H⁺-Pumping Driven by the Plasma Membrane ATPase in Membrane Vesicles from Radish: Stimulation by Fusicoccin

Abstract

ABSTRAC1The effect of fusicoccin on Mg:ATP-dependent H -pumping in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings was investigated by measuring the initial rate of decrease in the absorbance of the ApH probe acridine orange. Fusicoccin stimulated Mg:ATPdependent Hf-pumping when the pH of the assay medium was in the range 7.0 to 7.6 while no effect of fusicoccin was detected between pH 6.6 and pH 6.0. Both basal and fusicoccin-stimulated Hf-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccininduced stimulation of Mg:ATP-dependent H -pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated Hf-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of Hf-pumping was saturated by 100 nanomolar fusicoccin. These data indicate that fusicoccin activates the plasma membrane H -ATPase by acting at the membrane level independently of the involvement of other cell components. The percent stimulation by fusicoccin was the same at all ATP concentrations tested (0.5-5.0 millimolar), thus suggesting that with fusicoccin there is an increase in V.. of the plasma membrane H'-ATPase rather than a decrease in its apparent K. for Mg:ATP.All of the physiological responses to FC2 appear to be related to a stimulation of electrogenic H+ extrusion mediated by the plasma membrane H+-ATPase. Stimulation ofthe plasma membrane H+-ATPase has been proposed as a primary step in the action of the toxin on the basis of evidence such as lack of a lag in the stimulating effect of FC on H+ extrusion and on hyperpolarization of AI, insensitivity of FC effects to inhibitors of RNA and protein synthesis, identification of specific FC-binding proteins at the plasma membrane and some stimulation by FC of the ATPase activity measured in microsomal fractions (for a review, see Refs. 17 and 18).In contrast to this view, it has been recently reported that FC induces in root hairs and in coleoptiles an early acidification of the cytoplasm, evident before any hyperpolarization of AI or stimulation of H+ extrusion is detectable (5, 7, 15). Since it has been shown that electrogenic H+ extrusion in vivo (6, 15, 28) well as the plasma membrane H+-ATPase in vitro (10,14,20,29) A more direct insight into the mechanism of action of FC can be provided by the study of its effects in vitro. This approach allows FC effects on membrane bound activities to be studied in the absence of other cell components. Recently, Rasi-Caldogno and Pugliarello (25), showed that FC strongly and reproducibly stimulated the H+-pumping and, to a lesser extent, the hydrolytic activity of the plasma membrane H+-ATPase in membrane vesicles isolated from radish seedlings. In this paper, we present a more detailed characterization of the stimulating effect of FC on the H+-pumping activity ofthe plasma membrane H+-ATPase ofradish. The ...

Fusicoccin Activity and Binding in Arabidopsis thaliana

“…Evidence from both in vivo and in vitro experiments supports the hypothesis that FC2 primarily acts by stimulating the activity of ATPase proton pumps in the plasma membrane of plant cells (7,9,10). The putative receptor site for FC is not, however, the ATPase, but another protein located in the plant plasma membrane (13,14).…”

Comparison of the Effect of Indoleacetic Acid and Fusicoccin on the Breakdown of Phosphatidylinositol in Maize Coleoptiles

“…IAA is believed to act within the cytoplasm by promoting changes in the acid-producing metabolism (5). On the other hand, the postulated mechanism of FC action is the direct activation of the plasma membrane H+-ATPase (20) upon the binding to its receptor localized on the plasma membrane (7) and it is through this mechanism that the toxin exerts all its physiological effects (14). Moreover, a different mode of action of the (4) two substances has also been suggested for their capacity in lowering the cytosolic pH (10).…”

Purification and photoaffinity labeling of fusicoccin receptors from maize

“…(Ballio et al, 1964), interacts with high affinity and specificity to binding proteins localized at the plasma membrane of plant cells (Dohrmann et al, 1977;Ballio et al, 1980;Pesci et al, 1979a). Although evidence obtained in vitro in different systems (Rasi-Caldogno et al, 1986;Aducci et al, 1988;Blum et al, 1988;Marra et al, 1992) prove that the ultimate target of FC action is the H+-ATPase, the molecular mechanism of this activation is not understood.…”