The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy.

The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate. Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylcarbodiimide. Since substrate-level phosphorylation does not occur during malolactic fermentation, the generation of metabolic energy must originate from the uptake of L-malate and/or excretion of L-lactate. The initiation of malolactic fermentation is stimulated by the presence of L-lactate intracellularly, suggesting that L-malate is exchanged for L-lactate. Direct evidence for heterologous L-malate/L-lactate (and homologous L-malate/L-malate) antiport has been obtained with membrane vesicles of an L. lactis mutant deficient in malolactic enzyme. In membrane vesicles fused with liposomes, L-malate efflux and L-malate/L-lactate antiport are stimulated by a membrane potential (inside negative), indicating that net negative charge is moved to the outside in the efflux and antiport reaction. In membrane vesicles fused with liposomes in which cytochrome c oxidase was incorporated as a proton motive force-generating mechanism, transport of L-malate can be driven by a pH gradient alone, i.e., in the absence of L-lactate as countersubstrate. A membrane potential (inside negative) inhibits uptake of L-malate, indicating that L-malate is transported an an electronegative monoanionic species (or dianionic species together with a proton). The experiments described suggest that the generation of metabolic energy during malolactic fermentation arises from electrogenic malate/lactate antiport and electrogenic malate uptake (in combination with outward diffusion of lactic acid), together with proton consumption as result of decarboxylation of L-malate. The net energy gain would be equivalent to one proton translocated form the inside to the outside per L-malate metabolized.     

Uniport of anionic citrate and proton consumption in citrate metabolism generates a proton motive force in Leuconostoc oenos.

The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated. Resting cells of L. oenos generate both a membrane potential (delta psi) and a pH gradient (delta pH) upon addition of citrate. After a lag time, the internal alkalinization is followed by a continuous alkalinization of the external medium, demonstrating the involvement of proton-consuming reactions in the metabolic breakdown of citrate. Membrane vesicles of L. oenos were prepared and fused to liposomes containing cytochrome c oxidase to study the mechanism of citrate transport. Citrate uptake in the hybrid membranes is inhibited by a membrane potential of physiological polarity, inside negative, and driven by an inverted membrane potential, inside positive. A pH gradient, inside alkaline, leads to the accumulation of citrate inside the membrane vesicles. Kinetic analysis of delta pH-driven citrate uptake over a range of external pHs suggests that the monovalent anionic species (H2cit-) is the transported particle. Together, the data show that the transport of citrate is an electrogenic process in which H2cit- is translocated across the membrane via a uniport mechanism. Homologous exchange (citrate/citrate) was observed, but no evidence for a heterologous antiport mechanism involving products of citrate metabolism (e.g., acetate and pyruvate) was found. It is concluded that the generation of metabolic energy by citrate utilization in L. oenos is a direct consequence of the uptake of the negatively charged citrate anion, yielding a membrane potential, and from H(+)-consuming reactions involved in subsequent citrate metabolism, yielding a pH gradient. The uptake of citrate is driven by its own concentration gradient, which is maintained by efficient metabolic breakdown (metabolic pull).     

Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.

Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative delta psi and is stimulated by an inside positive delta psi. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.     

Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides.

