Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2003 Oct;165(2):707–720. doi: 10.1093/genetics/165.2.707

Limitations of allotopic expression of mitochondrial genes in mammalian cells.

Jose Oca-Cossio 1, Lesley Kenyon 1, Huiling Hao 1, Carlos T Moraes 1
PMCID: PMC1462783  PMID: 14573482

Abstract

The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Cavadini Patrizia, Gakh Oleksandr, Isaya Grazia. Protein import and processing reconstituted with isolated rat liver mitochondria and recombinant mitochondrial processing peptidase. Methods. 2002 Apr;26(4):298–306. doi: 10.1016/S1046-2023(02)00035-X. [DOI] [PubMed] [Google Scholar]
  2. Claros M. G. MitoProt, a Macintosh application for studying mitochondrial proteins. Comput Appl Biosci. 1995 Aug;11(4):441–447. doi: 10.1093/bioinformatics/11.4.441. [DOI] [PubMed] [Google Scholar]
  3. Claros M. G., Perea J., Shu Y., Samatey F. A., Popot J. L., Jacq C. Limitations to in vivo import of hydrophobic proteins into yeast mitochondria. The case of a cytoplasmically synthesized apocytochrome b. Eur J Biochem. 1995 Mar 15;228(3):762–771. [PubMed] [Google Scholar]
  4. Corral-Debrinski M., Belgareh N., Blugeon C., Claros M. G., Doye V., Jacq C. Overexpression of yeast karyopherin Pse1p/Kap121p stimulates the mitochondrial import of hydrophobic proteins in vivo. Mol Microbiol. 1999 Mar;31(5):1499–1511. doi: 10.1046/j.1365-2958.1999.01295.x. [DOI] [PubMed] [Google Scholar]
  5. Daley Daniel O., Clifton Rachel, Whelan James. Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase. Proc Natl Acad Sci U S A. 2002 Jul 25;99(16):10510–10515. doi: 10.1073/pnas.122354399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Giorgi F., Brini M., Bastianutto C., Marsault R., Montero M., Pizzo P., Rossi R., Rizzuto R. Targeting aequorin and green fluorescent protein to intracellular organelles. Gene. 1996;173(1 Spec No):113–117. doi: 10.1016/0378-1119(95)00687-7. [DOI] [PubMed] [Google Scholar]
  7. Farrell L. B., Gearing D. P., Nagley P. Reprogrammed expression of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Expression in vitro from a chemically synthesized gene and import into isolated mitochondria. Eur J Biochem. 1988 Apr 5;173(1):131–137. doi: 10.1111/j.1432-1033.1988.tb13976.x. [DOI] [PubMed] [Google Scholar]
  8. Fujiki M., Verner K. Coupling of cytosolic protein synthesis and mitochondrial protein import in yeast. Evidence for cotranslational import in vivo. J Biol Chem. 1993 Jan 25;268(3):1914–1920. [PubMed] [Google Scholar]
  9. Galanis M., Devenish R. J., Nagley P. Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency. FEBS Lett. 1991 May 6;282(2):425–430. doi: 10.1016/0014-5793(91)80529-c. [DOI] [PubMed] [Google Scholar]
  10. Galanis M., Law R. H., O'Keeffe L. M., Devenish R. J., Nagley P. Aberrant mitochondrial processing of chimaeric import precursors containing subunits 8 and 9 of yeast mitochondrial ATP synthase. Biochem Int. 1990 Dec;22(6):1059–1066. [PubMed] [Google Scholar]
  11. Gearing D. P., Nagley P. Yeast mitochondrial ATPase subunit 8, normally a mitochondrial gene product, expressed in vitro and imported back into the organelle. EMBO J. 1986 Dec 20;5(13):3651–3655. doi: 10.1002/j.1460-2075.1986.tb04695.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guy John, Qi Xiaoping, Pallotti Francesco, Schon Eric A., Manfredi Giovanni, Carelli Valerio, Martinuzzi Andrea, Hauswirth William W., Lewin Alfred S. Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy. Ann Neurol. 2002 Nov;52(5):534–542. doi: 10.1002/ana.10354. [DOI] [PubMed] [Google Scholar]
