Requirement for prolonged action in the cytosol for optimal protein synthesis inhibition by diphtheria toxin

Diphtheria toxin A-fragment enters the cytosol of target cells, where it in hibits protein synthesis by catalyzing ADP-ribosylation of elongation facto r 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibitio n in single cells by autoradiography and compared it with inhibition of pro tein synthesis in the whole cell culture. The data show that half-maximal p rotein synthesis inhibition in the whole cell population after a-short incu bation time is achieved by partially inhibiting protein synthesis in basica lly 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. Whil e the toxicity of the stable mutants increased with time, the unstable muta nts 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 t he cells. The data indicate that entry of toxin into a cell is not associat ed with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain compl ete protein synthesis inhibition at low toxin concentrations.