Cytochemical localization of ATPase activity in oat roots localizes a plasma membrane-associated soluble phosphatase, not the proton pump

Cytochemical techniques employing lead-precipitation of enzymically released inorganic phosphate have been widely used in attempts to localize the plasma membrane proton pump (H⁺-ATPase) in electron micrographs. Using Avena sativa root tissue we have performed a side-by-side comparison of ATPase activity observed in electron micrographs with that observed in in vitro assays using ATPases found in the soluble and plasma membrane fractions of homogenates. Cytochemical analysis of oat roots, which had been fixed in glutaraldehyde in order to preserve subcellular structures, identifies an ATPase located at or near the plasma membrane. However, the substrate specificity and inhibitor sensitivity of the in situ localized ATPase appear identical to those of an in vitro ATPase activity found in the soluble fraction, and are completely unlike those of the plasma membrane proton pump. Further studies demonstrated that the plasma membrane H⁺-ATPase is particularly sensitive to inactivation by the fixatives glutaraldehyde and formaldehyde and by lead. In contrast, the predominant soluble ATPase activity in oat root homogenates is less sensitive to fixation and is completely insensitive to lead. Based on these results, we propose a set of criteria for evaluating whether a cytochemically localized ATPase activity is, in fact, due to the plasma membrane proton pump.     

Mechanistic investigation on the temperature dependence and inhibition of corn root plasma membrane ATPase

The kinetics of corn root plasma membrane-catalyzed Mg-ATP hydrolysis may be satisfactorily described by a simple Michaelis-Menten scheme. It was found that the Km of the process was relatively insensitive to changes in temperature. This property allowed us to conveniently estimate the activation energy of the enzyme turnover process as approximately 14 kcal mol-1 in the temperature range of 10 to 45 degrees C. The enzyme activity was inhibited by the presence of diethystilbestrol (DES), miconazole, vanadate, and dicyclohexylcarbodiimide (DCCD). The inhibition caused by DES and miconazole was strictly uncompetitive and inhibition by vanadate was noncompetitive. The inhibition by DCCD showed a substrate concentration dependence, i.e., competitive at high and uncompetitive at low concentrations of Mg-ATP. The 1/V vs [I] plots suggested that there were different but unique binding sites for DES, vanadate, and miconazole. However, the modification of the plasma membrane by DCCD exhibited interaction with multiple sites. Unlike yeast plasma membrane ATPase, the enzyme of corn root cells was not affected by the treatment with N-ethylmaleimide. Although the enzyme activity was regulated by ADP, a product of the reaction, the presence of inorganic phosphate showed no inhibition to the hydrolysis of Mg-ATP.     

The effect of vanadate on proton-sucrose cotransport in ricinus cotyledons

The effects of orthovanadate on the uptake of sucrose by Ricinus cotyledons and on sucrose-coupled proton influx were measured in order to gain insight into the relationship to the plasma membrane proton pump. Vanadate had no effect on short-term sucrose uptake. In longterm experiments (>30 min) sucrose uptake was progressively inhibited, but only at high external sucrose concentrations. Vanadate did not affect proton efflux pumping in the absence of sucrose and neither did it change the initial rate of sucrose-coupled proton influx. However, it enhanced the maximal level of sucrose-induced alkalinization of the medium at all sucrose concentrations tested. This is interpreted as an inhibiting effect of vanadate on the proton pump that recycles protons during sucrose-proton cotransport. The sensitivity towards vanadate indicates that this proton pump is an ATPase. A second proton-translocating system, that is insensitive to vanadate, is postulated to function in the absence of sucrose.     

