Some Properties of a Functional Reconstituted Plasmalemma H-ATPase Activated by Fusicoccin

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H⁺-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V_max of the enzyme. Fusicoccin stimulation of maize H⁺-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).     

Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase

14-3-3 proteins interact with a novel phosphothreonine motif (Y(946)pTV) at the extreme C-terminal end of the plant plasma membrane H(+)-ATPase molecule. Phosphorylation-independent binding of 14-3-3 protein to the YTV motif can be induced by the fungal phytotoxin fusicoccin. The molecular basis for the phosphorylation-independent interaction between 14-3-3 and H(+)-ATPase in the presence of fusicoccin has been investigated in more detail. Fusicoccin binds to a heteromeric receptor that involves both 14-3-3 protein and H(+)-ATPase. Binding of fusicoccin is dependent upon the YTV motif in the H(+)-ATPase and, in addition, requires residues further upstream of this motif. Apparently, 14-3-3 proteins interact with the unusual epitope in H(+)-ATPase via its conserved amphipathic groove. This implies that very diverse epitopes bind to a common structure in the 14-3-3 protein.     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

Plant 14-3-3 proteins assist ion channels and pumps

Turgor pressure is a cellular parameter, important for a range of physiological processes in plants, like cell elongation, gas exchange and gravitropic/phototropic bending. Regulation of turgor pressure involves ion and water transport at the expense of metabolic energy (ATP). The primary pump in the plasma membrane (the H(+)-ATPase) is a key player in turgor regulation since it provides the driving force for ion uptake, followed by water influx through osmosis. Using the phytotoxin fusicoccin (a well-known activator of the ATPase) as a tool, 14-3-3 proteins were identified as regulators of the H(+)-ATPase. Since fusicoccin has a dramatic effect on K(+) accumulation and cellular respiration as well, we studied whether 14-3-3 proteins play a role in the regulation of the mitochondrial F(0)F(1)-ATP synthase and ion channels in the vacuolar and plasma membranes. Besides the plasma membrane H(+)-ATPase, we have identified thus far at least four other transport proteins that are regulated by 14-3-3 proteins. The mechanism of regulation will be described and the possibility that 14-3-3 proteins act as coordinators of ion transporters with varied but interdependent functions will be discussed.     

A plant plasma membrane H+-ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the absence of fusicoccin

The Nicotiana plumbaginifolia plasma membrane H(+)-ATPase isoform PMA2, equipped with a His(6) tag, was expressed in Saccharomyces cerevisiae and purified. Unexpectedly, a fraction of the purified tagged PMA2 associated with the two yeast 14-3-3 regulatory proteins, BMH1 and BMH2. This complex was formed in vivo without treatment with fusicoccin, a fungal toxin known to stabilize the equivalent complex in plants. When gel filtration chromatography was used to separate the free ATPase from the 14-3-3.H(+)-ATPase complex, the complexed ATPase was twice as active as the free form. Trypsin treatment of the complex released a smaller complex, composed of a 14-3-3 dimer and a fragment from the PMA2 C-terminal region. The latter was identified by Edman degradation and mass spectrometry as the PMA2 C-terminal 57 residues, whose penultimate residue (Thr-955) was phosphorylated. In vitro dephosphorylation of this C-terminal fragment prevented binding of 14-3-3 proteins, even in the presence of fusicoccin. Mutation of Thr-955 to alanine, aspartate, or a stop codon prevented PMA2 from complementing the yeast H(+)-ATPase. These mutations were also introduced in an activated PMA2 mutant (Gln-14 --> Asp) characterized by a higher H(+) pumping activity. Each mutation directly modifying Thr-955 prevented 14-3-3 binding, decreased ATPase specific activity, and reduced yeast growth. We conclude that the phosphorylation of Thr-955 is required for 14-3-3 binding and that formation of the complex activates the enzyme.     

