Abstract
Mitochondria are essential organelles in all eukaryotic cells where cellular ATP is generated through the process of oxidative phosphorylation. Protein components of the respiratory assembly are gene products of both mitochondrial and nuclear genes. The mitochondrial genome itself encodes several protein and nucleic acid components required for such oxidative phosphorylative processes, but the vast majority of genes encoding respiratory chain components are nuclear. Similarly, the processes of replication and transcription of mitochondrial DNA rely exclusively upon RNA and protein species encoded by nuclear genes. We have analyzed two key nuclear-encoded proteins involved in mitochondrial DNA replication and transcription as a function of the presence or absence of mitochondrial DNA. Mitochondrial DNA polymerase (DNA polymerase gamma), the nuclear-encoded enzyme which synthesizes mtDNA, is expressed and translated in cells devoid of mitochondrial DNA itself. In contrast, mitochondrial transcription factor A protein levels are tightly linked to the mtDNA status of the cell. These results demonstrate that the DNA polymerase gamma protein is stable in the absence of mitochondrial DNA, and that there appears to be no regulatory mechanism present in these cells to alter levels of this protein in the complete absence of mitochondrial DNA. Alternatively, it is possible that this enzyme plays an additional, as yet undefined, role in the cell, thereby mandating its continued production.
Full Text
The Full Text of this article is available as a PDF (125.9 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Antoshechkin I., Bogenhagen D. F. Distinct roles for two purified factors in transcription of Xenopus mitochondrial DNA. Mol Cell Biol. 1995 Dec;15(12):7032–7042. doi: 10.1128/mcb.15.12.7032. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Attardi G., Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol. 1988;4:289–333. doi: 10.1146/annurev.cb.04.110188.001445. [DOI] [PubMed] [Google Scholar]
- Bertazzoni U., Scovassi A. I., Brun G. M. Chick-embryo DNA polymerase gamma. Identity of gamma-polymerases purified from nuclei and mitochondria. Eur J Biochem. 1977 Dec 1;81(2):237–248. doi: 10.1111/j.1432-1033.1977.tb11945.x. [DOI] [PubMed] [Google Scholar]
- Bogenhagen D., Clayton D. A. Mouse L cell mitochondrial DNA molecules are selected randomly for replication throughout the cell cycle. Cell. 1977 Aug;11(4):719–727. doi: 10.1016/0092-8674(77)90286-0. [DOI] [PubMed] [Google Scholar]
- Bogenhagen D., Clayton D. A. The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells. Quantitative isolation of mitochondrial deoxyribonucleic acid. J Biol Chem. 1974 Dec 25;249(24):7991–7995. [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Clayton D. A. Replication and transcription of vertebrate mitochondrial DNA. Annu Rev Cell Biol. 1991;7:453–478. doi: 10.1146/annurev.cb.07.110191.002321. [DOI] [PubMed] [Google Scholar]
- Desjardins P., Frost E., Morais R. Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts. Mol Cell Biol. 1985 May;5(5):1163–1169. doi: 10.1128/mcb.5.5.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Flory P. J., Jr, Vinograd J. 5-bromodeoxyuridine labeling of monomeric and catenated circular mitochondrial DNA in HeLa cells. J Mol Biol. 1973 Feb 25;74(2):81–94. doi: 10.1016/0022-2836(73)90100-9. [DOI] [PubMed] [Google Scholar]
- Foury F. Cloning and sequencing of the nuclear gene MIP1 encoding the catalytic subunit of the yeast mitochondrial DNA polymerase. J Biol Chem. 1989 Dec 5;264(34):20552–20560. [PubMed] [Google Scholar]
- Grégoire M., Morais R., Quilliam M. A., Gravel D. On auxotrophy for pyrimidines of respiration-deficient chick embryo cells. Eur J Biochem. 1984 Jul 2;142(1):49–55. doi: 10.1111/j.1432-1033.1984.tb08249.x. [DOI] [PubMed] [Google Scholar]
- Habara A., Nagano H., Mano Y. Characterization of DNA polymerases in mature sperm of the sea urchin. Biochim Biophys Acta. 1980 Jul 29;608(2):287–294. doi: 10.1016/0005-2787(80)90174-4. [DOI] [PubMed] [Google Scholar]
- Hübscher U., Kuenzle C. C., Spadari S. Identity of DNA polymerase gamma from synaptosomal mitochondria and rat-brain nuclei. Eur J Biochem. 1977 Dec 1;81(2):249–258. doi: 10.1111/j.1432-1033.1977.tb11946.x. [DOI] [PubMed] [Google Scholar]
