Redox control of RNA synthesis in potato mitochondria.

ABSTRACT

This study shows that the incorporation of radiolabelled UTP into RNA in Percoll-gadient-purified potato mitochondria is regulated by the redox state of the mitochondrial electron transport chain. An early indication that there might be a redox effect on RNA synthesis was a decrease in UTP incorporation in incubates containing an oxidisable substrate, such as succinate or malate. Subsequent use of a variety of electron transport inhibitors acting at different points in the electron transport chain established that the redox state of the Rieske iron-sulphur protein was the major determinant of UTP incorporation. Inhibitors acting on the substrate side of the Rieske iron-sulphur protein, and causing oxidation of components on the oxygen side of their site of action, increased UTP incorporation into RNA. These included antimycin A, myxothiazole, and undecylhydroxydioxobenzothiazole at 500 nM. Inhibitors acting on the oxygen side of the Rieske iron-sulphur protein, and causing a reduction of components on the substrate side of the block, decreased UTP incorporation. These inhibitors were undecylhydroxydioxobenzothiazole at 25 nM and KCN. When phenazine methosulphate was present as an auto-oxidisable electron sink the effect of KCN was diminished. The conclusion from the inhibitor experiments that the redox state of the Rieske iron-suphur protein was important was supported when RNA synthesis was measured at a range of redox potentials. This gave a measured redox potential for the control of UTP incorporation into RNA of +270 mV and the slope of the curve indicated an n = 1 carrier. This value is close to the reported value of the Rieske iron-suphur protein. UTP incorporation was decreased by some 50% in the presence of low concentrations of okadaic acid (5 nM), an inhibitor of protein phosphatases PP1 and PP2A, and alpha-naphthyl acid phosphate, a broad-spectrum phosphatase inhibitor, indicating that the redox effect on RNA synthesis may be mediated via protein phosphorylation. We did not, however, detect an expected increase in RNA synthesis when protein kinase inhibitors were used, so the involvement of protein phosphorylation in the redox regulation of RNA synthesis is as yet uncertain.