The phytotoxin fusicoccin promotes platelet aggregation via 14-3-3-glycoprotein Ib-IX-V interaction

The fungal toxin fusicoccin induces plant wilting by affecting ion transport across the plasma membrane of plant cell. The activity of this toxin is so far unknown in humans. In the present study we show that fusicoccin is able to affect the platelet aggregation process. The toxin associates with platelet intracellular binding sites and induces aggregation in platelet-rich plasma in a dose-dependent manner. We identified the adhesion receptor glycoprotein Ib-IX-V as fusicoccin target. The toxin promotes the binding of the regulatory 14-3-3 proteins to glycoprotein Ibα and hampers that to glycoprotein Ibβ subunit. As a result, platelet adhesion to von Willebrand factor is stimulated, leading to platelet spreading and integrin αIIbβ3 activation. We anticipate the present study to be a starting point for future therapeutic use of fusicoccin in genetic bleeding diseases characterized by qualitative or quantitative abnormalities of the platelet membrane-adhesion receptors. Furthermore, the present study also sets the stage for future work to determine the potential pharmacological application of fusicoccin as a drug directed to other 14-3-3-target complexes.     

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

Fusicoccin stimulates the H+-ATPase of plasmalemma in isolated membrane vesicles from radish

The effect of fusicoccin on the plasmalemma H+-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H+-transport is mediated only by the plasmalemma H+-ATPase. Fusicoccin stimulated the plasmalemma H+-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of delta psi and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H+-ATPase was evident at pH 6.6.     

H-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin

The 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 ΔpH probe acridine orange. Fusicoccin stimulated Mg:ATP-dependent H⁺-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 H⁺-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccin-induced stimulation of Mg:ATP-dependent H⁺-pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated H⁺-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of H⁺-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_max of the plasma membrane H⁺-ATPase rather than a decrease in its apparent K_m for Mg:ATP.     

The Effects of Fusicoccin on Anion Fluxes in Isolated Guard Cells of Commelina communis L.

Clint, G. M. 1987. The effects of fusicoccin on anion fluxesin isolated guard cells of Commelina communis L.—J. exp.BoL 38: 863–876. The effects of 3?10^–2 mol m^–3 fusicoccin (FC) onbromide fluxes and contents in isolated guard cells of Commelinacommunis L. have been studied using K⁸²Br at pH 3?9 and pH 6?7.At pH 3?9 FC caused a reduction in both the influx and the effluxof ⁸²Br, whereas at pH 6?7 FC had no effect on the influx butcaused a transient increase in the efflux of ⁸²Br. There wasno obvious change in bromide content with FC treatment at eitherpH. The behaviour of the anion fluxes in response to FC suggeststhat FC does not act solely via a hyperpolarization at the plasmalemma.A redistribution of bromide between the intracellular compartmentssuggests that anion flux from the cytoplasm to the vacuole maybe stimulated by FC at pH 3?9. The failure of guard cells toincrease their anion content on treatment with FC despite anincrease in stomatal aperture and in cation content suggeststhat in FC-induced stomatal opening excess cation is balancedby organic acid synthesis within the guard cell. Key words: Fusicoccin, guard cells, ion fluxes, Commelina communis     

Topology and target interaction of the fusicoccin-binding 14-3-3 homologs of Commelina communis

Upon binding to a high-affinity plasma membrane (PM) protein (a member of the 14-3-3 family of regulatory proteins), the fungal phytotoxin fusicoccin (FC) activates the H⁺- ATPase by hindering the inhibitory interaction of the enzyme's C-terminus with its catalytic site. Protease protection experiments carried out with sealed PM vesicles of different orientation proved that the FC-binding site faces the cytoplasmic surface of the membrane. The in vivo induced activation of the H⁺-ATPase by FC was retained during solubilization of PM proteins. Two-dimensional gel systems combining a native separation of membrane protein complexes with a denaturing dimension as well as high-performance anion-exchange chromatography proved the existence of a labile ATPase:14-3-3 complex in plasma membranes. Stabilization of this complex could be achieved by FC treatment in vivo or in vitro . Mild proteolytic removal of the C-terminal auto-inhibitory domain of the H⁺ATPase liberated apparent hydrophobic 14-3-3 isoforms from the membrane in soluble form. During size exclusion chromatography of the proteolytically released proteins, co-elution of 14-3-3 dimers, protein-bound FC and the C-terminus of the H⁺ATPase was observed. Moreover, the data suggest that 14-3-3 dimers themselves are not able to bind FC. Based on these results, it is proposed that the 'FC receptor' is represented by a labile complex between a 14-3-3 dimer and the H⁺-ATPase whose formation is part of a mechanism regulating ATPase-activity under physiological conditions. In our working model, binding of FC stabilizes this labile complex, thus leading to a strong and persistent activation of the H⁺-ATPase in vivo . The possibility that the C-terminus of the enzyme represents the binding domain for 14-3-3 homologs is discussed.     