  13. Holmberg E., Olausson T., Hultman T., Rydström J., Ahmad S., Glavas N. A., Bragg P. D. Prediction and site-specific mutagenesis of residues in transmembrane alpha-helices of proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli and bovine heart mitochondria. Biochemistry. 1994 Jun 21;33(24):7691–7700. doi: 10.1021/bi00190a024. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Law R. H., Devenish R. J., Nagley P. Assembly of imported subunit 8 into the ATP synthase complex of isolated yeast mitochondria. Eur J Biochem. 1990 Mar 10;188(2):421–429. doi: 10.1111/j.1432-1033.1990.tb15419.x. [DOI] [PubMed] [Google Scholar]
  17. 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. 1988 Aug 29;236(2):501–505. doi: 10.1016/0014-5793(88)80086-3. [DOI] [PubMed] [Google Scholar]
  18. Law R. H., Nagley P. Import into mitochondria of precursors containing hydrophobic passenger proteins: pretreatment of precursors with urea inhibits import. Biochim Biophys Acta. 1990 Aug 24;1027(2):141–148. doi: 10.1016/0005-2736(90)90077-2. [DOI] [PubMed] [Google Scholar]
  19. Lithgow T. Targeting of proteins to mitochondria. FEBS Lett. 2000 Jun 30;476(1-2):22–26. doi: 10.1016/s0014-5793(00)01663-x. [DOI] [PubMed] [Google Scholar]
  20. Manfredi Giovanni, Fu Jin, Ojaimi Joseline, Sadlock James E., Kwong Jennifer Q., Guy John, Schon Eric A. Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus. Nat Genet. 2002 Feb 25;30(4):394–399. doi: 10.1038/ng851. [DOI] [PubMed] [Google Scholar]
  21. Ojaimi Joseline, Pan Junmin, Santra Sumana, Snell William J., Schon Eric A. An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit. Mol Biol Cell. 2002 Nov;13(11):3836–3844. doi: 10.1091/mbc.E02-05-0306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Owen R I. V., Lewin A. P., Peel A., Wang J., Guy J., Hauswirth W. W., Stacpoole P. W., Flotte T. R. Recombinant adeno-associated virus vector-based gene transfer for defects in oxidative metabolism. Hum Gene Ther. 2000 Oct 10;11(15):2067–2078. doi: 10.1089/104303400750001381. [DOI] [PubMed] [Google Scholar]
  23. Pérez-Martínez X., Antaramian A., Vazquez-Acevedo M., Funes S., Tolkunova E., d'Alayer J., Claros M. G., Davidson E., King M. P., González-Halphen D. Subunit II of cytochrome c oxidase in Chlamydomonad algae is a heterodimer encoded by two independent nuclear genes. J Biol Chem. 2000 Nov 27;276(14):11302–11309. doi: 10.1074/jbc.M010244200. [DOI] [PubMed] [Google Scholar]
  24. Pérez-Martínez X., Vazquez-Acevedo M., Tolkunova E., Funes S., Claros M. G., Davidson E., King M. P., González-Halphen D. Unusual location of a mitochondrial gene. Subunit III of cytochrome C oxidase is encoded in the nucleus of Chlamydomonad algae. J Biol Chem. 2000 Sep 29;275(39):30144–30152. doi: 10.1074/jbc.M003940200. [DOI] [PubMed] [Google Scholar]
  25. Rana M., de Coo I., Diaz F., Smeets H., Moraes C. T. An out-of-frame cytochrome b gene deletion from a patient with parkinsonism is associated with impaired complex III assembly and an increase in free radical production. Ann Neurol. 2000 Nov;48(5):774–781. [PubMed] [Google Scholar]
  26. Saccone Cecilia, Gissi Carmela, Reyes Aurelio, Larizza Alessandra, Sbisà Elisabetta, Pesole Graziano. Mitochondrial DNA in metazoa: degree of freedom in a frozen event. Gene. 2002 Mar 6;286(1):3–12. doi: 10.1016/s0378-1119(01)00807-1. [DOI] [PubMed] [Google Scholar]
  27. Wagstaff M. J., Smith J., Collaco-Moraes Y., de Belleroche J. S., Voellmy R., Coffin R. S., Latchman D. S. Delivery of a constitutively active form of the heat shock factor using a virus vector protects neuronal cells from thermal or ischaemic stress but not from apoptosis. Eur J Neurosci. 1998 Nov;10(11):3343–3350. doi: 10.1046/j.1460-9568.1998.00339.x. [DOI] [PubMed] [Google Scholar]
  28. Woischnik Markus, Moraes Carlos T. Pattern of organization of human mitochondrial pseudogenes in the nuclear genome. Genome Res. 2002 Jun;12(6):885–893. doi: 10.1101/gr.227202. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES