Activation of the H+-ATPase in the plasma membrane of cells of Saccharomyces cerevisiae grown under mild copper stress

Cells of Saccharomyces cerevisiae exibited a more active plasma membrane H⁺-ATPase during growth in media supplemented with CuSO₄ concentrations equal to or below 1 mM than did cells cultivated in the absence of copper stress. Maximal specific activities were found with 0.5 mM CuSO₄. ATPase activity declined when cells were grown with higher concentrations up to 1.5 mM (the maximal concentration that allowed growth), probably due to severe disorganization of plasma membrane. Cu²⁺-induced maximal activation was reflected in an increase of V _max (approximately threefold) and in the slight decrease of the K _m for MgATP (from 0.93 ± 0.13 to 0.65 ± 0.16 mM). The expression of the gene encoding the essential plasma membrane ATPase (PMA1) was reduced with a dose-dependent pattern in cells grown with inhibitory concentrations of copper, while the weakly expressed PMA2 gene promoter was moderately more efficient in cells cultivated under mild copper stress (1.5-fold maximal activation). ATPase was activated by copper despite the slightly lower content of ATPase protein in the plasma membrane of Cu²⁺-grown cells and the powerful inhibitory effect of Cu²⁺ in vitro. Received: 6 May 1998 / Accepted: 14 September 1998     

Comparative effects of Saccharomyces cerevisiae cultivation under copper stress on the activity and kinetic parameters of plasma-membrane-bound H+-ATPases PMA1 and PMA2

The major yeast plasma membrane H⁺-ATPase is encoded by the essential PMA ₁ gene. The PMA ₂ gene encodes an H⁺-ATPase that is functionally interchangeable with the one encoded by PMA ₁ , but it is expressed at a much lower level than the PMA ₁ gene and it is not essential. Using genetically manipulated strains of Saccharomyces cerevisiae that exclusively synthesize PMA₁ ATPase or PMA₂ ATPase under control of the PMA₁ promoter, we found that yeast cultivation under mild copper stress leads to a similar activation of PMA₂ and PMA₁ isoforms. At high inhibitory copper concentrations (close to the maximum that allowed growth), ATPase activity was reduced from maximal levels; this decrease in activity was less important for PMA₂ ATPase than for PMA₁ ATPase. The higher tolerance to high copper stress of the artificial strain synthesizing PMA₂ ATPase exclusively, as compared to that synthesizing solely PMA₁ ATPase, correlated both with the lower sensitivity of PMA₂ ATPase to the deleterious effects of copper in vivo and with its higher apparent affinity for MgATP, and suggests that plasma membrane H⁺-ATPase activity plays a role in yeast tolerance to copper. Received: 19 October 1998 / Accepted: 6 January 1999     

Activation of plasma membrane H(+)-ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at supraoptimal temperatures.

During exponential growth at temperatures of 30 to 39 degrees C, the specific activity of H(+)-ATPase in the plasma membrane of Saccharomyces cerevisiae (assayed at the standard temperature 30 degrees C) increased with increases in growth temperature. In addition, the optimal temperature for in vitro activity of this ATPase was 42 degrees C. Therefore, the maximum values of ATPase activity were expected to occur in cells that grew within the supraoptimal range of temperatures. Activation induced by supraoptimal temperatures was not the result of increased synthesis of this membrane enzyme. When the growth temperature increased from 30 to 40 degrees C, expression of the essential PMA1 gene, monitored either by the level of PMA1 mRNA or the beta-galactosidase activity of the lacZ-PMA1 fusion, was reduced. Consistently, quantitative immunoassays showed that the ATPase content in the plasma membrane decreased. Like ATPase activity, the efficiency of the PMA2 promoter increased with increases in growth temperature in cells that had been grown at 30 to 39 degrees C, but its level of expression was several hundred-fold lower than that of PMA1. These results suggest that the major PMA1 ATPase is activated at supraoptimal temperatures.     

