IRKUTSK, RUSSIA
Institute of Plant Physiology and Biochemistry

The effects of hydrogen peroxide and glutathione on protein phosphorylation in mitochondria

— Subota, IY; Konstantinov, YM; Arziev, AS; Tarasenko, VI

In spite of extensive studies of mitochondrial gene functioning, the molecular mechanism that governs the expression of plant mitochondrial genomes in response to changes of metabolic conditions in the cell and the whole organism has remained poorly understood.

Earlier we revealed that the redox state of particular respiratory complexes substantially affects the rate of mitochondrial translation (Konstantinov et al., Dokl. Acad. Nauk 354:699–701, 1997). The mechanism and level of this control is not clear. One of the mechanisms could be phosphorylation of mitochondrial proteins. It has been known that protein phosphorylation/dephosphorylation by protein kinases and protein phosphatases is a ubiquitous mechanism in eukaryotes and prokaryotes for modulating the activity of intracellular signals (McEntyre, Trends Biochem. Sci. 19:459–518, 1994).

In this work we investigated the influence of hydrogen peroxide and glutathione as physiological redox modulators on the activity of protein phosphorylation in maize mitochondria. The mitochondria were isolated from 3-day-old etiolated maize seedlings (hybrid VIR42MV) by a standard method of differential centrifugation. Protein phosphorylation assays were carried out according to Struglics et al. (FEBS Lett. 475:213–217, 2000) with the use of [γ32P] ATP (specific radioactivity was 6000 Ci/mmol).

Incubating mitochondria with [γ32P] ATP in the presence of hydrogen peroxide (0.5 mM and 1 mM) as oxidizing agent results in phosphorylation of more than 9 proteins as shown by SDS-PAGE and autoradiography (Fig. 1). The results obtained show that hydrogen peroxide (1 mM) caused stimulation of phosphorylation of polypeptide of 48 kDa and polypeptide of 22 kDa. It is interesting to note that the protein phosphorylation of polypeptide of 33 kDa in the presence of 0.5 mM hydrogen peroxide as compared with control is increased. Nevertheless, the level of its phosphorylation in the presence of 1 mM hydrogen peroxide is the same as in the control. Glutathione (GSH) in concentration of 5 mM as reducing agent decreased the rate of phosphorylation polypeptides of 66, 48, 39, 36, 33 and 22 kDa (Fig. 1).

These data correlate with our previous results (Konstantinov et al., Dokl. Acad. Nauk 354:699–701, 1997). The addition of potassium ferricyanide as an oxidizing agent enhanced translation in organello, and addition of sodium dithionite as a reducing agent suppressed this process. The results obtained suggest that changes in the redox state can stimulate or inhibit the protein kinase activity required for phosphorylation of some inducible proteins. These changes in the activity of protein phosphorylation evidently may affect the protein-synthesizing activity of mitochondria. In addition, the alteration of degree of phosphorylation of the one F0F1 – ATPase subunit of 22 kDa proteins (Struglics et al., Biochem. Biophys. Res. Commun. 243:664–668, 1998) in the presence of the redox modulators in our view might form part of a mechanism mediating the interrelation between redox state and genome expression in plant mitochondria.

 

Fig. 1. The effects of hydrogen peroxide and glutathione on the protein phosphorylation in maize mitochondria.


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