Pharmaceutical Research

, 28:2871 | Cite as

DNA Delivery to Mitochondria: Sequence Specificity and Energy Enhancement

  • Noha Ibrahim
  • Hirokazu Handa
  • Anne Cosset
  • Milana Koulintchenko
  • Yuri Konstantinov
  • Robert N. Lightowlers
  • André Dietrich
  • Frédérique Weber-Lotfi
Research Paper

ABSTRACT

Purpose

Mitochondria are competent for DNA uptake in vitro, a mechanism which may support delivery of therapeutic DNA to complement organelle DNA mutations. We document here key aspects of the DNA import process, so as to further lay the ground for mitochondrial transfection in intact cells.

Methods

We developed DNA import assays with isolated mitochondria from different organisms, using DNA substrates of various sequences and sizes. Further import experiments investigated the possible role of ATP and protein phosphorylation in the uptake process. The fate of adenine nucleotides and the formation of phosphorylated proteins were analyzed.

Results

We demonstrate that the efficiency of mitochondrial uptake depends on the sequence of the DNA to be translocated. The process becomes sequence-selective for large DNA substrates. Assays run with a natural mitochondrial plasmid identified sequence elements which promote organellar uptake. ATP enhances DNA import and allows tight integration of the exogenous DNA into mitochondrial nucleoids. ATP hydrolysis has to occur during the DNA uptake process and might trigger phosphorylation of co-factors.

Conclusions

Our data contribute critical information to optimize DNA delivery into mitochondria and open the prospect of targeting whole mitochondrial genomes or complex constructs into mammalian organelles in vitro and in vivo.

KEY WORDS

DNA import mitochondrial disease mitochondrial plasmid organelle transfection protein phosphorylation 

