The yeast plasma membrane H(+)-ATPase. An essential change of conformation triggered by H+.

The plasma membrane of Schizosaccharomyces pombe contains an H(+)-ATPase similar to the cation transport ATPases of other eukaryotic organisms. The fluorescence excitation and emission spectra of the purified H(+)-ATPase are characteristic of tryptophan residues. pH reduction from 7.5 to 5.7 produces a 4% decrease in fluorescence intensity, while a further reduction to pH 5.0 leads to an increase of fluorescence. A close correlation is observed between the pH dependence of the intrinsic fluorescence and the pH dependence of (i) ATPase activity, (ii) the fluorescence of Tb-formycin triphosphate bound to the active site, and (iii) inhibition by vanadate of ATPase activity. It is proposed that the effect of pH on intrinsic fluorescence reveals the existence of an H+ induced conformational change of the H(+)-ATPase similar to the E1----E2 transition of the other plasma membrane cation transport ATPases.     

Seed ageing-induced inhibition of germination and post-germination root growth is related to lower activity of plasma membrane H(+)-ATPase in maize roots

Seeds of most crops can be severely damaged and lose vigor when stored under conditions of high humidity and temperature. The aged seeds are characterized by delayed germination and slow post-germination growth. To date, little is known about the physiological mechanisms responsible for slow root growth of seedlings derived from aged seeds. Plasma membrane H(+)-ATPase is a universal H(+) pump in plant cells and is involved in various physiological processes including the elongation growth of plant cells. In the present study, we investigated the effect of a mild seed ageing treatment on plasma membrane H(+)-ATPase activity of seedling roots. Maize (Zea mays L.) seeds with 17% water content were aged at 45 degrees C for 30h. The aged seeds showed a 20% reduction in germination. Seedlings from aged seeds grew slowly during an experimental period of 120h after imbibition. Plasma membranes of maize seedling roots were isolated for investigation in vitro. Plasma membrane H(+)-ATPase (EC 3.6.3.6) activity was 14% lower for seedling roots developed from aged seeds as compared to control seeds. Protein gel immunoblotting analysis demonstrated that the reduced activity of plasma membrane H(+)-ATPase was attributed to a decrease in steady-state protein concentration of this enzyme. In conclusion, seed ageing causes a lower steady-state enzyme concentration of the H(+)-ATPase in the plasma membrane, which is related to slow germination and post-germination growth of seedling roots.     

Blepharismins, produced by the protozoan, Blepharisma japonicum, form ion-permeable channels in planar lipid bilayer membranes.

Blepharismins are polycyclic quinones found in the pigment granules of the ciliated protozoan, Blepharisma. Exposure to purified blepharismins results in lethal damage to several other ciliates. We here report that, at cytotoxic concentrations, blepharismins formed cation-selective channels in planar phospholipid bilayer membranes. The channels formed in a diphytanoylphosphatidylcholine bilayer had a K(+)/Cl(-) permeability ratio of 6.6:1. Single channel recordings revealed the conductance to be quite heterogeneous, ranging from 0.2 to 2.8 nS in solutions containing 0.1 M KCl, possibly reflecting different states of aggregation of blepharismin. Our observations suggest that channel formation is a cytotoxic mechanism of blepharismin's action against predatory protozoa.     

A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells

A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3'-dipropylthiadicarbocyanine iodide (DiSC(3)(5); DiSC(3)). In permeabilized cells, DiSC(3) fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K(+) and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H(+) transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential.     

Activation of H+-ATPase of the plasma membrane of Saccharomyces cerevisiae by glucose: the role of sphingolipid and lateral enzyme mobility

Activation of the plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae by glucose is a complex process that has not yet been completely elucidated. This study aimed to shed light on the role of lipids and the lateral mobility of the enzyme complex during its activation by glucose. The significance of H(+)-ATPase oligomerization for the activation of H(+)-ATPase by glucose was shown using the strains lcb1-100 and erg6, with the disturbed synthesis of sphyngolipid and ergosterol, respectively. Experiments with GFP-fused H(+)-ATPase showed a decrease in fluorescence anisotropy during the course of glucose activation, suggesting structural reorganization of the molecular domains. An immunogold assay showed that the incubation with glucose results in the spatial redistribution of ATPase complexes in the plasma membrane. The data suggest that (1) to be activated by glucose, H(+)-ATPase is supposed to be in an oligomeric state, and (2) glucose activation is accompanied by the spatial movements of H(+)-ATPase clusters in the PM.     

