The Leishmania ATP-binding cassette protein PGPA is an intracellular metal-thiol transporter ATPase

The Leishmania ATP-binding cassette (ABC) transporter PGPA is involved in m etal resistance (arsenicals and antimony), although the exact mechanism by which PGPA confers resistance to antimony, the first line drug against Leis hmania, is unknown. The results of co-transfection experiments, transport a ssays, and the use of inhibitors suggest that PGPA recognizes metals conjug ated to glutathione or trypanothione, a glutathione-spermidine conjugate pr esent in Leishmania, The HA epitope tag of the influenza hemagglutinin as w ell as the green fluorescent protein were fused at the COOH terminus of PGP A. Immunofluorescence, confocal, and electron microscopy studies of the ful ly functional tagged molecules clearly indicated that PGPA is localized in membranes that are close to the flagellar pocket, the site of endocytosis a nd exocytosis in this parasite. Subcellular fractionation of Leishmania tar entolae PG-PAHA transfectants was performed to further characterize this AB C transporter. The basal PGPA ATPase activity was determined to be 115 nmol /mg/min. Transport experiments using radioactive arsenite-glutathione conju gates clearly showed that PGPA recognizes and actively transports thiol-met al conjugates, Overall, the results are consistent with PGPA being an intra cellular ABC transporter that confers arsenite and antimonite resistance by sequestration of the metal-thiol conjugates.