ABBREVIATIONS

bp

base-pair

BSA

bovine serum albumin

CCCP

carbonyl cyanide m-chlorophenylhydrazone

DEAE-cellulose

diethylaminoethyl-cellulose

EDTA

ethylenediamine tetraacetic acid

EGTA

ethylene glycol tetraacetic acid

kb

kilobase-pair

mtDNA

mitochondrial DNA

OXPHOS

oxidative phosphorylation

PCR

polymerase chain reaction

PEI-cellulose

polyethylenimine-cellulose

PMSF

phenylmethylsulfonyl fluoride

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TLC

thin-layer chromatography

REFERENCES

  1. 1.
    Tuppen HA, Blakely EL, Turnbull DM, Taylor RW. Mitochondrial DNA mutations and human disease. Biochim Biophys Acta. 2010;1797:113–28.PubMedGoogle Scholar
  2. 2.
    Bonnefoy N, Remacle C, Fox TD. Genetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria. Methods Cell Biol. 2007;80:525–48.PubMedCrossRefGoogle Scholar
  3. 3.
    Zhou J, Liu L, Chen J. Mitochondrial DNA heteroplasmy in Candida glabrata after mitochondrial transformation. Eukaryot Cell. 2010;9:806–14.PubMedCrossRefGoogle Scholar
  4. 4.
    Kyriakouli DS, Boesch P, Taylor RW, Lightowlers RN. Progress and prospects: gene therapy for mitochondrial DNA disease. Gene Ther. 2008;15:1017–23.PubMedCrossRefGoogle Scholar
  5. 5.
    Koulintchenko M, Konstantinov Y, Dietrich A. Plant mitochondria actively import DNA via the permeability transition pore complex. EMBO J. 2003;22:1245–54.PubMedCrossRefGoogle Scholar
  6. 6.
    Koulintchenko M, Temperley RJ, Mason PA, Dietrich A, Lightowlers RN. Natural competence of mammalian mitochondria allows the molecular investigation of mitochondrial gene expression. Hum Mol Genet. 2006;15:143–54.PubMedCrossRefGoogle Scholar
  7. 7.
    Weber-Lotfi F, Ibrahim N, Boesch P, Cosset A, Konstantinov Y, Lightowlers RN, et al. Developing a genetic approach to investigate the mechanism of mitochondrial competence for DNA import. Biochim Biophys Acta. 2009;1787:320–7.PubMedCrossRefGoogle Scholar
  8. 8.
    Placido A, Gagliardi D, Gallerani R, Grienenberger JM, Maréchal-Drouard L. Fate of a larch unedited tRNA precursor expressed in potato mitochondria. J Biol Chem. 2005;280:33573–9.PubMedCrossRefGoogle Scholar
  9. 9.
    Boesch P, Ibrahim N, Paulus F, Cosset A, Tarasenko V, Dietrich A. Plant mitochondria possess a short-patch base excision DNA repair pathway. Nucleic Acids Res. 2009;37:5690–700.PubMedCrossRefGoogle Scholar
  10. 10.
    Boesch P, Ibrahim N, Dietrich A, Lightowlers RN. Membrane association of mitochondrial DNA facilitates base excision repair in mammalian mitochondria. Nucleic Acids Res. 2010;38:1478–88.PubMedCrossRefGoogle Scholar
  11. 11.
    Mileshina D, Koulintchenko M, Konstantinov Y, Dietrich A. Transfection of plant mitochondria and in organello gene integration. Nucleic Acids Res. 2011; doi:10.1093/nar/gkr517.
  12. 12.
    Katrangi E, D’Souza G, Boddapati SV, Kulawiec M, Singh KK, Bigger B, et al. Xenogenic transfer of isolated murine mitochondria into human rho0 cells can improve respiratory function. Rejuvenation Res. 2007;10:561–70.PubMedCrossRefGoogle Scholar
  13. 13.
    Rubino L, Burgyan J, Russo M. Molecular cloning and complete nucleotide sequence of Carnation Italian ringspot tombusvirus genomic and defective interfering RNAs. Arch Virol. 1995;140:2027–39.PubMedCrossRefGoogle Scholar
  14. 14.
    Weber-Lotfi F, Dietrich A, Russo M, Rubino L. Mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells. J Virol. 2002;76:10485–96.PubMedCrossRefGoogle Scholar
  15. 15.
    Leon P, Walbot V, Bedinger P. Molecular analysis of the linear 2.3 kb plasmid of maize mitochondria: apparent capture of tRNA genes. Nucleic Acids Res. 1989;17:4089–99.PubMedCrossRefGoogle Scholar
  16. 16.
    van Engelen FA, Molthoff JW, Conner AJ, Nap JP, Pereira A, Stiekema WJ. pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res. 1995;4:288–90.PubMedCrossRefGoogle Scholar
  17. 17.
    Handa H, Itani K, Sato H. Structural features and expression analysis of a linear mitochondrial plasmid in rapeseed (Brassica napus L.). Mol Genet Genomics. 2002;267:797–805.PubMedCrossRefGoogle Scholar
  18. 18.
    Neuburger M, Journet EP, Bligny R, Carde JP, Douce R. Purification of plant mitochondria by isopycnic centrifugation in density gradients of Percoll. Arch Biochem Biophys. 1982;217:312–23.PubMedCrossRefGoogle Scholar
  19. 19.
    Heins L, Mentzel H, Schmid A, Benz R, Schmitz UK. Biochemical, molecular, and functional characterization of porin isoforms from potato mitochondria. J Biol Chem. 1994;269:26402–10.PubMedGoogle Scholar
  20. 20.
    Meisinger C, Sommer T, Pfanner N. Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations. Anal Biochem. 2000;287:339–42.PubMedCrossRefGoogle Scholar