Plasma membrane proton pump inhibition and stalk cell differentiation in Dictyostelium discoideum

The choice of the stalk cell differentiation pathway in Dictyostelium is promoted by an endogenous substance, DIF-1, which is 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone. It is also favoured by weak acids and two inhibitors of the plasma membrane proton pumps of fungi and plants, diethylstilbestrol (DES) and zearalenone, and antagonised by ammonia and other weak bases, which promote spore differentiation. These observations led to the proposal that the choice of differentiation pathway is regulated by intracellular pH. They also prompted the conjecture that DIF-1 itself is a plasma membrane proton pump inhibitor. We report here experiments showing that DIF-1 is not a plasma membrane proton pump inhibitor. We demonstrate that diethylstilbestrol and zearalenone do inhibit the plasma membrane proton pump of Dictyostelium and we show that there is an excellent qualitative and quantitative correlation between the inhibitory activity of these agents, and of a number of other substances, and their ability to divert differentiation from the spore to the stalk pathway. We conclude that inhibition of the plasma membrane proton pump does shift the choice of differentiation pathway in Dictyostelium towards the stalk pathway, but that DIF does not act by this route, and we propose a model for the actions of DIF and plasma membrane proton pump inhibitors in which the differentiation pathway is controlled by the pH of intracellular vesicles rather than by intracellular pH itself. The model invokes a DIF- and proton-activated vesicular chloride channel whose opening permits acidification of the vesicles and lowers cytosolic Ca++ concentration.     

Calcium activates an electrogenic proton pump in neurospora plasma membrane

Calcium ionophoresis into coenocytic cells of Neurospora crassa activates the plasma membrane proton pump as measured by current-voltage analysis. This is direct evidence that intracellular calcium regulates the activity of a key transport enzyme found in higher plants and fungi.     

Light-Induced Proton Gradient Formation in Intact Cells of Dunaliella salina

A light-induced proton gradient (ΔpH) increase as exhibited by an increase of 9-aminoacridine fluorescence quenching is demonstrated between the external medium and the interior of the halophytic green alga Dunaliella salina. The formation and maintenance of the ΔpH is sensitive to electron transport inhibitors and to uncouplers. It is inhibited by p-chloromercuribenzenesulfonic acid (50% inhibition at 3 micromolar), which does not affect photosynthetic O₂ evolution. It is concluded that the observed ΔpH is located across the plasmalemma or the chloroplast envelope. The formation and maintenance of the light-induced proton gradient requires the presence of Na⁺. Substitution of NaCl by KCl or glycerol results in inhibition of the ΔpH formation. The proton gradient is also sensitive to ATPase and energy transfer inhibitors. It is suggested that a Na⁺/H⁺ pump mechanism may be involved in the formation of the proton gradient in intact Dunaliella cells.     

Loss of Vacuolar H+-ATPase Activity in Organelles Signals Ubiquitination and Endocytosis of the Yeast Plasma Membrane Proton pump Pma1p

Yeast mutants lacking the intracellular V-ATPase proton pump (vma mutants) have reduced levels of the Pma1p proton pump at the plasma membrane and increased levels in organelles including the vacuolar lumen. We examined the mechanism and physiological consequences of Pma1p mislocalization. Pma1p is ubiquitinated in vma mutants, and ubiquitination depends on the ubiquitin ligase Rsp5p and the arrestin-related adaptor protein Rim8p. vma mutant strains containing rsp5 or rim8 mutations maintain Pma1p at the plasma membrane, suggesting that ubiquitination is required for Pma1p internalization. Acute inhibition of V-ATPase activity with concanamycin A triggers Pma1p ubiquitination and internalization. In an endocytosis-deficient mutant (end4Δ) Pma1p is ubiquitinated but retained at the plasma membrane during concanamycin A treatment. Consistent with specificity in signaling loss of V-ATPase activity to Pma1p, another plasma membrane transporter, Mup1p, is not internalized in a vma mutant, and loss of the Mup1p adaptor Art1p does not prevent Pma1p internalization in a vma mutant. Very poor growth of vma2 rsp5-1 and vma2 rim8Δ double mutants suggests that Pma1p internalization benefits the vma mutants. We hypothesize that loss of V-ATPase-mediated organelle acidification signals ubiquitination, internalization, and degradation of a portion of Pma1p as a means of balancing overall pH homeostasis.     

