Functional asymmetry of the F0 motor in bacterial ATP synthases

F₁F₀ ATP synthases use the electrochemical potential of H⁺ or Na⁺ across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F₀ parts of a Na⁺- and H⁺-dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of ∼100 times higher Na⁺ or H⁺ concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F₀ parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo . In Escherichia coli , we observed respiratory chain-driven ATP production at pH 7–8, while P -site pH values < 6.5 were required for ATP synthesis in vitro . This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.     

The tonoplast H+-pyrophosphatase of radish seedlings: biochemical characteristics

The activity of the H⁺-pyrophosphatase (H⁺-PPase) was characterized in microsomes from 24-h-old radish ( Raphanus sativus L., ev. Tondo Rosso Quarantino) seedlings, which are virtually devoid of the tonoplast H⁺-ATPase. The H⁺-PPase was localized to membranes which roughly comigrated with the plasma membrane in a sucrose density gradient, but clearly separated from plasma membrane when microsomes were partitioned in an aqueous dextran-polyethylene glycol two-phase system. The H⁺-PPase activity was strictly dependent on Mg²⁺ and on the presence of a monovalent cation (K⁺=Rb⁺=NH₃⁺Cs⁺≫Na⁺Li⁺) and was insensitive to anions such as Cl−, Br−, NO₃− and SO₄^2-. It was inhibited by F−, imidodiphosphate and Ca²⁺. It had a pH optimum between pH 7.5 and 8.5 and was saturated by low concentrations of pyrophosphate (half saturation at 30 μ M pyrophosphate). All of these characteristics are identical to those reported for the tonoplast H⁺-PPase from various plant materials. The functional molecular weight of the H⁺-PPase, measured with the radiation-inactivation technique was 96 kDa.     

NaCl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato

NaCl-induced changes in the accumulation of message for the 70 kDa subunit of the tonoplast H⁺-ATPase and plasma membrane H⁺-ATPase were studied in hydroponically grown plants of Lycopersicon esculentum Mill. cv. Large Cherry Red. There was increased accumulation of message for the 70 kDa (catalytic) subunit of the tonoplast H⁺-ATPase in expanded leaves of tomato plants 24 h after final NaCl concentrations were attained. This was a tissue-specific response; levels of this message were not elevated in roots or in young, unexpanded leaves. The NaCl-induced accumulation of this message was transient in the expanded leaves and returned to control levels within 7 days. The temporal and spatial patterns of NaCl-induced accumulation of message for the plasma membrane H⁺-ATPase differed from the patterns associated with the 70 kDa subunit of the tonoplast H⁺-ATPase. NaCl-induced accumulation of the plasma membrane H⁺-ATPase message occurred in both roots and expanded leaves. Initially accumulation of the plasma membrane H⁺-ATPase message was greater in root tissue than in expanded leaves, but increased to higher levels in expanded leaves after 7 days. These results suggest that increased expression of the tonoplast H⁺-ATPase is an early response to salinity stress and may be associated with survival mechanisms, rather than with long-term adaptive processes.     

Tonoplast H+ pumps are activated during callus formation of tuber tissues of Jerusalem artichoke (Helianthus tuberosus)

Changes in tonoplast H⁺-ATPase (EC 3.6.1.3) and H⁺–PPase (EC 3.6.1.1) activities were examined during the early period of callus formation in tuber tissues of Jerusalem artichoke ( Helianthus tuberosus L.). In callus-forming tissues cultured on a medium containing 2,4-D, the ATP-dependent H⁺-translocation activity of tonoplast vesicles increased 3-fold after a 2-day lag phase, while the ATP-hydrolytic activity and amount of tonoplast H⁺-ATPase protein were relatively constant after the lag phase. In the control tissue disks cultured on a medium free of 2,4-D, large declines in ATP-hydrolytic and ATP-dependent H⁺-translocation activities were observed. By contrast, the PP-dependent H⁺-translocation activity of tonoplast vesicles increased about 8-fold during the first 3 days of culture without any lag phase, and regardless of the presence of 2,4-D in the culture medium. However, the PP-hydrolytic activity and amount of H⁺-PPase protein did not change during the culture period, independently of callus formation. Transfer of the control tissue disks to the 2,4-D-containing medium, however, resulted in a further rapid stimulation of PP-dependent H⁺-translocation as well as an activation of ATP-dependent H⁺-translocation. These results suggest that both tonoplast H⁺ pumps are involved in callus formation of tuber tissues of Jerusalem artichoke.     

