase subunits 8 and 9 encoded by artificial nuclear genes. FEBS Lett. 236, 501–505 (1988).

    Article  CAS  Google Scholar 

  4. DiMauro, S. & Andreu, A.L. Mutations in mtDNA: are we scraping the bottom of the barrel? Brain Pathol. 10, 431–441 (2000).

    Article  CAS  Google Scholar 

  5. Anderson, S. et al. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465 (1981).

    Article  CAS  Google Scholar 

  6. White, S.L. et al. Genetic counseling and prenatal diagnosis for the mitochondrial DNA mutations at nucleotide 8993. Am. J. Hum. Genet. 65, 474–482 (1999).

    Article  CAS  Google Scholar 

  7. de Vries, D.D., van Engelen, B.G.M., Gabreëls, F.J.M., Ruitenbeek, W. & van Oost, B.A. A second missense mutation in the mitochondrial ATPase6 gene in Leigh's syndrome. Ann. Neurol. 34, 410–412 (1993).

    Article  CAS  Google Scholar 

  8. Elston, T., Wang, H. & Oster, G. Energy transduction in ATP synthase. Nature 391, 510–513 (1998).

    Article  CAS  Google Scholar 

  9. Noji, H. & Yoshida, M. The rotary machine in the cell, ATP synthase. J. Biol. Chem. 276, 1665–1668 (2001).

    Article  CAS  Google Scholar 

  10. Rastogi, V.K. & Girvin, M.E. Structural changes linked to proton translocation by subunit c of the ATP synthase. Nature 402, 263–268 (1999).

    Article  CAS  Google Scholar 

  11. Hutcheon, M.L., Duncan, T.M., Ngai, H. & Cross, R.L. Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the F0 sector of Escherichia coli ATP synthase. Proc. Natl Acad. Sci. USA 98, 8519–8524 (2001).

    Article  CAS  Google Scholar 

  12. Garcia, J.J., Ogilvie, I., Robinson, B.H. & Capaldi, R.A. Structure, functioning, and assembly of the ATP synthase in cells from patients with the T8993G mitochondrial DNA mutation. Comparison with the enzyme in Rho0 cells completely lacking mtDNA. J. Biol. Chem. 275, 11075–11081 (2000).

    Article  CAS  Google Scholar 

  13. Manfredi, G. et al. Oligomycin induces a decrease in the cellular content of a pathogenic mutation in the human mitochondrial ATPase 6 gene. J. Biol. Chem. 274, 9386–9391 (1999).

    Article  CAS  Google Scholar 

  14. Tatuch, Y. & Robinson, B.H. The mitochondrial DNA mutation at 8993 associated with NARP slows the rate of ATP synthesis in isolated lymphoblast mitochondria. Biochem. Biophys. Res. Commun. 192, 124–128 (1993).

    Article  CAS  Google Scholar 

  15. Vazquez-Memije, M.E. et al. Comparative biochemical studies in fibroblasts from patients with different forms of Leigh syndrome. J. Inher. Metab. Dis. 19, 43–50 (1996).

    Article  CAS  Google Scholar 

  16. Chinnery, P.F. et al. Peptide nucleic acid delivery to human mitochondria. Gene Ther. 6, 1919–1928 (1999).

    Article  CAS  Google Scholar 

  17. Geromel, V. et al. Mitochondria transfection by oligonucleotides containing a signal peptide and vectorized by cationic liposomes. Antisense Nucleic Acid Drug Dev. 11, 175–180 (2001).

    Article  CAS  Google Scholar 

  18. Nakada, K. et al. Inter-mitochondrial complementation: mitochondria-specific system preventing mice from expression of disease phenotypes by mutant mtDNA. Nature Med. 7, 934–939 (2001).

    Article  CAS  Google Scholar 

  19. Seibel, M. et al. Processing of artificial peptide-DNA-conjugates by the mitochondrial intermediate peptidase (MIP). Biol. Chem. 380, 961–967 (1999).

    Article  CAS  Google Scholar 

  20. Gray, R.E., Law, R.H.P., Devenish, R.J. & Nagley, P. Allotopic expression of mitochondrial ATP synthase genes in nucleus of Saccharomyces cerevisiae. Methods Enzymol. 264, 369–389 (1996).

    Article  CAS  Google Scholar 

  21. Herlitze, S. & Koenen, M. A general and rapid mutagenesis method using polymerase chain reaction. Gene 91, 143–147 (1990).

    Article  CAS  Google Scholar 

  22. Sutherland, L., Davidson, J., Glass, L.L. & Jacobs, H.T. Multisite oligonucleotide-mediated mutagenesis: application to the conversion of a mitochondrial gene to universal genetic code. Biotechniques 18, 458–464 (1995).

    CAS  PubMed  Google Scholar 

  23. Rizzuto, R. et al. A gene specifying subunit VIII of human cytochrome c oxidase is localized to chromosome 11 and is expressed in both muscle and non-muscle tissues. J. Biol. Chem. 264, 10595–10600 (1989).

    CAS  PubMed  Google Scholar 

  24. Rizzuto, R., Simpson, A.W.M., Brini, M. & Pozzan, T. Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358, 325–327 (1992).

    Article  CAS  Google Scholar 

  25. Higuti, T., Kawamura, Y., Kuroiwa, K., Miyazaki, S. & Tsujita, H. Molecular cloning and sequence of two cDNAs for human subunit c of H+-ATP synthase in mitochondria. Biochim. Biophys. Acta 1173, 87–90 (1993).

