Activation and significance of vacuolar H+-ATPase in Saccharomyces cerevisiae adaptation and resistance to the herbicide 2,4-dichlorophenoxyacetic acid

The stimulation of the activity of the H(+)-ATPase present in the vacuolar membrane (V-ATPase) of Saccharomyces cerevisiae is here described in response to a moderate stress induced by 2,4-dichlorophenoxyacetic acid (2,4-D). This in vivo activation (up to 5-fold) took place essentially during the adaptation period, preceding cell division under herbicide stress, in coordination with a marked activation of plasma membrane H(+)-ATPase (PM-ATPase) (up to 30-fold) and the decrease of intracellular and vacuolar pH values, suggesting that activation may be triggered by acidification. Single deletion of VMA1 and genes encoding other V-ATPase subunits led to a more extended period of adaptation and to slower growth under 2,4-D stress. Results suggest that a functional V-ATPase is required to counteract, more rapidly and efficiently, the dissipation of the physiological H(+)-gradient across vacuolar membrane registered during 2,4-D adaptation.     

Blue light activates the plasma membrane H(+)-ATPase by phosphorylation of the C-terminus in stomatal guard cells.

The opening of stomata, which is driven by the accumulation of K(+) salt in guard cells, is induced by blue light (BL). The BL activates the H(+) pump; however, the mechanism by which the perception of BL is transduced into the pump activation remains unknown. We present evidence that the pump is the plasma membrane H(+)-ATPase and that BL activates the H(+)-ATPase via phosphorylation. A pulse of BL (30 s, 100 micromol/m(2)/s) increased ATP hydrolysis by the plasma membrane H(+)-ATPase and H(+) pumping in Vicia guard cell protoplasts with a similar time course. The H(+)-ATPase was phosphorylated reversibly by BL, and the phosphorylation levels paralleled the ATP hydrolytic activity. The phosphorylation occurred exclusively in the C-termini of H(+)-ATPases on both serine and threonine residues in two isoproteins of H(+)-ATPase in guard cells. An endogenous 14-3-3 protein was co-precipitated with H(+)-ATPase, and the recombinant 14-3-3 protein bound to the phosphorylated C-termini of H(+)-ATPases. These findings demonstrate that BL activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus by a serine/threonine protein kinase, and that the 14-3-3 protein has a key role in the activation.     

Fusicoccin Activates the Plasma Membrane H+-ATPase by a Mechanism Involving the C-Terminal Inhibitory Domain

Plasma membrane vesicles isolated from spinach leaves incubated with the fungal toxin fusicoccin showed a twofold increase in ATP hydrolytic activity and a threefold increase in H+ pumping compared to controls. This increase in H+-ATPase activity was largely completed within 4 min of incubation and was not due to de novo synthesis of H+-ATPase as demonstrated by immunoblotting. Incubation with fusicoccin also resulted in a decrease in the apparent Km for ATP of the H+-ATPase from 0.22 to 0.10 mM. The fusicoccin-mediated activation of H+-ATPase activity and the accompanying decrease in the Km for ATP are changes very similar to those observed upon trypsin activation of the H+-ATPase, where an autoinhibitory domain in the C-terminal region of the H+-ATPase is removed. Thus, trypsin treatment of plasma membrane vesicles from control leaves gave a twofold increase in ATP hydrolytic activity and a threefold increase in H+ pumping, as well as a decrease in the apparent Km for ATP of the H+-ATPase from 0.22 to 0.10 mM. Trypsin treatment of plasma membranes from fusicoccin-incubated leaves did not further enhance the H+-ATPase activity, however, and neither was the Km for ATP further decreased. That trypsin really removed a small segment from the fusicoccin-activated H+-ATPase was confirmed by immunoblotting, which showed the appearance of a 90-kD band in addition to the native 100-kD H+-ATPase band upon trypsin treatment. Taken together, our data suggest that in vivo activation of the H+-ATPase by fusicoccin proceeds by a mechanism involving a displacement of the C-terminal inhibitory domain.     