Intracellular acidification does not account for inhibition of Saccharomyces cerevisiae growth in the presence of ethanol

Abstract Intracellular acidification has been considered one of a number of mechanisms underlying the inhibition of growth and fermentation by ethanol in yeast. However, most of the studies on the effect of ethanol on yeast intracellular pH (pH_i) were carried out by using unadapted cells to which ethanol was added. In this paper we show that the pH_i of exponential cells of Saccharomyces cerevisiae IGC 3507 III grown in a medium with glucose and inhibitory concentrations of ethanol only decreased to values below those in unstressed cells (6.9) for concentrations equal to or above 7% (v/v). Only at these supracritical levels (7–10% (v/v)) was pH homeostasis in ethanol-adapted yeast affected. This is consistent with the significant increase of plasma membrane permeability and decrease of plasma membrane H⁺-ATPase in comparison with the corresponding values in unstressed cells. These deleterious effects were only observed with those high concentrations of toxin. These results indicate that intracellular acidification does not account for inhibition of yeast growth in the presence of ethanol. In fact, growth was inhibited by ethanol concentrations (3–6% (v/v)) that did not lead to the decrease of pH_i. Furthermore, even for supracritical concentrations, close to the maximal that allowed growth (10% (v/v)), the dedrease of pH_i was not important reaching, at the most, values of 6.5–6.6.     

过氧化氢诱导酿酒酵母细胞膜透性和组成的变化

以下简述了过氧化氢(H2O2)作为一种信号分子诱导并调节酿酒酵母(Saccharomyces cerevisiae)细胞膜的变化。H2O2是一种强氧化剂,可以跨膜扩散进入细胞中,形成跨膜梯度;当外源H2O2达到亚致死剂量时,酿酒酵母的细胞膜透性和流动性降低,产生跨膜梯度,从而限制H2O2向细胞内的扩散速率,保护细胞免受氧化胁迫的伤害。研究表明,由H2O2引起的膜透性和流动性的变化与膜的组成有关:当酵母细胞对H2O2产生适应时,与膜组成和微区域变化有关的几个基因的表达发生了改变。膜组成的变化和微区域的调整还可能与H2O2依赖的信号途径有关,即以H2O2为信号分子,调节膜的变化并赋予细胞对氧化压力更高的适应性,但这种信号分子的具体传递途径及机制还需要进一步研究。     

Evaluation of the antimicrobial activity of ebselen: role of the yeast plasma membrane H+-ATPase

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a selenium-containing antioxidant demonstrating anti-inflammatory and cytoprotective properties in mammalian cells and cytotoxicity in lower organisms. The mechanism underlying the antimicrobial activity of ebselen remains unclear. It has recently been proposed that, in lower organisms like yeast, the plasma membrane H+-ATPase (Pma1p) could serve as a potential target for this synthetic organoselenium compound. Using yeast and bacteria, the present study found ebselen to inhibit microbial growth in a concentration- and time-dependent manner, and yeast and Gram-positive bacteria to be more sensitive to this action (IC50 approximately 2-5 microM) than Gram-negative bacteria (IC50 < 80 microM). Washout experiments and scanning electron microscopic analysis revealed ebselen to possess fungicidal activity. In addition, ebselen was found to inhibit medium acidification by PMA1-proficient haploid yeast in a concentration-dependent manner. Additional studies comparing PMA1 (+/-) and PMA1 (+/+) diploid yeast cells revealed the mutant to be more sensitive to treatment with ebselen than the wild type. Ebselen also inhibited the ATPase activity of Pma1p from S. cerevisiae in a concentration-dependent manner. The interaction of ebselen with the sulfhydryl-containing compounds L-cysteine and reduced glutathione resulted in the complete and partial prevention, respectively, of the inhibition of Pma1p ATPase activity by ebselen. Taken together, these results suggest that the fungicidal action of ebselen is due, at least in part, to interference with both the proton-translocating function and the ATPase activity of the plasma membrane H+-ATPase.     

