Evidence for a Cl⁻-Stimulated MgATPase Proton Pump in Oat Root Membranes

Abstract

The possibility that plant membrane-bound MgATPases may act as electrogenic proton pumps has been investigated. Using an oat (Avena sativa L. cv. Victory) root membrane preparation which is partially enriched in tightly sealed vesicles, we have shown that MgATP stimulates the uptake of the membrane-permeable anion [¹⁴C]SCN⁻ by the vesicles; this indicates that an electrical potential (interior positive) is generated across the membrane. Both Cl⁻ ions and the proton ionophore trifluoromethoxy(carbonyl-cyanide)phenylhydrazone inhibit the MgATP-driven [¹⁴C]SCN⁻ uptake, presumably by collapsing the MgATP-generated membrane potential. The uptake of the pH gradient probe [¹⁴C]imidazole into the vesicles is also greatly stimulated by MgATP, indicating the presence of a transmembrane proton gradient (interior acid). MgATP-driven [¹⁴C]imidazole uptake is temperature sensitive, Cl⁻-stimulated, substrate specific for MgATP, sensitive to the MgATPase inhibitors vanadate and N,N′-dicyclohexylcarbodiimide, and completely eliminated by trifluoromethoxy(carboxyl-cyanide)phenylhydrazone. The mitochondrial ATPase inhibitor oligomycin has little effect on the MgATPase activity and on the MgATP-dependent [¹⁴C]SCN⁻ and [¹⁴C]imidazole uptake. These data indicate that a class of oat root membrane-bound MgATPases, stimulated primarily by Cl ions, is capable of using the free energy of ATP-hydrolysis to generate an apparent electrochemical proton gradient in vitro.