Effects of inhibitors and uncouplers in the synthesis of 52kD heat shock protein by mitochondria

Corn seedling mitochondria respond to a 10-degree increase in incubation temperature by in organello synthesis of a 52kD protein (Nebiolo and White, Plant Phys. 79:1129, 1985). Mitochondrial translation of this protein is insensitive to cycloheximide and erythromycin and sensitive to chloramphenicol (MNL 61:73, 1987). Sodium arsenite, an inducer of the heat shock response in intact seedlings, induces synthesis of the 52kD protein at concentrations greater than 50µM (Nebiolo and Walden, J. Cell Biol. 79:258a, 1986). In our hands, seedling mitochondria purified on both sucrose and Percoll gradients are free of bacterial contamination, measured by plate counts of mitochondrial pellets and minimal protein synthesis in the presence of acetate as carbon source, and synthesize the 52kD protein only when "stressed" by temperature or the mitochondrial uncoupler arsenite.

To extend our investigation of this mitochondrial response to stress, we treated purified seedling mitochondria isolated under sterile conditions with various inhibitors and uncouplers of mitochondrial function. We measured 35S-methionine incorporation under optimum conditions for detecting only mitochondrial protein synthesis: succinate/ADP as energy source, erythromycin and cycloheximide to inhibit plastid and 80S ribosome translation respectively, and vigorous shaking to maintain sufficient oxygen concentrations for efficient electron transport.

The general protonophoric uncoupler 2,4-dinitrophenol inhibits 35S-methionine incorporation by mitochondria by 65-70% at concentrations of 10µM but has no apparent effect at 50 and 100µM. No change was observed in protein profiles; the 52kD protein was not induced at any concentration. The uncoupler valinomycin, which acts as a mobile ion carrier of K+, also had no effect on incorporation or on protein profiles.

The mitochondrial inhibitor rotenone had no effect on mitochondrial translation at treatments of 10, 25 and 100µM rotenone. Similar results were found using 10-100µM KCN, a potent mitochondrial electron transport inhibitor.

Methomyl, a carbamate insecticide, uncouples corn seedling mitochondria of T male sterile cytoplasm at concentrations as low as 4mM (Klein and Koeppe, Plant Phys. 77:912, 1985). Lannate, a commercial product with methomyl as the active ingredient, induces heat shock protein synthesis in corn seedlings of both T and N cytoplasm at concentrations as low as 20mM (MNL 61:69, 1987). We treated purified mitochondria from T and N seedlings with various concentrations of lannate and methanol, the solvent in which methomyl is dissolved to produce lannate.

At concentrations of 13 and 26mM lannate inhibits 35S-methionine incorporation by N mitochondria by 35% while showing no significant effect on T mitochondria at the same concentrations. At lower concentrations (1.3 and 2.6mM lannate) T mitochondria incorporation is inhibited while N mitochondria are not affected. This may imply 2 different mechanisms of action of lannate on mitochondria, one toxic effect to which N mitochondria are more susceptible and a second effect at lower concentrations, approximately those which uncouple the mitochondria, which affect T cytoplasm differentially.

Methanol at concentrations found in lannate treatments slightly inhibits incorporation of both N and T mitochondria.

We are currently investigating alterations of in organello translation products by lannate and methanol in both T and N seedling mitochondria, specifically looking for induction of the 52kD protein produced in response to heat and arsenite "stress".

David B. Walden and Christine M. Nebiolo


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