A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans

The H(+) proton-translocating inorganic pyrophosphatase (H(+)-PPase) family is composed of two phylogenetically distinct types of enzymes: K(+)-dependent and K(+)-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H(+)-PPase from the thermophilic bacterium Carboxydothermus hydrogenoformans. Both PP(i)-hydrolyzing and PP(i)-energized H(+) translocation activities of the recombinant enzyme in Escherichia coli inner membrane vesicles are strictly K(+)-dependent. Here we deduce the K(+) requirement of all available H(+)-PPase sequences based on the K(+) dependence of C. hydrogenoformans H(+)-PPase in conjunction with phylogenetic analyses. Our data reveal that K(+)-independent H(+)-PPases possess conserved Lys and Thr that are absent in K(+)-dependent H(+)-PPases. We further demonstrate that a A460K substitution in C. hydrogenoformans H(+)-PPase is sufficient to confer K(+) independence to both PP(i) hydrolysis and PP(i)-energized H(+) translocation. In contrast, a A463T mutation does not affect the K(+) dependence of H(+)-PPase.     

Elucidating the role of conserved glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

H(+)-pyrophosphatase (H(+)-PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H(+)-PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu(197), Glu(202), Glu(550), and Glu(649) in the H(+)-PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP(i) hydrolysis and PP(i)-energized proton translocation, indicating that the negative charge of Glu(202) is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg(2+) concentrations but were only slightly affected at high Mg(2+) concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two- and three-metal pathways. Substitution of Glu(197), Glu(202), and Glu(649) resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu(550) and Glu(649), R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP(i) hydrolysis to proton transport.     

Expression of functional Streptomyces coelicolor H+-pyrophosphatase and characterization of its molecular properties

H(+)-translocating pyrophosphatases (H(+)-PPases) are proton pumps that are found in many organisms, including plants, bacteria and protozoa. Streptomyces coelicolor is a soil bacterium that produces several useful antibiotics. Here we investigated the properties of the H(+)-PPase of S. coelicolor by expressing a synthetic DNA encoding the amino-acid sequence of the H(+)-PPase in Escherichia coli. The H(+)-PPase from E. coli membranes was active at a relatively high pH, stable up to 50 degrees C, and sensitive to N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide and acylspermidine. Enzyme activity increased by 60% in the presence of 120 mM K(+), which was less than the stimulation observed with plant vacuolar H(+)-PPases (type I). Substitutions of Lys-507 in the Gly-Gln-x-x-(Ala/Lys)-Ala motif, which is thought to determine the K(+) requirement of H(+)-PPases, did not alter its K(+) dependence, suggesting that other residues control this feature of the S. coelicolor enzyme. The H(+)-PPase was detected during early growth and was present mainly on the plasma membrane and to a lesser extent on intracellular membranous structures.     

Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity

Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.     

Roles of histidine residues in plant vacuolar H(+)-pyrophosphatase

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H(+)-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H(+)-translocation of vacuolar H(+)-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H(+)-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H(+)-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T(1/2) (pretreatment temperature at which half enzymatic activity is observed) for PP(i) hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H(+)-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F(-) and Na(+), but not that of Ca(2+). Single substitution of H704, H716 and H758 by alanine partially released the effect of K(+) stimulation, indicating possible location of K(+) binding in the vicinity of domains surrounding these residues.     

Na+-translocating membrane pyrophosphatases are widespread in the microbial world and evolutionarily precede H+-translocating pyrophosphatases

Membrane pyrophosphatases (PPases), divided into K(+)-dependent and K(+)-independent subfamilies, were believed to pump H(+) across cell membranes until a recent demonstration that some K(+)-dependent PPases function as Na(+) pumps. Here, we have expressed seven evolutionarily important putative PPases in Escherichia coli and estimated their hydrolytic, Na(+) transport, and H(+) transport activities as well as their K(+) and Na(+) requirements in inner membrane vesicles. Four of these enzymes (from Anaerostipes caccae, Chlorobium limicola, Clostridium tetani, and Desulfuromonas acetoxidans) were identified as K(+)-dependent Na(+) transporters. Phylogenetic analysis led to the identification of a monophyletic clade comprising characterized and predicted Na(+)-transporting PPases (Na(+)-PPases) within the K(+)-dependent subfamily. H(+)-transporting PPases (H(+)-PPases) are more heterogeneous and form at least three independent clades in both subfamilies. These results suggest that rather than being a curious rarity, Na(+)-PPases predominantly constitute the K(+)-dependent subfamily. Furthermore, Na(+)-PPases possibly preceded H(+)-PPases in evolution, and transition from Na(+) to H(+) transport may have occurred in several independent enzyme lineages. Site-directed mutagenesis studies facilitated the identification of a specific Glu residue that appears to be central in the transport mechanism. This residue is located in the cytoplasm-membrane interface of transmembrane helix 6 in Na(+)-PPases but shifted to within the membrane or helix 5 in H(+)-PPases. These results contribute to the prediction of the transport specificity and K(+) dependence for a particular membrane PPase sequence based on its position in the phylogenetic tree, identity of residues in the K(+) dependence signature, and position of the membrane-located Glu residue.     

Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase

Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.     

H+-PPases: yesterday, today and tomorrow

Suggestions by Calvin about a role of inorganic pyrophosphate (PPi) in early photosynthesis and by Lipmann that PPi may have been the original energy-rich phosphate donor in biological energy conversion, were followed in the mid-1960s by experimental results with isolated chromatophore membranes from the purple photosynthetic bacterium Rhodospirillum rubrum. PPi was shown to be hydrolysed in an uncoupler stimulated reaction by a membrane-bound inorganic pyrophosphatase (PPase), to be formed at the expense of light energy in photophosphorylation and to be utilized as an energy donor for various energy-requiring reactions, as a first known alternative to ATP. This direct link between PPi and photosynthesis led to increasing attention concerning the role of PPi in both early and present biological energy transfer. In the 1970s, the PPase was shown to be a proton pump and to be present also in higher plants. In the 1990s, sequences of H(+)-PPase genes were obtained from plants, protists, bacteria and archaea and two classes of H(+)-PPases differing in K(+) sensitivity were established. Over 200 H(+)-PPase sequences have now been determined. Recent biochemical and biophysical results have led to new progress and questions regarding the H(+)-PPase family, as well as the families of soluble PPases and the inorganic polyphosphatases, which hydrolyse inorganic linear high-molecular-weight polyphosphates (HMW-polyP). Here we will focus attention on the H(+)-PPases, their evolution and putative active site motifs, response to monovalent cations, genetic regulation and some very recent results, based on new methods for obtaining large quantities of purified protein, about their tertiary and quaternary structures.     

Immunological cross-reactivity between proton-pumping inorganic pyrophosphatases of widely phylogenic separated species.

Immunological cross-reactivity among three types of inorganic pyrophosphatases, that is, the proton pumping inorganic pyrophosphate synthase (H(+)-PPi synthase) and the soluble inorganic pyrophosphatase, both from Rhodospirillum rubrum, and the vacuolar membrane inorganic pyrophosphatase (H(+)-PPase) from mung bean (Vigna radiata), were examined by means of immunoblot analyses. Antibodies raised against the mung bean H(+)-PPase cross-reacted with the H(+)-PPi synthase from R. rubrum but not with the soluble PPase from R. rubrum. N,N'-dicyclohexylcarbodiimide (DCCD), which inhibits both synthesis and hydrolysis of PPi catalysed by purified and chromatophore H(+)-PPi synthase, binds to the enzyme as shown by fluorography of [14C]DCCD labelling. These results suggest that the R. rubrum H(+)-PPase share close structural similarities with the vacuolar H(+)-PPase from Mung bean.     

Oligomerization of H(+)-pyrophosphatase and its structural and functional consequences

The H(+)-pyrophosphatase (H(+)-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H(+)-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H(+)-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H(+)-PPase is present as an oligomer made up of at least two or three sets of dimers.     

H+ -PPases: a tightly membrane-bound family.

The earliest known H+-PPase (proton-pumping inorganic pyrophosphatase), the integrally membrane-bound H+-PPi synthase (proton-pumping inorganic pyrophosphate synthase) from Rhodospirillum rubrum, is still the only alternative to H+-ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+-PPase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton-pumping enzymes. The bacterial H+-PPi synthase and two algal vacuolar H+-PPases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+-PPases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+-PPases are reviewed and compared with H+-ATPases and soluble PPases.     

