title: Reconstitution of the lysosomal proton pump.
abstract: Lysosomal membrane proteins solubilized with octyl beta-D-glucopyranoside were reconstituted into proteoliposomes using acetone/ether-washed phospholipids from Escherichia coli. Assays of the quenching of acridine orange fluorescence showed that addition of both ATP and valinomycin to K+-loaded proteoliposomes led to the formation of a pH gradient that was acidic inside. ATP-driven acidification took place in the absence of permeant anions and was inhibited by the "protonophore", carbonylcyanide p-trifluoromethoxyphenylhydrazone, indicating that only H+ was transported actively. Proton translocation was readily blocked by N-ethylmaleimide (10 microM gave 50% inhibition of fluorescence quenching) but was unaffected by oligomycin (50 nM), orthovanadate (50 microM), or ouabain (0.5 mM); similarly, only N-ethylmaleimide affected ATP hydrolysis by proteoliposomes (88% inhibition). Other work showed that reconstitution of ATP-driven proton translocation required the presence of glycerol during protein solubilization and that optimal recovery depended on the use of both glycerol and phospholipid at this stage. We conclude that acidification of the lysosome is mediated by an ATPase capable of electrogenic H+ translocation without molecular coupling to other ionic species.
mesh_terms: D001693:Biological Transport, Active / Q000187:drug effects; D007425:Intracellular Membranes / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008563:Membrane Lipids / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism; D011522:Protons; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D008081:Liposomes; D008563:Membrane Lipids; D010743:Phospholipids; D011522:Protons; D014634:Valinomycin; D006180:Proton-Translocating ATPases; D011188:Potassium