The cytotoxic action of diphtheria toxin and its degradation in intact Vero cells are inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase.

T Umata,Y Moriyama,M Futai,E Mekada

doi:10.1016/s0021-9258(18)45829-2

Abstract

The role of vacuolar-type H(+)-ATPase (V-ATPase) in the cytotoxic action of diphtheria toxin (DT) was studied by using bafilomycin A1, a specific inhibitor of V-ATPase. Studies with acridine orange showed that the acidification of intracellular acidic compartments was inhibited strongly when Vero cells were treated with 500 nM bafilomycin A1, indicating that bafilomycin effectively inhibits V-ATPase when it is added to the culture medium. The toxicity of DT to Vero cells, which was determined by the inhibition of protein synthesis by DT, was inhibited partially by bafilomycin at 10 nM and inhibited completely at 500 nM. Therefore, V-ATPase is involved in the expression of the toxicity of DT. Studies using 125I-labeled DT showed that bafilomycin inhibited the degradation of internalized DT, indicating that V-ATPase is also involved in this step. Subcellular fractionation revealed that 125I-DT accumulated mainly in the endosome fraction, and not in the lysosome fraction, when the cells were incubated with 125I-DT in the presence of bafilomycin. Under the cell fractionation conditions similar to those used for the DT-treated cells, we determined the location of 125I-labeled epidermal growth factor in the degradation pathway. The result suggests that bafilomycin A1 does not inhibit the transport of epidermal growth factor to lysosome.

Highlights

The Cytotoxic Action of Diphtheria Toxin and Its Degradation in Intact Vero Cells Are Inhibited by Bafilomycin Al, a Specific Inhibitor of Vacuolar-type H+-ATPase*
Effect of Bafilomycin Al on the Cytotoxic Action of diphtheria toxin (DT)-To determine if bafilomycin Al inhibits the cytotoxic action of DT, Vero cells were incubated with various concentrations of bafilomycin Al at 37 “C for 30 min, and DT was added to the medium
Inhibition of Acidification of Intracellular Compartments with Bafilomycin A 1-As it was not clear whether bafilomycin Al can permeate the plasma membrane, we examined whether bafilomycin effectively inhibited V-ATPase in intact cells when this drug was added to the medium

Summary

PROCEDURES

Materials-DT was produced and purified as described previously [34]. Bafilomycin Al was kindly given by Prof. Measurement of the Amount of DT Associating with Cell SurfaceVero cells incubated with ‘*“I-DT were washed three times with icecold PBS and treated with protease solution (0.5 mg/ml trypsin, 0.5 mz/ml chvmotrvnsin in PBS) for 6 min at 37 “C. Effect of Bafilomycin Al on the Cytotoxic Action of DT-To determine if bafilomycin Al inhibits the cytotoxic action of DT, Vero cells were incubated with various concentrations of bafilomycin Al at 37 “C for 30 min, and DT was added to the medium. The toxicity of DT was assayed by measuring the protein synthesis rate of Vero cells. The concentration of bafilomycin Al required to inhibit the cytotoxic action of DT in intact Vero cells completely was similar to that required to inhibit V-ATPase in cell-free experiments [32] This concentration is at least three orders of magnitude lower than the minimum concentration showing an effect on other ATPases.

RESULTS

Time after DT washing

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DISCUSSION