IRKUTSK,RUSSIA
Institute of Plant Physiology and Biochemistry

Protein synthesis in mitochondria under different redox conditions
--Konstantinov, YM; Subota, IV and Arziev, AS

It is known that gene expression can be efficiently regulated at the level of translation along with transcriptional and posttranscriptional levels. However specific molecular mechanisms of such regulation, especially regarding translation in mitochondria, are poorly known.

As previously shown, redox conditions can provide a profound effect on the template activity of the mitochondrial genome regarding RNA and DNA syntheses in organello (Konstantinov et al., Biochem. Mol. Biol. 36:319-326, 1995). Moreover, the activation of transcription in mitochondria under oxidising conditions and its inhibition under reducing conditions can indicate possible redox regulation of genetic processes in mitochondria. With consideration for the existence of multi-level regulation of gene expression, a question arises at what levels such regulation can exist during functioning of mitochondrial genes.

The aim of the present work was to examine the mitochondrial protein synthesis in organello under changes of redox conditions by the addition of potassium ferricyanide as an oxidising agent and sodium dithionite as a reducing agent.

The mitochondria were isolated from 3-day-old etiolated seedlings of hybrid VIR 46MV by a standard method of differential centrifugation. Protein was determined by the Lowry method, protein synthesis was measured in mitochondria according to the method of Bhat et al. (Biochemistry 21:2452-2460, 1982) with the use of [14C]-leucine (specific radioactivity was 1760 GBq/mol). Protein synthesis reactions in seedling mitochondria were highly sensitive to chloramphenicol (50 ug/ml). In order to study the effect of oxidation phosphorylation uncoupler on in organello protein synthesis carbonyl cyanide chlorophenylhydrazone (CCCP) at a final concentration of 1 µM was used.

The effect of redox conditions on the kinetics of protein synthesis in maize seedling mitochondria is shown in Table 1.

Table 1. Kinetics of protein synthesis in isolated maize mitochondria in the absence and the presence of potassium ferricyanide or sodium dithionite.
 
 
Incorporation of [14C]-leucine, counts/min/mg protein
Conditions
5 min
10 min
15 min
20 min
Control
554
1432
2833
4986
Ferricyanide (5 mM)
1098
2221
3931
6039
Dithionite (5mM)
180
132
310
1190

The activity of protein synthesis is seen to increase in the presence of potassium ferricyanide used as an oxidising agent, while this process is strongly inhibited when mitochondria were supplemented by sodium dithionite as a reducing agent. Thus, redox conditions used affected pronouncedly the activity of the protein synthesizing system in isolated plant mitochondria.

In addition, the effect of redox conditions on protein synthesis has been examined in the presence of carbonyl cyanide chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation. It is seen in Table 2 that the effect of potassium ferricyanide on the protein synthesis is negligible in the presence of CCCP, while the treatment with simultaneous addition of sodium dithionite and CCCP resulted in more profound inhibition of translation. Since the addition of CCCP alone caused a significant decrease in the activity of mitochondrial protein synthesis, apparently due to development of an energy deficient state in mitochondria, the changes in the redox conditions' influence in the presence of the uncoupler is related, in our opinion, mainly to disturbances in energy supply of this process.

Table 2. The effect of potassium ferricyanide and sodium dithionite on protein synthesis in maize mitochondria in the presence of CCCP.
 
Conditions Incorporation of [14C]-leucine, counts/min/mg protein
Control 7380
CCCP 4005
Ferricyanide 11863
Ferricyanide + CCCP 8044
Dithionite 2804
Dithionite + CCCP 1411

The effect of potassium ferricyanide on the translation activity has been examined in the presence of such an inhibitor of template RNA synthesis as ethidium bromide in order to elucidate whether changes at the transcriptional level are the main reason for changes in mitochondrial protein synthesis in the presence of redox agents (Table 3). It is expected from data given in Table 3 that redox conditions can affect the expression of mitochondrial proteins directly at the level of translation.

Table 3. The effect of potassium ferricyanide on protein synthesis in maize mitochondria in the presence of ethidium bromide.
 
Conditions Activity of protein synthesis in mitochondria (% of control)
Control 100
Ferricyanide 167
Ethidium bromide 28
Ferricyanide + ethidium bromide 54

As a whole, we assume that translation along with other genetic processes in maize mitochondria can be subjected to redox control. 


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