In Leuconostoc mesenteroides subsp. mesenteroides 19D, citrate is transported by a secondary citrate carrier (CitP). Previous studies of the kinetics and mechanism of CitP performed in membrane vesicles of L. mesenteroides showed that CitP catalyzes divalent citrate HCit2-/H+ symport, indicative of metabolic energy generation by citrate metabolism via a secondary mechanism (C. Marty-Teysset, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Biol. Chem. 270:25370-25376, 1995). This study also revealed an efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate cometabolism, suggesting that under physiological conditions, CitP may function as a precursor/product exchanger rather than a symporter. In this paper, the energetic consequences of citrate metabolism were investigated in resting cells of L. mesenteroides. The generation of metabolic energy in the form of a pH gradient (delta pH) and a membrane potential (delta psi) by citrate metabolism was found to be largely dependent on cometabolism with glucose. Furthermore, in the presence of glucose, the rates of citrate utilization and of pyruvate and lactate production were strongly increased, indicating an enhancement of citrate metabolism by glucose metabolism. The rate of citrate metabolism under these conditions was slowed down by the presence of a membrane potential across the cytoplasmic membrane. The production of D-lactate inside the cell during cometabolism was shown to be responsible for the enhancement of the electrogenic uptake of citrate. Cells loaded with D-lactate generated a delta psi upon dilution in buffer containing citrate, and cells incubated with citrate built up a pH gradient upon addition of D-lactate. The results are consistent with an electrogenic citrate/D-lactate exchange generating in vivo metabolic energy in the form of a proton electrochemical gradient across the membrane. The generation of metabolic energy from citrate metabolism in L. mesenteroides may contribute significantly to the growth advantage observed during cometabolism of citrate and glucose.     

The role of transport processes in survival of lactic acid bacteria, Energy transduction and multidrug resistance

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural an d functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist.     

A respiration-dependent primary sodium extrusion system functioning at alkaline pH in the marine bacterium Vibrioalginolyticus

The membrane potential generated at pH 8.5 by K⁺-depleted and Na⁺-loaded $\text{Vibrio}\text{alginolyticus}$ is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na⁺-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na⁺ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na⁺-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na⁺ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na⁺ chemical gradient.     

Genetic organization and expression of citrate permease in lactic acid bacteria

Citrate is present in many natural substrates, such as milk, vegetables and fruits, and its metabolism by lactic acid bacteria (LAB) plays an important role in food fermentation. The industrial importance of LAB stems mainly from their ability to convert carbohydrates into lactic acid and, in some species, like Lactococcus lactis and Leuconostoc mesenteroides, to produce C4 flavor compounds (diacetyl, acetoin) through citrate metabolism. Three types of genetic organization and gene locations, involving citrate metabolism, have been found in LAB. Citrate uptake is mediated by a citrate permease, which leads to a membrane potential upon electrogenic exchange of divalent citrate and monovalent lactate. The internal citrate is cleaved into acetate and oxaloacetate by a citrate lyase, and oxaloacetate is decarboxylated into pyruvate by an oxaloacetate decarboxylase, yielding a pH gradient through the consumption of scalar protons.     

Growth and Energy Generation by Lactococcus lactis subsp. lactis biovar diacetylactis during Citrate Metabolism

Growth of Lactococcus lactis subsp. lactis biovar diacetylactis was observed on media with citrate as the only energy source. At pH 5.6, steady state was achieved in a chemostat on a citrate-containing medium in the absence of a carbohydrate. Under these conditions, pyruvate, acetate, and some acetoin and butanediol were the main fermentation products. This indicated that energy was conserved in L. lactis subsp. lactis biovar diacetylactis during citrate metabolism and presumably during the conversion of citrate into pyruvate. The presumed energy-conserving step, decarboxylation of oxaloacetate, was studied in detail. Oxaloacetate decarboxylase was purified to homogeneity and characterized. The enzyme has a native molecular mass of approximately 300 kDa and consists of three subunits of 52, 34, and 12 kDa. The enzyme is apparently not sodium dependent and does not contain a biotin moiety, and it seems to be different from the energy-generating oxaloacetate decarboxylase from Klebsiella pneumoniae. Energy-depleted L. lactis subsp. lactis biovar diacetylactis cells generated a membrane potential and a pH gradient immediately upon addition of citrate, whereas ATP formation was slow and limited. In contrast, lactose energization resulted in rapid ATP formation and gradual generation of a proton motive force. These data were confirmed during studies on amino acid uptake. α-Aminoisobutyrate uptake was rapid but glutamate uptake was slow in citrate-energized cells, whereas lactose-energized cells showed the reverse tendency. These data suggest that, in L. lactis subsp. lactis bv. diacetylactis, a proton motive force could be generated during citrate metabolism as a result of electrogenic citrate uptake or citrate/product exchange together with proton consumption by the intracellular oxaloacetate decarboxylase.     

