The lysolipid sphingosine modulates pyrophosphatase activity in tonoplast vesicles and isolated vacuoles from a heterotrophic cell suspension culture of Chenopodium rubrum

The proton pumping activity of the tonoplast (vacuolar membrane) H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) has been studied on a tonoplast-enriched microsomal fraction and on intact vacuoles isolated from a heterotrophic cell suspension culture of Chenopodium rubrum L. in the presence of the lysosphingolipids D-sphingosine, psychosine (galactosylsphingosine) and lysosulfatide (sulfogalactosyl-sphingosine). Sphingosine strongly stimulates (K_a= 0.16 μ M ) the PPase activity, assayed both as ΔpH formation across the tonoplast vesicle membrane, and as reversible clamp current measured by the whole-vacuolar mode of the patch-clamp technique. Psychosine showed a minor, and lysosulfatide no stimulatory effect. No effect upon the ATPase activity has been observed. No sphingosine-induced change could be observed in the affinity of the PPase for its substrate (apparent K_m= 10 μ M MgPPi). We tentatively conclude that sphingosine, which is known as a potent inhibitor of the protein kinase C in animal cells, may be a regulator of the plant vacuolar PPase.     

Does a proton-pumping ATPase exist in the tonoplast?

In order to account for the accumulation of metabolites in plant vacuoles, the existence of a proton-pumping ATPase has been widely suggested in the literature. The demonstration of such a tonoplast-bound ATPase was merely based on the characterization of a nitrate-sensitive microsomal fraction. In some examples, this ATPase activity has been evidenced on vacuole preparations obtained under conditions which were criticized by Boller. The application of the reverse phase high-performance liquid chromatography method (RP-HPLC) to the simultaneous separation of adenine nucleotides, in the presence of tonoplast vesicles isolated from Catharanthus roseus, showed results not necessarily correlated with the ATPase hypothesis. Moreover, in light of the H+-quenching of quinacrine fluorescence observed during ATP hydrolysis by vacuoles or tonoplast vesicles, the existence of a proton-pumping ATPase may be questioned.     

Na+/H+-transporter, H+-pumps and an aquaporin in light and heavy tonoplast membranes from organic acid and NaCl accumulating vacuoles of the annual facultative CAM plant and halophyte Mesembryanthemum crystallinum L.

Crassulacean acid metabolism (CAM) was induced in Mesembryanthemum crystallinum L. by either NaCl- or high light (HL)- stress. This generated in mesophyll cells predominantly of NaCl-stressed plants two different types of vacuoles: the generic acidic vacuoles for malic acid accumulation and additionally less acidic (“neutral”) vacuoles for NaCl sequestration. To examine differences in the tonoplast properties of the two types of vacuoles, we separated microsomal membranes of HL- and NaCl-stressed M. crystallinum plants by centrifugation in sucrose density gradients. Positive immunoreactions of a set of antibodies directed against tonoplast specific proteins and tonoplast specific ATP- and PP_i-hydrolytic activity were used as markers for vacuolar membranes. With these criteria tonoplast membranes were detected in both HL- and NaCl-stressed plants in association with the characteristic low sucrose density but also at an unusual high sucrose density. In HL-stressed plants most of the ATP- and PP_i-hydrolytic activity and cross reactivity with antibodies including that directed against the Na⁺/H⁺-antiporter from Arabidopsis thaliana was detected with light sucrose density. This relationship was inverted in NaCl-stressed plants; they exhibited most pump activity and immunoreactivity in the heavy fraction. The relative abundance of the heavy membrane fraction reflects the relative occurrence of “neutral” vacuoles in either HL- or NaCl-stressed plants. This suggests that tonoplasts of the “neutral” vacuoles sediment at high sucrose densities. This is consistent with the view that this type of vacuoles serves for Na⁺ sequestration and is accordingly equipped with a high capacity of proton pumping and Na⁺ uptake via the Na⁺/H⁺-antiporter.     

