Bafilomycin inhibits proton flow through the H+ channel of vacuolar proton pumps

Abstract

Vacuolar-type proton-translocating ATPases are complex heterooligomers that are characterized by a specific inhibition by bafilomycin A1. These enzymes have a peripheral ATP hydrolytic domain as well as a transmembranous sector. The transmembranous sector has been isolated by glycerol gradient centrifugation, and this subcomplex is composed of polypeptides of 116, 39, and 17 kDa. Both this sector and native holoenzyme were reconstituted into potassium-loaded (150 mM KCl) liposomes prepared from pure lipids. When diluted into potassium-free buffer, a valinomycin-induced membrane potential did not drive proton uptake, as assessed by acridine orange quenching. In contrast, pretreatment of both the reconstituted proton pump and isolated transmembranous sector at pH 4.2 activated a latent proton conductance. Bafilomycin A1 (1 nM) inhibited ATP-energized proton pumping catalyzed by the proton pump, as well as membrane potential-driven proton flow through both the acid-activated proton pump and the isolated proton pore. Thus bafilomycin A1 inhibits vacuolar proton pumps by blocking proton conduction through the proton pore, which we term VB.