Isolation and characterization of Zygosaccharomyces rouxii mutants defective in proton pumpout activity and salt tolerance

Two mutants defective in salt tolerance were identified among hygromycin B (HygB)-resistant mutants of Zygosaccharomyces rouxii. These mutants showed different phenotypes in terms of sensitivity towards high concentrations of glucose and KCl. Recovery of salt tolerance by the addition of KCl and CaCl₂ or by lowering pH (pH 4.0) was different for the two mutants. Moreover, both mutants showed lowered plasma membrane (PM-) ATPase activity and proton pumpout activity. They exhibited neither growth nor proton pumpout activity in a medium containing 5% NaCl. The proton pumpout activity was inhibited by vanadate, an inhibitor of PM-ATPase, only when cells were incubated in the presence of more than 1% NaCl. Damage of the proton pumpout activity seems to be the reason for the salt sensitivity of both mutants. We showed that it was essential for Z. rouxii cells to pump out protons under a high salt environment using mutants defective in this ability.     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa₃) is coupled to translocation of H⁺ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa₃ is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational “strain” in the cytochrome aa₃ complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

Abscisic Acid-Induced Membrane Potential Changes in Barley Aleurone Protoplasts: a Possible Relevance for the Regulation of rab Gene Expression

The effect of ABA on the membrane potential of barley (Hordeumvulgare cv. Himalaya) aleurone protoplasts was studied by measuringthe distribution of the lipophilic cation tetraphenylphosphonium(TPP⁺). The resting membrane potential (E_m) according to ourmeasurements with TPP⁺ is about –53 mV and is in agreementwith membrane potential values as measured with intracellularmicroelectrodes (about –55 mV). The TPP⁺-measurementscould demonstrate a clear dependence of the resting E_m on theexternal pH (pH_e). Stimulation of the protoplasts with ABA induced a transienthyperpolarization of the membrane to –62 mV as measuredwith TPP⁺. The hyperpolarization was ABA-concentration dependent. Inhibition of the H⁺-ATPases with the specific proton pump inhibitorsdiethylstilbestrol (DES) or Micanozole effectively preventedhyperpolarization. This indicates that the hyperpolarizationis consistent with the activation of plasma membrane H⁺-ATPases.The K⁺-inward rectifier inhibitor BaCl₂ was able to prolongthe hyperpolarization. This result suggests that the hyperpolarizationcauses the opening of K⁺-channels. The ABA-induced proton-pump activation may be involved in ABA-inducedgene-expression, as DES was able to inhibit this gene expression.BaCl₂ did only show a slight inhibitory effect on ABA-inducedgene-expression. (Received January 4, 1994; Accepted April 12, 1994)     

An inward proton transport using anabaena sensory rhodopsin

ATP is synthesized by an enzyme that utilizes proton motive force and thus nature creates various proton pumps. The best understood proton pump is bacteriorhodopsin (BR), an outward-directed light-driven proton pump in Halobacterium salinarum. Many archaeal and eubacterial rhodopsins are now known to show similar proton transport activity. Proton pumps must have a specific mechanism to exclude transport in the reverse direction to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Although an inward proton pump has neither been created naturally nor artificially, we recently reported that an inward-directed proton transport can be engineered from a bacterial rhodopsin by a single amino acid replacement Anabaena sensory rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria, possessing little proton transport activity. When we replace Asp217 at the cytoplasmic domain (distance ～15 Å from the retinal chromophore) to Glu, ASR is converted into an inward proton transport, driven by absorption of a single photon. FTIR spectra clearly show an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to normal proton pumps.     

The Role of the Plasma Membrane Proton Pump in Short-term pH Regulation in the Aquatic Liverwort Riccia fluitans L.