Immunological Evidence for the Existence of a Carrier Protein for Sucrose Transport in Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Root Storage Tissue

Monoclonal antibodies were raised in mice against a highly purified tonoplast fraction from isolated red beet (Beta vulgaris L. ssp. conditiva) root vacuoles. Positive hybridoma clones and sub-clones were identified by prescreening using an enzyme-linked immunosorbent assay (ELISA) and by postscreening using a functional assay. This functional assay consisted of testing the impact of hybridoma supernatants and antibody-containing ascites fluids on basal and ATP-stimulated sugar uptake in vacuoles, isolated from protoplasts, as well as in tonoplast vesicles, prepared from tissue homogenates of red beet roots. Antibodies from four clones were particularly positive in ELISAs and they inhibited sucrose uptake significantly. These antibodies were specific inhibitors of sucrose transport, but they exhibited relatively low membrane and species specificity since uptake into red beet root protoplasts and sugarcane tonoplast vesicles was inhibited as well. Fast protein liquid chromatography assisted size exclusion chromatography on Superose 6 columns yielded two major peaks in the 55 to 65-kD regions and in the 110- to 130-kD regions of solubilized proteins from red beet root tonoplasts, which reacted positively in immunoglobulin-M(IgM)-specific ELISAs with anti-sugarcane tonoplast monoclonal IgM antibodies. Only reconstituted proteoliposomes containing polypeptides from the 55- to 65-kD band took up [14C]-sucrose with linear rates for 2 min, suggesting that this fraction contains the tonoplast sucrose carrier.     

Proton-transport activity,sidedness, and morphometry of tonoplast and plasma-membrane vesicles purified by free-flow electrophoresis from roots of Lepidium sativum L. and hypocotyls of Cucurbita pepo L.

Large-scale preparations of highly purified tonoplast and plasma-membrane vesicles were obtained from roots (garden cress, Lepidium sativum L.) and shoots (etiolated zucchini hypocotyl, Cucurbita pepo L.) of representative dicotyledonous seedlings. When tonoplast-enriched fractions of cress roots were prepared by centrifugation and then subjected to free-flow electrophoresis a highly purified tonoplast fraction was obtained. This fraction from cress roots was characterized by morphometry of filipin-treated freeze-fractured preparations and by enzymology to be about 90% homogeneous. Using latency of nitrate-inhibited ATPase and H⁺-pumping as criteria we found that the majority of the tonoplast vesicles from both sources were oriented right(cytoplasmic)-side-out. Plasma-membrane vesicles were first purified by two-phase partitioning and then subjected to free-flow electrophoresis for further purification. From cress roots, the fraction of highest purity contained 89% plasma-membrane vesicles as judged by morphometry of filipin-treated, freeze-fractured preparations and by enzymology. From both sources, the major plasma-membrane subfraction in the upper phase after two-phase partitioning was shown to have the least electrophoretic mobility in free-flow electrophoresis and to be oriented right(extracytoplasmic)-side-out a slightly more mobile plasma-membrane subfraction was oriented inside-out and originated after freezing thawing from outside-out plasma-membrane vesicles.Part of the doctoral thesis (D5) of B. vom DorpWe thank the Bundesministerium für Forschung und Technologie for financial support.     

Separation of tonoplast and plasma membrane H+-ATPase from Zucchini hypocotyls by consecutive sucrose and glycerol gradient centrifugation

Summary In order to isolate tonoplast and plasma membrane vesicles involved in ATP-dependent proton transport we devised a preparative procedure with two consecutive centrifugations. Three fractions were obtained on a sucrose step gradient: light microsomes, heavy microsomes, and a mitochondria-rich fraction. The light and heavy microsomal fractions were each recentrifuged on an isopycnic glycerol density gradient. Recentrifugation of light microsomes resulted in two fractions with H⁺-ATPase activity, one equilibrating at a density less than 1.11 g/cm³ and one equilibrating at a density of about 1.17g/cm³. Comparison with marker enzyme activities suggests that the upper fraction was enriched in tonoplast, and the dense fraction with plasma membrane. In addition to marker enzyme content, H⁺ transport in the H⁺-ATPase-containing fractions was further characterized with respect to pH dependence, cation and anion dependence, and uncouplers and inhibitors. H⁺ transport in all fractions was strongly dependent on the presence of halides but no specific stimulation by potassium or any other monovalent cation was found. Of the anions tested, malate and fumarate preferentially stimulated H⁺ transport in the tonoplast-enriched fraction. It is suggested that a Ca²⁺/H⁺ antiporter is present in all fractions. Only H⁺-ATPase in the plasma membrane-enriched fractions was sensitive to nystatin, an uncoupler, and to orthovanadate, an inhibitor. The tonoplast fraction was more sensitive to nitrate than the plasma membrane-enriched fraction, and all fractions showed some sensitivity to high concentrations of oligomycin. Oligomycin sensitivity was not due to the presence of mitochondria.     