Na+/H+ antiport activity in tonoplast vesicles from roots of the salt-tolerant Plantago maritima and the salt-sensitive Plantago media

Plantago species differ in their strategy towards salt stress, a major difference being the uptake and distribution of Na⁺ ions. A salt-sensitive ( Plantago media L.) and a salt-tolerant ( P. maritima L.) species were compared with respect to Na⁺/H⁺ antiport activities at the tonoplast. After exposure of the plants to 50 m M NaCl for 6 days isolated tonoplast vesicles of P. maritima showed Na⁺/H⁺ antiport activity with saturation kinetics and a K_m of 2.4 m M Na⁺, NaCl-grown P. media and the control plants of both species showed no antiport activity. Selectivity of the antiport system for Na⁺ was high and was determined by adding different chloride salts after formation of a Δ pH in the vesicles. Specific tonoplast ATPase activities were similar in the two species and did not alter after exposure to NaCl stress.     

Pyrophosphate import and synthesis by plant mitochondria

The matrix level of pyrophosphate (PPi) in mitochondria isolated from etiolated pea ( Pisum sativum L. cv. Alaska) stems was evaluated, on the basis of an enzymatic assay, to be approx. 0.2 m M . Pyrophosphate could enter from the cytoplasm to the mitochondria via adenine nucleotide translocase (ANT), because F^– and Ca²⁺ (two penetrating PPiase inhibitors) and atractylate (ANT inhibitor) inhibited PPiase activity in isolated mitochondria supplied with PPi. This result was also confirmed by measuring oxygen consumption and membrane potential (ΔΨ) in succinate-energized mitochondria. In a medium free of phosphate (Pi), the addition of PPi before the substrate rendered possible an ADP-stimulated oxygen consumption that was inhibited by F^– or Ca²⁺. In a similar experiment, ADP induced the dissipation of ΔΨ when it was added after the succinate-generated ΔΨ had reached a steady state and, again, F^– inhibited this dissipation. These results imply that PPi enters the mitochondria where it is hydrolyzed to 2 Pi which become available for the H⁺-ATPase (EC 3.6.1.34). In addition, PPi may be synthesized by the H⁺-PPiase (EC 3.6.1.1), acting as a synthase. This evidence arises from the observation that Pi stimulated an oxygen consumption (respiratory control ratio of 1.7) that was inhibited by F^– or Ca²⁺. The physiological role of the mitochondrial H⁺-PPiase is discussed in the light of the consideration that this enzyme can catalyse a readily reversible reaction.     

Proton pumping pyrophosphatase from higher plant mitochondria

In higher plant cells, there are some enzymes capable of utilizing pyrophosphate (PP_i) as an energy donor. Among these, membrane-bound proton pumping pyrophosphatases (H⁺-PP_iase) have been identified. In addition to the well-known vacuolar H⁺-PP_iase (V-PP_iase), there is evidence for the presence of a mitochondrial H⁺-PP_iase. This enzyme is localized on the inner surface of the inner membrane and catalyzes the specific hydrolysis of PP_i, coupled to proton transport, with a H⁺/PP_i stoichiometry of ca 2. This activity is Mg²⁺-requiring, is stimulated by monovalent cations, and is inhibited by Ca²⁺, F⁻ and diphosphonates. The H⁺-PP_iase contains a catalytic head which is constituted by a 35-kDa protein which is loosely bound to the inner membrane. This protein exhibits a PP_iase activity, stimulated by phospholipids, with characteristics very similar to the membrane-bound enzyme. The mitochondrial PP_iase is distinct from the V-PP_iase, because an antibody raised against the 35-kDa protein does not react with tonoplast membranes. The mitochondrial H⁺-PP_iase seems to have an F-type structure, similar to the F-ATP synthase and the membrane-bound PP_iases from mammalian and yeast mitochondria. It is suggested that, beside synthesizing PP_i, this enzyme may act as a buffer for the electrochemical proton gradient, by hydrolyzing PP_i, during conditions of oxygen deprivation.     

The F1F0-ATPase of Bifidobacterium animalis is involved in bile tolerance

Adaptation and tolerance to bile stress are important factors for the survival of bifidobacteria in the intestinal tract. Bifidobacterium animalis is a probiotic microorganism which has been largely applied in fermented dairy foods due to its technological properties and its health-promoting effects for humans. The effect of the presence of bile on the activity and expression of F₁F₀-ATPase, the pool of ATP and the intracellular pH of B. animalis IPLA 4549 and its mutant with acquired resistance to bile B. animalis 4549dOx was determined. The bile-resistant mutant tolerated the acid pH better than its parent strain. Bile induced the expression of the F₁F₀-ATPase and increased the membrane-bound H⁺-ATPase activity, in both parent and mutant strains. In acidic conditions (pH 5.0), the expression and the activity of this enzyme were higher in the mutant than in the parent strain when cells were grown in the absence of bile. Total ATP content was higher for the mutant in the absence of bile, whereas the presence of bile induced a decrease of intracellular ATP levels, which was much more pronounced for the parent strain. At pH 4.0, and independently on the presence or absence of bile, the mutant showed a higher intracellular pH than its parent strain. These findings suggest that the bile-adapted B. animalis strain is able to tolerate bile by increasing the intracellular ATP reserve, and by inducing proton pumping by the F₁F₀-ATPase, therefore tightly regulating the internal pH, and provide a link between the physiological state of the cell and the response to bile.     

