Phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don

 Transport of inorganic orthophosphate (Pi) across the tonoplast membrane was studied using intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus. Orthophosphate uptake was strongly stimulated in the presence of Mg-ATP and Mg-pyrophosphate and inhibited by bafilomycin and concanamycin which are potent inhibitors of the vacuolar H⁺-ATPase. These results indicated that the build-up of an electrochemical gradient by the H⁺ pumps was essential for the uptake of Pi. Potassium thiocyanate, which dissipates the membrane potential across the tonoplast, strongly inhibited the Mg-ATP-stimulated uptake of Pi, while only a weak inhibition was observed in the presence of NH₄Cl, which dissipates the pH gradient. These results indicate that, as observed for other anions like malate or chloride, the electrical component is the driving force of Pi uptake, whereas the ΔpH plays only a minor role. Possible competitive inhibitors of Pi, MoO²⁻ ₄, VO³⁻ ₄ and CrO²⁻ ₄ were tested. Among them, CrO²⁻ ₄ strongly inhibited Pi uptake into the vacuoles. Various inhibitors of anion transport were also tested. Only 4,4-diisothiocyanostilbene-2,2′-disulfonic acid strongly inhibited Pi uptake into the vacuoles. The function of the vacuolar Pi transporters for cytoplasmic Pi homeostasis is discussed. Received: 20 September 1999 / Accepted: 28 January 2000     

The function of tonoplast ATPase in intact vacuoles of red beet is governed by direct and indirect ion effects

The pH gradient and the electric potential across the tonoplast in mechanically isolated beetroot vacuoles has been studied by following the uptake of [¹⁴C]methylamine and [¹⁴C]triphenyl-methylphosphoniumchloride. In response to Mg-ATP, the vacuolar interior is acidified by 0.8 units. This strong acidification is accompanied by a slight hyperpolarization of the membrane potential, which is probably caused by a proton diffusion potential. In preparations where only a small acidification (0.4 units) occurred, the membrane potential was depolarized by the addition of Mg-ATP. Different monovalent cations and anions were tested concerning their effect on the pH gradient and ATPase activity in proton-conducting tonoplasts. Chloride stimulation and NO ₃ ^- inhibition were clearly present. The observed decline of the pH gradient upon the addition of Na⁺ salts is probably caused by an Na⁺/H⁺ antiport system.Abbreviations and symbol CCCP carbonylcyanide-m-chlorophenylhydrazone - Mes 2(N-morpholino)ethanesulfonic acid - TPMP⁺ triphenylmethylphosphoniumchloride - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - membrane potential Dedicated to Professor A. Betz on the occasion of his 65th birthday     

Two Types of Channels Involved in the Malate Ion Transport across the Tonoplast of a Crassulacean Acid Metabolism Plant

Ion channels in tonoplast of leaf cells of a Crassulacean acid metabolism plant, Graptopetalum paraguayense, using the patch clamp technique were investigated. Results showed the existence of two types of channels involved in the malate ion transport across the tonoplast. One type corresponded to the slow-activating vacuolar-type (R Hedrich, E Neher [1987] Nature 329: 833-836), probably taking part in the malate efflux from vacuoles. Another showed the membrane potential-dependent channel current of malate flux over a wide range of cytoplasmic free Ca²⁺ concentration (10⁻⁸-10⁻⁵ molar), a property favoring the malate uptake. This type seems to be different from the fast-activating vacuolar-type.     

Evidence for a Malate Transport into Vacuoles Isolated from Catharanthus roseus Cells

Malate uptake was investigated with vacuoles isolated from Catharanthus roseus cells. The uptake process showed saturation kinetics, was inhibited by organic anions, and was very strongly dependent on the pH of the medium. These data support the classical concept of an anion carrier or channel mechanism and suggest that the Hmal^? form was the transported species. Moreover, malate transport was stimulated by the proton gradient across the tonoplast. The H⁺ translocating enzymes ATPase and PPiase are able to favour malate uptake and, in combination, exert a synergistic effect on this transfer.     

Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells

The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K_m of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30°C. Several effectors known to inhibit ATPase (N,N′-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H⁺ electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN⁻ and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH—, OH—, and NH₂— groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions.     

l-Glutamate effects on electrical potentials of synaptic plasma membrane vesicles

The electrogenic nature of the l-glutamate-stimulated Na⁺ flux was examined by measuring the distribution of the lipophilic anion [³⁵S]thiocyanate (SCN^?) into synaptic membrane vesicles that were incubated in a NaCl medium. Concentrations of l-glutamate from 10^?7 to 10^?4 M added to the incubation medium caused an enhanced intravesicular accumulation of SCN^?. Based on the SCN^? distribution in synaptic membrane vesicles it was calculated that 10 μM l-glutamate induced an average change in the membrane potential of + 13 mV. l-Glutamate enhanced both the Na⁺ and K⁺ conductance of these membranes as determined by increases in SCN^? influx. Other neuroexcitatory amino acids and amino acid analogs (d-glutamate, l-aspartate, l-cysteine sulfinate, kainate, ibotenate, quisqualate, $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{aspartate}$, and dl-homocysteate) also increased SCN^? accumulation in synaptic membrane vesicles. These observations are indicative of the activation by l-glutamate and some of its analogs of excitatory amino acid receptor ion channel complexes in synaptic membranes.     

Identification with a photoaffinity reagent of a tonoplast protein involved in vacuolar malate transport of Catharanthus roseus

The effect of N-(4-azido-salicylyl) aspartic acid (AzSA), a photolysable analogue of malate, was tested on the malate transport activity of tonoplast vesicles isolated from Catharanthus roseus cell suspension cultures. AzSA inhibited malate uptake in a competitive manner with a K_ti of 1.7 millimolar. When iodinated, the malate analogue was found to be still photolysable and a competitive inhibitor of malate uptake. Photolysis of ¹²⁵I-labelled AzSA in the presence of purified tonoplast vesicles led to label incorporation into several polypeptides after analysis by gel electrophoresis. Only one polypeptide, with an apparent molecular mass of 37 kDa, was totally protected by the inclusion of 50 millimolar malate, the original substrate, in the photolysis medium. The labelled polypeptide is therefore apparently a specific malate-binding protein. Diethylpyrocarbonate (DEPC), a very potent inhibitor of malate transport acting at the active site of the transporter, also protected the 37 kDa polypeptide from labelling. Citrate and, to a lesser extent, quinate afforded protection from labelling whilst other organic acids or aspartic acid (100 millimolar) did not. These photoprotection results are in good agreement with the data concerning the specificity of malate transport across the tonoplast. Polyclonal antibodies against the 37 kDa polypeptide strongly inhibited malate uptake both in tonoplast vesicles and in isolated vacuoles. These results suggest the involvement of the 37 kDa polypeptide in vacuolar malate transport.     

Proton transport in isolated vacuoles from corn coleoptiles

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 α-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 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. We conclude that the microsomal proton pump previously characterized in corn coleoptiles (Mettler et al. 1982 Plant Physiol 70: 1738-1742) is derived from the tonoplast.     

Vacuolar malate uptake is mediated by an anion-selective inward rectifier

Electrophysiological studies using the patch‐clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current–voltage relations of the tonoplast were dominated by a strongly inward‐rectifying anion‐selective channel that was active at cytoplasmic‐side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage‐dependent gating of a 3‐pS malate‐selective ion channel, which showed a half‐maximal open probability at ?43 mV. Also, the time‐averaged unitary currents following a step to a negative voltage corresponded to the time‐dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion‐selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent K_m of 2.5 mm malate^2? activity), a selectivity sequence of fumarate^2? > malate^2? > Cl^? > maleate^2– ≈ citrate^3–, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 µm niflumate reversibly blocked the activity of the 3‐pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 µm^?2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3‐pS, inward‐rectifying, anion‐selective channel represents the principal pathway for malate influx into the vacuole.     