Lateral and Rotational Mobilities of Lipids in Specific Cellular Membranes of Eucalyptus gunnii Cultivars Exhibiting Different Freezing Tolerance

Two cell lines of Eucalyptus gunnii have been shown to keep their differential frost tolerance at the cellular level after long-term culture. They have been used to investigate the fluidity of specific cell membranes in relation with frost tolerance. Protoplasts and isolated vacuoles were obtained from both cell lines. In addition, purified plasma membrane and tonoplast (the vacuolar membrane) were separated from a crude microsomal fraction through free-flow electrophoresis. The lateral and rotational mobilities of lipids in these different membranes were studied by two biophysical techniques: fluorescence recovery after photobleaching (FRAP) and fluorescence polarization. After labeling the vacuoles isolated from the frost-sensitive cells with 1-oleoyl-2-(7-nitro-2,1,3-benz-oxadiazol-4-yl)aminocaproyl phosphatidylcholine, a single mobile component was observed with a diffusion coefficient of 2.4 × 10⁻⁹ cm² s⁻¹ and a mobile fraction close to 100% at a temperature of 23°C. When using isolated vacuoles from the frost tolerant line, a higher lateral diffusion of tonoplast lipids was found with a diffusion coefficient of 3.2 × 10⁻⁹ cm² s⁻¹, still with a mobile fraction close to 100%. No convincing data were obtained when performing fluorescence recovery after photobleaching experiments on protoplasts. Fluorescence polarization experiments confirmed the differential behavior of the two cell lines for tonoplast and also for plasma membrane. In addition, they showed that intrinsically tonoplast exhibited a higher fluidity than plasma membrane. Our results provide the first information on the fluidity of tonoplast and on the compared properties of two important plant membranes—tonoplast and plasma membrane—through the use of two complementary biophysical approaches. In addition, they suggest there is a correlation between membrane fluidity and cold tolerance. The potential interest of plant vacuole as a natural model system in membrane studies is emphasized.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

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.     

Monoclonal antibody TOP 71 recognizes a tonoplast protein of wide distribution in the plant kingdom

Highly purified tonoplast and plasma-membrane vesicles isolated from roots of Lepidium sativum L. (garden cress) were used as a starting material for generating a monoclonal antibody against plant tonoplast. Tonoplast vesicles were isolated by discontinuous-sucrose-gradient centrifugation followed by free-flow electrophoresis. The deglycosylated tonoplast fraction was used to generate monoclonal antibodies by immunization of Balb/c-mice and by fusion of their -lymphocytes with the mouse cell line X 63 Ag 8.653. Using plasma membrane purified by two-phase partitioning and freeflow electrophoresis to define the negative signal in screening, and purified tonoplast to define the positive signal in screening, a monoclonal antibody (TOP 71) was obtained which recognized a tonoplast protein of 71 kDa by immunoblotting in cress-root membrane fractions. Two-dimensional gel electrophoresis, affinoblotting and binding to concanavalin A showed that the TOP 71-antigen was a glycosylated protein and had an isoelectric point (pI) of 4.5. The TOP 71-antigen was found in the different tissues of organs of several higher plants (Glycine max L., Curcurbita pepo L., Zea mays L.) where it did not cross-react with the purified plasma-membrane fractions of these plants. Additionally, TOP 71 recognized its antigen in microsomal fractions of two lower plants (Chara globularis Thuili., Matteucia struthiopteris Tod.).Abbreviations ELISA enzyme-linked immunosorbent assay - FFE free-flow electrophoresis - IEF isoelectric focusing - MAB monoclonal antibody - P_FFE purified plasma membrane after FFE - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - T_gr tonoplast-enriched fraction (gr = gradient) - T_FFE purified tonoplast after FFE We thank I. Hartmann for technical assistance, R. Görlich (Institut für Landwirtschaftliche Botanik, Universität Bonn, Bonn, FRG) for advice on hybridoma techniques, M.F. Manolson (Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pa., USA) for the gift of the anti-A subunit-ATPase antibody, and R. Liedtke, H. Geithmann, and A. Heppekausen for preparation of figures. This work was financially supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium für Forschung und Technologie.     

