Abstract
Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C4 plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane α-helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity-purified. The antibody against the recombinant protein cross-reacted with proteins of 31–32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.
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References
Arnon DI: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1–15 (1949).
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. John Wiley, New York (1987).
Bensadoun A, Weinstein D: Assay of proteins in the presence of interfering materials. Anal Biochem 70: 241–250 (1976).
Bisaccia F, Capobianco L, Brandolin G, Palmieri F: Transmembrane topography of the mitochondrial oxoglutarate carrier assessed by peptide-specific antibodies and enzymatic cleavage. Biochemistry 33: 3705–3713 (1994).
Bisaccia F, Indiveri C, Palmieri F: Purification of reconstitutively active α-oxoglutarate carrier from pig heart mitochondria. Biochim Biophys Acta 810: 362–369 (1985).
Fiermonte G, Walker JE, Palmieri F: Abundant bacterial expression and reconstitution of an intrinsic membrane-transport protein from bovine mitochondria. Biochem J 294: 293–299 (1993).
Furbank RT, Agostino A, Hatch MD: C4 acid decarboxylation and photosynthesis in bundle sheath cells of NAD-malic enzyme-type C4 plants; mechanism and the role of malate and orthophosphate. Arch Biochem Biophys 276: 374–381 (1990).
Genchi G, Santis AD, Ponzone C, Palmieri F: Partial purification and reconstitution of the α-ketoglutarate carrier from corn (Zea mays L.) mitochondria. Plant Physiol 96: 1003–1007 (1991).
Hanley BA, Schuler MA: Plant intron sequences: evidence for distinct groups of introns. Nucl Acids Res 16: 7159–7176 (1988).
Hartl F-U, Pfanner N, Nicholson DW, Neupert W: Mitochondrial protein import. Biochim Biophys Acta 988: 1–45 (1989).
Indiveri C, Palmieri F, Bisaccia F, Krämer R: Kinetics of the reconstituted 2-oxoglutarate carrier from bovine heart mitochondria. Biochim Biophys Acta 890: 310–318 (1987).
Kaplan RS, Mayor JA, Wood DO: The mitochondrial tricarboxylate transport protein. cDNA cloning, primary structure, and comparison with other mitochondrial transport proteins. J Biol Chem 268: 13682–13690 (1993).
Kyte J, Doolittle RF: A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105–132 (1982).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Loddenkötter B, Kammerer B, Fischer K, Flüger U-I: Expression of the functional mature chloroplast triose phosphate translocator in yeast internal membranes and purification of the histidine-tagged protein by a single metal-affinity chromatography step. Proc Natl Acad Sci USA 90: 2155–2159 (1993).
Martinoia E, Rentsch D: Malate compartmentation-responses to a complex metabolism. Annu Rev Plant Physiol Plant Mol Biol 45: 447–467 (1994).
McGookin R: RNA extraction by the guanidine thiocyanate procedure. In: Walker JM (ed) Methods in Molecular Biology, Vol. 2, pp. 113–116. Humana Press, Clifton, NJ (1984).
McIntosh CA, Oliver DJ: Isolation and characterization of the tricarboxylate transporter from pea mitochondria. Plant Physiol 100: 2030–2034 (1992).
Menzlaff E, Flügge U-I: Purification and functional reconstitution of the 2-oxoglutarate/malate translocator from spinach chloroplasts. Biochim Biophys Acta 1147: 13–18 (1993).
Mozo T, Fischer K, Flügge U-I, Schmitz UK: The N-terminal extension of the ADP/ATP translocator is not involved in targeting to plant mitochondria in vivo. Plant J 7: 1015–1020 (1995).
Palmieri F: Mitochondrial carrier proteins. FEBS Lett 346: 48–54 (1994).
Palmieri F, Bisaccia F, Capobianco L, Dolce V, Fiermonte G, lacobazzi V, Zara V: Transmembrane topology, genes, and biogenesis of the mitochondrial phosphate and oxoglutarate carriers. J Bioenerg Biomembr 25: 493–501 (1993).
Runswick MJ, Powell SJ, Nyren P, Walker JE: Sequence of the bovine mitochondrial phosphate carrier protein: structural relationship to ADP/ATP translocase and the brown fat mitochondria uncoupling protein. EMBO J 6: 1367–1373 (1987).
Runswick MJ, Walker JE, Bisaccia F, Iacobazzi V, Palmieri F: Sequence of the bovine 2-oxoglutarate/malate carrier protein: structural relationship to other mitochondrial transport proteins. Biochemistry 29: 11033–11040 (1990).
Sauer N, Stadler R: A sink-specific H+/monosaccharide co-transporter from Nicotiana tabacum: cloning and heterologous expression in baker's yeast. Plant J 4: 601–610 (1993).
Slater RJ: The purification of poly(A)-containing RNA by affinity chromatography. In: Walker JM (ed) Methods in Molecular Biology, Vol. 2. pp. 117–120. Humana Press, Clifton, NJ (1984).
Stolz J, Stadler R, Opekarová M, Sauer N: Functional reconstitution of the solubilized Arabidopsis thaliana STP1 monosaccharide-H+ symporter in lipid vesicles and purification of the histidine tagged protein from transgenic Saccharomyces cerevisiae. Plant J 6: 225–233 (1994).
Taniguchi M, Mori J, Sugiyama T: Structure of genes that encode isozymes of aspartate aminotransferase in Panicum miliaceum L., a C4 plant. Plant Mol Biol 26: 723–734 (1994).
Taniguchi M, Sawaki H, Sasakawa H, Hase T, Sugiyama T: Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant. Eur J Biochem 204: 611–620 (1992).
Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 (1979).
von Heijne G, Steppuhn J, Herrmann RG: Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535–545 (1989).
Walker JE: The mitochondrial transporter family. Curr Opinion Struct Biol 2: 519–526 (1992).
Weber A, Menzlaff E, Arbinger B, Gutensohn M, Eckerskorn C, Flügge U-I: The 2-oxoglutarate/malate translocator of chloroplast envelope membranes: molecular cloning of a transporter containing a 12-helix motif and expression of the functional protein in yeast cells. Biochemistry 34: 2621–2627 (1995).
Winning BM, Sarah CJ, Purdue PE, Day CD, Leaver CJ: The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria. Plant J 2: 763–773 (1992).
Yonetani T: cytochrome oxidase: beef heart. Meth Enzymol 10: 332–335 (1967).
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Taniguchi, M., Sugiyama, T. Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L.. Plant Mol Biol 30, 51–64 (1996). https://doi.org/10.1007/BF00017802
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DOI: https://doi.org/10.1007/BF00017802