Overproduction and purification of a functional Na+/H+ antiporter coded by nhaA (ant) from Escherichia coli.

A Na+/H+ antiporter coded by the nhaA (ant) gene of Escherichia coli has been overproduced and purified. The amino-terminal sequence of the protein has been determined and shown to correlate with initiation at a GUG codon, 75 bases upstream from the previously suggested AUG initiation codon. The purified protein, when reconstituted into proteoliposomes, has Na+/H+ antiport activity. It can mediate sodium uptake when a transmembrane pH gradient is applied. Downhill sodium efflux is shown to be highly dependent on pH and is accelerated by a transmembrane pH gradient. An imposed membrane potential negative inside accelerates Na+ efflux at all pH values tested. These findings suggest that the antiporter is electrogenic both at acid and alkaline pH. The activation at alkaline pH values (2000-fold increase) is consistent with the proposed role of the antiporter in regulation of internal pH at the alkaline pH range.     

The effect of trypsin treatment on the incorporation and energy-transducing properties of bacteriorhodopsin in liposomes

Bacteriorhodopsin and trypsin-modified bacteriorhodopsin have been reconstituted into liposomes by means of a low pH-sonication procedure. The incorporation of bacteriorhodopsin in these proteoliposomes is predominantly in the same direction as in vivo and the direction of proton pumping is from inside to outside the liposomes. The direction of proton translocation and electrical potential generation was studied as a function of the reconstitution pH. Light-dependent proton extrusion and generation of a Δp, interior negative and alkaline was observed at a reconstitution pH below 3.0 using bacteriorhodopsin, and at a pH below 3.5 using trypsin-modified bacteriorhodopsin. The shift in inflection point is explained in terms of differences between bacteriorhodopsin and trypsin-modified bacteriorhodopsin in a specific protein-phospholipid interaction which depends on the surface charge density of the cytoplasmic side of bacteriorhodopsin. The magnitude of the protonmotive force (Δp) generated by trypsin-modified bacteriorhodopsin in liposomes was quantitated. Illumination of the proteoliposomes resulted in the generation of a high Δp (135 mV, inside negative and alkaline), with a major contribution of the pH gradient. The ionophores nigericin and valinomycin induced, respectively, a compensatory interconversion of ΔpH into Δψ and vice versa. If no endogenous proton permeability of the membrane would exist, a protonmotive force could be generated of − 143 mV as electrical potential alone or − 162 mV as pH gradient alone.     

The proton motive force generated in Leuconostoc oenos by L-malate fermentation.

In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi. Synthesis of ATP was observed in cells performing malolactic fermentation.     

Transport of Charged Dipeptides by the Intestinal H+/Peptide Symporter PepT1 Expressed in Xenopus laevis Oocytes

The cloned intestinal peptide transporter is capable of electrogenic H⁺-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K_0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K_0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5–8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H⁺/H₃O⁺ cotransport may vary depending on pH. Received: 19 August 1996/Revised: 10 October 1996     

Proton-coupledl-lysine uptake by renal brush border membrane vesicles from mullet (Mugil cephalus)

The uptake of the basic amino acid, L-lysine, was studied in brush border membrane vesicles isolated from the kidney of the striped mullet (Mugil cephalus). The uptake of L-lysine was not significantly stimulated by a Na+ gradient and no overshoot was observed. However, when a proton gradient (pHo = 5.5; pHi = 8.3) was imposed across the membrane in the absence of Na+, uptake was transiently stimulated. When the proton gradient was short circuited by the proton ionophore, carbonylcyanide p-triflouromethoxyphenyl hydrazone, proton gradient-dependent uptake of lysine was inhibited. Kinetics of lysine uptake determined under equilibrium exchange conditions indicated that the Vmax increased as available protons increased (2.1 nmol/min/mg protein at pH 7.5 to 3.7 nmol/min/mg at pH 5.5), whereas the apparent Km (4.9 +/- 0.6 mM) was not altered appreciably. When membrane potential (inside negative) was imposed by K+ diffusion via valinomycin, a similar (but smaller) stimulation of lysine uptake was observed. When the membrane potential and the proton gradient were imposed simultaneously, a much higher stimulation in lysine uptake was shown, and the uptake of lysine was approximately the sum of the components measured separately. These results indicate that the uptake mechanism for basic amino acids is different from that of neutral or acidic amino acids and that the proton-motive force can provide the driving force for the uptake of L-lysine into the isolated brush border membrane vesicles.     

