A recent report (M.I. Ikeda and M.W. Gray, Mol Cell Biol 19:8113-8122, 1999) suggests that transcription in wheat mitochondria also involves special DNA-binding protein. There is a great body of evidence that eukaryotic transcriptional activators (c-Jun, c-Fos, c-Myb, Egr-1, AP-2) and their DNA-binding activity may be the target for special redox regulation (Y. Sun and L.W. Oberley, Free Radic Biol Med 21:335-348, 1996). It is clear now that redox status plays an important role in the control of gene expression, both in the nucleus and in mitochondria. Alterations of the redox status in the cell can lead to changes in DNA binding and trans-activating abilities of many transcription factors, and thus lead to changes in gene expression which may affect cellular metabolism and adaptive responses (Y. Huang, F.E. Domann, Bioch Biophys Res Commun 249: 307-312, 1998). But little is known about DNA-binding proteins in maize mitochondria and the sensitivity of the DNA-binding activity of these proteins to redox conditions. The aim of the present work was to investigate the influence of redox conditions on DNA-binding activity in mitochondrial protein extracts.
The mitochondria were isolated from 3-day-old etiolated maize seedlings of hybrid VIR42 MV by a standard method of differential centrifugation. Mitochondrial protein was determined by the Lowry method. Mitochondrial protein extracts were prepared by a salt lysis procedure performed in a buffer containing 25 mM Tris-HCl (pH 7.8), 600 mM, 1 mM EDTA, 10% glycerol, 13.5 mM 2-mercaptoethanol, 0.5 mM PMSF, 100 µg/ml BSA under stirring for 15 minutes at +4 C. The lysate was centrifuged for 15 minutes under 15,000 rpm. The supernatant obtained was subjected to dialysis against EMSA buffer. DNA-binding activity in mitochondrial extracts was detected in a gel mobility shift assay (EMSA). A DNA probe containing the yeast DNA topoisomerase I binding site was prepared as described (Kagoshima et al, J Biol Chem 271:33074-33082, 1996).
The effects of chemical and physiological redox agents on DNA-binding activity in mitochondrial protein extracts is shown in Figure 1. The mitochondrial DNA-binding activity is seen to decrease in the presence of sodium dithionite. In contrast, the treatment of potassium ferricyanide and the oxidized form of glutathione (GSSG) leads to a significant increase in DNA-binding activity. At the moment we could not explain similar effects both oxidized and reduced forms of glutathione on DNA-binding activity in mitochondrial protein extracts. Possibly, GSH converted rapidly in GSSG form as a result of high oxidant content in non-purified mitochondrial extract.
Figure 1. The DNA binding activity of mitochondrial extracts is modulated by reduction-oxidation with chemical and physiological redox agents in vitro. 1, free probe; 2, mitochondrial extract; 3, mitochondrial extract + potassium ferricyanide (5 mM); 4, mitochondrial extract + sodium dithionite (5 mM); 5, mitochondrial extract + glutathione in oxidized form (GSSG) (5 mM); 6, mitochondrial extract + glutathione in reduced form (GSH) (5 mM).
The results obtained suggest that DNA binding activity of proteins potentially implicated in transcription in maize mitochondria is redox-modulated in vitro, presumably by GSSG-mediated oxidation of the appropriate regulatory cysteine residues in transcription factors.
Financial support from the RFBR (Project Number 01-04-48162) is acknowledged.
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