Proton Transport in Isolated Vacuoles from Corn Coleoptiles

Abstract

Vacuoles were isolated from corn coleoptile protoplasts and ATP-dependent proton transport was measured by quinacrine fluorescence quenching or by the uptake of [14C]methylamine. Intact vacuoles were judged to be free of a surrounding plasma membrane based on fluorescent staining with fluoroscein-diacetate. Essentially all of the detectable ATP-stimulated methylamine uptake and .alpha.-mannosidase activities present in intact protoplasts were recovered in isolated vacuoles. The activities of marker enzymes for plasma membranes, Golgi, endoplasmic reticulum, and mitochondria were reduced to 5 to 17% in vacuolar preparations. The characteristics of proton pumping by isolated vacuoles were compared to those of light microsomal membranes possibly derived from the tonoplast. ATP-dependent proton pumping by both isolated vacuoles and light microsomal vesicles was stimulated by Cl-, and inhibited by NO3-, carbonyl cyanide-m-chlorophenylhydrazone, N,N''-dicyclohexylcarbodiimide, N-ethylmaleimide, 4,4''-diisothiocyano-2,2''-stilbene disulfonic acid, diethylstilbestrol, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but not by vanadate. Both activities also showed substrate specificity for Mg-ATP. Finally, proton transport activities of vacuolar and microsomal fractions exhibited similar profiles after flotation in linear dextran gradients. The microsomal proton pump previously characterized in corn coleoptiles is derived from the tonoplast.