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.     

Transport processes of solutes across the vacuolar membrane of higher plants

The central vacuole is the largest compartment of a mature plant cell and may occupy more than 80% of the total cell volume. However, recent results indicate that beside the large central vacuole, several small vacuoles may exist in a plant cell. These vacuoles often belong to different classes and can be distinguished either by their contents in soluble proteins or by different types of a major vacuolar membrane protein, the aquaporins. Two vacuolar proton pumps, an ATPase and a PPase energize vacuolar uptake of most solutes. The electrochemical gradient generated by these pumps can be utilized to accumulate cations by a proton antiport mechanism or anions due to the membrane potential difference. Uptake can be catalyzed by channels or by transporters. Growing evidence shows that for most ions more than one transporter/channel exist at the vacuolar membrane. Furthermore, plant secondary products may be accumulated by proton antiport mechanisms. The transport of some solutes such as sucrose is energized in some plants but occurs by facilitated diffusion in others. A new class of transporters has been discovered recently: the ABC type transporters are directly energized by MgATP and do not depend on the electrochemical force. Their substrates are organic anions formed by conjugation, e.g. to glutathione. In this review we discuss the different transport processes occurring at the vacuolar membrane and focus on some new results obtained in this field.     

1-Chloro-2,4-Dinitrobenzene-Elicited Increase in Vacuolar Glutathione-S-Conjugate Transport Activity

Unlike most other characterized organic solute transport in plants, uptake of the model compound S-(2,4-dinitrophenyl)glutathione (DNP-GS) and related glutathione-S-conjugated by vacuolar membranes is directly energized by MgATP. Here we show that exogenous application of the DNP-GS precursor 1-chloro-2,4-dinitrobenzene (CDNB) to seedlings of Vigna radiata (mung bean) increases the capacity of vacuolar membrane vesicles isolated from hypocotyls for MgATP-dependent DNP-GS transport in vitro. Our findings are 4-fold: (a) Pretreatment of seedlings with CDNB causes a progressive increase in MgATP-dependent DNP-GS uptake by vacuolar membrane vesicles, whereas the same range of CDNB concentrations causes only marginal stimulation when the compound benoxacor [4-(dichloroacetyl)-3,4-dihydro-3-methyl-2H-1,4-benzoxazine] is included in the pretreatment solution. (b) Increased DNP-GS uptake is accompanied by a proportionate and selective increase in Vmax(DNP-GS) but not in Km(DNP-GS) or Km(MgATP). (c) CDNB-enhanced DNP-GS uptake is not accompanied by a change in the density profile or sidedness of the vacuolar membrane fraction. (d) Basal and CDNB-enhanced DNP-GS uptake are indistinguishable in terms of their inhibitor profiles. On the basis of these findings, it is inferred that pretreatment with CDNB increases the amount or recruitment of functional transporter into the vacuolar membrane and that agents such as benoxacor antagonize the effects otherwise seen with CDNB in this sytem.     

Some properties of membrane-bound, solubilized and reconstituted into liposomes H+-ATPase of vacuoles of Saccharomyces carlsbergensis

Vacuoles of yeast grown in peptone medium possessed high ATPase activity (up to 1 mumol X mg protein-1 X min-1). Membrane-bound and solubilized ATPase activities were insensitive to vanadate and azide, but were inhibited by NO-3 . K+ and cyclic AMP stimulated both membrane-bound and solubilized ATPase activities. Dio-9 activated the membrane form of vacuolar ATPase 1.5-2-fold and did not affect the solubilized enzyme. Solubilized and partially purified vacuolar ATPase was reconstituted with soy-bean phospholipids by a freeze-thaw procedure. ATPase activities in native vacuoles and proteoliposomes were stimulated effectively by Dio-9, the protonophore FCCP and ionophores valinomycin and nigericin. ATP-dependent H+ transport into proteoliposomes was also shown by quenching of ACMA fluorescence. Vacuolar and partially purified ATPase preparations possessed also GTPase activity. Unlike ATPase, however, GTPase was not incorporated as a proton pump into liposomes.     

