Abstract
A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5′-terminal and 3′-terminal were obtained by RACE technique. The full-length cDNAthat encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.
Similar content being viewed by others
References
Huang, S., Lin, Q., Functional expression and processing of rat choline dehydrogenase precursor, Biochem. Biophys. Res. Commun., 2003, 309: 344–350.
Ruzicka, F. J., Beinert, H., A new iron-sulfur flavoprotein of the respiratory chain, A component of the fatty acid beta oxidation pathway, J. Biol. Chem., 1977, 252: 8440–8445.
Beckmann, J. D., Frerman, F. E., Electron-transfer flavoproteinubiquinone oxidoreductase from pig liver: Purification and molecular, redox, and catalytic properties, Biochemistry, 1985, 24: 3913–3921.
Johnson, M. K., Morningstar, J. E., Oliver, M., Frerman, F. E., Electron paramagnetic resonance and magnetic circular dichroism studies of electron-transfer flavoprotein-ubiquinone oxidoreductase from pig liver, FEBS Lett., 1987, 226: 129–133.
Frerman, F. E., Reaction of electron-transfer flavoprotein ubiquinone oxidoreductase with the mitochondrial respiratory chain, Biochim. Biophys. Acta, 1987, 893: 161–169.
Frerman, F. E., Acyl-CoA dehydrogenases, electron transfer flavoprotein and electron transfer flavoprotein dehydrogenase, Biochem. Soc. Trans., 1988, 16:416–418.
Simkovic, M., Degala, G. D., Eaton, S. S., Frerman, FE. Expression of human electron transfer flavoprotein-ubiquinone oxidoreductase from a baculovirus vector: kinetic and spectral characterization of the human protein, Biochem. J., 2002, 364: 659–667.
Finocchiaro, G., Ito, M., Tanaka, K., Purification and properties of short chain acyl-CoA, medium chain acyl-CoA, and isovaleryl-CoA dehydrogenases from human liver, J. Biol. Chem., 1987, 262: 7982–7989.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 1951, 193:265–275.
Wu Rudan, Lin Chishui, Solubilization of mitochondrial membrane bound choline dehydrogenase by detergents, Acta Biochimica et Biophysica Sinica, 1984, 16: 610v614
Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989.
Ausubel, F. M. et al., Short Protocols in Molecular biology, 3rd. ed., New York: Johe Wiley & Sons, Inc., 1995.
Murdza-Inglis, D. L., Patel, H. V., Freeman, K. B., Jezek, P., Orosz, D. E., Garlid, K. D., Functional reconstitution of rat uncoupling protein following its high level expression in yeast, J. Biol. Chem., 1991, 266: 11871–11875.
Daum, G., Bohni, P. C., Schatz, G., Import of proteins into mitochondria, Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria, J. Biol. Chem., 1982, 257: 13028–13033.
Neupert, W., Protein import into mitochondria, Annu. Rev. Biochem., 1997, 66:863–917.
Muhlenhoff, U., Lill, R., Biogenesis of iron-sulfur proteins in eukaryotes: A novel task of mitochondria that is inherited from bacteria, Biochim. Biophys. Acta, 2000, 1459: 370–382.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Huang, S., Song, W. & Lin, Q. cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein. Sci. China Ser. C.-Life Sci. 48, 357–367 (2005). https://doi.org/10.1360/03yc0258
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1360/03yc0258