Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Summary Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells ofChenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa.

The Chenopodiaceae Suaeda salsa L. was grown under different salt concentrations and under osmotic stress. The fresh weight was markedly stimulated by 0.1 M NaCl, 0.4 M NaCl and 0.1 M KCl and reduced by osmotic stress (PEG iso-osmotic to 0.1 M NaCl). Treatment with 0.4 M KCl severely damaged the plants. Membrane vesicle fractions containing tonoplast vesicles were isolated by sucrose gradient from leaves of the S. salsa plants and modulations of V-ATPase and V-PPase depending on the growth conditions were determined. Western blot analysis revealed that V-ATPase of S. salsa consists of at least nine subunits (apparent molecular masses 66, 55, 52, 48, 36, 35, 29, 18, and 16 kDa). This polypeptide pattern did not depend on culture conditions. V-PPase is composed of a single polypeptide (69 kDa). An additional polypeptide (54 kDa) was detected in the fractions of NaCl-, KCl- and PEG-treated plants. It turned out that the main strategy of salt-tolerance of S. salsa seems to be an up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, while V-PPase plays only a minor role. The increase in V-ATPase activity is not obtained by structural changes of the enzyme, but by an increase in V-ATPase protein amount.     

Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings

Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata) were successfully isolated using Ficoll gradient and two-phase (polyethylene glycol-dextran) partition. The ER-enriched vesicles contained inorganic pyrophosphate (PPi) hydrolysis and its associated proton translocating activities. Antiserum prepared against vacuolar H+-pyrophosphatase (V-PPase, EC 3.6.1.1) did not inhibit this novel pyrophosphatase-dependent proton translocation, excluding the possible contamination of tonoplast vesicles in the ER-enriched membrane preparation. The optimal ratios of Mg2+/PPi (inorganic pyrophosphate) for enzymatic activity and PPi-dependent proton translocation of ER-enriched vesicles were higher than those of vacuolar membranes. The PPi-dependent proton translocation of ER-enriched vesicles absolutely required the presence of monovalent cations with preference for K+, but could be inhibited by a common PPase inhibitor, F-. Furthermore, ER H+-pyrophosphatase exhibited some similarities and differences to vacuolar H+-PPases in cofactor/substrate ratios, pH profile, and concentration dependence of F-, imidodiphosphate (a PPi analogue), and various chemical modifiers. These results suggest that ER-enriched vesicles contain a novel type of proton-translocating PPase distinct from that of tonoplast from higher plants.     

Differential energization of the tonoplast in suspension cells and seedlings from Picea abies

 Vacuolar ATPase (EC 3.6.1.3) and PPase (EC 3.6.1.1) were studied in suspension cells and seedlings from spruce [Picea abies (L.) Karst. Proton transport activity and uncoupler (1 μM nigericin) stimulated substrate hydrolysis were measured in tonoplast enriched membrane vesicles. In suspension cells the vacuolar PPase exhibited 1.8-fold activity of the ATPase. In roots and needles from 12-week-old spruce seedlings the vacuolar PPase was inactive, whereas the ATPase was active. Therefore, we investigated whether the preparation of spruce tonoplast vesicles from roots and needles inactivates the vacuolar PPase but not the ATPase. For this purpose, maize (Zea mays L.) tonoplast membranes exhibiting vacuolar PPase as well as ATPase activity were used as a probe and added to the homogenization medium prior to the preparation of spruce vesicles. The preparation of spruce vesicles was more inhibitory to the vacuolar ATPase than to the PPase. The comparison of vacuolar PPases from spruce suspension cells and maize roots revealed similar enzymatic properties. After isopycnic centrifugation on continuous sucrose gradients the vacuolar PPase from spruce suspension cells co-purified with the vacuolar ATPase. Together, these data show: (1) vacuolar PPases from spruce suspension cells and maize roots are similar, (2) the preparation of tonoplast vesicles from spruce roots and needles does not inactivate the vacuolar PPase, (3) tonoplasts of suspension cultured cells and seedlings from spruce are differentially energized by the vacuolar pyrophosphatase that may indicate a difference in pyrophosphate metabolism between embryogenic and differentiated spruce cells, and (4) tonoplast vesicles from spruce seedlings may allow investigations of the effect of pyrophosphate on the vacuolar ATPase in the absence of vacuolar PPase activity. Received: 2 July 1998 / Accepted: 14 September 1998     