An active proton pump of intact vacuoles isolated from Tulipa petals

Anthocyanin pigments within Tulipa petal vacuoles provide the means for real-time spectrophotometric monitoring of vacuolar sap pH and for studying ATP-dependent proton transport in isolated, intact vacuoles. Spectra of petal extracts were used to select empirically those wavelengths giving an approximately linear variation in anthocyanin absorbance with pH over a pH range of interest. A sensitive single-beam spectrophotometer with vertical optics was used to minitor absorbance changes of intact, settled vacuoles. Substrates and inhibitors of vacuolar ATPase (Lin, W., Wagner, G.J., Siegelman, H.W. and Hind, Q. (1977) Biochim. Biophys. Acta 465, 110–117) were added to probe proton transport. Acidification of the vacuole sap occurred following addition of MgATP, but not CaATP. Proton accumulation was inhibited by 10 μM Dio 9, an inhibitor of tonoplast ATPase in vitro, and the proton gradient established by addition of MgATP was dissipated after addition of 10 μM CCCP. No pumping response was observed with intact protoplasts. Potential differences across the tonoplast were directly measured by impaling vacuoles with glass microelectrodes. Potential differences of 10–20 mV (inside positive) were recorded when vacuoles were suspended in 0.7 M mannitol/10 mM Hepes buffer (adjusted to pH 8.0 with KOH), and 0.5 mM dithiothreitol. Addition of MgATP increased the potential difference by 2–5 mV.     

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.     

Functional Specialization of Vacuoles in Sugarcane Leaf and Stem

Plant vacuoles are frequently targeted as a storage site for novel products. We have used environment-sensitive fluorescent dyes and the expression of vacuolar marker proteins to characterize the vacuoles in different organs and cell types of sugarcane. The results demonstrated that the lumen of the vacuole in the parenchyma cells of the stem is acidic (<pH 5) and contains active proteases, characteristic of lytic vacuoles. Western blots and tissue labelling with antibodies to vacuolar H⁺-ATPase suggest that this proton pump is involved in acidification of the vacuolar lumen. Quantitative real-time PCR was used to show that the expression of vacuolar proteases and a vacuolar sorting receptor is also coordinately regulated. In contrast to the stem parenchyma cells, the cells of sugarcane leaves contain diverse types of vacuoles. The pH of these vacuoles and their capacity to hydrolyze protease substrates varies according to cell type and developmental stage. Sugarcane suspension-cultures contain cells with vacuoles that resemble those of stem parenchyma cells and are thus a useful model system for investigating the properties of the vacuole. Understanding the growth and development of storage capacity will be useful in designing strategies to maximize the production of sucrose or alternative bioproducts.     

Intravacuolar proteolysis in Plasmodium falciparum digestive vacuoles is similar to intralysosomal proteolysis in mammalian cells

Hemoglobin of intact human erythrocytes was labelled with [125I]iodide and these cells were inoculated with late-stage cultures of Plasmodium falciparum. Subcellular parasite particulate material containing intact digestive vacuoles filled with 125I-labelled hemoglobin was incubated in sucrose-buffer medium and degradation of labelled intravacuolar hemoglobin was measured by precipitation with trichloroacetic acid. Proteolysis was maximal at pH 5.0 or in the presence of MgATP at pH 8.0. The stimulatory effect of MgATP was probably due to energization of a proton pump activity as reported by others (Krogstad, D.J., Schlesinger, P.H. and Gluzman, I.Y. (1984) J. Cell Biol. 101, 2302-2309). Proteolysis was also inhibited by ionophores and antimalarials. These results suggest that P. falciparum digestive vacuoles have an ATP-dependent acidification mechanism similar to mammalian lysosomes but with some exceptions. The properties of this intravacuolar proteolysis were remarkably similar to intralysosomal proteolysis in mouse liver or kidney preparations.     

欧洲山杨液泡膜Na+/H+ 反向运输体的性质鉴定

植物液泡膜Na /H 反向运输体可将细胞质中的Na 转运到液泡内储存,以减少胞内Na 的毒性.但木本植物如杨树是否有同样的机制目前还不清楚.以欧洲山杨的愈伤组织为材料,捣碎破碎愈伤组织细胞,经过差速离心和不连续蔗糖梯度离心得到纯化的欧洲山杨液泡微囊.通过液泡V-ATPase建立质子梯度,该液泡能够利用此梯度调控Na 的转运,表明液泡膜上存在Na /H 反向运输体活性(表观米氏常数Km是11.4mmol/L).Na /H 反向运输体的抑制剂——氨氯吡嗪咪能明显抑制转运体的活性.该Na /H 反向运输体也可以转运K ,但亲和能力比Na 低30%.该结果首次证明木本植物的液泡膜上存在Na /H 反向运输体.初步功能研究表明,愈伤组织在盐胁迫条件下,Na /H 反向运输体活性明显下降,提示该机制可能与山杨不耐盐有关.     

