Essential role of calcium in cellular internalization of Pseudomonas toxin

Pseudomonas exotoxin (PE) has been shown previously to enter mouse LM cells by receptor-mediated endocytosis, to block protein synthesis, and to cause cell death. The requirement for the divalent cation calcium in the binding and internalization of PE was examined. Biochemical studies showed that depletion of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid protected cells from the action of PE when the chelator was present during the internalization step. Extracellular calcium was not required for binding. We observed with immunoelectron microscopy that, in the cold, toxin bound to cell surfaces equally well in the presence or absence of chelator. In the presence of chelator, toxin was not cleared from the cell surface when cells were warmed to 37 degrees C. Replenishment of calcium (2 mM CaCl2), however, allowed normal rapid clearance of PE to occur. We suggest that internalization, but not binding, of PE by LM cells requires extracellular calcium.