The role of vacuolar malate-transport capacity in crassulacean acid metabolism and nitrate nutrition. Higher malate-transport capacity in ice plant after crassulacean acid metabolism-induction and in tobacco under nitrate nutrition

Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H(+)-transport by H(+)-pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, Deltamu(H+), across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, J(mal)(rel), of apparent ATP-dependent malate-stimulated H(+)-transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. J(mal)(rel) was high in the obligate crassulacean acid metabolism (CAM) species Kalancho? daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO(3)(-) nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher J(mal)(rel). It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO(3)(-) nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins.     

Changes in H+-Pumps and a Tonoplast Intrinsic Protein of Vacuolar Membranes during the Development of Pear Fruit

Vacuolar H⁺-ATPase (V-ATPase) was purified from pear fruit andantibodies were raised against the subunits of 55 and 33 kDa.Antibodies against mung bean H⁺-pyro-phosphatase (V-PPase) andradish VM23, which is a tonoplast intrinsic protein (TIP) anda water channel, cross-reacted with the vacuolar membrane proteinsof pear fruit. To clarify the roles of these proteins in developmentof pear fruit, we determined their levels relative to the totalamount of protein by immunoblot analysis. The levels of subunitsof the V-ATPase increased with fruit development. By contrast,the level of V-PPase was particularly high at the cell-divisionstage and remained almost the same at other stages. The changesin the activities of V-ATPase and V-PPase corresponded to thosein their protein levels. The ratio of V-PPase activity to V-ATPaseactivity indicated that V-PPase is a major H⁺-pump of the vacuolarmembranes of young fruit and that the contribution of V-ATPaseincreases with fruit development, finally, V-ATPase becomesthe major H⁺-pump during the later stages of fruit development.The level of a protein analogous to VM23 (VM23P) was especiallyhigh during the active cell-expansion stage in young fruit,and VM23P might, therefore, play an important role in the rapidexpansion of cells as a vacuolar water channel. Our resultsshow that the levels of V-ATPase, V-PPase and VM23P change differentlyand reflect the roles of the respective proteins in the developmentof pear fruit. ³Research Fellow of the Japan Society for the Promotion of Science ⁴Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan     

Molecular Evidence for the Occurrence of H+-Transporting V-ATPase Subunit D and Two Different Forms of Subunit E in Leaves of the Obligate CAM Species Kalanchoë daigremontiana

Membrane proteins of purified tonoplast vesicles from leaves of Kalanchoë daigremontiana Hamet et Perrier were solubilized by the non-ionic detergent Triton X-114 and subsequently separated by MonoQ® anion-exchange chromatography. Special attention was given to the range of molecular masses around 30 kDa comprising the central stalk subunit peptides of the H⁺-transporting V-ATPase. Three polypeptides of apparent molecular masses of 32, 33 and 34 kDa were separated. Proteolytic fragments were obtained by trypsin digestion. Analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry of tryptic fragments of the 32 and 33 kDa peptides and protein data- bank comparisons showed that they are two different forms of subunit E. N-terminal amino acid sequencing of tryptic fragments of the 34 kDa peptide showed that it is subunit D. This work provides for the first time unequivocal molecular evidence that the central stalk of the V-ATPase of the obligate CAM plant K. daigremontiana includes subunit D and different forms of subunit E.     