The hexose transporters at the plasma membrane and the tonoplast of higher plants

The uptake of hexoses in higher plant cells is thought to be catalyzed by an H⁺/hexose contrasporter in the plasma membrane. Transport studies with isolated plant vacuoles indicate that, at the tonoplast, a second hexose transporter is located with properties different from the plasma membrane transporter. Recently membrane vesicles of high purity and defined orientation have been used for a more rigorous individual characterization of these two carriers. Concomitantly, a cDNA for the inducible H⁺/hexose cotransporter of the green alga Chlorella has been sequenced and shown to exhibit homology to a group of hexose transporters (for facilitated diffusion) of other eukaryotic and prokaryotic organisms. With a probe derived from the Chlorella sequence, the first plant gene for an H⁺/hexose contransporter ( Arabidopsis thaliana ) has been isolated, opening the route to molecular studies of structure, function and evolution of the hexose transporters of higher plants. The present review discusses recent work on the kinetic characterization and identification of the higher plant plasma membrane and tonoplast hexose transporters as well as their respective cellular functions. Furthermore, perspectives for future research on the plant hexose transporters are outlined.     

Demonstration of a K+/H+ exchange activity in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Abstract Washed cells of Rhodopseudomonas sphaeroides forma sp. denitrificans , grown under photodenitrifying conditions, exhibited K⁺ uptake dependent on the transmembrane proton gradient (Δ pH). These cells also acidified the suspension medium in response to K⁺ pulses both aerobically and anaerobically in light and in the dark. The results indicate that the photodenitrifier has a reversible K⁺/H⁺ exchange activity which reflects its role in regulating the intracellular K⁺ concentration, as well as intracellular pH. The acidification of the external medium resulting from K⁺ pulses was inhibited by carbonyl cyanide- m -chlorophenylhydrazone (CCCP) indicating that the antiporter is energy-dependent. Addition of KCl to washed cells depolarized the membrane potential (Δψ) with a concomitant increase in ΔpH, indicating that the K⁺/H⁺ antiporter was electrogenic.     

Two Na^＋ and Cl^- Hyperaccumulators of the Chenopodiaceae

The authors found five sodium (Na^ ) and chloride (Cl^-) hyperaccumulating halophytes in the Temperate Desert of Xinjiang, China and studied two of them (Suaeda salsa (L.) Pall. and Kalidium folium (Pall.) Moq.). K. folium and S. salsa had a NaCl content of 32.1% and 29.8%, respectively, on a dry weight basis. X-ray microanalysis of the Na in the vacuole, apoplasts and cytoplasm of the two plants indicated a ratio of 7.3:5.6:1.0 in K. folium and 7.3:6.6:1.0 in S. salsa. These data show that K. folium and S. salsa both have a high Na and Cl^- accumulating capacity, which is related to high activity of tonoplast H^ -ATPase and H^ -PPase.     

The role of Mg2+ in proton transport by the tonoplast pyrophosphatase in Riccia fluitans vacuoles

The Mg²⁺-dependent activity of the tonoplast pyrophosphatase (PPase) was investigated by measuring proton transport and by using the acridine orange technique on intact vacuoles of the aquatic liverwort Riccia fluitans L. In solutions with both Mg²⁺ and pyrophosphate present, a number of complexes are formed, which could all influence the enzymatic and hence the transport activity of the PPase. Therefore, the individual concentrations of these complexes were calculated and their contributions to proton transport across the tonoplast were tested. From these experiments we conclude that Mg²⁺ has three different roles: (i) Mg²⁺ stimulates transport activity of the PPase. (ii) Mg₂PP_i inhibits PPase-mediated H⁺ transport, (iii) MgPP_i* (= MgPP_i^2-+ MgHPP_i^-) is the substrate with an apparent K_1/2= 5–10 μM, with no discrimination between MgPP_i^2- and MgHPP_i^-.     

Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast

Transport across the plasma membrane is driven by an electrochemical gradient of H⁺ ions generated by the plasma membrane proton pump (H⁺-ATPase). Random mutants of Arabidopsis H⁺-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H⁺-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H⁺ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H⁺ extrusion and in their ability to sustain an electrochemical H⁺ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H⁺-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H⁺ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters.     