Exocytosis in bovine chromaffin cells: studies with patch-clamp capacitance and FM1-43 fluorescence

In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension.     

Membrane potential changes after infection of monocytes by Toxoplasma gondii.

Membrane potential changes in host cell plasma membrane were analyzed and the parasitophorous vacuole membrane (PVM) potential was characterized after infection by Toxoplasma gondii. Human monocytes infested by T. gondii were stained with two membrane potential sensitive dyes, DiOC(6)(3) carbocyanine and DiSBAC(2)(3) bis-oxonol, before fluorescence emission analysis by confocal laser scanning microscopy. After 24 and 48 h of infection, 34 and 39%, respectively, of monocytes showed several parasites (from two to six) per cell. At these infection times, significant decreases in cytoplasmic emissions were observed for both DiOC(6)(3) and DiSBAC(2)(3). Thus, hyperpolarisation of the host plasma membrane would occur consecutively to infection. Inside the parasitophorous vacuole, the fluorescence intensity of DiOC(6)(3) and DiSBAC(2)(3) increased significantly from 6 to 24 h after infection and the PVM became less polarised. Involvement of different ATPases in the membrane potential of infected monocytes was evaluated with ouabain, DCCD, omeprazole and sodium orthovanadate, ATPase inhibitors. All inhibitors induced a depolarisation of the plasma membrane. In the parasitophorous vacuole compartment, DCCD, omeprazole and sodium orthovanadate but not ouabain caused a significant depolarisation of the PVM, suggesting that H(+), H(+)/K(+) and P-type ATPases were at the origin of the PVM potential. This is the first report showing the presence of ion transporters in the T. gondii PVM and the existence of at least two members of the P-type family of ion pumps: an electrogenic H(+)ATPase and an electroneutral H(+)/K(+) ATPase.     

A novel method to quantify H+-ATPase-dependent Na+ transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na(+) is excluded from the cytosol to the apoplast or the vacuole by Na(+)/H(+) antiporters. The secondary active transport of Na(+) to apoplast against its electrochemical gradient is driven by plasma membrane H(+)-ATPases that hydrolyze ATP and pump H(+) across the plasma membrane. Current methods to determine Na(+) flux rely either on the use of Na-isotopes ((22)Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H(+) gradient due to H(+)-ATPase in the presence or absence of Na(+) by pH-sensitive probes. To date, there are no methods that can directly quantify H(+)-ATPase-dependent Na(+) transport in plasma membrane vesicles. We developed a method to measure bidirectional H(+)-ATPase-dependent Na(+) transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H(+)-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na(+) sealed for the study of H(+)-ATPase-dependent Na(+) transport. Our data implicate that Na(+) movement across vesicle membranes is highly dependent on H(+)-ATPase activity requiring ATP and Mg(2+) and displays optimum rates of 2.50 microM Na(+) mg(-1) membrane protein min(-1) at pH 6.5 and 25 degrees C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H(+)-ATPase-dependent Na(+) transport. The results demonstrate that the Na(+) content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H(+)-ATPase-dependent Na(+) transport with membrane vesicles.     

