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Plant Molecular Biology

, 62:951 | Cite as

A plant mitochondrial phospholipid hydroperoxide glutathione peroxidase: its precise localization and higher enzymatic activity

  • Xiao-Dong Yang
  • Chun-Juan Dong
  • Jin-Yuan Liu
Original Paper

Abstract

A novel cDNA of phospholipid hydroperoxide glutathione peroxidase (PHGPx), which encodes a functional protein capable of complementing the yeast PHGHX-deletion mutant, was recently discovered in radish (Raphanus sativus) and designated as RsPHGPx [Yang X-D, Li W-J, Liu J-Y (2005) Biochim Biophys Acta 1728:199–205]. Sequence alignment suggested that RsPHGPx contains a targeting peptide required for transport to mitochondria, but the experimental evidence for the exact intracellular distribution of RsPHGPx remains to be elucidated. To uncover the cellular localization of plant PHGPx, we first investigated RsPHGPx’s intracellular distribution. Western blot analysis of subcellular fractions using the RsPHGPx antiserum clearly indicated the distribution of RsPHGPx in the radish mitochondrial fraction. Furthermore, a construct expressing the RsPHGPx precursor tagged with green fluorescent protein was introduced into tobacco and yeast cells, and the fusion protein was transported into both mitochondria, indicating that RsPHGPx was indeed localized in mitochondria. To explore the biochemical functions of this enzyme, we tested the enzymatic activity of the recombinant RsPHGPx protein. It displayed GSH-dependent peroxidase activity and exhibited the largest affinity to and the highest catalytic efficiency on phosphatidylcholine hydroperoxide, suggesting that phospholipid hydroperoxide is probably the optimum substrate for RsPHGPx. Furthermore, RsPHGPx showed a much higher V max value, by two orders of magnitude, than those of all other known plant PHGPxs. Taken together, these results showed evidence for the first time of mitochondrial localization and higher activity of PHGPx in plants and provided a framework for continued studies on the physiological functions of RsPHGPx.

Keywords

Radish Phospholipid hydroperoxide glutathione peroxidase Mitochondrial localization Enzymatic activity Biochemical function 

Notes

Acknowledgements

We are grateful to Y. Inoue for his providing the yeast strains used in this study, A. Duchene for the gift of the pCK-GFP vector, and G. Bonnard for the gifts of NAD9 and LSU RuBisCo rabbit polyclonal antibodies and his help with immunolocalization. This work was supported by grants from the National Natural Science Foundation of China (30170080, 39770078), the State Key Basic Research and Development Plan of China (2004CB117303), and the Hi-Tech Research and Development Program of China (2004AA222100, 2002AA212051 and 2002AA07006).

