A plant mitochondrial phospholipid hydroperoxide glutathione peroxidase: its precise localization and higher enzymatic activity
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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.
KeywordsRadish Phospholipid hydroperoxide glutathione peroxidase Mitochondrial localization Enzymatic activity Biochemical function
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).
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