Membrane translocation of diphtheria toxin carrying passenger protein domains

For diphtheria toxin to be cytotoxic, the enzymatically active part (fragment A) must be translocated to the cytosol. We here demonstrate that additional proteins linked as N-terminal extensions can be translocated along with fragment A across the plasma membrane of toxin-sensitive cells. Thus, an extra fragment A of diphtheria toxin and some of apolipoprotein AI were translocated as passenger proteins along with mutant diphtheria toxin fragment A. Translocation was monitored by the cytotoxic effect of the additional fragment A as well as by the translocation of [35S]methionine-labelled protein to a compartment protected from externally added pronase. Cytotoxicity experiments indicated that double A fragments can also be translocated across the membrane of intracellular vesicles. The results demonstrate that the translocation apparatus used for toxin translocation is not limited to a single A fragment but can accommodate additional proteins as well. The fact that proteins as large as 20 kDa can be brought into cells by way of diphtheria toxin under both in vitro and in vivo conditions opens up the possibility of using diphtheria toxin mutants for introducing molecules with biological activity into cells.