title: Requirement for prolonged action in the cytosol for optimal protein synthesis inhibition by diphtheria toxin.
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.
authors.affiliation: Institute for Cancer Research, Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
authors.forename: P O
authors.initials: PO
authors.lastname: Falnes
authors.forename: S
authors.initials: S
authors.lastname: Ariansen
authors.forename: K
authors.initials: K
authors.lastname: Sandvig
authors.forename: S
authors.initials: S
authors.lastname: Olsnes
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000235:genetics / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology*; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D014709:Vero Cells
chemical_list: D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D010446:Peptide Fragments; D011500:Protein Synthesis Inhibitors; C029795:diphtheria toxin fragment A