Magnesium Adenosine 5[prime]-Triphosphate-Energized Transport of Glutathione-S-Conjugates by Plant Vacuolar Membrane Vesicles

By characterization of the uptake of glutathione-S-conjugates, principally dinitrophenyl-S-glutathione (DNP-GS), by vacuolar membrane vesicles, we demonstrate that a subset of energy-dependent transport processes in plants are not H+-coupled but instead are directly energized by MgATP. The most salient features of this transport pathway are: (a) its specific, obligate requirement for MgATP as energy source; (b) the necessity for hydrolysis of the [gamma]-phosphate of MgATP for uptake; (c) the insensitivity of uptake to uncouplers of the transtonoplast H+ gradient (carbonylcyanide 4-trifluoromethoxyphenylhydrazone, gramicidin-D, and NH4Cl); (d) its pronounced sensitivity to vanadate and partial inhibition by vinblastine and verapamil; (e) the lack of chemical modification of DNP-GS either during or after transport; (f) the capacity of S-conjugates of chloroacetanilide herbicides, such as metolachlor-GS, but not free herbicide, to inhibit uptake; and (g) the ability of vacuolar membrane vesicles purified from a broad range of plant species, including Arabidopsis, Beta, Vigna, and Zea, to mediate MgATP-dependent, H+-electrochemical potential difference-independent DNP-GS uptake. On the basis of these findings it is proposed that the transport of DNP-GS across the vacuolar membrane of plant cells is catalyzed by a glutathione-conjugate transporter that directly employs MgATP rather than the energy contained in the transtonoplast H+-electrochemical potential difference to drive uptake. The broad distribution of the vacuolar DNP-GS transporter and its inhibition by metolachlor-GS are consistent with the notion that it plays a general role in the vacuolar sequestration of glutathione-conjugable cytotoxic agents.     

Transport of 1-aminocyclopropane-1-carboxylic acid into isolated maize mesophyll vacuoles

Intracellular transport of the ethylene precursor, I-aminocyclopropane-1-carboxylic acid (ACC) can change the ACC concentration in cell compartments and impact ethylene biosynthesis. Transport of ACC into isolated maize ( Zea mays L.) mesophyll vacuoles was studied by silicon layer flotation filtering. The transport of ACC across the tonoplast was stimulated 2. 4- to 8. 1-fold by 5 m M MgATP, showed saturation kinetics with an apparent K_m for ACC of 20 μ M , and was optimal at 25°C. Transport of ACC was sensitive to the pH of the medium, falling as external pH rose. Effectors known to inhibit proton-translocating ATPases (N, N-dicyclohexylcarbodiimide) and to collapse the electrical (thiocyanate, valinomycin) and chemical (carbonylcyanide m -chlorophenylhydrazone, gramicidin) potential gradients for protons across the tonoplast all reduced ACC transport. The nonhydrolyzable MgATP analog. Mg adenylyl-imidodiphosphate, stimulated ACC transport as effectively as MgATP. Other nucleotides (MgADP, MgCTP, MgUTP, MgGTP) and MgPP_i had little or no effect. These results suggest that ACC uptake into isolated maize mesophyll vacuoles is carrier mediated, is dependent upon an electrochemical potential gradient for protons and is specifically regulated, but not necessarily energized, by MgATP     

The structure of sialyl-glycopeptides of the O-glycosidic type, isolated from sialidosis (mucolipidosis I) urine

Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H+-translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean-acid-metabolism plant Kalancho? daigremontiana and compare it with other phosphohydrolases. Vacuoles were isolated by polybase-induced lysis of mesophyll-cell protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50% inhibition at about 0.5 mmol m-3), but was not completely inhibited even at much higher ammonium-molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 mmol m-3 ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed at appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 nmol h-1 (10(3) vacuoles)-1, an average 13% of which was attributable to residual acid-phosphatase activity. Inorganic-pyrophosphatase activity could not be demonstrated unambiguously. The vacuolar ATPase activity was Mg2+-dependent, had an apparent Km for MgATP2- of 0.31 mol m-3, and was 32% stimulated by 50 mol m-3 KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO-3 were both markedly inhibitory. Dicyclohexylcarbodiimide and tributyltin were also strongly inhibitory. Tributyltin caused a 50% inhibition at about 0.3 mmol m-3. This is taken as evidence that the vacuolar ATPase might function as an H+-translocating ATPase. It is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic-acid accumulation in K. daigremontiana.     