玉米根V-ATPase的A亚基磷酸化位点的鉴定

以玉米 (Zea mays L.) 根的高纯度液泡膜为材料进行的磷酸化反应表明,液泡膜蛋白的磷酸化可明显提高V型H -ATPase (V-ATPase) 的ATP水解活性和H 转运活性。进一步研究表明,纯化的液泡膜蛋白能被硫代磷酸化,用V-ATPase的A亚基抗体将一条约69 kD的条带鉴定为A亚基。为了测定V-ATPase的A亚基的磷酸化位点,从硫代磷酸化的凝胶中切下A亚基条带并用胰蛋白酶彻底消化。用RP-HPLC分离纯化酶解片断,收集纯化的硫代磷酸化肽段进行质谱分析所测定的分子量为573.83 Da。A亚基胰蛋白酶彻底消化后能产生61个肽段,只有F56肽段的分子量573.66 Da与573.83 Da最接近,而且F56肽段上只有第525位的丝氨酸可以被磷酸化。因此可以确定,玉米根V-AT-Pase A亚基的潜在磷酸化位点为Ser525。就我们所知,这是首次确定植物V-ATPase A亚基的磷酸化位点。     

Chilling-induced changes of vacuolar proton pumps in hypocotyls of <Emphasis Type="Italic">Vigna unguiculata</Emphasis>

Vigna unguiculata (cowpea) is a legume adapted to high temperatures and is sensitive to low temperatures. Temperature is one of the limiting factors of growth and yield for many crops but its effect on cowpea metabolism is not known. We investigated the effect of chilling on activity of vacuolar proton pumps (V-ATPase and V-PPase) and their protein content in tonoplast vesicles of cowpea hypocotyls. Seedlings grown for 7 days at 10 or 4°C were used for experiments. Chilling treatment at 10 or 4°C markedly suppressed growth of cowpea seedlings. Following chilling at 10 and 4°C, activity of both proton pumps and the relative amount of V-PPase and subunit A of V-ATPase were significantly increased. Both substrate hydrolysis and H⁺ transport activities of V-PPase remained at relatively high levels during chilling treatment. For V-ATPase, treatment at 10°C for 6 days increased the ATP hydrolysis activity. However, the H⁺ transport activity of the enzyme was increased when treated for 4 days but was markedly decreased when treated for 6 days. Our results provide evidence for different regulation for these vacuolar proton pumps, indicating that V-PPase is the more stable proton pump throughout chilling stress.     

玉米根V-ATPase的A亚基磷酸化位点的鉴定

以玉米(Zea mays L)根的高纯度液泡膜为材料进行的磷酸化反应表明,液泡膜蛋白的磷酸化可明显提高v型H -ATPase(V-ATPase)的ATP水解活性和H 转运活性.进一步研究表明,纯化的液泡膜蛋白能被硫代磷酸化,用V-ATPase的A亚基抗体将一条约69 kD的条带鉴定为A亚基.为了测定V-ATPase的A亚基的磷酸化位点,从硫代磷酸化的凝胶中切下A亚基条带并用胰蛋白酶彻底消化.用RP-HPLC分离纯化酶解片断,收集纯化的硫代磷酸化肽段进行质谱分析所测定的分子量为573.83 Da.A亚基胰蛋白酶彻底消化后能产生61个肽段,只有F56肽段的分子量573.66 Da与573.83 Da最接近,而且F56肽段上只有第525位的丝氨酸可以被磷酸化.因此可以确定,玉米根V-AT-Pase A亚基的潜在磷酸化位点为Ser525.就我们所知,这是首次确定植物V-ATPase A亚基的磷酸化位点.     

Aminomethylenediphosphonate: A Potent Type-Specific Inhibitor of Both Plant and Phototrophic Bacterial H+-Pyrophosphatases