Fusicoccanes: diterpenes with surprising biological functions

Fusicoccin is the best-studied member of a class of diterpenes sharing a 5-8-5 ring structure, called fusicoccanes. Fusicoccin was and still is a 'tool in plant physiology', targeting the main engine of plasma membrane transport, the P-type H(+)-ATPase, assisted by members of the 14-3-3 family. The key position of 14-3-3 proteins in cell biology, combined with a broader specificity of other fusicoccanes as shown by crystallography studies, make fusicoccanes a versatile tool in plant and animal biology. In this review, we examine recent evidence that fusicoccanes act on animal cells, describe the discovery of the fungal biosynthetic pathway and emphasize that lower (liverworts) and higher plants produce fusicoccanes with intriguing biological activities.     

Structural view of a fungal toxin acting on a 14-3-3 regulatory complex

The fungal phytotoxin fusicoccin stabilizes the interaction between the C-terminus of the plant plasma membrane H(+)-ATPase and 14-3-3 proteins, thus leading to permanent activation of the proton pump. This results in an irreversible opening of the stomatal pore, followed by wilting of plants. Here, we report the crystal structure of the ternary complex between a plant 14-3-3 protein, fusicoccin and a phosphopeptide derived from the C-terminus of the H(+)-ATPase. Comparison with the corresponding binary 14-3-3 complexes indicates no major conformational change induced by fusicoccin. The compound rather fills a cavity in the protein-phosphopeptide interaction surface. Isothermal titration calorimetry indicates that the toxin alone binds only weakly to 14-3-3 and that peptide and toxin mutually increase each others' binding affinity approximately 90-fold. These results are important for herbicide development but might have general implications for drug development, since rather than inhibiting protein-protein interactions, which is difficult to accomplish, it might be easier to reverse the strategy and stabilize protein-protein complexes. As the fusicoccin interaction shows, only low-affinity interactions would be required for this strategy.     

Phytochrome-mediated closure of Albizzia lophantha leaflets as affected by sorbitol, mannitol and fusicoccin

The effect of sorbitol and mannitol and their interaction with the effect of fusiccocin has been investigated in phytochrome-mediated nyctinastic closure of Albizzia lophantha leaflets. Treatments were commenced 2 h into the photoperiod. Pairs of leaflets were excised, floated on test solutions, and transferred to darkness immediately after red or far red irradiation. Leaflet angles were measured in darkness for 3 h. Fusicoccin (1 to 100 μ M ) inhibited nyctinastic closure and enhanced reopening in the presence of P_ft (far-red-absorbing form of phytochrome). Fusicoccin effects on far red treated leaflets were not constant and whichever fusicoccin concentration was applied, the effects were small. These results confirm the role of the plasma membrane proton pump in extensor cells during opening. Sorbitol and mannitol (25–100 m M ) increased nyctinastic closure for about 2 h and then enhanced reopening in the presence of P_fr. When sugar alcohols were applied together with fusicoccin, sorbitol and mannitol enhanced the fusicoccin inhibition of closure and a marked synergistic effect was observed. These results indicate that both fusiccocin and the reduction of external osmotic potential caused by sorbitol and mannitol supply may stimulate the activity of the extensor H⁺ pump promoting the reopening.     

Fusicoccin, an inhibitor of brassinosteroid-induced ethylene production

Fusicoccin was evaluated for its effects on brassinosteroid (BR), indole-3-acetic acid (IAA) and BR + IAA-induced ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC-synthase production by etiolated mung bean ( Vigna radiata L. Rwilez cv. Berken) hypocotyl segments. Fusicoccin inhibition of ethylene and ACC production induced by 2 μ M BR started at concentrations as low as 0.05 μ M . Maximum inhibition occurred at a 1 μ M concentration with no further inhibition at higher concentrations tested. Fusicoccin (1 μ M ) was effective in the inhibition of BR-induced ethylene, ACC and ACC-synthase production at low and high concentrations of BR.
Fusicoccin at concentrations as high as 2 μ M had no effect on ethylene and ACC production promoted by low concentrations of IAA (1 to 10 μ M ). When higher concentrations (100–1000 μ M ) of IAA were used, fusicoccin (1 μ M ) had an inhibitory effect on ethylene and ACC production. Interestingly, fusicoccin (1 μ M ) had little or no effect on ACC-synthase promoted by high concentrations of IAA (1000 μ M ).
When BR and IAA were used in combination, fusicoccin inhibited ethylene and ACC production at concentrations as low as 0.05 μ M with maximum inhibition occurring at 0.5 μ M . At a 1 μ M concentration, fusicoccin was effective in inhibiting the synergistic stimulation of ACC-synthase promoted by BR and IAA.     