Stabilization and Transport Capacity of Cowpea and Barley Vacuoles

Subjecting either cowpea or barley protoplasts to a combinedosmotic and pH shock provides the optimum conditions for theisolation of cowpea and barley vacuoles. Incubation of vacuolesin a defined medium resulted in 50% lysis after 30 min (cowpea)and 20 min (barley). The addition of 1 mM EDTA resulted in increasedstability of vacuoles with 50% lysis occurring after 50 min(cowpea) and 120 min (barley). Other compounds were tested fortheir effects on the stability of vacuoles. The longer life of vacuoles in the presence of EDTA allowedtransport studies to be carried out using radiolabeled tracers.The uptake of [¹⁴C]sucrose (10 mM) by cowpea vacuoles was stimulatedapproximately two-fold by the presence of MgATP (10 mM); theK_m for [¹⁴G]sucrose uptake by cowpea vacuoles was 12.5 mM. Uptakeof [³H]GA₁ ([³H]gibberellin A₁) by cowpea vacuoles was alsostimulated two-to-four fold in the presence of 10 mM MgATP comparedto untreated vacuoles. No MgATP stimulation of [³H]GA₁ or [¹⁴C]sucroseuptake could be observed in barley vacuoles. The effect of pHon uptake of [³H]GA₁ was studied in both cowpea and barley vacuoles.Uptake was optimal at about neutral pH which also coincidedwith the optimum pH for maximum stability of vacuoles. ¹ Dedicated to the memory of Prof. J. Ashida. ² Present address: International Plant Research Institute, 853Industrial Road, San Carlos, CA 94070, U.S.A. ³ Present address: Institute de Agroquimica y Tecnológiade Alimentos, Jaíme Roig, 11, Valencia 10, Spain. (Received December 11, 1982; Accepted February 24, 1983)     

Alternative mechanisms of vacuolar acidification in H(+)-ATPase-deficient yeast

Acidification of the endosomal/lysosomal pathway by the vacuolar-type proton translocating ATPase (V-ATPase) is necessary for a variety of essential eukaryotic cellular functions. Nevertheless, yeasts lacking V-ATPase activity (Deltavma) are viable when grown at low pH, suggesting alternative methods of organellar acidification. This was confirmed by directly measuring the vacuolar pH by ratio fluorescence imaging. When Deltavma yeasts were cultured and tested in the acidic conditions required for growth of V-ATPase-deficient mutants, the vacuolar pH was 5.9. Fluid-phase pinocytosis of acidic extracellular medium cannot account for these observations, because the V-ATPase-independent vacuolar acidification was unaffected in mutants deficient in endocytosis. Similarly, internalization of the plasmalemmal H(+)-ATPase (Pma1p) was ruled out, because overexpression of Pma1p failed to complement the Deltavma phenotype and did not potentiate the vacuolar acidification. To test whether weak electrolytes present in the culture medium could ferry acid equivalents to the vacuole, wild-type and the Deltavma yeasts were subjected to sudden changes in extracellular pH. In both cell types, the vacuoles rapidly alkalinized when external pH was raised from 5.5 (the approximate pH of the culture medium) to 7.5 and re-acidified when the yeasts were returned to a medium of pH 5.5. Importantly, these rapid pH changes were only observed when NH(4)(+), routinely added as a nitrogen source, was present. The NH(4)(+)-dependent acidification was not due to efflux of NH(3) from the vacuole, as cells equilibrated to pH 7.5 in the absence of weak electrolytes rapidly acidified when challenged with an acidic medium containing NH(4)(+). These findings suggest that although NH(3) can act as a cell-permeant proton scavenger, NH(4)(+) may function as a protonophore, facilitating equilibration of the pH across the plasma and vacuolar membranes of yeast. The high concentration of NH(4)(+) frequently added as a nitrogen source to yeast culture media together with effective NH(4)(+) transporters thereby facilitate vacuolar acidification when cells are suspended in acidic solutions.     