References

  1. Arai M, Imai H, Koumura T, Yoshida M, Emoto K, Umeda M, Chiba N, Nakagawa Y (1999) Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells. J Biol Chem 274:4924–4933PubMedCrossRefGoogle Scholar
  2. Arthur JR (2000) The glutathione peroxidases. Cell Mol Life Sci 57:1825–1835PubMedCrossRefGoogle Scholar
  3. Avsian-Kretchmer O, Eshdat Y, Gueta-Dahan Y, Ben-Hayyim G (1999) Regulation of stress-induced phospholipid hydroperoxide glutathione peroxidase expression in citrus. Planta 209:469–477PubMedCrossRefGoogle Scholar
  4. Avsian-Kretchmer O, Gueta-Dahan Y, Lev-Yadun S, Gollop R, Ben-Hayyim G (2004) The salt-stress signal transduction pathway that activates the gpx1 promoter is mediated by intracellular H2O2, different from the pathway induced by extracellular H2O2. Plant Physiol 135:1685–1696PubMedCrossRefGoogle Scholar
  5. Bao YP, Chambers SJ, Williamson G (1995) Direct separation of hydroperoxy- and hydroxy-phosphatidylcholine derivatives: application to the assay of phospholipid hydroperoxide glutathione peroxidase. Anal Biochem 224:395–399PubMedCrossRefGoogle Scholar
  6. Boris H, Martin M, Sabine G, Ekkehard R, Thomas B, Andreas W (1999) Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plants in vivo. Plant J 17:557–561CrossRefGoogle Scholar
  7. Bradford M (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of dye-binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  8. Chen S, Vaghchhipawala Z, Li W, Asard H, Dickman MB (2004) Tomato phospholipids hydroperoxide glutathione peroxidase inhibits cell death induced by bax and oxidative stresses in yeast and plants. Plant Physiol 135:1630–1641PubMedCrossRefGoogle Scholar
  9. Chew O, Whelan J, Millar AH (2003) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants J Biol Chem 278:46869–46877PubMedCrossRefGoogle Scholar
  10. Churin Y, Schilling S, Borner T (1999) A gene family encoding glutathione peroxidase homologues in Hordeum vulgare (barley). FEBS Lett 459:33–38PubMedCrossRefGoogle Scholar
  11. Claros MG, Vincens P (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem 241:779–786PubMedCrossRefGoogle Scholar
  12. Criqui MC, Jamet E, Parmentier Y, Marbach J, Fleck J (1992) Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases. Plant Mol Biol 18:623–627PubMedCrossRefGoogle Scholar
  13. Day DA, Neuburger M, Douce R (1985) Biochemical characterization of chlorophyll-free mitochondria from pea leaves. Aust J Plant Physiol 12:219–228Google Scholar
  14. Depège N, Drevet J, Boyer N (1998) Molecular cloning and characterization of tomato cDNAs encoding glutathione peroxidase-like proteins. Eur J Biochem 253:445–451PubMedCrossRefGoogle Scholar
  15. Duchen MR (2004) Mitochondria in health and disease: perspectives on a new mitochondrial biology. Mol Aspects Med 25:365–451PubMedGoogle Scholar
  16. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016PubMedCrossRefGoogle Scholar
  17. Eshdat Y, Holland D, Faltin Z, Ben-Hayyim G (1997) Plant glutathione peroxidases. Physiol Plant 100:234–240CrossRefGoogle Scholar
  18. Flohé L, Günzler WA (1984) Assays of glutathione peroxidase. Methods Enzymol 105:114–121PubMedGoogle Scholar
  19. Grebenok RJ, Pierson E, Lambert GM, Gong FC, Afonso CL, Haldeman-Cahill R, Carrington JC, Galbraith DW (1997) Green-Fluorescent Protein fusions for efficient characterization of nuclear localization signals. Plant J 11:573–586PubMedCrossRefGoogle Scholar
  20. Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile PZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994PubMedCrossRefGoogle Scholar
  21. Hazebrouck S, Camoin L, Faltin Z, Strosberg AD, Eshdat Y (2000) Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli. J Biol Chem 275:28715–28721PubMedCrossRefGoogle Scholar
  22. Herbette S, Lenne C, Leblanc N, Julien JL, Drevet JR, Roeckel-Drevet P (2002) Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities. Eur J Biochem 269:2414–2420PubMedCrossRefGoogle Scholar
  23. Holland D, Ben-Hayyim G, Faltin Z (1993) Molecular characterization of salt-stress associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases. Plant Mol Biol 21:923–927PubMedCrossRefGoogle Scholar
  24. Hurst R, Korytowski W, Kriska T, Esworthy RS, Chu FF, Girotti AW (2001) Hyperresistance to cholesterol hydroperoxide-induced peroxidative injury and apoptotic death in a tumor cell line that overexpresses glutathione peroxidase isotype-4. Free Radic Biol Med 31:1051–1065PubMedCrossRefGoogle Scholar
  25. Imai H, Nakagawa Y (2003) Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) in mammalian cells. Free Radic Biol Med 34:145–169PubMedCrossRefGoogle Scholar
  26. Johnson RR, Cranston HJ, Chaverra ME, Dyer WE (1995) Characterization of cDNA clones for differentially expressed genes in embryos of dormant and nondormant Avena fatua L. caryopses. Plant Mol Biol 28:113–122PubMedCrossRefGoogle Scholar
  27. Jung BG, Lee KO, Lee SS, Chi YH, Jang HH, Kang SS, Lee K, Lim D, Yoon SC, Yun DJ, Inoue Y, Cho MJ, Lee SY (2002) A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase. J Biol Chem 277:12572–12578PubMedCrossRefGoogle Scholar