Cytochemical demonstration of NPPase activity for detecting proton-translocating ATPase of Golgi complex in rat pancreatic acinar cells

An attempt at cytochemical demonstration of acidification proton-translocating ATPase (H(+)-ATPase) of Golgi complex in rat pancreatic acinar cells has been made by using p-nitrophenylphosphatase (NPPase) cytochemistry which is used for detecting of Na(+)-K(+)-ATPase (Mayahara et al. 1980) and gastric H(+)-K(+)-ATPase (Fujimoto et al. 1986). K(+)-independent NPPase activity was observed on the membrane of the trans cisternae of Golgi complex, but not inside of cisternae. The localization of NPPase activity is different from that of acid phosphatase activity where reaction products were seen on the inside of the trans Golgi cisternae. Since this activity was insensitive to vanadate, ouabain and independent of potassium ions, it was distinct from plasma membranous ATPases such as Na(+)-K(+)-ATPase and Ca2(+)-ATPase. The K(+)-independent NPPase activity was diminished by the inhibitors of H(+)-ATPase such as N-ethylmaleimide (NEM) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The NPPase reaction products were also seen on the membranes of other acidic organelles, i.e., lysosomes, endosomes, autophagosomes and coated vesicles. These results suggest that NPPase activity on the membrane of the Golgi complex and other acidic organelles corresponds with H(+)-ATPase which plays a role in acidification.     

茉莉酸甲酯处理对绿豆下胚轴质膜H+-ATPase水解活力及磷酸化水平的影响

运用γ-32P示踪、蛋白激酶和磷酸酶抑制剂药理实验探讨茉莉酸甲酯(MeJA)对质膜H -ATP酶水解活力及磷酸化水平的影响.结果如下:MeJA可促进H -ATP酶水解活力30%;斑蝥素和岗田酸促进了MeJA对质膜H -ATP酶的刺激作用;星形孢菌素和白屈菜红碱削弱了MeJA对质膜H -ATP酶的刺激作用.H -ATP酶活力变化同时,其上的γ-32P标记量发生变化.Ca2 对H -ATP酶水解活力有很大的刺激作用,但对MeJA促进H -ATP酶活力的作用没有进一步的影响.根据这些结果可以得出结论:MeJA刺激质膜H -ATP酶水解活力的变化与H -ATP酶磷酸化水平呈正相关,并且催化这一作用的蛋白激酶可能不依赖于Ca2 ,而蛋白磷酸酶依赖于Ca2 .     

C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane.     

The involvement of calcium carriers and of the vacuole in the glucose-induced calcium signaling and activation of the plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells

Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H(+)-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H(+)-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca(2+)-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H(+)-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H(+)-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP(3). Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H(+)-ATPase activation.     

Proteolytic activation of the plant plasma membrane H(+)-ATPase by removal of a terminal segment

Incubation of oat root plasma membrane vesicles in the presence of ATP with trypsin or chymotrypsin increased the rate of ATP hydrolysis and ATP-dependent proton pumping by the plasma membrane H(+)-ATPase. Proton pumping was stimulated more than 200%, whereas ATP hydrolytic activity was stimulated about 30%. The Km (ATP) for both proton pumping and ATP hydrolysis was lowered from about 0.3 mM to below 0.1 mM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trypsin-treated plasma membranes revealed a decrease in a 100-kDa band and the appearance of a 93-kDa band. Western blot analysis using antibodies against the H(+)-ATPase showed that both of these bands represented the H(+)-ATPase and suggested that a 7-kDa segment was released. Extensive treatment with carboxypeptidase A also activated the H(+)-ATPase indicating that the 7-kDa segment originated from the C terminus.     

Localization of the plasma membrane and mitochondrial H(+)-ATPases in Leishmania donovani promastigotes.

Immunochemical methods were used to characterize the proton-translocating ATPases (H(+)-ATPases) of the plasma membrane and mitochrondrion of Leishmania donovani promastigotes. Antisera directed against the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae reacted with a 66 kDa membrane protein of L. donovani promastigotes. By immunocytochemistry, the antiserum was shown to label the cell and flagellar surface of promastigotes as well as the Golgi apparatus and the membrane of intracellular organelles. The target antigen was shown to possess ATPase activity resembling the leishmanial H(+)-ATPase activity. Antisera raised against the beta-subunit of the F0F1-ATPase of Escherichia coli reacted with a 56 kDa protein in L. donovani promastigotes. Ultrastructurally, the anti-beta-subunit antibody was exclusively associated with the mitochondrion in these cells. This antiserum immunoprecipitates ATP hydrolytic activity typical of the F1 beta-subunit activity of the mitochondria of higher eukaryotes.     