Sucrose uptake into vacuoles of sugarcane suspension cells

Uptake of sucrose into vacuoles of suspension cells of Saccharum sp. (sugarcane) was investigated using a vacuole-isolation method based on osmotic- and pH-dependent lysis of protoplasts. Vacuoles took up sucrose at high rates without the influence of tonoplast energization on sucrose transport. Neither addition of ATP or pyrophosphate nor dissipation of the membrane potential or the pH gradient by ionophores changed uptake rates appreciably. Generation of an ATP-dependent pH gradient across the tonoplast was measured in vacuoles and tonoplast vesicles by fluorescence quenching of quinacrine. No H⁺ efflux could be measured by addition of sucrose to energized vacuoles or vesicles so that there was no evidence for a sucrose/H⁺ antiport system. Uptake rates of glucose and other sugars were similar to those of sucrose indicating a relatively non-specific sugar uptake into the vacuoles. Sucrose uptake was concentration-dependent, but no clear saturation kinetics were found. Strict dependence on medium pH and inhibition of sucrose transport by p-chloromercuriphenylsulfonic acid (PCMBS) indicate that sucrose uptake into sugarcane vacuoles is a passive, carrier-mediated process.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - Mes 2-(N-morpholino)ethanesulfonic acid - Mops 3-(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuriphenylsulfonic acid - PPi pyrophosphate This research was supported by the Deutsche Forschungsgemeinschaft. The technical assistance of H. Schroer is gratefully acknowledged.     

Localization of the proton pump of corn coleoptile microsomal membranes by density gradient centrifugation

Previous studies characterizing an ATP-dependent proton pump in microsomal membrane vesicles of corn coleoptiles led to the conclusion that the proton pump was neither mitochondrial nor plasma membrane in origin (Mettler, Mandala, Taiz 1982 Plant Physiol 70: 1738-1742). To facilitate positive identification of the vesicles, corn coleoptile microsomal membranes were fractionated on linear sucrose and dextran gradients, with ATP-dependent [¹⁴C]methylamine uptake as a probe for proton pumping. On sucrose gradients, proton pumping activity exhibited a density of 1.11 grams/cubic centimeter and was coincident with the endoplasmic reticulum (ER). In the presence of high magnesium, the ER shifted to a heavier density, while proton pumping activity showed no density shift. On linear dextran gradients, proton pumping activity peaked at a lighter density than the ER. The proton pump appears to be electrogenic since both [¹⁴C]SCN⁻ uptake and ³⁶Cl⁻ uptake activities coincided with [¹⁴C] methylamine uptake on dextran gradients. On the basis of density and transport properties, we conclude that the proton pumping vesicles are probably derived from the tonoplast. Nigericin-stimulated ATPase activity showed a broad distribution which did not coincide with any one membrane marker.     

Citrate transport into barley mesophyll vacuoles — comparison with malate-uptake activity

Citrate uptake into barley (Hordeum vulgare L.) mesophyll vacuoles was found to be saturable with a K _m of about 200 M. Uptake appears to occur via the citrate^3- form, as indicated by concentration-dependent uptake studies at different pHs. Free citrate and not the Mg-citrate complex was taken up by the vacuoles, even though slow transport of the Mg complex could not be excluded. Citrate transport into vacuoles was competitively inhibited by malate (K _i=0.68 mM). Various organic acids and protein-modifying agents affected the uptake of malate and citrate to a similar extent. These results indicate that both organic acids cross the tonoplast by means of the same carrier. Accumulation of citrate was ATP-dependent and could be inhibited by ionophores. Bovine serum albumin strongly stimulated citrate uptake, but other proteins tested did not show a similar stimulatory effect.Abbreviation BSA bovine serum albumin We wish to thank Esther Vogt for her help with the experiments and Professor N. Amrhein (ETH, Zürich, Switzerland) and Dr. Michael Kertesz (ETH, Zürich) for helpful discussions. This work was supported by the Swiss National Foundation grant No. 31-25196.88.     

