The redox control of genetic functions in isolated mitochondria
--Yuri M. Konstantinov, Galina N. Lutsenko and Vladimir A. Podsosonny

The problem of regulation of genetic functions of plant mitochondria in response to change of metabolic conditions in the cells and the whole organism remains poorly understood. The hypothesis has recently been advanced on the redox control of gene expression and functions of genomes of chloroplasts and mitochondria (Allen, J. Theor. Biol. 165:609-631, 1993). According to the hypothesis, two types of regulatory protein factors (so called "redox sensors" and "redox response regulators") form the two-component regulatory system. This system may place expression of mitochondrial and chloroplast genes under redox regulatory control (Allen, 1993). The aim of the present work was to study a possible redox control of the mitochondrial genome functions in isolated mitochondria. The kinetics of the RNA and DNA syntheses in mitochondria has been examined in the presence of potassium ferricyanide, an oxidising agent, and sodium dithionite, a reducing agent. In addition, the effect of particular inhibitors of protein kinases and phosphatases on the activity of nucleic acid synthesis in mitochondria has been examined, since hypothetical redox sensors and redox response regulators perform their regulatory functions in the form of phosphorylated protein molecules (Allen, 1993) and serve as a target of protein kinase and protein phosphatase action.

The mitochondria were isolated from 3-day-old etiolated maize seedlings (hybrid VIR42 MV) by a standard method of differential centrifugation. Mitochondrial protein was determined by the Lowry method. DNA synthesis in mitochondria was determined according to the method of Schegget and Borst (Biochim. Biophys. Acta 95:235-248, 1971) with the use of 32P-dATP with specific radioactivity 185 PBq mol-1. The conditions for measuring RNA synthesis in mitochondria were essentially similar to those described elsewhere (Carlson et al. Curr. Genet. 11:151, 1986). The kinetics of the mtRNA sysnthesis was recorded by 32P-UTP with specific activity > 74 pBq mol-1. The kinetic data were obtained from at least 3 to 4 experiments.

The observed specific changes of the transcription of the mitochondrial genome following the addition of ferricyanide and dithionite to mitochondria (Table 1) suggest the existence of the redox control of template functions of the mitochondrial genome. A significant activation of the transcription was observed in the presence of ferricyanide, while the addition of dithionite was followed by the inhibition of the RNA synthesis in mitochondria.

The changes in redox conditions in the mitochondrial system caused by the addition of ferricyanide or dithionite were also followed by changes in the activity of the DNA synthesis in these organelles (Table 2). Furthermore, a significant decrease in the DNA synthesis activity was observed in the presence of both oxidising and reducing agents, which was more prominent in the case of dithionite. The effect of the repression of the DNA synthesis was promoted in the presence of sodium fluoride, an inhibitor of protein phosphatases.

The effect of sodium fluoride on the level of the DNA and RNA syntheses in mitochondria (Tables 2 and 3) can presumably be accounted for by dependence of the activity of redox regulatory phosphoprotein factor(s) (Allen, 1993) on their phosphorylation state. Such influence of sodium fluoride on the genetic processes in mitochondria (activation of transcription and repression of the DNA synthesis activity) can presumably result from the competitive interrelationships between RNA and DNA syntheses at the level of genetic templates under the redox control, as well as from prevailing of transcription over replication under a redox potential decrease.

Table 1. The effect of potassium ferricyanide and sodium dithionite on kinetics of RNA synthesis in mitochondria of maize hybrid VIR42 MV.
 
Incorporation of 32P-UTP into acid insoluble mitochondrial fraction, counts/min mg protein
Conditions 5 min 10 min 15 min 20 min
Control 6810 8889 16300 37463
Ferricyanide (5 mM) 9247 12041 19809 41245
Dithionite (5 mM) 5020 6712 15014 31486

Table 2. The effect of potassium ferricyanide and sodium dithionite on kinetics of DNA synthesis in mitochondria of maize hybrid VIR42 MV.
 
Incorporation of 32P-dATP into acid insoluble mitochondrial fraction, counts/min mg protein
Conditions 5 min 10 min 15 min 20 min
Control 21825 31791 41668
Ferricyanide (5 mM) 17043 21090 27951 31103
Dithionite (5 mM) 11033 13750 22721 26053
NaF (40 mM) 20830 21623 26593 30016
Ferricyanide + NaF 8611 17115 25105 27826
Dithionite + NaF 6213 12106 13526 19243

Table 3. The effect of protein phosphatase inhibitor sodium fluoride on kinetics of RNA synthesis in mitochondria of maize hybrid VIR42 MV.
 
Incorporation of 32P-UTP into acid insoluble mitochondrial fraction, counts/min mg protein
Conditions 5 min 10 min 15 min 20 min
Control 5865 6832 10926 20908
NaF (40 mM) 6807 12359 12740 30441

The kinetics of RNA and DNA syntheses in mitochondria in the presence of different concentrations (10, 25, 50, 100 and 200 nM) of staurosporine, an inhibitor of protein kinases, showed an increase in the activity of transcription and a decrease in the activity of DNA synthesis (data not shown). The changes in kinetic parameters of nucleic acid synthesis correlated with the staurosporine concentrations used. The results of the inhibitor analysis performed with sodium fluoride and staurosporine suggest the existence of at least two phosphoproteins regulating the transcription and replication in mitochondria, which affects differently the activity of these processes. It could not be ruled out that these proteins perform their regulatory action by binding with the corresponding polymerase complex.

As a whole, the changes in transcription and DNA synthesis under the influence of reducing and oxidising agents in isolated mitochondria suggest that the system of the redox control of genetic functions of mitochondria may also exist under in vivo conditions. 


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