Plant Molecular Biology

, Volume 36, Issue 5, pp 691–698 | Cite as

Nad+-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits

  • Robert H. Behal
  • David J. Oliver


Two cDNA clones which appear to encode different subunits of NAD+-dependent isocitrate dehydrogenase (IDH; EC were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD+-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.

NAD+-dependent isocitrate dehydrogenase Arabidopsis thaliana 


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  1. 1.
    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 215: 403–410 (1990).Google Scholar
  2. 2.
    Behal RH, DeBuysere MS, Demeler B, Hansen JC, Olson MS: Pyruvate dehydrogenase multienzyme complex. Characterization of assembly intermediates by sedimentation velocity analysis. J Biol Chem 269: 31372–31377 (1994).Google Scholar
  3. 3.
    Cupp JR, McAlister-Henn L: NAD+-dependent isocitrate dehydrogenase: cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae. J Biol Chem 266: 22199–22205 (1991).Google Scholar
  4. 4.
    Cupp JR, McAlister-Henn L: Cloning and characterization of the gene encoding the IDH1 subunit of NAD+-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae. J Biol Chem 267: 16417–16423 (1992).Google Scholar
  5. 5.
    Cupp JR, McAlister-Henn L: Kinetic analysis of NAD+-isocitrate dehydrogenase with altered isocitrate binding sites: contribution of IDH1 and IDH2 subunits to regulation and catalysis. Biochemistry 32: 9323–9328 (1993).Google Scholar
  6. 6.
    Daum G, Bohni PC, 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 257: 13028–13033 (1982).Google Scholar
  7. 7.
    Dayhoff MO, Schwartz RM, Orcutt BC: In Dayhoff MO (eds) Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3. pp. 345–352. National Biomedical Research Foundation, Washington (1978).Google Scholar
  8. 8.
    Doyle JJ, Doyle, JL: Isolation of plant DNA from fresh tissue. Focus 12: 13–15 (1990).Google Scholar
  9. 9.
    Ehrlich RS, Colman RF: Separation, recombination, and characterization of dissimilar subunits of the DPN-dependent isocitrate dehydrogenase from pig heart. J Biol Chem 258: 7079–7086 (1983).Google Scholar
  10. 10.
    Ehrlich RS, Colman RF: The role of dissimilar subunits of NAD-specific isocitrate dehydrogenase from pig heart. Evaluation using affinity labeling. J Biol Chem 259: 11936–11942 (1984).Google Scholar
  11. 11.
    Etesham NZ, Hasnain SE: Direct in-gel hybridization, without blotting, using nicktranslated cloned DNA probe. Bio Techniques 11: 718–721 (1991).Google Scholar
  12. 12.
    Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791 (1985).Google Scholar
  13. 13.
    Gasser SM: Import of polypeptides into isolated yeast mitochondria. Meth Enzymol 97: 329–336 (1983).Google Scholar
  14. 14.
    Gasser SM, Daum G, Schatz G: Import of proteins into mitochondria. Energy-dependent uptake of precursors by isolated mitochondria. J Biol Chem 257: 13034–13041 (1982).Google Scholar
  15. 15.
    Hartl FU, Schmidt B, Wachter E, Weiss H, Neupert W: Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinolcytochrome c Reductase. Cell 47: 939–951 (1986).Google Scholar
  16. 16.
    Haselbeck RJ, McAlister-Henn L: Function and expression of yeast mitochondrial NAD-and NADP-specific isocitrate dehydrogenases. J Biol Chem 268: 12116–12122 (1993).Google Scholar
  17. 17.
    Hill SA, Grof CPL, Bryce JH, Leaver CJ: Regulation of mitochondrial function and biogenesis in cucumber cotyledons during early seedling growth. Plant Physiol 99: 60–66 (1992).Google Scholar
  18. 18.
    Huang YC, Colman RF: Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase. Biochemistry 29: 8266–8273 (1990).Google Scholar
  19. 19.
    Hurley JH, Dean AM, Sohl JL, Koshland DE Jr., Stroud RM: Regulation of an enzyme by phosphorylation at the active site. Science 249: 1012–1016 (1990).Google Scholar
