Involvement of ATP-dependentPseudomonasExotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Abstract

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal 125 I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after Ͼ 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4-and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocationcompetent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect 125 I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when 125

Acid-triggered Membrane Insertion of Pseudomonas Exotoxin A Involves an Original Mechanism Based on pH-regulated Tryptophan Exposure

“…Internalization and ADP Ribosylation Assays-Mutant internalization by mouse lymphocytes was assayed as described (6). The intracellular pathway of the mutants was studied by immunofluorescence (20).…”

“…First, domain I binds to the ␣ 2 -macroglobulin/low density lipoprotein receptor-related protein (5), enabling internalization via receptor-mediated endocytosis. Domain II will then mediate translocation into the cytosol of the entire toxin (6) or of a carboxyl-terminal fragment generated by furin proteolysis and encompassing domain III and most of domain II (1). Finally, domain III will catalyze the ADP ribosylation of elongation factor 2, thereby inhibiting protein synthesis and killing the cell (1).…”

“…Because cell intoxication by PE requires low endosomal pH (1,6), it is also clear that this membrane insertion process is an essential step of the intoxication procedure. Nevertheless, the molecular bases for acid-triggered PE membrane insertion are not known.…”

Identification of the Ricin Lipase Site and Implication in Cytotoxicity

“…This assay did not reveal any difference between control and mutant ricin (not shown). Ricin intracellular routing was examined by immunofluorescence confocal microscopy using fluorescent transferrin, TGN46, and Lamp-1 as markers of early endosomes (22), the trans-Golgi network (29), late endosomes and lysosomes (30), respectively. Both control and mutant ricins were efficiently endocytosed by A431 cells (Fig.…”

“…The rabbit anti-ricin antibodies (Sigma) were revealed using TRITClabeled swine anti-rabbit IgG; sheep anti-TGN46 (Serotec) and the H4A3 anti-Lamp-1 monoclonal antibody (Iowa Developmental Studies Hybridoma Bank) were detected using fluorescein isothiocyanate-conjugated donkey anti-sheep and goat anti-mouse antibodies, respectively. After mounting, cells were examined under a Leica confocal microscope (22).…”