  28. Köhler RH, Zipfel WR, Webb WW, Hanson MR (1997) The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J 11:613–621PubMedCrossRefGoogle Scholar
  29. Kvaratskhelia M, Winkel C, Thorneley RNF (1997) Purification and characterization of a novel class III peroxidase isoenzyme from tea leaves. Plant Physiol 114:1237–1245PubMedCrossRefGoogle Scholar
  30. Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–593PubMedCrossRefGoogle Scholar
  31. Li WJ, Feng H, Fan JH, Zhang RQ, Zhao NM, Liu JY (2000) Molecular cloning and Expression of A phospholipid hydroperoxide glutathione peroxidase homolog in Oryza sativa. Biochim Biophys Acta 1493:225–230PubMedGoogle Scholar
  32. Li W, Liu JY, Zhao N (2001) Cloning and characterization of phospholipid hydroperoxide glutathione peroxidase gene from Momordica charantia. Prog Biochem Biophys 28:908–911Google Scholar
  33. Liu JY, Li W, Yang X, Zhao N (2002) Cloning and initial characterization of a rasPHGPX, a Raphanus sativus homolog of mammalian PHGPXs. Plant Cell Physiol 43:197Google Scholar
  34. Maiorino M, Aumann KD, Brigelius-Flohe R, Doria D, van den Heuvel J, McCarthy J, Roveri A, Ursini F, Flohe L (1995) Probing the presumed catalytic triad of selenium-containing peroxidases by mutational analysis of phospholipid hydroperoxide glutathione peroxidase (PHGPx). Biol Chem Hoppe-Seyler 376:651–660PubMedGoogle Scholar
  35. Maiorino M, Gregolin C, Ursini F (1990) Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol 186:448–457PubMedGoogle Scholar
  36. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) The reactive oxygen gene network of plants. Trends Plant Sci 9:490–498PubMedCrossRefGoogle Scholar
  37. Mullineaux PM, Karpinski S, Jimenez A, Cleary SP, Robinson C, Creissen GP (1998) Identification of cDNA encoding plastid-targeted glutathione peroxidase. Plant J 13:375–379PubMedCrossRefGoogle Scholar
  38. Nakagawa Y (2004) Role of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx) as an antiapoptotic factor. Biol Pharm Bull 27:956–960PubMedCrossRefGoogle Scholar
  39. Rodriguez Milla MA, Maurer A, Rodriguez Huete A, Gustafson JP (2003) Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways. Plant J 36:602–615PubMedCrossRefGoogle Scholar
  40. Roeckel-Drevet P, Gagne G, Labrouhe TD, Dufaure J-P, Nicolas P, Drevet JR (1998) Molecular characterization, organ distribution and stress mediated induction of two glutathione peroxidase encoding mRNAs in sunflower (Helianthus annuus). Physiol Plant 103:385–394CrossRefGoogle Scholar
  41. Sakamoto W, Spielewoy N, Bonnard G, Murata M, Wintz H (2000) Mitochondrial localization of AtOXA1, an Arabidopsis homologue of yeast Oxa1p involved in the insertion and assembly of protein complexes in mitochondrial inner membrane. Plant Cell Physiol 41:1157–1163PubMedCrossRefGoogle Scholar
  42. Schock I, Gregan J, Steinhauser S, Schweyen R, Brennicke A, Knoop V (2000) A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant. Plant J 24:489–501PubMedCrossRefGoogle Scholar
  43. Sreenivasulu N, Miranda M, Prakash HS, Wobus U, Weschke W (2004) Transcriptome changes in foxtail millet genotypes at high salinity: identification and characterization of a PHGPx gene specifically upregulated by NaCl in a salt-tolerant line. J Plant Physiol 161:467–477PubMedCrossRefGoogle Scholar
  44. Sugimoto M, Furui S, Suzuki Y (1997) Molecular cloning and characterization of A cDNA encoding putative phospholipid hydroperoxide Glutathione peroxidase from spinach. Biosci Biotechnol Biochem 61:1379–1381PubMedCrossRefGoogle Scholar
  45. Sugimoto M, Sakamoto W (1997) Putative phospholipid hydroperoxide glutathione peroxidase gene from Arabidopsis thaliana induced by oxidative stress. Genes Genet Syst 72:311–316PubMedCrossRefGoogle Scholar
  46. Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552:335–344PubMedCrossRefGoogle Scholar
  47. Ursini F, Mariorino M, Brigelius-Flohé R, Aumann KD, Roveri A, Schomburg D, Flohé L (1995) Diversity of glutathione peroxidase. Methods Enzymol 252:38–53PubMedCrossRefGoogle Scholar
  48. Utomo A, Jiang X, Furuta S, Yun J, Levin D S, Wang Y-C, Desai KV, Green JE, Chen P-L, Lee W-H (2004) Identification of a novel putative non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx) essential for alleviating oxidative stress generated from polyunsaturated fatty acids in breast cancer cells. J Biol Chem 279:43522–43529PubMedCrossRefGoogle Scholar
  49. Wang HP, Qian SY, Schafe FQ, Domann FE, Oberley LW, Buettner GR (2001) Phospholipid hydroperoxide glutathione peroxidase protects against the singlet oxygen-induced cell damage of photodynamic therapy. Free Radic Biol Med 30:825–835PubMedCrossRefGoogle Scholar
  50. Yang XD, Li WJ, Liu JY (2005) Isolation and characterization of a novel PHGPx gene in Raphanus sativus. Biochim Biophys Acta 1728:199–205PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  1. 1.Laboratory of Molecular Biology and MOE Laboratory of Protein Science, Department of Biological Sciences and BiotechnologyTsinghua UniversityBeijingChina

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