Energy metabolism in wheat root cells under modification of plasma membrane permeability by antibiotic nystatin

This paper reports changes in ion transport and energy metabolism of plant cells during short- and long-term expositions, resp., to antibiotic nystatin, which is known to specifically bind with plasma membrane sterols to form channels. The excised roots of 5 days old wheat seedlings were used as a model system in this research. It has been shown that treatment of excised roots with nystatin leads to activation of energy metabolism expressed as an increase of respiration and heat production by root cells. Furthermore, in the presence of nystatin increased pH of incubation medium, plasma membrane depolarization and a significant loss of potassium ions were observed. Nystatin-induced stimulation of respiration was prevented by malonate, an inhibitor of succinate dehydrogenase, electron acceptor dichlorophenolindophenol, and AgNO3, an inhibitor of H(+)-ATPase. Based on the data obtained it can be suggested that nystatin-induced stimulation of respiration is related to electron transport activation via mitochondrial respiratory chain, and is connected with activation of plasmalemma proton pump. Moreover, nystatin-induced increase of oxygen consumption was prevented by cerulenin, an inhibitor of fatty acid and sterol synthesis. This indicates that additional sterols and phospholipids may be synthesized in root cells to "heal" nystatin-caused damage of plasma membrane. A supposed chain of events of cell response to nystatin action may by as following: formation of nystatin channels-influx of protons--depolarization of plasmalemma-efflux of potassium ions-disturbance of ion homeostasis--activation of H(+)-ATPase work-increase in energy "requests" for H(+)-ATPase function--increase in the rate of oxygen consumption and heat production. The increased energy production under the action of nystatin, may provide the work of proton pump and synthesis of sterols and phospholipids, which are necessary for membrane regeneration.     

在耐热性诱导中豌豆叶片过氧化氢和水杨酸含量与质膜ATP酶活性的变化及相互关系

在高温锻炼(37℃,2h)过程中,豌豆(Pisum sativum L.)叶片过氧化氢(H_2O_2)和游离态水杨酸(SA)含量与质膜ATP酶(H~ -ATPase)活性都有一个高峰,H_2O_2的迸发早于游离态SA的积累,而质膜H~ -ATPase活性高峰的出现则迟于SA高峰;活性氧清除剂、抗氧化剂、质膜NADPH氧化酶抑制剂和H_2O_2的淬灭剂预处理均可有效地阻止高温下H_2O_2和SA的积累以及质膜H~ -ATPase活性的增加。根据以上结果推测,H_2O_2、质膜H~ -ATPase和SA均参与耐热性诱导相关的信号传递,前者作用于SA的上游,而后者在SA下游起作用。     

The action of adaptogenic preparations on vacuolar proton pumps activity in corn root cells under salt stress conditions

The function of two electrogenic H+ -pumps in plant vacuolar membrane (tonoplast) and their response to the salt stress conditions and adaptogenic preparations have been studied. Experiments were carried out on tonoplast fraction isolated from the roots of corn seedlings grown in water culture which were exposed at 7-day age in the presence of 0.1 M NaCl during 1 or 10 days. The role of every H+ -pump in potential generation was elucidated by assaying transport and hydrolytic activity of enzymes--V-type H+ -ATPase and H+ pyrophosphatase represented their mechanisms. It was found in the control variant that transport activity H+ -PPase exceeded considerably H+ -ATPase one in the tonoplast from 7-day seedlings. However this situation was changed in 18-day seedlings by H+ -ATPase transport activity increasing with age whereas its hydrolytic one was decreased. Both NaCI expositions caused the progressive decrease of transport and hydrolytic activity of H+ -PPase whereas H+ -ATPase responded to this factor by increasing transport activity, while its hydrolytic one fell. Bioactive preparations Methyure and Ivine (10(-7) M) used by seed soaking caused a further increase of H+ -ATPse transport activity especially in the presence of NaCl whereas H+ -PPase one was not changed. Methyure effect in these experiments was more pronounced. Obtained results demonstrated participation of tonoplast H+ -pumps in plant adaptation to NaCI which can be realized by amplification of Na+ -H(+) antiporter energization. An important role of vacuolar H+ -ATPase in growth and adaptation processes in plants has been proved.     