The suitability of different pyrophosphate (PPi) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) of tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata was investigated. Five 1,1-diphosphonates and imidodiphosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the Km of the enzyme. The order of inhibitory potency (apparent inhibition constants, Kiapp values, [mu]M, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7) [almost equal to] ethane-1-hydroxy-1,1-diphosphonate (6.5) > imidodiphosphate (12) > methylenediphosphonate (68) >> dichloromethylenediphosphonate (>500). The specificity of three of these compounds, aminomethylenediphosphonate, imidodiphosphate, and methylenediphosphonate, was determined by comparing their effects on the V-PPase and vacuolar H+-ATPase from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi synthase of chromatophores prepared from Rhodospirillum rubrum, soluble PPase from Saccharomyces cerevisiae, alkaline phosphatase from bovine intestinal mucosa, and nonspecific monophosphoesterase from Vigna at a PPi concentration equivalent to 10 times the Km of the V-PPase. Although all three PPi analogs inhibited the plant V-PPase and bacterial H+-PPi synthase with qualitatively similar kinetics, whether substrate hydrolysis or PPi-dependent H+-translocation was measured, neither the vacuolar H+-ATPase nor plasma membrane H+-ATPase nor any of the non-V-PPase-related PPi hydrolases were markedly inhibited under these conditions. It is concluded that 1, 1-diphosphonates, in general, and aminomethylenediphosphonate, in particular, are potent type-specific inhibitors of the V-PPase and its putative bacterial homolog, the H+-PPi synthase of Rhodospirillum.     

Purification of an h-translocating inorganic pyrophosphatase from vacuole membranes of red beet

An H⁺-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150-fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase.     

Tonoplast inorganic pyrophosphatase in Vicia faba guard cells

Lysed guard-cell protoplasts of Vicia faba L. exhibited hydrolytic activity characteristic of tonoplast inorganic pyrophosphatase (V-PPase; EC 3.6.1.1). Activity was inhibited by the specific V-PPase inhibitor aminomethylenediphosphonate, stimulated by K⁺ (K _m = 51 mM) and inhibited by Ca²⁺ (80 nM free Ca²⁺ was required for 50% inhibition at 0.27 mM free Mg²⁺). Patch-clamp measurements of electrogenic activity confirmed enzyme localisation at the tonoplast. This is the first report of V-PPase activity in guard cells; its possible involvement in stomatal opening is discussed. Received: 12 February 1998 / Accepted: 24 April 1998     

Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.     

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Solubilization of microsomal-associated phosphatidylserine synthase and phosphatidylinositol synthase from Saccharomyces cerevisiae

Membrane-associated cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC2.7.8.8.) and CDP-diacylglycerol:myo-inositol phosphatidyltransferase (phosphatidylinositol synthase, EC 2.7.8.11) were solubilized from the microsomal fraction of Saccharomyces cerevisiae. A variety of detergents were examined for their ability to release phosphatidylserine synthase and phosphatidylinositol synthase activities from the microsome fraction. Both enzymes were solubilized from the microsome fraction with Renex 690 in yield over 80% with increase to specific activity of 1.6-fold. Both solubilized enzymatic activities were dependent on manganese ions and Triton X-100 for maximum activity. The pH optimum for each reaction was 8.0. The apparent Km values for CDP-diacylglycerol and serine for the phosphatidylserine synthase reaction were 0.1 and 0.25 mM, respectively. The apparent Km values for CDP-diacylglycerol and inositol for the phosphatidylinositol synthase reaction were 70 microM and 0.1 mM, respectively. Thioreactive agents inhibited both enzymatic activities. Both solubilized enzymatic activities were thermally inactivated at temperatures above 30 degrees C.     

Limited proteolysis of calf thymus terminal deoxynucleotidyl transferase

Controlled, limited proteolysis of homogeneous calf thymus terminal deoxynucleotidyl transferase (EC 2.7.7.31) using immobilized Staphylococcus aureus V-8 protease results in a low molecular weight form of the enzyme which possesses unaltered catalytic activity. Analysis of the products of limited proteolysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that only the large subunit, β, is modified from a molecular weight of 30,500 to 25,500. The small subunit, α, which has a molecular weight of 9500, is unchanged. A shift in the apparent isoelectric pH of the calf enzyme following proteolysis is observed from pI = 8.2 to 7.8. Both forms of the enzyme are homogeneous in the isoelectric focusing gel system, as determined by coincidence of single protein bands with terminal transferase activity on the gel. The specific activities of cleaved and uncleaved terminal transferase proteins, as well as their thermal stabilities, are comparable. These results suggest that the polypeptide domain involved in terminal transferase enzymatic activity can be probed further by novel methods involving limited proteolysis without concomitant loss in enzymatic function.