Na+/HCO3-co-transport in basolateral membrane vesicles isolated from rabbit renal cortex

Recent studies suggest that the major pathway for exit of HCO3- across the basolateral membrane of the proximal tubule cell is electrogenic Na+/HCO3- co-transport. We therefore evaluated the possible presence of Na+/HCO3- co-transport in basolateral membrane vesicles isolated from the rabbit renal cortex. Imposing an inward HCO3- gradient induced the transient uphill accumulation of Na+, and imposing an outward Na+ gradient caused HCO3- -dependent generation of an inside-acid pH gradient as monitored by quenching of acridine orange fluorescence, findings consistent with the presence of Na+/HCO3- co-transport. In the absence of other driving forces, generating an inside-positive membrane potential by imposing an inward K+ gradient in the presence of valinomycin caused net Na+ uptake via a HCO3- -dependent pathway, indicating that Na+/HCO3- co-transport is electrogenic and associated with a flow of negative charge. Imposing transmembrane Cl- gradients did not appreciably affect HCO3- gradient-stimulated Na+ influx, suggesting that Na+/HCO3- co-transport is not Cl- -dependent. The rate of HCO3- gradient-stimulated Na+ influx was a simple, saturable function of the Na+ concentration (Km = 9.7 mM, Vmax = 160 nmol/min/mg of protein), was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (I50 = 100 microM), but was inhibited less than 10% by up to 1 mM amiloride. We could not demonstrate a HCO3- -dependent or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive component of Na+ influx in microvillus membrane vesicles. This study thus indicates the presence of a transport system mediating electrogenic Na+/HCO3- co-transport in basolateral, but not luminal, membrane vesicles isolated from the rabbit renal cortex. Analogous to the use of renal microvillus membrane vesicles to study Na+/H+ exchange, renal basolateral membrane vesicles may be a useful model system for examining the kinetics and possible regulation of Na+/HCO3- co-transport.     

Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH

Measurement of the flux through the citrate fermentation pathway in resting cells of Lactococcus lactis CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH. The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells. The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism. The results clearly demonstrate that citrate metabolism in L. lactis is a secondary proton motive force-generating pathway. Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of L. lactis in rich media. However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium. Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition. The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity. It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.     

The cell membrane and the struggle for life of lactic acid bacteria

The major life-threatening event for lactic acid bacteria (LAB) in their natural environment is the depletion of their energy sources and LAB can survive such conditions only for a short period of time. During periods of starvation LAB can exploit optimally the potential energy sources in their environment usually by applying proton motive force generating membrane transport systems. These systems include in addition to the proton translocating F_oF₁-ATPase: a respiratory chain when hemin is present in the medium, electrogenic solute uptake and excretion systems, electrogenic lactate/proton symport and precursor/ product exchange systems. Most of these metabolic energy-generating systems offer as additional bonus the prevention of a lethal decrease of the internal and external pH. LAB have limited biosynthetic capacities and rely heavily on the presence of essential components such as sources of amino acids in their environment. The uptake of amino acids requires a major fraction of the available metabolic energy of LAB. The metabolic energy cost of amino acid uptake can be reduced drastically by accumulating oligopeptides instead of the individual amino acids and by proton motive force-generating efflux of excessively accumulated amino acids. Other life-threatening conditions that LAB encounter in their environment are rapid changes in the osmolality and the exposure to cytotoxic compounds, including antibiotics. LAB respond to osmotic upshock or downshock by accumulating or releasing rapidly osmolytes such as glycine-betaine. The life-threatening presence of cytotoxic compounds, including antibiotics, is effectively counteracted by powerful drug extruding multidrug resistance systems. The number and variety of defense mechanisms in LAB is surprisingly high. Most defense mechanisms operate in the cytoplasmic membrane to control the internal environment and the energetic status of LAB. Annotation of the functions of the genes in the genomes of LAB will undoubtely reveal additional defense mechanisms.     