The mechanism of oleanolic acid monoglycosides transport into vacuoles isolated from Calendula officinalis leaf protoplasts

The uptake of [3-³H]-oleanolic acid 3-O-monoglucoside and 3-O-monoglucuronide into vacuoles isolated from Calendula officinalis leaf protoplasts was investigated under various conditions. The transport of monoglucoside was stimulated by ATP and pyrophosphate, and sensitive to protein-modifying agent as well as dissipaters of the membrane potential. On the contrary, the transport of monoglucuronide was not dependent on tonoplast energization and was affected only by a protein-modifying factor. Moreover, the kinetics of tonoplast transport of both monoglycosides was characterized as concentration-dependent with saturation phase. The obtained results indicate that oleanolic acid monoglucoside is transported to isolated vacuoles by an active, carrier-mediated and energy-dependent mechanism, whereas the transport of monoglucuronide is a passive, carrier-mediated process.     

Vacuolar ethylene formation does not depend on membrane potential

Enzymatic synthesis of ethylene in the vacuole is assumed to require membrane integrity. The possibility that this reflects dependence on the vacuolar membrane potential was investigated. Vacuoles were released from protoplasts isolated from leaves of Vicia faba L. cv. Cyprus. The dependence of the ethylene-forming activity on tonoplast integrity was re-examined by immobilization of the vacuoles in a cross-linked polymeric matrix and subsequent permeabilization of the tonoplast with toluene, a pore-forming reagent. The relationship between the vacuolar ethylene formation and the membrane potential of free vacuoles was investigated by following the uptake of thiocyanate using permeabilized, depolarized and hyperpolarized vacuoles. Toluene and the proton conductor carbonyl cyanide m -chlorophenylhydrazone (CCCP) caused loss of ethylene-forming activity and depolarized the vacuolar membrane potential. However, depolarization of the membrane potential with choline chloride and hyperpolarization by ATP did not affect ethylene biosynthesis. These conflicting results lead to the conclusion that vacuolar ethylene biosynthesis is not dependent on the vacuolar membrane potential. The possibility that the inhibition of ethylene biosynthesis by toluene and CCCP may result from direct hydrophobic interactions between these compounds and hydrophobic components of the ethylene-forming enzyme is discussed.     

Characteristics,partial purification and reconstitution of the vacuolar malate transporter of the CAM plant Kalanchoë daigremontiana Hamet et Perrier de la Bâthie

When native tonoplast vesicles of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie were energized by an artificial K⁺ gradient establishing only an inside-positive electrical membrane potential (), it was shown that was sufficient as the sole driving force and that a proton gradient (pH) is not required for malate uptake. Following [¹⁴C]malate uptake, K _m-malate of the malate transporter was estimated as 2.7–3.0 mM, a value that would allow malate synthesis via phosphoenolpyruvate carboxylase and malate accumulation in vivo in view of the feed-back inhibition of cytosolic phosphoenolpyruvate carboxylase by malate. The maximum reaction velocity (V _max) was found to be between 30 and 85 nmol malate·min^–1·mg _protein ^–1 , a value that would explain nocturnal malate accumulation in K. daigremontiana even if the transporter were operating below substrate saturation. Citrate (50 mM at pH 7) inhibited transport by 78%. The malate-transport protein of the tonoplast of K. daigremontiana may be a carboxylate uniporter with strong affinities for malate and citrate. From total tonoplast proteins solubilized from native tonoplast vesicles the malate transporter was functionally reconstituted into phospholipid liposomes. The malate transporter was purified and separated from the tonoplast H⁺-ATPase by hydroxyapatite chromatography, but not from the tonoplast H⁺-pyrophosphatase. The partially purified malate-transport protein was functionally reconstituted into phospholipid liposomes. In these final proteoliposomes, 0.6% of the protein of the initial tonoplast-vesicle preparation used for solubilization of membrane proteins was recovered. Using the specific rates of malate transport as a reference, i.e. rates of transport related to protein in the preparations, enrichment of the malate transporter in the final proteoliposomes obtained with the reconstitution of the hydroxyapatite eluate was 44-fold compared to the initial native tonoplast vesicles and 2000-fold compared to the liposomes reconstituted from solubilized tonoplast proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the peptides from the final proteoliposomes, which were functional in malate transport, showed only a few polypeptide bands among which the malate transporter must be found.Abbreviations and Symbols CAM Crassulacean acid metabolism - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - Triton X-100 polyoxyethylene(9,10)p-t-octylphenol - pH proton gradient at the tonoplast - membrane potential at the tonoplast This work was supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie and is now funded in SFB 199 (Teilprojekt B2) of the Deutsche Forschungsgemeinschaft. We thank Dr. Elke Fischer-Schliebs for valuable discussions and Dr. E. Martinoia for making us acquainted with his experimental approaches in his laboratory in Zürich, Switzerland, and for much valuable exchange. Dr. D.P.S. Verma, Ohio, USA, kindly provided Nod-26 antibodies, and the tonoplast H⁺-pyrophosphatase antibodies were a generous gift of Dr. M. Maeshima, Sapporo, Japan.     