Vacuolar acidification in Saccharomyces cerevisiae induced by elevated hydrostatic pressure is transient and is mediated by vacuolar H+-ATPase

We analyzed the vacuolar acidification in response to elevated hydrostatic pressure in Saccharomyces cerevisiae. The vacuolar pH, defined using 6-carboxyfluorescein, was directly measured in a hyperbaric chamber with a transparent window under high hydrostatic pressure. The vacuole of strain X2180 became acidified at the onset of pressurization to an extent dependent on the magnitude of pressure applied. A pressure of 40–60 MPa transiently reduced the vacuolar pH by about 0.33 within 4 min. The transient acidification was observed in the presence of D-glucose, D-fructose, or D-mannose as a carbon source, but not 3-o-methyl-D-glucose, ethanol, or glycerol, suggesting that the generation of CO₂ was involved in the process. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH when hydrostatic pressure was applied. This result indicates that the transient vacuolar acidification induced by elevated hydrostatic pressure is mediated through the function of the vacuolar H+-ATPase. Received: August 21, 1996 / Accepted: November 11, 1996     

The Na+,K+/H+ -antiporter Nha1 influences the plasma membrane potential of Saccharomyces cerevisiae

There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.     

Characteristics of non-specific permeability and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by excessive Cu2+

Effects of Cu²⁺ on a non-specific conductance and H⁺-ATPase activity in the plasma membrane of the freshwater alga Nitella flexilis L. Agardh was studied using a conventional microelectrode voltage-clamp technique. We show that a Cu²⁺-induced increase in the non-specific conductance is related to the formation of pores in the plasma membrane. Pore formation is the result of unidentified chemical reactions, since the Q₁₀ for the rate of increase of conductance over time was about 3. Various oxidants and antioxidants (10 mmol/l H₂O₂, 10 mmol/l ascorbate, 100 μg/ml superoxide dismutase, and 100 μg/ml catalase) did not alter Cu²⁺-induced changes in the plasma membrane conductance, suggesting that the effect of Cu²⁺ was unrelated to peroxidation of plasma-membrane lipids. In contrast, organic and inorganic Ca²⁺-channel antagonists (nifedipine, Zn²⁺, Cd²⁺, Fe²⁺, Ni²⁺) inhibited the Cu²⁺-induced non-specific conductance increase. This suggests that changes in Ca²⁺ influx underlie this effect of Cu²⁺. Decreasing the pH or the ionic strength of external solutions also inhibited the Cu²⁺-induced plasma-membrane conductance increase. Copper was also found to inhibit plasma-membrane H⁺-ATPase activity with half-maximal inhibition occurring at about 5–20 μmol/l and full inhibition at about 100–300 μmol/l. The Hill coefficient of Cu²⁺ inhibition of the H⁺-ATPase was close to two. Received: 8 December 1999 / Accepted: 16 August 2000     

Calcium signaling and sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells

In this work, we show that glucose-induced activation of plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is strongly dependent on calcium metabolism and that the glucose sensor Snf3p works in a parallel way with the G protein Gpa2p in the control of the pathway. The role of Snf3p is played by the Snf3p C-terminal tail, since in a strain with the deletion of the SNF3 gene, but also expressing a chimera protein formed by Hxt1p (a glucose transporter) and the Snf3p C-terminal tail, a normal glucose-activation process can be observed. We present evidences indicating that Snf3p would be the sensor for the internal signal (phosphorylated sugars) of this pathway that would connect calcium signaling and activation of the plasma membrane ATPase. We also show that Snf3p could be involved in the control of Pmc1p activity that would regulate the calcium availability in the cytosol.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

The plasma membrane proteome of Saccharomyces cerevisiae and its response to the antifungal calcofluor

