DIPHTHERIA-TOXIN TRANSLOCATION ACROSS ENDOSOME MEMBRANES - A NOVEL CELL PERMEABILIZATION ASSAY REVEALS NEW DIPHTHERIA-TOXIN FRAGMENTS IN ENDOCYTIC VESICLES

By using cells overexpressing diphtheria toxin (DT) receptor and a nov el method of permeabilizing the plasma membrane with a bacterial pore- forming toxin, specific translocation of fragment A to the cytosol was observed, whereas full-size DT and other minor species of DT-derived fragments were left in intracellular vesicles, The translocation compe tence of DT proteins with mutations in the transmembrane domain is con sistent with their cytotoxicities. The charge-reversal mutants E349K a nd D352K do not translocate their fragment A to the cytosol, whereas D 352N is partially competent. ADP-ribosyltransferase activity of fragme nt A is not required for translocation. Novel fragments of DT with app arent molecular masses of 28 and 35 kDa were detected in endocytic ves icles, The 28-kDa fragment consists of fragment A and an N-terminal pi ece of fragment B, whereas the 35-kDa fragment contains part of fragme nt B and may be complementary to the 28-kDa fragment. Time course stud ies show that the 28-kDa fragment appears in endocytic vesicles prior to translocation of fragment A to the cytosol, raising the possibility that the 28-kDa fragment is an intermediate in translocation. We pres ent a model for translocation of fragment A that incorporates the obse rvations made using the novel permeabilization method.