Electrogenic L-malate transport by Lactobacillus plantarum: a basis for energy derivation from malolactic fermentation.

L-Malate transport in Lactobacillus plantarum was inducible, and the pH optimum was 4.5. Malate uptake could be driven by an artificial proton gradient (delta pH) or an electroneutral lactate efflux. Because L-lactate efflux was unable to drive L-malate transport in the absence of a delta pH, it did not appear that the carrier was a malate-lactate exchanger. The kinetics of malate transport were, however, biphasic, suggesting that the external malate concentration was also serving as a driving force for low-affinity malate uptake. Because the electrical potential (delta psi, inside negative) inhibited malate transport, it appeared that the malate transport-lactate efflux couple was electrogenic (net negative) at high concentrations of malate. De-energized cells that were provided with malate only generated a large proton motive force (greater than 100 mV) when the malate concentration was greater than 5 mM, and malate only caused an increase in cell yield (glucose-limited chemostats) when malate accumulated in the culture vessel. The use of the malate gradient to drive malate transport (facilitated diffusion) explains how L. plantarum derives energy from malolactic fermentation, a process which does not involve substrate-level phosphorylation.     

Calcium transport driven by a proton gradient and inverted membrane vesicles of Escherichia coli.

Calcium transport into inverted vesicles of Escherichia coli was observed to occur without an exogenous energy source when an artificial proton gradient was used. The orientation of the proton gradient was acid inside and alkaline outside. Either phosphate or oxalate was necessary for transport, as was found for respiratory-driven or ATP-driven uptake (Tsuchiya, T., and Rosen, B.P. (1975) J. Biol. Chem. 250, 7687-7692). Phosphate accumulation was found to occur in conjunction with calcium accumulation. Calcium transport driven by an artificial proton gradient was stimulated by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ATPase (EC 3.6.1.3). Valinomycin, which catalyzes electrogenic potassium movement, stimulated calcium accumulation, while nigericin, which catalyzes electroneutral exchange of potassium and protons, inhibited both artificial proton gradient-driven transport and respiratory-driven transport. Other properties of the proton gradient-driven system and the previously reported energy-linked calcium transport system are similar, indicating that calcium is transported by the same carrier whether energy is supplied through an artificial proton gradient or an energized membrane state. These results suggest the existence of a calcium/proton antiport.     

Ion-dependent generation of the electrochemical proton gradient delta muH+ in reconstituted plasma membrane vesicles from the yeast Metschnikowia reukaufii

Plasma membrane vesicles were reconstituted by freezing and thawing of purified plasma membrane fraction from the yeast Metschnikowia reukaufii and phosphatidylcholine (type II-S from Sigma). The reconstituted plasma membrane vesicles generated a proton gradient (acidic inside) upon addition of ATP in presence of alkali cations. delta pH generation was most efficient when K+ was present both outside and inside the plasma membrane vesicles. Both ATPase activity and proton translocation in plasma membrane vesicles were inhibited by orthovanadate (50% inhibition at 100 microM). Plasma membrane vesicles reconstituted without added phosphatidylcholine generated in addition to delta pH, also an electrical potential difference delta psi (inside positive). Delta psi generation exhibited no K+ specificity. 50 microM dicyclohexylcarbodiimide inhibited completely delta psi generation whereas the K+-channel blocker quinine (5 microM) caused an 8-fold increase of delta psi. The proton gradient was much less affected by the agents. Taking into account the K+-dependent stimulation of the plasma membrane ATPase of M. reukaufii, these results further support the conclusion that the ATPase operates as a partially electrogenic H+/K+ exchanger, as was also suggested for other yeast plasma membrane ATPases.