Properties of mutated Rhodospirillum rubrum H+-pyrophosphatase expressed in Escherichia coli

The membrane-bound proton pumping inorganic pyrophosphate synthase/pyrophosphatase (H(+)-PPi synthase/H(+)-PPase) from the photosynthetic bacterium Rhodospirillum rubrum was functionally expressed in Escherichia coli C43(DE3) cells. Based on a new topology model of the enzyme, charged residues predicted to be located near or within the membrane were selected for site-directed mutagenesis. Several of these mutations resulted in an almost complete inactivation of the enzyme. Four mutated residues appear to show a selective impairment of proton translocation and are thus likely to be involved in coupling pyrophosphate hydrolysis with electrogenic proton pumping. Two of these mutations, R176K and E584D, caused increased tolerance to salt. In addition, the former mutation caused an increased K(m) of one order of magnitude for the hydrolysis reaction. These results and their possible implications for the enzyme function are discussed.     

Acylspermidine derivatives isolated from a soft coral,Sinularia sp,inhibit plant vacuolar H(+)-pyrophosphatase

H(+)-pyrophosphatase (H(+)-PPase), which pumps H(+) across membranes coupled with PP(i) hydrolysis, is found in most plants, and some parasitic protists, eubacteria and archaebacteria. We assayed a number of extracts derived from 145 marine invertebrates as to their inhibitory effect on plant vacuolar H(+)-PPase. Acylspermidine derivatives [RCONH(CH(2))(3)N(CH(3))(CH(2))(4)N(CH(3))(2)] from a soft coral (Sinularia sp.) inhibited the PPi-hydrolysis activity of purified H(+)-PPase and the PP(i)-dependent H(+) pump activity (half inhibition concentration, 1 micro M) of vacuolar membranes of mung bean. The apparent K(i) was determined to be 0.9 micro M. Acylspermidines did not affect the activity of vacuolar H(+)-ATPase, plasma membrane H(+)-ATPase, mitochondrial ATPase or cytosolic PPase. Acylspermidines inhibited the acidification of vacuoles in protoplasts, as found on monitoring by the acridine orange fluorescent method. These results indicate that acylspermidine derivatives represent new inhibitors of H(+)-PPase with relatively high specificity.     

A lysine residue involved in the inhibition of vacuolar H(+)-pyrophosphatase by fluorescein 5'-isothiocyanate

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a central role in the electrogenic translocation of protons from cytosol to the vacuole lumen at the expense of PP(i) hydrolysis. A fluorescent probe, fluorescein 5'-isothiocyanate (FITC), was used to modify a lysine residue of vacuolar H(+)-PPase. The enzymatic activity and its associated H(+) translocation of vacuolar H(+)-PPase were markedly decreased by FITC in a concentration-dependent manner. The inhibition of enzymatic activity followed pseudo-first-order rate kinetics. A double-logarithmic plot of the apparent reaction rate constant against FITC concentration yielded a straight line with a slope of 0.89, suggesting that the alteration of a single lysine residue on the enzyme is sufficient to inhibit vacuolar H(+)-PPase. Changes in K(m) but not V(max) values of vacuolar H(+)-PPase as inhibited by FITC were obtained, indicating that the labeling caused a modification in affinity of the enzyme to its substrate. FITC inhibition of vacuolar H(+)-PPase could be protected by its physiological substrate, Mg(2+)-PP(i). These results indicate that FITC might specifically compete with the substrate at the active site and the FITC-labeled lysine residue locates probably in or near the catalytic domain of the enzyme. The enhancement of fluorescence intensity and the blue shift of the emission maximum of FITC after modification of vacuolar H(+)-PPase suggest that the FITC-labeled lysine residue is located in a relatively hydrophobic region.     

Vacuolar proton-pumping pyrophosphatase in Beta vulgaris shows vectorial activation by potassium.

Previous work with membrane vesicles has demonstrated an absolute dependence on K+ for proton translocation by the inorganic pyrophosphatase (H(+)-PPase: EC 3.6.1.1) from the vacuolar membrane (tonoplast) of higher plants. Using intact vacuoles from sugar beet (Beta vulgaris) storage tissue, we have monitored PP1-dependent currents by patch clamp in 'whole vacuole' mode. Serial K+ substitutions were made at both tonoplast faces. The results show that K+ activation occurs only at the cytosolic face.     