Cytochemistry of Ca(++)-dependent adenosine triphosphatase (Ca-ATPase) in rat anterior pituitary cells

In the present study, we demonstrate the localization of Ca(++)-ATPase in the anterior pituitary of the male rat. Ca(++)-ATPase was mainly distributed on the membrane system of the granular cells, which included the plasma membrane, the outer mitochondrial membrane, the enveloping membrane of secretory granules, the smooth endoplasmic reticulum and some components of the Golgi complex. No reaction product was detected on the membrane of the rough endoplasmic reticulum or that surrounding the lysosomes. A positive reaction was clearly observed on the membranes surrounding 'large' secretory granules, while that present on the membranes of the 'small' granules was comparatively weak. The cells which contained the 'large' granules were interpreted as growth hormone-secreting cells and those in which the 'small' granules were located as gonadotrophs. There were either no reaction or one that was barely detectable on the plasma membrane of the folliculo-stellate cells. These data along with our previous findings (Soji, 1982, 1984) suggest that the membranous enzymes are not uniformly distributed over all pituitary cells but rather are specific for a given cell population(s).     

The dual mechanism of the antifungal effect of new lysosomotropic agents on the Saccharomyces cerevisiae RXII strain

Quinacrine was used to visualize the intracellular pH changes in the yeast strain Saccharomyces cerevisiae RXII occurring after exposure to four recently-synthesized lysosomotropic drugs: DM-11, PY-11, PYG-12s and DMAL-12s. The cells took up quinacrine, mostly accumulating it in their vacuoles. DM-11 and PY-11 gave rise to diffuse quinacrine fluorescence throughout the cells, with the vacuoles staining to a somewhat greater extent than the cytosol. This quinacrine-detected overall acidification of the cell interior is very probably caused by blocking of plasma membrane H(+)-ATPase. PYG-12s gave rise to a strong vacuolar accumulation of the dye. Like the vacuolar ATPase inhibitor bafilomycin A(1), DMAL-12s strongly lowered the intensity of quinacrine fluorescence. Owing to its low pK(a), it can penetrate rapidly into the cells and may inhibit vacuolar H(+)-ATPase and prevent quinacrine-detectable vacuolar acidification without causing strong cell acidification. Since these drugs were found to penetrate into the cells, their lack of effect may reflect a higher resistance of both plasma membrane H(+)-ATPase and vacuolar ATPase to the drugs. Our data indicate that the lysosomotropic drugs under study have a dual action. On entering the cell, they cause intracellular acidification, very probably by inhibiting plasma membrane H(+)-ATPase and curtailing active proton pumping from the cells. Furthermore, they interfere with the function of V-type ATPase, causing vacuolar alkalinization and eventually cell death.     

Vesicular ATPase inserted into the plasma membrane of motor terminals by exocytosis alkalinizes cytosolic pH and facilitates endocytosis

Key components of vesicular neurotransmitter release, such as Ca(2+) influx and membrane recycling, are affected by cytosolic pH. We measured the pH-sensitive fluorescence of Yellow Fluorescent Protein transgenically expressed in mouse motor nerve terminals, and report that Ca(2+) influx elicited by action potential trains (12.5-100 Hz) evokes a biphasic pH change: a brief acidification (～ 13 nM average peak increase in [H(+)]), followed by a prolonged alkalinization (～ 30 nM peak decrease in [H(+)]) that outlasts the stimulation train. The alkalinization is selectively eliminated by blocking vesicular exocytosis with botulinum neurotoxins, and is prolonged by the endocytosis-inhibitor dynasore. Blocking H(+) pumping by vesicular H(+)-ATPase (with folimycin or bafilomycin) suppresses stimulation-induced alkalinization and reduces endocytotic uptake of FM1-43. These results suggest that H(+)-ATPase, known to transfer cytosolic H(+) into prefused vesicles, continues to extrude cytosolic H(+) after being exocytotically incorporated into the plasma membrane. The resulting cytosolic alkalinization may facilitate vesicular endocytosis.     

Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation

The ultrastructural changes in electropermeabilized bovine platelets that accompany the Ca2(+)-induced secretion of serotonin were investigated in ultra-thin sections of chemically fixed cells. Such preparations permitted us to study both the localization of and the structures associated with serotonin-containing dense granules. Localization of dense granules within cells was examined by measuring the shortest distances between the granular membranes and the plasma membrane. About 40% of total granules were located close to the plasma membrane at an average distance of 10.8 +/- 1.6 nm. 71% of the total number of granules were localized at a similar average distance of 12.5 +/- 2.7 nm in intact platelets. The percentage of granules apposed to the plasma membrane corresponded closely to the percentage of total serotonin that was maximally secreted after stimulation of the permeabilized (38 +/- 4.9%) and the intact platelets (72 +/- 3.6%). Furthermore, the percentage of granules anchored to the membrane, but not of those in other regions of permeabilized cells, decreased markedly when cells were stimulated for 30 s by extracellularly added Ca2+. The decrease in the numbers of granules in the vicinity of the plasma membrane corresponded to approximately 22% of the total number of dense granules that were used for measurements of the distances between the two membranes and corresponded roughly to the overall decrease (15%) in the average number of the granules per cell. Most dense granules were found to be associated with meshwork structures of microfilaments. Upon secretory stimulation, nonfilamentous, amorphous structures found between the plasma membrane and the apposed granules formed a bridge-like structure that connected both membranes without any obvious accompanying changes in the microfilament structures. These results suggest that the dense granules that are susceptible to secretory stimulation are anchored to the plasma membrane before stimulation, and that the formation of the bridge-like structure may participate in the Ca2(+)-regulated exocytosis.     

The membrane potential of the intraerythrocytic malaria parasite Plasmodium falciparum

The membrane potential (Deltapsi) of the mature asexual form of the human malaria parasite, Plasmodium falciparum, isolated from its host erythrocyte using a saponin permeabilization technique, was investigated using both the radiolabeled Deltapsi indicator tetraphenylphosphonium ([(3)H]TPP(+)) and the fluorescent Deltapsi indicator DiBAC(4)(3) (bis-oxonol). For isolated parasites suspended in a high Na(+), low K(+) solution, Deltapsi was estimated from the measured distribution of [(3)H]TPP(+) to be -95 +/- 2 mV. Deltapsi was reduced by the specific V-type H(+) pump inhibitor bafilomycin A(1), by the H(+) ionophore CCCP, and by glucose deprivation. Acidification of the parasite cytosol (induced by the addition of lactate) resulted in a transient hyperpolarization, whereas a cytosolic alkalinization (induced by the addition of NH(4)(+)) resulted in a transient depolarization. A decrease in the extracellular pH resulted in a membrane depolarization, whereas an increase in the extracellular pH resulted in a membrane hyperpolarization. The parasite plasma membrane depolarized in response to an increase in the extracellular K(+) concentration and hyperpolarized in response to a decrease in the extracellular K(+) concentration and to the addition of the K(+) channel blockers Ba(2+) or Cs(+) to the suspending medium. The data are consistent with Deltapsi of the intraerythrocytic P. falciparum trophozoite being due to the electrogenic extrusion of H(+) via the V-type H(+) pump at the parasite surface. The current associated with the efflux of H(+) is countered, in part, by the influx of K(+) via Ba(2+)- and Cs(+)-sensitive K(+) channels in the parasite plasma membrane.     

THE EFFECT OF STRYCHNINE AND LIGHT ON PIGMENTATION IN BLEPHARISMA UNDULANS STEIN

The effect of strychnine sulfate and light on pigmentation in the ciliate protozoan Blepharisma undulans has been determined. Upon exposure of cells to strychnine, the pigment granules become loosened from their surrounding membranes. Eventually these membranes break and the granules are simultaneously released from the cell. At the cell surface, a fusion occurs between adjacent membraneless granules with the incorporation of membrane fragments. This fusion of granules and membrane fragments results in the formation of a pigmented "capsule" around the organism. After elimination of the pigment, the granule membranes remaining in the cytoplasm fuse to form apparently empty vesicles. Other cell organelles are generally undisturbed. A similar situation occurs upon exposure of cells to artificial light for 12 to 18 hr, however, the slow elimination of granules from the cells under these conditions does not result in the formation of a pigmented "capsule." The possible mechanisms of these reactions are discussed.     

Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae.

We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae. Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH. Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane. This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell. Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole. Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1. The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH. This was followed by slow acidification. Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1. The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH. Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose. Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes. Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase.     