Calcofluor is an antifungal compound known to induce structural perturbations of the cell wall by interfering with the synthesis of chitin microfibril. Proteins from a stripped plasma membrane fraction were solubilized with the neutral and non-denaturing detergent, the n-dodecyl beta-D-maltoside. Proteins were then resolved using a recently described ion-exchange chromatography (IEC)/lithium dodecyl sulfate (LDS)-PAGE procedure. Nearly 90 proteins were identified and clustered, based on their pI, molecular weight, abundance and/or hydrophobicity. This method was then applied to profile the plasma membrane response to calcofluor. The LDS-PAGE patterns obtained from whole plasma membrane proteins were similar for the non-treated and calcofluor-treated samples. However, IEC/LDS-PAGE analysis revealed subtle changes in the expression of several proteins of low abundance, in response to calcofluor. These proteins include Pil1p and Lsp1p, two sphingolipid long-chain base-responsive inhibitors of protein kinases involved in signaling pathways for cell wall integrity and Rho1p, a small GTPase. It was recently hypothesized that Pil1p and Lsp1p could associate with, and regulate, the plasma membrane beta-1-3-glucan synthase, responsible for the synthesis of another major microfibril for yeast cell wall. Results are discussed with respect to both calcofluor effects on the plasma membrane proteins and the power of the IEC/LDS-PAGE procedure in the search for new potential therapeutics targets.     

Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance

K+ is one of the cations (besides protons) whose transport across the plasma membrane is believed to contribute to the maintenance of membrane potential. To ensure K+ transport, Saccharomyces cerevisiae cells possess several types of active and passive transporters mediating the K+ influx and efflux, respectively. A diS-C3(3) assay was used to compare the contributions of various potassium transporters to the membrane potential changes of S. cerevisiae cells in the exponential growth phase. Altogether, the contributions of six K+ transporters to the maintenance of a stable membrane potential were tested. As confirmed by the observed hyperpolarization of trk1 trk2 deletion strains, the diS-C3(3) assay is a suitable method for comparative studies of the membrane potential of yeast strains differing in the presence/absence of one or more cation transporters. We have shown that the presence of the Tok1 channel strongly influences membrane potential: deletion of the TOK1 gene results in significant plasma membrane depolarization, whereas strains overexpressing the TOK1 gene are hyperpolarized. We have also proved that plasma membrane potential is not the only parameter determining the hygromycin B sensitivity of yeast cells, and that the role of intracellular transporters in protecting against its toxic effects must also be considered.     

Evaluation of the antifungal and plasma membrane H+-ATPase inhibitory action of ebselen and two ebselen analogs in S. cerevisiae cultures

The plasma membrane H⁺-ATPase pump (Pma1p) has been proposed as a viable target for antifungal drugs since this high capacity proton pump plays a critical role in the intracellular regulation of pH and in nutrient uptake of yeast and other fungi. In recent years, this and other laboratories have verified that the antifungal activity of 2-phenylbenzisoselenazol-3(2H)-one, an organoselenium compound commonly referred to as ebselen (1), stems, at least in part, from its inhibitory action on the fungal Pma1p. In the present study, the antifungal efficacy of 2-(3-pyridinyl)-benzisoselenazol-3(2H)-one (2) and 2-phenylbenzisoselenazol-3(2H)-one 1-oxide (3), two ebselen analogs, was evaluated using a strain of S. cerevisiae and compared against that of 1. In addition, the study also examined the inhibitory potential of these three compounds toward the Pma1p of S. cerevisiae. Based on mean IC₅₀ values, the antifungal potency was found to decrease in the order 3?>?1?>?2. However, in terms of inhibitory action on Pma1p, the potency decreased in the order 1?>?3?>?2. The magnitude of these activities appears to be correlated with the corresponding log P values, with compound 2 being the most hydrophilic and the least active of the three.     

The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains

According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from l(d) to l(o) can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca(2+)-ATPase (PMCA), in lipid rafts (l(o) phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid l(o) phase is more compact than in lipid l(d) phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l(o) phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.