Effect of Hormones on Sucrose Uptake and on ATPase Activity of Citrus sinensis L. Osbeck Leaves

Carbohydrate accumulation in young, fully expanded leaves ofCitrus sinensis L. Osbeck is affected by the presence of thefruitlet on the shoot. Previous work gave evidence that gibberellinsmay be involved in this 'fruit effect'. In the present workwe have studied the effect of gibberellic acid (GA₃) on ¹⁴C-sucroseuptake by leaf discs and whether its action could be due toa modulation of the plasma membrane ATPase, which maintainsthe H⁺ gradient that drives H⁺/sucrose co-transport. The effect of GA₃ on ¹⁴C-sucrose uptake depended on the osmolarityof the assay medium. At 300 mOsm a reduction in the uptake ratewas observed. The inhibitory effect of the hormone disappearedafter preincubating the leaf discs with para-chloromercuri-phenylsulphonicacid (PCMPS), a sulphydril binding inhibitor. ATPase activityof isolated plasma membrane vesicles was inhibited by IAA treatments,while GA₃ or ABA did not affect this enzyme, even after a 3h preincubation period. However, in the absence of a surfactantin the assay medium, GA₃, together with turgor pressure, modulatedplasma membrane ATPase activity, possibly through modificationsof membrane permeability. The hormone effect on ¹⁴ C-sucroseuptake may involve action on the sucrose carrier.Copyright 1994,1999 Academic Press Abscisic acid, Citrus sinensis, gibberellic acid, indoleacetic acid, orange, osmotic pressure, plasma membrane ATPase, ¹⁴C-sucrose uptake     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K_d of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K_d 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H⁺-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H⁺-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H⁺-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.     

Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants

Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.     

Characterization, Functional Reconstitution and Activation by Fusicoccin of a Ca2+-ATPase from Corydalis sempervirens Pers. Cell Suspension Cultures

Cell suspension cultures of Corydalis sempervirens have provenideal for the study of fusicoccin action [Schulz et al. (1990)Planta 183: 83] and express the fusicoccin-binding protein aswell as a plasma membrane H⁺-ATPase which is activated by thefungal toxin. Microsomal vesicles prepared from these cellsaccumulate Ca²⁺ in the presence of Mg-ATP. The protonophorecar-bonylcyanide m-chlorophenylhydrazone did not inhibit theMg-ATP dependent Ca²⁺-transport into the vesicles. This processis thus due to the activity of at least one primary active,ATP-driven, Ca²⁺-pump. The enzyme was characterized in detail.It has a pH optimum of 7.2, an apparent K_m of 0.3 mu (ATP),12pm (Ca²⁺), accepts ATP>ITP GTP>CTP UTP, and is strongly(Kⁱ, app 0.75 µmM) inhibited by erythrosine B but lessso (Kⁱ, app 95 µM) by or-thovanadate. These characteristicsare typical for the plasma membrane Ca²⁺-ATPase characterizedfrom differentiated tissues [Graf and Weiler (1990) Physiol.Plant. 75: 634]. Fusicoccin activates the erythrosine-sensitiveCa²⁺-pump by lowering its K_m for ATP, when added to living cellsprior to tissue homogenization. Thus, fusicoccin appears toactivate at least two ion-translocating ATPases in one and thesame tissue, suggesting that the toxin's mechanism of actionis complex and not restricted to activation of the H⁺-ATPase.FC has no effect when administered to microsomes. The microsomalenzyme was solubilized and reconstituted into asolec-tin liposomesin functional form. The reconstituted, erythrosine sensitiveCa²⁺-ATPase was insensitive to fusicoccin. Thus, componentsessential for toxin action are either lost or inactivated duringsubcellular fractionation. It is likely that FC action requiressoluble components. (Received April 22, 1991; Accepted July 24, 1991)