Fusicoccin affects cytochrome c leakage and cytosolic 14-3-3 accumulation independent of H-ATPase activation

Fusicoccin (FC) is a well known toxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H(+)-ATPase and the biochemical and physiological changes induced in the cell by this toxin have, up to now, been ascribed to the increased rate of proton extrusion by this pump leading to external acidification and cell hyperpolarization. In a recent work (Malerba M et al. 2003, Physiologia Plantarum, 119: 480-488) it was shown that, besides the previously well studied changes, FC induces a large stimulation of H(2)O(2) production, an activation of alternative respiration and a leakage of cytochrome c from mitochondria. In this article further studies on the relation between the H(2)O(2) overproduction and medium acidification are reported. The increase in the rate of H(2)O(2) accumulation is particularly evident when high concentrations of the toxin ensure a rapid acidification of the medium, but it is not obtained when the time-course of acidification is reproduced by external acid additions. The FC-dependent H(2)O(2) overproduction is strongly inhibited by inhibitors of the H(+)-ATPase activity, such as vanadate and erythrosin B, and it does not occur when the activation of the H(+)-ATPase is prevented by phenylarsine oxide (PAO), an inhibitor of the activating interaction between the enzyme and its regulative 14-3-3 protein. Interestingly, all these inhibitors only partially prevent the leakage of cytochrome c from the mitochondria. A kinetic analysis of FC-dependent changes of 14-3-3s shows that the initial increase in the plasma membrane level of these proteins, presumably due to translocation of free cytosolic forms, is followed by a remarkable increase in the level of the 14-3-3 proteins located in the cytosol. This latter change is not prevented by inhibitors of the activity or activation of the H(+)-ATPase. These results suggest that, besides the H(+)-ATPase activation, FC can induce other cell changes possibly mediated by changes of the regulative 14-3-3 proteins.     

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.     

Glucose-induced activation of plasma membrane H(+)-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis.

Addition of glucose-related fermentable sugars or protonophores to derepressed cells of the yeast Saccharomyces cerevisiae causes a 3- to 4-fold activation of the plasma membrane H(+)-ATPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP synthesis, incubation at the restrictive temperature reduced the extent of H(+)-ATPase activation. Incubation of non-temperature-sensitive strains, however, at such temperatures also caused reduction of H(+)-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H(+)-ATPase activation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H(+)-ATPase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAMP-protein kinase A signaling pathway is not required for glucose activation of the H(+)-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phosphorylating enzymes hexokinase PI and PII and glucokinase showed that activation of the H(+)-ATPase with glucose or fructose was completely dependent on the presence of a kinase able to phosphorylate the sugar. These and other data concerning the role of initial sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H(+)-ATPase have a common initiation point.     

Requirement of translocated lysosomal V1 H(+)-ATPase for activation of membrane acid sphingomyelinase and raft clustering in coronary endothelial cells

Acid sphingomyelinase (ASM) mediates the formation of membrane raft (MR) redox signalosomes in a process that depends on a local acid microenvironment in coronary arterial endothelial cells (CAECs). However, it is not known how this local acid microenvironment is formed and maintained. The present study hypothesized that lysosomal V1 H(+)-ATPase provides a hospitable acid microenvironment for activation of ASM when lysosomes traffic and fuse into the cell membrane. Confocal microscopy showed that local pH change significantly affected MRs, with more fluorescent patches under low pH. Correspondingly, the ASM product, ceramide, increased locally in the cell membrane. Electron spin resonance assay showed that local pH increase significantly inhibited NADPH oxidase-mediated production of O(2)(-.) in CAECs. Direct confocal microscopy demonstrated that Fas ligand resulted in localized areas of decreased pH around CAEC membranes. The inhibitors of both lysosomal fusion and H(+)-ATPase apparently attenuated FasL-caused pH decrease. V1 H(+)-ATPase accumulation and activity on cell membranes were substantially suppressed by the inhibitors of lysosomal fusion or H(+)-ATPase. These results provide the first direct evidence that translocated lysosomal V1 H(+)-ATPase critically contributes to the formation of local acid microenvironment to facilitate activation of ASM and consequent MR aggregation, forming MR redox signalosomes and mediating redox signaling in CAECs.