Membrane topology of the H+-pyrophosphatase of Streptomyces coelicolor determined by cysteine-scanning mutagenesis

The H+-translocating pyrophosphatase (H+-PPase) is a proton pump that is found in a wide variety of organisms. It consists of a single polypeptide chain that is thought to possess between 14 and 17 transmembrane domains. To determine the topological arrangement of its conserved motifs and transmembrane domains, we carried out a cysteine-scanning analysis by determining the membrane topology of cysteine substitution mutants of Streptomyces coelicolor H+-PPase expressed in Escherichia coli using chemical reagents. First, we prepared a synthetic DNA that encoded the enzyme and constructed a functional cysteine-less mutant by substituting the four cysteine residues. We then introduced cysteine residues individually into 42 sites in its hydrophilic regions and N- and C-terminal segments. Thirty-six of the mutant enzymes retained both pyrophosphatase and H+-translocating activities. Analysis of 29 of these mutant forms using membrane-permeable and -impermeable sulfhydryl reagents revealed that S. coelicolor H+-PPase contains 17 transmembrane domains and that several conserved segments, such as the substrate-binding domains, are exposed to the cytoplasm. Four essential serine residues that were located on the cytoplasmic side were also identified. A marked characteristic of the S. coelicolor enzyme is a long additional sequence that includes a transmembrane domain at the C terminus. We propose that the basic structure of H+-PPases has 16 transmembrane domains with several large cytoplasmic loops containing functional motifs.     

Proton-Pumping Inorganic Pyrophosphatases in Some Archaea and Other Extremophilic Prokaryotes

Comparative studies between the proton-pumping, membrane-bound inorganic pyrophosphatases (H(+)-PPases) from hyperthermophilic and thermophilic prokaryotes and those from mesophilic organisms can now be performed because of very recent sequence data. Typical overall factors that contribute to protein thermostability are found in H(+)-PPases from extremophiles; nevertheless, putative active site motifs of this class of enzymes may be identical over the whole range of average growth temperatures of the compared prokaryotes. Heterologous expression in yeast of H(+)-PPases from organisms spanning a wide range of thermal habitats has allowed the biochemical comparison among these proteins within the same system, ensuring that differences observed are due to intrinsic characteristics of the proteins and not to their interactions with different cellular environments. On the other hand, the availability of H(+)-PPase sequences from a variety of sources have permitted molecular phylogenetic studies of this class of proton pumps, thus providing information about their general structural and functional properties. A great step forward may be expected when one of the several groups now attempting crystallization and 3D structural determination of H(+)-PPases will be successful.     

The H(+)-pyrophosphatase of Rhodospirillum rubrum is predominantly located in polyphosphate-rich acidocalcisomes

Acidocalcisomes are acidic, calcium storage compartments with a H(+) pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H(+)-pyrophosphatase (H(+)-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H(+)-PPase with pyrophosphate (PPi) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane. polyP was also localized to the acidocalcisomes by using 4',6'-diamino-2-phenylindole staining and identified by using 31P NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PPi and polyP increased when bacteria were grown in the light. Taken together, these results suggest that the H(+)-PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PPi synthesis in the chromatophore membranes.     

Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance

An H(+)-pyrophosphatase (PPase) gene named TsVP involved in basic biochemical and physiological mechanisms was cloned from Thellungiella halophila. The deduced translation product has similar characteristics to H(+)-PPases from other species, such as Arabidopsis and rice, in terms of bioinformation. The heterologous expression of TsVP in the yeast mutant ena1 suppressed Na(+) hypersensitivity and demonstrated the function of TsVP as an H(+)-PPase. Transgenic tobacco overexpressing TsVP had 60% greater dry weight than wild-type tobacco at 300 mM NaCl and higher viability of mesophyll protoplasts under salt shock stress conditions. TsVP and AVP1, another H(+)-PPase from Arabidopsis, were heterologously expressed separately in both the yeast mutant ena1 and tobacco. The salt tolerance of TsVP or AVP1 yeast transformants and transgenic tobacco were improved to almost the same level. The TsVP transgenic tobacco lines TL3 and TL5 with the highest H(+)-PPase hydrolytic activity were studied further. These transgenic tobacco plants accumulated 25% more solutes than wild-type plants without NaCl stress and 20-32% more Na(+) under salt stress conditions. Although transgenic tobacco lines TL3 and TL5 accumulated more Na(+) in leaf tissues, the malondialdehyde content and cell membrane damage were less than those of the wild type under salt stress conditions. Presumably, compartmentalization of Na(+) in vacuoles reduces its toxic effects on plant cells. This result supports the hypothesis that overexpression of H(+)-PPase causes the accumulation of Na(+) in vacuoles instead of in the cytoplasm and avoids the toxicity of excessive Na(+) in plant cells.