Abstract
We attached human transferrin to Pseudomonas exotoxin A (PE) to specifically localize this toxin to the endosomal compartment and study its translocation from purified endosomes using a cell-free assay. Transferrin was linked to PE via a disulfide bond. Chemical derivatization inactivated the PE cell-binding domain, and transferrin-PE was found to be endocytosed via the transferrin receptor only. Transferrin was also conjugated to a truncated PE with no receptor-binding domain (PE46). After labeling mouse lymphocytes with radiolabeled transferrin-PE or transferrin-PE46 and endosome isolation, selective translocation of the full-sized toxin portion of the conjugate was observed in a cell-free system. This translocation was strictly dependent upon ATP hydrolysis and was not affected when the acidity of the endosome lumen was neutralized using weak bases, protonophores, or bafilomycin A1. Nevertheless, when present during cell labeling, inhibitors of endosome acidification prevented PE from acquiring translocation competence. Similar inhibition was observed when endocytosis was performed in the presence of brefeldin A, a drug known to interfere with the delivery of endocytic tracers to acidic endosomes. Our data indicate that full-length PE can be transferred to the cytosol directly from endosomes during intoxication by PE conjugates and that, although exposure to acidic pH is a prerequisite for translocation, ATP hydrolysis directly provides the energy required for PE translocation.