Evidence for an indirect coupling mechanism for the nitrate-sensitive proton pump from corn root tonoplast membranes

The nitrate-sensitive proton-translocating adenosine triphosphatase (H⁺-ATPase) of tonoplast membranes plays an important role in regulating the flow of nutrients and metabolic waste between the cytoplasm and vacuole in the cells of plant roots. Relatively little information is available regarding the coupling between ATP hydrolysis and proton pumping by the nitrate-sensitive, tonoplast H⁺-ATPase. The coupling may be achieved either directly, i. e. the two reaction pathways share at least one common molecular step, or indirectly, i. e. the two reaction pathways do not share an intermediate step. These coupling mechanisms may be differentiated by the responses of the two events to external perturbation. The effects of the presence of nitrate in the assay medium on the rates of ATP hydrolysis and proton transport catalyzed by the tonoplast H⁺-ATPase from maize ( Zea mays L. cv. FRB 73) were investigated. The presence of nitrate inhibited proton transport activity of the tonoplast H⁺-ATPase to a much greater degree than ATP hydrolysis. This differential response of the two activities to nitrate is the basis for a proposed reaction model for the tonoplast H⁺-ATPase that features an indirect coupling mechanism between ATP hydrolysis and proton transport.     

Cation-stimulated H+ efflux by intact roots of barley

Abstract. Rates of proton extrusion and potassium (⁸⁶Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K⁺ concentrations (0.01 to 1 mol m⁻³), H⁺ efflux and K⁺ influx were strongly correlated in both low- and high-K⁺ roots, although K⁺: H⁺ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m⁻³), H⁺ efflux was either reduced or remained unchanged while K⁺ influxes increased. In the presence of Na₂SO₄, rates of H⁺ extrusion demonstrated similar cation dependence, although below 10 mol m⁻³ Na₂SO₄, H⁺ fluxes were generally 50% lower than in equivalent concentrations of K₂SO₄. These observations are considered in the context of current hypotheses regarding the mechanisms of k⁺/H⁺ exchange.     

Measurement of intracellular pH in maize root tissue with α-methyl fluorinated alanines and in-vivo 19F NMR spectroscopy

Methods for the simultaneous measurement of vacuolar and cytoplasmic pH in plant tissues currently have significant limitations. This study demonstrates the usefulness of methyl difluoro alanine (F₂ALA) and methyl trifluoro alanine (F₃ALA) with in-vivo ¹⁹F NMR spectroscopy to measure vacuolar and cytoplasmic pH in maize root tissue. The pH dependence of the chemical shift of F₂ALA and F₃ALA is greater than either the commonly used ³¹P NMR signal of inorganic phosphate or the ¹³C NMR signals of trans-aconitic acid, which is also found in some plant cells. F₂ALA and F₃ALA were also able to detect changes over a greater range of pH. When maize root tissue was incubated in the presence of 0.35 m m F₂ALA or F₃ALA, these accumulated to significant concentrations in two compartments of different pH with no significant effect on growth rate of root tips. The time course of accumulation and the pH of the two compartments were consistent with one being the cytoplasm and the other the vacuole. The chemical shift of both C₂ of trans-aconitic acid and vacuolar F₃ALA indicated that the mean vacuolar pH of maize root cells was 4.6 and that the pH gradient across the tonoplast membrane was about 2.8 units. Under a variety of conditions, there was considerable heterogeneity in the pH of the vacuoles in maize root tissue as indicated by the peak width of the signal from F₃ALA. The significance of these values is discussed in terms of the bioenergetics of proton transport across the tonoplast membrane in vivo.     

H+/PPi stoichiometry of a membrane‐bound pyrophosphatase of plant mitochondria

The H⁺/PP_i stoichiometry of the mitochondrial H⁺‐PP_iase from pea ( Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H⁺/substrate stoichiometries of the mitochondrial H⁺‐ATPase, and the vacuolar H⁺‐PP_iase and H⁺‐ATPase. Using sub‐mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate‐dependent H⁺‐transport were evaluated: by a mathematical model, describing the time‐course of H⁺‐gradient (ΔpH) formation; or by determining the rate of H⁺‐leakage following H⁺‐pumping inhibition by EDTA at the steady‐state ΔpH. When the H⁺‐transport rates were divided by those of PP_i or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H⁺‐PP_iase and H⁺‐ATPase, and for the vacuolar H⁺‐ATPase. The stoichiometry of the vacuolar H⁺‐PP_iase was found to be ca 1. From these results, it is suggested that the mitochondrial H⁺‐PP_iase may, in theory, function as a primary H⁺‐pump poised towards synthesis of PP_i and, therefore, acting in parallel with the main H⁺‐ATPase.