Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L.
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 alpha-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.
Taniguchi
M
M
Department of Applied Bioloical Sciences, School of Agricultural Sciences, Nagoya University, Japan.
Sugiyama
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002451:Cell Compartmentation; D018076:DNA, Complementary / Q000235:genetics; D029382:Dicarboxylic Acid Transporters; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006639:Histidine; D008293:Malates / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D008897:Panicum / Q000235:genetics*; D010455:Peptides / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002352:Carrier Proteins; D018076:DNA, Complementary; D029382:Dicarboxylic Acid Transporters; D008293:Malates; D024101:Mitochondrial Proteins; D010455:Peptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C098791:mitochondrial malate translocator, Panicum miliaceum; C066909:2-oxoglutarate-malate carrier protein; C033223:polyhistidine; D006639:Histidine; C030298:malic acid