Differential redox regulation by glutathione of translation in isolated mitochondria
--Konstantinov, YM, Subota, IY, Arziev, AS

Recent studies have shown that transcription of some genes (so-called 'redox sensitive genes') is regulated by cellular redox status, especially by thiol-disulfide balance (Sen and Packer, FASEB Journal, 10:709-720, 1996). We have previously reported (MNL 69:63-64, 1995; MNL 70:29-30, 1996; MNL 71:40-41, 1997) on the effect of different redox conditions on mitochondrial genome expression regarding DNA, RNA and protein syntheses in organello. Significant activation of transcription and translation in mitochondria under oxidising conditions and its profound repression under reducing conditions can indicate possible multi-level redox regulation of mitochondrial gene expression.

The aim of the present work was to verify our hypothesis about possible involvement of the glutathione system in the regulation of translational activity in mitochondria.

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. Preparation of mitoplasts was performed using digitonin treatment according to Cristophe et al. (Plant Sci. Lett. 21:181-192, 1981). Protein synthesis in mitochondria and mitoplasts was measured 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-1). Protein synthesis reactions in maize seedling mitochondria were highly sensitive to chloramphenicol (50 ug/ml). The kinetic data were obtained from at least 3 to 4 experiments.

The effect of reduced and oxidized forms of glutathione on the kinetics of protein synthesis in maize seedling mitochondria is shown in Table 1.

Table 1. The effect of reduced (GSH) and oxidized (GSSG) forms of glutathione on translational activity in mitochondria of maize hybrid VIR42 MV.
 

	Incorporation of [14C]-leucine, % of control
Conditions	5 min	10 min	15 min	20 min
Control	100	100	100	100
Ferricyanide (5 mM)	166	164	164	161
GSSG (5 mM)	108	101	150	145
GSH (5 mM)	65	87	84	80
GSH + GSSG	28	69	56	86

The mitochondrial translational activity is seen to increase in the presence of the oxidized form of glutathione while this process is strongly repressed when mitochondria were supplemented by the reduced form of glutathione. Thus, the redox system of such an endogenous thiol compound as glutathione affected pronouncedly the activity of the protein synthesizing system in isolated plant mitochondria.

Table 2 shows the kinetics of protein synthesis in mitochondria and mitoplasts (i.e. mitochondria without outer membrane). We used this model system to study the possible involvement of outer mitochondrial membrane components in the redox signal transduction in mitochondria. It is seen in Table 2 that mitoplasts are also capable of synthesizing the protein but with a lower rate then intact mitochondria.

Table 2. The kinetics of protein synthesis in mitochondria and mitoplasts from maize hybrid VIR42 MV.
 

	Incorporation of [14C]-leucine, counts/min/mg protein
Conditions	5 min	10 min	15 min	20 min
Mitochondria	49	679	1241	1635
Mitoplasts	4	202	717	919

The data on the influence of redox conditions on the translational activity in mitoplasts are summarized in Table 3. The effects of potassium ferricyanide and sodium dithionite on the activity of protein synthesis in mitoplasts were similar to those observed for the intact mitochondria. As for the influence of GSH and GSSG on mitoplast translational activity we showed the decrease of such effects in comparison with the whole mitochondria. It is expected from the data given in Table 3 that the outer mitochondrial membrane is involved somehow in redox signal transduction in the case of the glutathione system.

Table 3. The effect of reduced and oxidized forms of glutathione on translational activity in mitoplasts from maize hybrid VIR42 MV.
 

	Incorporation of [14C]-leucine, % of control
Conditions	5 min	10 min	15 min	20 min
Control	100	100	100	100
Ferricyanide	184	136	131	118
Dithionite	95	80	73	77
GSSG	140	97	107	108
GSH	129	98	73	77

As a whole, the data obtained suggests that the redox state of glutathione plays an important role in the in vivo regulation of mitochondrial translational activity in plants. We assume also that both transcription and translation factors in plant mitochondria are subject to thiol-mediated redox regulation.

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