- Kalf G. F., Maguire R. F., Metrione R. M., Koszalka T. R. DNA replication by isolated rat trophoblast nuclei. Characterization of the system and the product. Dev Biol. 1980 Jun 15;77(2):253–270. doi: 10.1016/0012-1606(80)90474-1. [DOI] [PubMed] [Google Scholar]
- King M. P., Attardi G. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science. 1989 Oct 27;246(4929):500–503. doi: 10.1126/science.2814477. [DOI] [PubMed] [Google Scholar]
- Larsson N. G., Oldfors A., Holme E., Clayton D. A. Low levels of mitochondrial transcription factor A in mitochondrial DNA depletion. Biochem Biophys Res Commun. 1994 May 16;200(3):1374–1381. doi: 10.1006/bbrc.1994.1603. [DOI] [PubMed] [Google Scholar]
- Lestienne P., Bataillé N. Mitochondrial DNA alterations and genetic diseases: a review. Biomed Pharmacother. 1994;48(5-6):199–214. doi: 10.1016/0753-3322(94)90134-1. [DOI] [PubMed] [Google Scholar]
- Li K., Neufer P. D., Williams R. S. Nuclear responses to depletion of mitochondrial DNA in human cells. Am J Physiol. 1995 Nov;269(5 Pt 1):C1265–C1270. doi: 10.1152/ajpcell.1995.269.5.C1265. [DOI] [PubMed] [Google Scholar]
- Liao X. S., Small W. C., Srere P. A., Butow R. A. Intramitochondrial functions regulate nonmitochondrial citrate synthase (CIT2) expression in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jan;11(1):38–46. doi: 10.1128/mcb.11.1.38. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liao X., Butow R. A. RTG1 and RTG2: two yeast genes required for a novel path of communication from mitochondria to the nucleus. Cell. 1993 Jan 15;72(1):61–71. doi: 10.1016/0092-8674(93)90050-z. [DOI] [PubMed] [Google Scholar]
- Nass M. M. Differential effects of ethidium bromide on mitochondrial and nuclear DNA synthesis in vivo in cultured mammalian cells. Exp Cell Res. 1972 May;72(1):211–222. doi: 10.1016/0014-4827(72)90583-6. [DOI] [PubMed] [Google Scholar]
- Parikh V. S., Morgan M. M., Scott R., Clements L. S., Butow R. A. The mitochondrial genotype can influence nuclear gene expression in yeast. Science. 1987 Jan 30;235(4788):576–580. doi: 10.1126/science.3027892. [DOI] [PubMed] [Google Scholar]
- Parisi M. A., Clayton D. A. Similarity of human mitochondrial transcription factor 1 to high mobility group proteins. Science. 1991 May 17;252(5008):965–969. doi: 10.1126/science.2035027. [DOI] [PubMed] [Google Scholar]
- Randahl H., Elliott G. C., Linn S. DNA-repair reactions by purified HeLa DNA polymerases and exonucleases. J Biol Chem. 1988 Sep 5;263(25):12228–12234. [PubMed] [Google Scholar]
- Ropp P. A., Copeland W. C. Characterization of a new DNA polymerase from Schizosaccharomyces pombe: a probable homologue of the Saccharomyces cerevisiae DNA polymerase gamma. Gene. 1995 Nov 7;165(1):103–107. doi: 10.1016/0378-1119(95)00412-y. [DOI] [PubMed] [Google Scholar]
- Shadel G. S., Clayton D. A. Mitochondrial transcription initiation. Variation and conservation. J Biol Chem. 1993 Aug 5;268(22):16083–16086. [PubMed] [Google Scholar]
- Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
- Tiranti V., Rocchi M., DiDonato S., Zeviani M. Cloning of human and rat cDNAs encoding the mitochondrial single-stranded DNA-binding protein (SSB). Gene. 1993 Apr 30;126(2):219–225. doi: 10.1016/0378-1119(93)90370-i. [DOI] [PubMed] [Google Scholar]
- Virbasius C. A., Virbasius J. V., Scarpulla R. C. NRF-1, an activator involved in nuclear-mitochondrial interactions, utilizes a new DNA-binding domain conserved in a family of developmental regulators. Genes Dev. 1993 Dec;7(12A):2431–2445. doi: 10.1101/gad.7.12a.2431. [DOI] [PubMed] [Google Scholar]
- Wang H., Parent M., Morais R. Cloning and characterization of a cDNA encoding elongation factor 1 alpha from chicken cells devoid of mitochondrial DNA. Gene. 1994 Mar 25;140(2):155–161. doi: 10.1016/0378-1119(94)90539-8. [DOI] [PubMed] [Google Scholar]
- Wang S. S., Brandriss M. C. Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product. Mol Cell Biol. 1987 Dec;7(12):4431–4440. doi: 10.1128/mcb.7.12.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wiseman A., Attardi G. Reversible tenfod reduction in mitochondria DNA content of human cells treated with ethidium bromide. Mol Gen Genet. 1978 Nov 16;167(1):51–63. doi: 10.1007/BF00270321. [DOI] [PubMed] [Google Scholar]