Biochemical characterization of the yeast vacuolar H(+)-ATPase

The yeast vacuolar proton-translocating ATPase was isolated by two different methods. A previously reported purification of the enzyme (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095) was repeated. This procedure consisted of isolation of vacuoles, solubilization with the zwitterionic detergent ZW3-14, and glycerol gradient centrifugation of the solubilized vacuoles. The fraction with the highest specific activity (11 mumol of ATP hydrolyzed mg-1 min-1) included eight polypeptides of apparent molecular masses of 100, 69, 60, 42, 36, 32, 27, and 17 kDa, suggesting that the enzyme may be more complex than the three-subunit composition proposed from the original purification. The 69-kDa polypeptide was recognized by antisera against the catalytic subunits of two other vacuolar ATPases and labeled with the ATP analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, indicating that it contains all or part of the catalytic site. A monoclonal antibody was prepared against this subunit. Under nondenaturing conditions, the antibody immunoprecipitated eight polypeptides, of the same molecular masses as those seen in the glycerol gradient fraction, from solubilized vacuolar vesicles. All eight of these polypeptides are therefore good candidates for being genuine subunits of the enzyme. The structure and function of the yeast vacuolar H+-ATPase were further characterized by examining the inhibition of ATPase activity by KNO3. In the presence of 5 mM MgATP, 100 mM KNO3 inhibited 71% of the ATPase activity of vacuolar vesicles, and the 69- and 60-kDa subunits (and possibly the 42-kDa subunit) were removed from the vacuolar membrane to a similar extent. At concentrations of less than 200 mM KNO3, the stripping of the ATPase subunits and the inhibition of ATPase activity were dependent on the presence of MgATP, suggesting that this is a conformation-specific disassembly of the enzyme. The yeast vacuolar H+-ATPase is a multisubunit enzyme, consisting of a combination of peripheral and integral membrane subunits. Its structure and subunit composition are very similar to other vacuolar ATPase, and it shares some characteristics with the F1F0-ATPases.     

A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.     

An integrative view on vacuolar pH homeostasis in Arabidopsis thaliana: Combining mathematical modeling and experimentation

The acidification of plant vacuoles is of great importance for various physiological processes, as a multitude of secondary active transporters utilize the proton gradient established across the vacuolar membrane. Vacuolar-type H⁺-translocating ATPases and a pyrophosphatase are thought to enable vacuoles to accumulate protons against their electrochemical potential. However, recent studies pointed to the ATPase located at the trans-Golgi network/early endosome (TGN/EE) to contribute to vacuolar acidification in a manner not understood as of now. Here, we combined experimental data and computational modeling to test different hypotheses for vacuolar acidification mechanisms. For this, we analyzed different models with respect to their ability to describe existing experimental data. To better differentiate between alternative acidification mechanisms, new experimental data have been generated. By fitting the models to the experimental data, we were able to prioritize the hypothesis in which vesicular trafficking of Ca²⁺/H⁺-antiporters from the TGN/EE to the vacuolar membrane and the activity of ATP-dependent Ca²⁺-pumps at the tonoplast might explain the residual acidification observed in Arabidopsis mutants defective in vacuolar proton pump activity. The presented modeling approach provides an integrative perspective on vacuolar pH regulation in Arabidopsis and holds potential to guide further experimental work.     

A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364).     

Abnormal acidification of melanoma cells induces tyrosinase retention in the early secretory pathway.

In tyrosinase-positive amelanotic melanoma cells, inactive tyrosinase accumulates in the endoplasmic reticulum. Based on studies described here, we propose that aberrant vacuolar proton ATPase (V-ATPase)-mediated proton transport in melanoma cells disrupts tyrosinase trafficking through the secretory pathway. Amelanotic but not melanotic melanoma cells or normal melanocytes display elevated proton export as observed by the acidification of the extracellular medium and their ability to maintain neutral intracellular pH. Tyrosinase activity and transit through the Golgi were restored by either maintaining the melanoma cells in alkaline medium (pH 7.4-7.7) or by restricting glucose uptake. The translocation of tyrosinase out of the endoplasmic reticulum and the induction of cell pigmentation in the presence of the ionophore monensin or the specific V-ATPase inhibitors concanamycin A and bafilomycin A1 supported a role for V-ATPases in this process. Because it was previously shown that V-ATPase activity is increased in solid tumors in response to an acidified environment, the appearance of hypopigmented cells in tyrosinase-positive melanoma tumors may indicate the onset of enhanced glycolysis and extracellular acidification, conditions known to favor metastatic spread and resistance to weak base chemotherapeutic drugs.     

Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of V-type ATPase from Arabidopsis

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.