Isolation and partial characterization of vacuoles from tobacco protoplasts

Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5′-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.     

Reconstitution of the tonoplast amino-acid carrier into liposomes

The tonoplast amino-acid transporter of barley (Hordeum vulgare L.) mesophyll cells was functionally reconstituted by incorporating solubilized tonoplast membranes, vacuoplast membranes or tonoplast-enriched microsomal vesicles into phosphatidylcholine liposomes. (i) Time-, concentration- and ATP-dependence of amino-acid uptake were similar to results with isolated vacuoles. Although the orientation of incorporation could not be controlled, the results indicate that the transporter functions as a uniport system which allows regulated equilibration by diffusion between the cytosolic and vacuolar amino-acid pools. (ii) The ATP-modulated amino-acid carrier was also successfully reconstituted from barley epidermal protoplasts and Valerianella or Tulipa vacuoplasts, indicating its general occurrence. (iii) Fractionation of solubilized tonoplasts by size-exclusion chromatography followed by reconstitution of the fractions for glutamine transport gave two activity peaks: the first eluted in the region of high-molecular-mass vesicles and the second at a size of 300 kDa for the Triton-protein micelle.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis This work was part of our research efforts within the Sonderforschungsbereich 176 of the University. We gratefully acknowledge experimental support by Marion Betz and valuable discussions with Professors U. Heber and U.-I. Flügge and Dr. Armin Gross (University of Würzburg) and Dr. E. Martinoia (ETH, Zürich, Switzerland).     

Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana

A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the molecular level. In this study, proteomic analysis was used to identify new tonoplast proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high. Using suspension-cultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles. No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS. Using this approach, it was possible to identify 163 proteins. These included well-characterized tonoplast proteins such as V-type H+ -ATPases and V-type H+ -PPases, and others with functions reasonably expected to be related to the tonoplast. There were also a number of proteins for which a function has not yet been deduced.     

Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle proteins in crassulacean Acid metabolism plants

Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.     

Tonoplast vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis

Tonoplast vesicles were purified from a microsomal fraction isolated from etiolated soybean hypocotyls (Glycine max L.) by preparative free-flow electrophoresis. Marker enzyme determinations and immunoblot analysis against the vacuolar-ATPase confirmed the nature and the purity of the isolated membranes. A purified tonoplast fraction also was obtained by consecutive sucrose and glycerol centrifugation which was further resolved into two different populations of vesicles (T_A and T_B) by free-flow electrophoresis. The determination of the sidedness of these different vesicles included concanavalin A binding as an imposed label, NADH-ferricyanide oxidoreductase cytochemistry, and ATPase latency. The tonoplast fractions, obtained by consecutive sucrose and glycerol gradient centrifugations, were found to consist of a mixture of two populations of vesicles of opposite sidedness. The least electronegative fraction obtained by free-flow electrophoresis (T_B) consisted predominantly of cytoplasmic side out tonoplast vesicles while a fraction of greater electronegativity (T_A) contained the cytoplasmic side in tonoplast vesicles. The relative amounts of each type of vesicle varied with the method of homogenization. Razor blade chopping, Polytron, and Waring Blendor homogenization gave predominantly cytoplasmic side out vesicles, whereas mashing with a mortar and pestle gave nearly equal amounts of the two populations of membrane vesicles of different orientation.     

Isolation and characterization of plasma membrane from lactating bovine mammary gland

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5′-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg²⁺-ATPase, (Na⁺ + K⁺)-ATPase, γ-glutamyl transpeptidase, phosphodiesterase I, akaline phosphatase and xanthine oxidase were also high (12–18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide compositions in both light and heavy membranes were similar upon SDS-polyacrylamide gel electrophoresis.     

Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.     

Reconstitution of vacuolar ion channels into planar lipid bilayers.

Vacuolar ion channels were characterized after reconstitution into planar lipid bilayers. (1) Channel activity was observed after incorporation of tonoplast-enriched microsomal membranes, purified tonoplast membranes or of solubilized tonoplast proteins. (2) Channels of varying single-channel conductances were detected after reconstitution. In symmetrical 100 mmol l-1 KCl, conductances between 1 and 110 pS were frequently measured; the largest number of independent reconstitution events was seen for single-channel conductances of 16-25 pS (28 experiments), 30-42 pS (26), 49-56 pS (15) and 64-81 pS (15). Channel current usually increased linearly with voltage. (3) In asymmetrical solutions, cation-, non-selective and, for the first time for the tonoplast, anion-selective channels were detected. Ca(2+)-dependent regulation of channel opening was not observed in our reconstitution system. (4) Permeability was also observed for Cl-, NO3-, SO4(2-) and phosphate. (5) After fractionation of tonoplast proteins by size exclusion chromatography, ion channel activity was recovered in specific fractions. (6) Some of these fractions catalyzed sulfate transport after reconstitution into liposomes. The results suggest that different channels are active at the tonoplast membrane at a larger number than has been concluded from previous work.     

Phenotypic changes in the fluidity of the tonoplast membrane of crassulacean-acid-metabolism plants in response to temperature and salinity stress

Electron paramagnetic resonance-spectroscopic studies on spin-labeled purified tonoplast membranes showed that in the obligate crassulacean-acid-metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perr. the fluidity of the tonoplast decreased during acclimation to higher temperatures. This phenotypic change in tonoplast fluidity was paralleled by a decrease in the mobilization of malic acid from the vacuoles during CAM in the light. The shift from the C₃ to the CAM mode of photosynthesis in the facultative CAM plant Mesembryanthemum crystallinum L. also led to a decrease in the fluidity of the tonoplast membrane. The results are consistent with the hypothesis that the ability to store malic acid during CAM in the vacuoles depends largely on the actual fluidity of the tonoplast membrane.     

Further Evidence for Stachyose and Sucrose/H+ Antiporters on the Tonoplast of Japanese Artichoke (Stachys sieboldii) Tubers

Vacuoles of Japanese artichoke (Stachys sieboldii) tubers accumulate up to 180 mM stachyose ([alpha]-galactose-[1->6]-[alpha]-galactose-[1->6]-[alpha]-glucose-[1 <->2]-[beta]-fructose) against a concentration gradient, probably by means of an active stachyose/H+ antiporter situated on the tonoplast. The goal of this study was to use isolated tonoplast vesicles to provide further evidence for the existence of such a transport mechanism. Therefore, vesicles were prepared from purified vacuoles of dormant tubers. ATP- and pyrophosphate (PPi)-dependent fluorescence quenching of the [delta]pH probe 9-amino-6-chloro-2-methoxyacridine (ACMA) indicated that these vesicles were capable of building up a pH gradient ([delta]pH, inside acid). The potent V-type H+-ATPase inhibitor bafilomycin prevented the formation of a [delta]pH in the vesicles. Bafilomycin (as well as nitrate, but not vanadate) also inhibited ATP hydrolysis, confirming the tonoplast origin of the isolated vesicles. Addition of stachyose (or sucrose, but not of mannitol) to energized vesicles caused a recovery of ACMA fluorescence, indicating a sugar-dependent dissipation of [delta]pH. The rate of fluorescence recovery was dependent on the external sugar concentration used. It displayed a single saturable response to increasing sugar concentrations. Apparent Km values of 52 and 25 mM were computed for stachyose and sucrose antiporter activities, respectively. It was also demonstrated that energized vesicles showed a much higher rate of [14C]stachyose (3 mM) and [14C]sucrose (1 mM) uptake than deenergized vesicles. The results obtained with isolated tonoplast vesicles were very similar to those obtained earlier with intact vacuoles and, therefore, confirm the existence of active stachyose and sucrose/H+ antiporters on the tonoplast of Stachys tuber vacuoles.