Evidence for an electrogenic ATPase in microsomal vesicles from pea internodes

The transmembrane electropotential of microsomal vesicles from pea internode segments, monitored by equilibrium distribution of the permeant anion SCN^?, is strongly hyperpolarized when ATP is present in the incubation medium.The stimulation of SCN^? uptake by ATP is rather specific with respect to the other nucleoside di- and triphosphates tested: ADP, GTP, CTP and UTP. ATP-stimulated SCN^? uptake is strongly inhibited by ATPase inhibitors such as $\text{p-}\text{chloromercuribenzenesulphonate}$ and $\text{N,N}{}^{\mathrm{\prime }}\text{-}\text{dicyclohexylcarbodiimide}$ and by 2.5% toluene/ethanol (1 : 4, v/v), the latter being a treatment which makes the vesicles permeable. On the contrary, oligomycin is almost ineffective in influencing ATP-induced SCN^? uptake. The proton conductor carbonyl cyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$ strongly inhibits ATP-stimulated SCN^? uptake. The effect of ATP on SCN^? uptake depends on the pH of the medium, the maximum being reached at about pH 7.0.These data support the view that microsomal fractions from pea internodes contain membrane vesicles endowed with a membrane-bound ATPase coupling ATP hydrolysis to electrogenic transport of ions, probably H⁺.     

Evidence for an electrogenic adenosine-triphosphatase in Hevea tonoplast vesicles

The function of the Mg-dependent ATPase of Hevea tonoplast in active proton transport was investigated by using a purified tonoplast fraction containing tightly sealed vesicles. In the used experimental conditions, the uptake of [¹⁴C]triphenylmethyl-phosphonium ion ([¹⁴C]TPMP⁺) and [³H]tetraphenyl-phosphonium ion ([³H]TPP⁺) by the vesicles indicated a transmembrane potential difference, negative inside. In parallel, the uptake of [¹⁴C]methylamine into the vesicles monitored a transmembrane pH gradient, interior acid. The addition of 5 mM Mg-ATP markedly depolarized the membrane and increased the magnitude of trnasmembrane pH gradient. These ATP-driven events were substrate specific for Mg-ATP. They were strongly inhibited by ATPase inhibitors such as N, N′-dicyclohexylcar-bodiimide. They were completely eliminated by proton conductors such as carbonylcyanide-p-trifluoromethoxy-phenylhydrazone and 5-chloro-3-tert-butyl-2′-chloro-4-nitro-salicylanilide. They depended on the pH of the medium, the maximum being reached at about pH 7.0. These data provide in vitro evidence that the Mg-ATPase localized at tonoplast level is an electrogenic pump. They are consistent with the hypothesis that an electrogenic H⁺ pump is catalyzed by the tonoplast ATPase of higher plants.