The question is raised, to what extent is the plasma membraneproton pump involved in short-term pH regulation of plant cells?For this purpose the cytosolic pH (pH_c) of Riccia fluitans rhizoidand thallus cells has been measured continuously using pH-sensitivemicroelectrodes (Felle and Bertl, 1986a). It is demonstratedthat pH perturbations (light, weak acids, external pH) in bothdirections are completely or at least partly eliminated withinminutes. The pH_c recovery occurs regardless of the activationstate of the proton pump. The proton pump reacts to changesin cytosolic pH as expected, namely with increased proton extrusionto decreased pH_c; however, changes in pump activity (fusicoccin,CCCP, cyanide) do not necessarily result in cytosolic pH shifts.These results suggest that several proton transport mechanisms(including the proton pump) co-operate in the restoration ofa perturbed cytosolic pH. It is concluded, however, that theproton pump, although most important for the energization ofthe plasma membrane, does not regulate cytosolic pH.     

A channel mechanism for electrogenic ion pumps

A model of active ion transport is analyzed in which an essential part of the pump molecule is an ion channel. Ion translocation in the channel is described as a series of jumps between binding sites which are separated by energy barriers. Pumping action results from a transient energy-dependent modification of the barrier structure of the channel and requires only minor conformational changes of the pump molecule. This model is applied to the lightdriven proton pump of Halobacterium and to redox-coupled proton pumps in the mitochondrial respiratory chain. Similar considerations may be used to describe ATP-dependent ion transport.     

Phase Shifting of the Circadian Clock by Diethylstilbestrol and Related Compounds in Neurospora crassa

Phase shifts of the circadian conidiation rhythm in Neurospora crassa were induced by 3-hour treatments of mycelia in liquid medium with diethylstilbestrol (DES), dienestrol (DIE), hexestrol (HEX), diethylstilbestroldipropionate (DESP), and dienestroldiacetate (DIEA). Over a 24-hour period beginning 24 hours after the transition from light to constant dark, maximum phase shifts occurred about 36 hours. DES was the most effective of the drugs tested, giving 10-hour phase advances at 20 micromolar. DIE and HEX caused similar phase shifts as DES at 40 micromolar. The two derivatives of the last, DESP and DIEA, were much less effective in shifting phase; only a few hours of phase advance result from treatments at 80 micromolar concentrations.

The activity of isolated plasma membrane ATPase was inhibited by DES and partially by HEX, but not by DIE, DESP, or DIEA. O₂ consumption of the mycelia was inhibited equally by DES, DIE, and HEX, while DIEA and DESP had little effect. Phase-shifts by DES cannot be interpreted as evidence that plasma membrane ATPase is a component of the circadian clock.

     

The membrane potential of the cellular slime mold Dictyostelium discoideum is mainly generated by an electrogenic proton pump

Trans membrane potential or ionic current changes may play a role in signal transduction and differentiation in the cellular slime mold dictyostelium discoideum. Therefore, the contribution of electrogenic ion pumps to the membrane potential of D. discoideum cells was investigated. the (negative) peak-value of the rapid potential transient, seen upon microelectrode impalement, was used to detect membrane potential changes upon changes in the external pH in the range of 5.5 to 8.0. The membrane potential was close to the Nernstian potential for protons over the pH range 5.5 to 7.5. The acid-induced changes in membrane potential were consistent with outward-proton pumping. The maximal membrane potential was at pH 7.5. Furthermore, the proton pump inhibitors diethylstilbestrol, miconazole and zearalenone directly depolarize the membrane. Cyanide and temperature decrease cause membrane depolarization as well. During recovery from cyanide poisoning a H+ efflux is present. From these measurements we conclude that the membrane potential of d. discoideum cells is mainly generated by an electrogenic proton pump. Measurements in cells with different extracellular potassium and H+ concentrations suggest a role for potassium in the function of the electrogenic proton pump. These results provide a framework for future research towards a possible role for the proton pump in signal transduction and differentiation.     