  20. 20.
    Kaiser C, Michaelis S, Mitchell A: Methods in yeast genetics, 1994 Edition, pp. 201–203. Cold Spring Harbor Laboratory Press, Plainview, NY (1994).Google Scholar
  21. 21.
    Kelly SM, Duncan D, Price NC: Unfolding and refolding of the NAD+-dependent isocitrate dehydrogenase from yeast. Int J Biol Macromol 15: 75–79 (1993).Google Scholar
  22. 22.
    Kim YO, Oh IU, Park HS, Jeng J, Song BJ, Huh TL: Characterization of a cDNA clone for human NAD+-specific isocitrate dehydrogenase α subunit and structural comparison with its isoenzymes from different species. Biochem J 308: 6368 (1995).Google Scholar
  23. 23.
    McIntosh CA, Oliver DJ: NAD+-linked isocitrate dehydrogenase: isolation, purification, and characterization of the protein from pea mitochondria. Plant Physiol 100: 69–75 (1992).Google Scholar
  24. 24.
    Millhouse J, Wiskich JT, Beevers H: Metabolite oxidation and transport in mitochondria of endosperm from germinating castor bean. Aust J Plant Phys 10: 167–177 (1983).Google Scholar
  25. 25.
    Newman T, de Bruijn FJ, Green P, Keegstra K, Kende H, McIntosh L, Ohlrogge J, Raikhel N, Somerville S, Thomashow M, Retzel E Somerville C: Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol 106: 1241–1255 (1994).Google Scholar
  26. 26.
    Nichols BJ, Hall L, Perry AC, Denton RM: Molecular cloning and deduced amino acid sequences of the gamma subunits of rat and monkey NAD+-isocitrate dehydrogenases. Biochem J 295: 347–350 (1993).Google Scholar
  27. 27.
    Nichols BJ, Perry AC, Hall L, Denton RM: Molecular cloning and deduced amino acid sequences of the α and β subunits of mammalian NAD+-isocitrate dehydrogenase. Biochem J 310: 917–922 (1995).Google Scholar
  28. 28.
    Ramachandran N, Colman RF: Chemical characterization of distinct subunits of pig heart DPN-specific isocitrate dehydrogenase. J Biol Chem 255: 8859–8864 (1980).Google Scholar
  29. 29.
    Rushbrook JI, Harvey RA: Nicotinamide adenine dinucleotide dependent isocitrate dehydrogenase from beef heart: subunit heterogeneity and enzyme dissociation. Biochemistry 17: 5339–5346 (1978).Google Scholar
  30. 30.
    Saitou N, Nei M: The neighborjoining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425 (1987).Google Scholar
  31. 31.
    Schuler GD, Altschul SF, Lipman DJ: A workbench for multiple alignment construction and analysis. Proteins structure function genetics 9: 180–190 (1991).Google Scholar
  32. 32.
    Stoddard BL, Koshland DE, Jr.: Structure of isocitrate dehydrogenase with a-ketoglutarate at 2.7-È resolution: conformational changes induced by decarboxcylation of isocitrate. Biochemistry 32: 9317–9322 (1993).Google Scholar
  33. 33.
    Stoddard BL, Dean A, Koshland DE, Jr.: Structure of isocitrate dehydrogenase with isocitrate, nicotinamide adenine dinucleotide phosphate, and calcium at 2.5-È resolution: a pseudo-Michaelis ternary complex. Biochemistry 32: 9310–9316 (1993).Google Scholar
  34. 34.
    Swofford DL: PAUP: Phylogenetic Analysis Using Parsimony (PAUP), Version 3.0s. Illinois Natural History Survey, Champaign, IL. (1990).Google Scholar
  35. 35.
    Thorsness PE, Koshland DE Jr: The inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. J Biol Chem 262: 10422–10425 (1987).Google Scholar
  36. 36.
    Zeng Y, Weiss C, Yao TT, Huang J, Siconolfi-Baez L, Hsu P, Rushbrook JI: isocitrate dehydrogenase from bovine heart: Primary structure of subunit 3/4. Biochem J 310: 507–516 (1995).Google Scholar
  37. 37.
    Zhao W-N, McAlister-Henn L: Assembly and function of a cytosolic form of NADH-specific isocitrate dehydrogenase in yeast. J Biol Chem 271: 10347–10352 (1996).Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Robert H. Behal
    • 1
  • David J. Oliver
    • 1
  1. 1.Department of BotanyIowa State UniversityAmesUSA

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