Electrogenic nature of lysosomal proton pump as revealed with a cyanine dye

Studies were carried out on the electrogenicity of the lysosomal proton pump using dipropyithiadicarbocyanine iodide (diS-C₃-(5)) as a membrane potential probe. Pure lysosome preparations (tritosomes) quenched the fluorescence of diS-C₃-(5). The quenching correlated well with the potassium ion diffusion potential (inside negative) generated by K⁺ with or without valinomycin. The quenching caused by lysosomes was reversed by lipophilic cations, tetraphenylarsonium (TPA) or triphenylmethyiphosphonium (TPMP). Mg-ATP also reversed the quenching, which was inhibited by a protonophore, 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (SF-6847). The properties of the ATP-induced recovery of the quenching were exactly the same as those of ATP-induced acidification, as measured with fluoresceinisothiocyanate-dextran (FD) (Ohkuma, S., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762) and acridine orange (Moriyama, Y., et al. (1982) J. Biochem. 92, 1333-1336), except replacement of the anion by an impermeable one enhanced ATPinduced recovery of quenching, but reduced ATP-induced acidification. Amines which dissipate △pH across the lysosomal membrane also enhanced the Mg-ATPinduced fluorescence recovery. These results suggest that isolated lysosomes exhibit an inside negative membrane potential, especially in low K⁺ medium, mostly due to the K⁺-diffusion potential, and that the Mg-ATP-driven proton pump causes membrane depolarization (in the direction of inside positive). These possibilities were supported by results on the uptake of the radioactive membrane-permeant ions [³H]TPMP and [¹⁴C]SCN. The present results provide evidence for the electrogenic nature of the lysosomal proton pump.