  21. 21.
    Weinstock GM, McEntee K, Lehman IR. Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Characterization of ATP hydrolysis. J Biol Chem. 1981;256:8829–34.PubMedGoogle Scholar
  22. 22.
    Sato H, Saito C, Handa H. Mitochondrial DNA decreases during pollen development in rapeseed (Brassica napus L.), but mitochondrial linear-plasmid-encoded RNA polymerase persists in mature pollen. Protoplasma. 2004;224:179–85.PubMedCrossRefGoogle Scholar
  23. 23.
    Turpen T, Garger SJ, Marks MD, Grill LK. Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid. Mol Gen Genet. 1987;209:227–33.PubMedCrossRefGoogle Scholar
  24. 24.
    da Silva LP, Lindahl M, Lundin M, Baltscheffsky H. Protein phosphorylation by inorganic pyrophosphate in yeast mitochondria. Biochem Biophys Res Commun. 1991;178:1359–64.PubMedCrossRefGoogle Scholar
  25. 25.
    Ito J, Taylor NL, Castleden I, Weckwerth W, Millar AH, Heazlewood JL. A survey of the Arabidopsis thaliana mitochondrial phosphoproteome. Proteomics. 2009;9:4229–40.PubMedCrossRefGoogle Scholar
  26. 26.
    Horobin RW, Trapp S, Weissig V. Mitochondriotropics: a review of their mode of action, and their applications for drug and DNA delivery to mammalian mitochondria. J Control Release. 2007;121:125–36.PubMedCrossRefGoogle Scholar
  27. 27.
    Weissig V, Lasch J, Erdos G, Meyer HW, Rowe TC, Hughes J. DQAsomes: a novel potential drug and gene delivery system made from Dequalinium. Pharm Res. 1998;15:334–7.PubMedCrossRefGoogle Scholar
  28. 28.
    Boddapati SV, Tongcharoensirikul P, Hanson RN, D’Souza GG, Torchilin VP, Weissig V. Mitochondriotropic liposomes. J Liposome Res. 2005;15:49–58.PubMedGoogle Scholar
  29. 29.
    D’Souza GG, Rammohan R, Cheng SM, Torchilin VP, Weissig V. DQAsome-mediated delivery of plasmid DNA toward mitochondria in living cells. J Control Release. 2003;92:189–97.PubMedCrossRefGoogle Scholar
  30. 30.
    D’Souza GG, Boddapati SV, Weissig V. Mitochondrial leader sequence–plasmid DNA conjugates delivered into mammalian cells by DQAsomes co-localize with mitochondria. Mitochondrion. 2005;5:352–8.PubMedCrossRefGoogle Scholar
  31. 31.
    Clark MA, Shay JW. Mitochondrial transformation of mammalian cells. Nature. 1982;295:605–7.PubMedCrossRefGoogle Scholar
  32. 32.
    Ber R, Stauver MG, Shay JW. Use of isolated mitochondria to transfer chloramphenicol resistance in hamster cells. Isr J Med Sci. 1984;20:244–8.PubMedGoogle Scholar
  33. 33.
    Pinkert CA, Irwin MH, Johnson LW, Moffatt RJ. Mitochondria transfer into mouse ova by microinjection. Transgenic Res. 1997;6:379–83.PubMedCrossRefGoogle Scholar
  34. 34.
    Irwin MH, Johnson LW, Pinkert CA. Isolation and microinjection of somatic cell-derived mitochondria and germline heteroplasmy in transmitochondrial mice. Transgenic Res. 1999;8:119–23.PubMedCrossRefGoogle Scholar
  35. 35.
    Takeda K, Tasai M, Akagi S, Matsukawa K, Takahashi S, Iwamoto M, et al. Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes. Mitochondrion. 2010;10:137–42.PubMedCrossRefGoogle Scholar
  36. 36.
    Spelbrink JN. Functional organization of mammalian mitochondrial DNA in nucleoids: history, recent developments, and future challenges. IUBMB Life. 2010;62:19–32.PubMedGoogle Scholar
  37. 37.
    Kaufman BA, Kolesar JE, Perlman PS, Butow RA. A function for the mitochondrial chaperonin Hsp60 in the structure and transmission of mitochondrial DNA nucleoids in Saccharomyces cerevisiae. J Cell Biol. 2003;163:457–61.PubMedCrossRefGoogle Scholar
  38. 38.
    Bacman SR, Williams SL, Moraes CT. Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks. Nucleic Acids Res. 2009;37:4218–26.PubMedCrossRefGoogle Scholar
  39. 39.
    Fukui H, Moraes CT. Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons. Hum Mol Genet. 2009;18:1028–36.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Noha Ibrahim
    • 1
    • 2
  • Hirokazu Handa
    • 3
  • Anne Cosset
    • 1
  • Milana Koulintchenko
    • 1
    • 2
    • 4
  • Yuri Konstantinov
    • 4
  • Robert N. Lightowlers
    • 2
  • André Dietrich
    • 1
  • Frédérique Weber-Lotfi
    • 1
  1. 1.Institut de Biologie Moléculaire des PlantesCNRS and Université de StrasbourgStrasbourgFrance
  2. 2.Mitochondrial Research Group, Institute for Aging & Health Medical SchoolNewcastle UniversityNewcastle upon TyneUK
  3. 3.Plant Genome Research UnitNational Institute of Agrobiological SciencesTsukubaJapan
  4. 4.Siberian Institute of Plant Physiology and BiochemistryRussian Academy of SciencesIrkutskRussia

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