Gloeobacter Rhodopsin,Limitation of Proton Pumping at High Electrochemical Load

We studied the photocurrents of a cyanobacterial rhodopsin Gloeobacter violaceus (GR) in Xenopus laevis oocytes and HEK-293 cells. This protein is a light-driven proton pump with striking similarities to marine proteorhodopsins, including the D121-H87 cluster of the retinal Schiff base counterion and a glutamate at position 132 that acts as a proton donor for chromophore reprotonation during the photocycle. Interestingly, at low extracellular pH_o and negative voltage, the proton flux inverted and directed inward. Using electrophysiological measurements of wild-type and mutant GR, we demonstrate that the electrochemical gradient limits outward-directed proton pumping and converts it into a purely passive proton influx. This conclusion contradicts the contemporary paradigm that at low pH, proteorhodopsins actively transport H⁺ into cells. We identified E132 and S77 as key residues that allow inward directed diffusion. Substitution of E132 with aspartate or S77 with either alanine or cysteine abolished the inward-directed current almost completely. The proton influx is likely caused by the pK_a of E132 in GR, which is lower than that of other microbial ion pumping rhodopsins. The advantage of such a low pK_a is an acceleration of the photocycle and high pump turnover at high light intensities.     

Light- and dark-induced action potentials in Physcomitrella patens

Glass microelectrodes were inserted into Physcomitrella patens gametophyte leaves and action potentials (APs) were recorded in response to sudden illumination as well as after darkening, i.e., when the dark-induced membrane depolarization crossed a threshold. Application of 5 mM La³⁺ (a calcium channel inhibitor), 10 mM TEA⁺ (a potassium channel inhibitor) and increased free Ca²⁺ resulted in a loss of excitability. Lack of Ca²⁺ in the external medium did not prevent APs from occurring. It was concluded that during light- dark-induced excitation of Physcomitrella patens, APs might rely upon calcium influxes from the intracellular compartments. APs were not blocked by the proton pump inhibitors (DES, DCCD), although the resting potential (RP) diminished significantly.Key words: action potential, calcium, moss, Physcomitrella patens, plant     

Gloeobacter Rhodopsin,Limitation of Proton Pumping at High Electrochemical Load

We studied the photocurrents of a cyanobacterial rhodopsin Gloeobacter violaceus (GR) in Xenopus laevis oocytes and HEK-293 cells. This protein is a light-driven proton pump with striking similarities to marine proteorhodopsins, including the D121-H87 cluster of the retinal Schiff base counterion and a glutamate at position 132 that acts as a proton donor for chromophore reprotonation during the photocycle. Interestingly, at low extracellular pH_o and negative voltage, the proton flux inverted and directed inward. Using electrophysiological measurements of wild-type and mutant GR, we demonstrate that the electrochemical gradient limits outward-directed proton pumping and converts it into a purely passive proton influx. This conclusion contradicts the contemporary paradigm that at low pH, proteorhodopsins actively transport H⁺ into cells. We identified E132 and S77 as key residues that allow inward directed diffusion. Substitution of E132 with aspartate or S77 with either alanine or cysteine abolished the inward-directed current almost completely. The proton influx is likely caused by the pK_a of E132 in GR, which is lower than that of other microbial ion pumping rhodopsins. The advantage of such a low pK_a is an acceleration of the photocycle and high pump turnover at high light intensities.     

Mechanism of proton transport by plant plasma membrane proton ATPases

The mechanism of proton translocation by P-type proton ATPases is poorly defined. Asp684 in transmembrane segment M6 of the Arabidopsis thaliana AHA2 plasma membrane P-type proton pump is suggested to act as an essential proton acceptor during proton translocation. Arg655 in transmembrane segment M5 seems to be involved in this proton translocation too, but in contrast to Asp684, is not essential for transport. Asp684 may participate in defining the E₁ proton-binding site, which could possibly exist as a hydronium ion coordination center. A model of proton translocation of AHA2 involving the side chains of amino acids Asp684 and Arg655 is discussed.     

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14α-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.