Abstract
Diphtheria toxin A-fragment enters the cytosol of target cells, where it inhibits protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibition in single cells by autoradiography and compared it with inhibition of protein synthesis in the whole cell culture. The data show that half-maximal protein synthesis inhibition in the whole cell population after a short incubation time is achieved by partially inhibiting protein synthesis in basically all the cells, while half-maximal protein synthesis inhibition after a long incubation time is due to a complete protein synthesis block in about half the cells in the population. We have also compared stable and unstable A-fragment mutants with respect to the kinetics of cell intoxication. While the toxicity of the stable mutants increased with time, the unstable mutants showed a similar toxicity at early and late time points. When studying the kinetics of cell intoxication by toxins with short cytosolic half-life, we could not detect any recovery of protein synthesis at late time points when all the mutant A-fragments should be degraded. This indicates that the ADP-ribosylation of EF-2 cannot be reversed by an endogenous activity in the cells. The data indicate that entry of toxin into a cell is not associated with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations.
Highlights
Several plant and bacterial toxins are able to enter the cytosol of target cells where they inhibit cellular protein synthesis through catalytic action
We have previously shown that when a short peptide with an N-terminal Asp residue was fused to the N terminus of diphtheria toxin A-fragment (DTA), the resulting protein (Asp-FLAG-DTA) was unstable in the cytosol [11]
We have studied the kinetics of cell intoxication by such mutants, and the results support the notion that a prolonged action of the diphtheria toxin A-fragment in the cytosol is required for optimal toxicity
Summary
Several plant and bacterial toxins are able to enter the cytosol of target cells where they inhibit cellular protein synthesis through catalytic action. When diphtheria toxin is bound to the cell surface and subsequently briefly exposed to acidic medium, thereby mimicking the conditions in the endosome, a direct and synchronous translocation of A-fragments to the cytosol is induced [7, 8].
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