    Article  CAS  Google Scholar 

  26. Ryan, M.T., Voos, W. & Pfanner, N. Assaying protein import into mitochondria. Methods Cell Biol. 65, 189–215 (2001).

    Article  CAS  Google Scholar 

  27. Branda, S.S. & Isaya, G. Prediction and identification of new natural substrates of the yeast mitochondrial intermediate peptidase. J. Biol. Chem. 270, 27366–27373 (1995).

    Article  CAS  Google Scholar 

  28. Breen, G.A.M., Miller, D.M., Holmans, P.L. & Welch, G. Mitochondrial DNA of two independent oligomycin-resistant chinese hamster ovary cell lines contains a single nucleotide change in the ATPase 6 gene. J. Biol. Chem. 261, 11680–11685 (1986).

    CAS  PubMed  Google Scholar 

  29. John, U.P. & Nagley, P. Amino acid substitutions in mitochondrial ATPase subunit 6 of Saccharomyces cerevisiae leading to oligomycin resistance. FEBS Lett. 207, 79–83 (1986).

    Article  CAS  Google Scholar 

  30. Strub, A., Lim, J.H., Pfanner, N. & Voos, W. The mitochondrial protein import motor. Biol. Chem. 381, 943–949 (2000).

    Article  CAS  Google Scholar 

  31. Galanis, M., Devenish, R.J. & Nagley, P. Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency. FEBS Lett. 282, 425–430 (1991).

    Article  CAS  Google Scholar 

  32. Altendorf, K., Stalz, W., Greie, J. & Deckers-Hebestreit, G. Structure and function of the F(0) complex of the ATP synthase from Escherichia coli. J. Exp. Biol. 203, 19–28 (2000).

    CAS  PubMed  Google Scholar 

  33. Jäger, H., Birkenhäger, R., Stalz, W.D., Altendorf, K. & Deckers-Hebestreit, G. Topology of subunit a of the Escherichia coli ATP synthase. Eur. J. Biochem. 251, 122–132 (1998).

    Article  Google Scholar 

  34. Schon, E.A., Santra, S., Pallotti, F. & Girvin, M.E. Pathogenesis of primary defects in mitochondrial ATP synthesis. Semin. Cell Dev. Biol. 12, 441–448 (2001).

    Article  CAS  Google Scholar 

  35. Mizushima, S. & Nagata, S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 18, 5322 (1990).

    Article  CAS  Google Scholar 

  36. Rees, S. et al. Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein. Biotechniques 20, 102–110 (1996).

    Article  CAS  Google Scholar 

  37. Zolotukhin, S., Potter, M., Hauswirth, W.W., Guy, J. & Muzyczka, N. A “humanized” green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J. Virol. 70, 4646–4654 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Hauswirth, W.W., Lewin, A.S., Zolotukhin, S. & Muzyczka, N. Production and purification of recombinant adeno-associated virus. Methods Enzymol. 316, 743–761 (2000).

    Article  CAS  Google Scholar 

  39. Isaya, G., Kalousek, F. & Rosenberg, L.E. Sequence analysis of rat mitochondrial intermediate peptidase: similarity to zinc metallopeptidases and to a putative yeast homologue. Proc. Natl Acad. Sci. USA 89, 8317–8321 (1992).

    Article  CAS  Google Scholar 

  40. Sciacco, M. & Bonilla, E. Cytochemistry and immunocytochemistry of mitochondria in tissue sections. Methods Enzymol. 264, 509–521 (1996).

    Article  CAS  Google Scholar 

  41. Pallotti, F. & Lenaz, G. Isolation and subfractionation of mitochondria from animal cells and tissue culture lines. Methods Cell Biol. 65, 1–35 (2001).

    Article  CAS  Google Scholar 

  42. Klement, P., Nijtmans, L.G., Van den Bogert, C. & Houstek, J. Analysis of oxidative phosphorylation complexes in cultured human fibroblasts and amniocytes by blue-native-electrophoresis using mitoplasts isolated with the help of digitonin. Anal. Biochem. 231, 218–224 (1995).

    Article  CAS  Google Scholar 

  43. Manfredi, G., Spinazzola, A., Checcarelli, N. & Naini, A. Assay of mitochondrial ATP synthesis in animal cells. Methods Cell Biol. 65, 133–145 (2001).

    Article  CAS  Google Scholar 

  44. Mariottini, P. et al. Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA. Proc. Natl Acad. Sci. USA 83, 1563–1567 (1986).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Goff and E. Bacharach for advice and H. Du for technical assistance. This work was supported by grants from the US National Institutes of Health (to G.M., E.A.S. and J.G.) and the Muscular Dystrophy Association (to E.A.S. and G.M.).

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Author notes

  1. Jin Fu

    Present address: University of California, Irvine, California, USA

  2. James E. Sadlock

    Present address: Roche Molecular Biochemicals, Indianapolis, Indiana, USA

Authors and Affiliations

  1. Department of Neurology and Neuroscience, Cornell University, New York, New York, USA

    Giovanni Manfredi & Jennifer Q. Kwong

  2. Department of Neurology, Room 4-431, Columbia University, 630 West 168th Street, New York, 10032, New York, USA

    Jin Fu, Joseline Ojaimi, James E. Sadlock & Eric A. Schon

  3. Department of Ophthalmology, University of Florida College of Medicine, Gainesville, Florida, USA

    John Guy

  4. Department of Genetics and Development, Columbia University, New York, New York, USA

    Eric A. Schon

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Correspondence to Eric A. Schon.

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Manfredi, G., Fu, J., Ojaimi, J. et al. Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus. Nat Genet 30, 394–399 (2002). https://doi.org/10.1038/ng851

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