Blue light inactivates plasma membrane H(+)-ATPase in pulvinar motor cells of Phaseolus vulgaris L

Unilateral blue light irradiation induces bending of pulvini of Phaseolus vulgaris towards the source of light. The pulvinar bending is caused by a decrease in turgor pressure of motor cells that are irradiated with blue light. Decrease in the turgor pressure is caused by the net efflux of K(+) and counter anions, accompanying membrane depolarization. In the present study the effect of blue light on the activity of plasma membrane H(+)-ATPase was studied in relation to the membrane depolarization. The activity of the plasma membrane H(+)-ATPase was measured using protoplast suspensions prepared from laminar pulvini from primary leaves. A pulse of blue light under continuous red light irradiation induced both a transient increase in the external pH and transient inhibition of the vanadate-sensitive ATPase. Continuous blue light irradiation under continuous red light irradiation induced both a sustained increase in the external pH and sustained inhibition of the vanadate-sensitive ATPase. These results show that blue light inhibits the activity of the plasma membrane H(+)-ATPase. Inactivation of the plasma membrane H(+)-ATPase supports the membrane depolarization induced by the blue light irradiation.     

Heterodimerization of y(+)LAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy

y(+)LAT-1 and 4F2hc are the subunits of a transporter complex for cationic amino acids, located mainly in the basolateral plasma membrane of epithelial cells in the small intestine and renal tubules. Mutations in y(+)LAT-1 impair the transport function of this complex and cause a selective aminoaciduria, lysinuric protein intolerance (LPI, OMIM #222700), associated with severe, complex clinical symptoms. The subunits of an active transporter co-localize in the plasma membrane, but the exact process of dimerization is unclear since direct evidence for the assembly of this transporter in intact human cells has not been available. In this study, we used fluorescence resonance energy transfer (FRET) microscopy to investigate the interactions of y(+)LAT-1 and 4F2hc in HEK293 cells expressing y(+)LAT-1 and 4F2hc fused with ECFP or EYFP. FRET was quantified by measuring fluorescence intensity changes in the donor fluorophore (ECFP) after the photobleaching of the acceptor (EYFP). Increased donor fluorescence could be detected throughout the cell, from the endoplasmic reticulum and Golgi complex to the plasma membrane. Therefore, our data prove the interaction of y(+)LAT-1 and 4F2hc prior to the plasma membrane and thus provide evidence for 4F2hc functioning as a chaperone in assisting the transport of y(+)LAT-1 to the plasma membrane.     

Respiration-Linked Proton Transport, Changes in External pH, and Membrane Energization in Cells of Escherichia coli

The kinetics of respiration-dependent proton efflux and membrane energization have been studied in intact cells of logarithmic-phase Escherichia coli. Parallel measurements of the rate and extent of proton efflux into the external medium (half-time, about 10 s; ratio of H(+) to O, about 0.5) and the oxidation of E. coli cytochrome b (half-time, 相似文献   

Polarization of the Surface Membrane and Cortical Layer of Sea Urchin Blastomeres, and its Inhibition by Cytochalasin B

Sea-urchin blastomeres have two domains of the plasma membrane which can be distinguished immunocytochemically. An egg-surface antibody (anti-ES), which binds to the membrane of the entire surface region of eggs before cleavage, binds to the membrane of the outer surface region of blastomeres after cleavage, but not to that of the cleavage furrow region or interblastomeric surface region.
The anti-ES binding sites on the egg membrane were chased after cleavage by labeling the egg plasma membrane with FITC conjugated monovalent anti-ES (FITC-Fab anti-ES) before the first cleavage, and then allowing the eggs to cleave. The surface fluorescence increased in intensity in the cleavage furrow region with progress of furrowing, but after completion of the furrowing, the fluorescence became uniform and finally decreased in the interblastomeric surface region.
The distributions of pigment granules and NBD-phallacidin stainable microfilaments in the cortex after completion of furrowing were polarized in the same way as the anti-ES binding area. As cytochalasin B completely inhibited the polarization in both the surface and cortical layer but colchicine did not, polarization of the anti-ES binding area was concluded to be due to the post-cleavage polarized distribution of submembranous microfilaments in the cortical layer.