Proton Transport in Isolated Vacuoles from Corn Coleoptiles

Abstract

Vacuoles were isolated from corn coleoptile protoplasts and ATPdependent proton transport was measured by quinacrine fluorescence quenching or by the uptake of l'4Clmethylamine. Intact vacuoles were judged to be free of a surrounding plasma membrane based on fluorescent staining with fluoroscein-diacetate. Essentially all of the detectable ATPstimulated methylamine uptake and a-mannosidase activities present in intact protoplasts were recovered in isolated vacuoles. In contrast, 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 Cl1, 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. We conclude that the microsomal proton pump previously charcterized in com coleoptiles Plant Physiol 70: 1738-1742) is derived from the tonoplast.The importance of electrogenic proton pumping ATPases in the transport of solutes across the plasma membrane and tonoplast of plant cells is being increasingly recognized. A cationstimulated Mg2+-ATPase on the plasma membrane is thought to be an electrogenic proton pump (25), and recent attempts to purify and reconstitute plasma membrane proteins have supported this idea (28). The presence of a proton-pumping ATPase on the tonoplast has been more controversial, although recent studies with isolated, intact vacuoles have led to the identification of a tonoplast ATPase in several systems (2-4, 7, 17, 19, 29-31). Investigations of the transport properties of sealed microsomal membranes have indicated that vesicles capable of in vitro ATPdependent proton transport can be isolated from a number of different plant tissues (26 and references therein). The bulk of this activity appears to be due to a novel ATPase which is distinguishable from other known ATPases (plasma membrane, mitochondrial, and chloroplast), but is similar to the recently characterized tonoplast ATPase.We have previously shown that microsomal membranes from corn coleoptiles have ATP-dependent proton pumping activity which is stimulated by Cl-, has a slightly alkaline pH optimum,

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

“…2-3). A lare peak of activity at 1.08 to 1.13 g.cm-3 had characteristics in common with the tonoplast-type ATPase (3,5,9,20,21,26,31) and a smaller peak of activity at 1.14 to 1.18 gcm-3 had characteristics in common with the plasma membrane ATPase (10,15,31). Glucan synthase I, a marker enzyme for Golgi membranes, overlapped both peaks of transport activity.…”

Electrogenic H+-Pumping Pyrophosphatase in Tonoplast Vesicles of Oat Roots

“…Fig. 1 (2,14). (c) A H+-pumping PPase has been demonstrated in low density vesicles from oat roots (6), red beets (19), and corn coleoptiles (5), and this activity is similar to the PPase found in isolated vacuolar membranes of red beets and tulip petals (25,27).…”

Calcium Transport in Tonoplast and Endoplasmic Reticulum Vesicles Isolated from Cultured Carrot Cells

“…If the active Ca2+ transport system is associated with the tonoplast, the Cl-stimulated, N03--inhibited ATPase (a tonoplast associated enzyme [15,31]) should comigrate with Ca2' transport in vesicles separated on a sucrose density gradient. Calcium transport activity peaked at 1.10 g/cm3, as did the Cl--stimulated, NO3-inhibited ATPase (Fig.…”