Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid.

Plasma membrane ATPase activity of Saccharomyces cerevisiae IGC 3507III grown in the presence of the lipophilic acid octanoic acid [4-50 mg l-1 (0.03-0.35 mM), pH 4.0] was 1.5-fold higher than that in cells grown in its absence. The Km for ATP, the pH profile and the sensitivity to orthovanadate of the basal and the activated forms of the membrane ATPase were identical. This activation was closely associated with a decrease in the biomass yield and an increase in the ethanol yield, and was rapidly reversed in vivo after removal of the acid. However, the activated level was preserved when membranes were extracted and subjected to manipulations which eliminated or decreased octanoic acid incorporation in the plasma membrane. The activity of the basal plasma membrane ATPase in the total membrane fraction was slightly increased by incubation at pH 6.5 with octanoic acid at 100 mg l-1 or less (2.4 mg acid form plus 97.6 mg octanoate ion l-1). However, destruction of the permeability barrier between the enzyme and its substrate could not explain the in vivo activation. A role for plasma membrane ATPase activation in the regulation of the intracellular pH (pHi) of cells grown with octanoic acid was not proven.     

Modification of plasma membrane lipid order and H+-ATPase activity as part of the response of Saccharomyces cerevisiae to cultivation under mild and high copper stress

Plasma membrane lipid disorganization takes place in cells of Saccharomyces cerevisiae grown under copper stress, as shown by fluorescence anisotropy measurements with the lipid reporter probe 1,6-diphenyl-1,3,5-hexatriene. The extent of plasma membrane disorganization, presumably due to copper-induced lipid peroxidation, was discontinuous when measured in cells grown in media supplemented with different concentrations of CuSO4. Results suggested the existence of adaptive mechanisms that cells employ to protect themselves against the deleterious effects of copper. The adaptive mechanisms examined in this study included the coordinate increase in the activities of Cu,Zn-superoxide dismutase (up to five-fold), glutathione reductase (up to 1.7-fold), and plasma membrane H+-ATPase (up to three-fold). These enzyme activities showed maximal levels in cells grown with copper supplied at intermediate concentrations, within the range that allowed growth. Significantly, at these concentrations, plasma membrane disorganization did not increase when increasing amounts of CuSO4 were supplied. However, at copper concentrations close to the maximal that allowed growth, the capacity of the yeast cell response to cope with the deleterious effects of copper was exceeded; plasma membrane lipid organization and plasma-membrane-bound H+-ATPase activity drastically declined in response to the increased levels of copper stress and the consequences on growth kinetics were even more severe. Our results clearly suggest that modification of plasma membrane H+-ATPase activity is either part of or the result of the global response of yeast to mild or high copper stress.     

Hsp30, the integral plasma membrane heat shock protein of Saccharmyces cerevisiae, is a stress-inducible regulator of plasma membrane H+-ATPase

Saccharomyces cerevisiae has a single integral plasma membrane heat shock protein (Hsp). This Hsp30 is induced by several stresses, including heat shock, ethanol exposure, severe osmostress, weak organic acid exposure and glucose limitation, Plasma membrane H⁺-ATPase activities of heat shocked and weak acid-adapted, hsp30 mutant and wild-type cells, revealted that Hsp30 induction leads to a downregulation of the stress-stimulation of this H⁺-ATPase. Plasma membrane H⁺-ATPase activity consumes a substantial fraction of the ATP generated by the cell, a usage that will be increased by the H⁺-ATPase stimulation occurring with several Hsp30-inducing stresses. Hsp30 might therefore provide an energy conservation role, limiting excessive ATP consumption by plasma membrance H⁺-ATPase during prolonged stress exposure or glucose limitation, Consistent with the role of Hsp30 being energy conservation, Hsp30 null cultures give lower final biomass yields. They also have lower ATP levels, consistent with higher H⁺-ATPase activity, at the glucose exhaustion stage of batch fermentations (diauxic lag), when Hsp30 is normally induced. Loss of Hsp30 does not affect several strees tolerances but it extends the time needed for cells to adapt to growth under several stressful conditions where the maintenance of homeostasis will demand an unusually high usage of energy. hsp30 is the first yeast gene identified as both weak organic acid-inducible and assisting the adaptation to growth in the presence of these acids.     

Intracellular pH is a major factor in the induction of tolerance to acid and other stresses in Lactococcus lactis.

This study demonstrates that exposure of log-phase Lactococcus lactis subsp. cremoris 712 cells to mildly acid conditions induces resistance to normally lethal intensities of environmental stresses such as acid, heat, NaCl, H2O2, and ethanol. The intracellular pH (pHi) played a major role in the induction of this multistress resistance response. The pHi was dependent on the extracellular pH (pHo) and on the specific acid used to reduce the pHo. When resuspended in fresh medium, cells were able to maintain a pH gradient even at pHo values that resulted in cell death. Induction of an acid tolerance response (ATR) coincided with an increase in the ability of cells to resist change to an unfavorable pHi; nevertheless, a more favorable pHi was not the sole reason for the increased survival at acid pHo. Cells with an induced ATR survived exposure to a lethal pHo much better than did uninduced cells with a pHi identical to that of the induced cells. Survival following lethal acid shock was dependent on the pHi during induction of the ATR, and the highest survival was observed following induction at a pHi of 5.9, which was the lowest pHi at which growth occurred. Increased acid tolerance and the ability to maintain a higher pHi during lethal acid stress were not acquired if protein synthesis was inhibited by chloramphenicol during adaptation.     

Internal substrate concentrations during biotransformation of octanoic acid into 2-heptanone by spores ofPenicillium roquefortii

Internal substrate concentrations were monitored during biotransformation of octanoic acid into 2-heptanone by spores ofPenicillium roquefortii. The fatty acid rapidly enters the spores in its undissociated form, and a Collander-type relation shows that it strongly accumulates in the spore wall and membrane; this accumulation is reversible. The reaction takes place with cytoplasmic substrate concentrations that quickly fall to zero, and the process is limited by octanoic acid penetration into the cells. This entry is accompanied by proton efflux and involves an active transport process with a H⁺-ATPase system that exhibits Michaelian behavior. The driving force is postulated to be pH, which takes a value set by the initial substrate concentration through the stoichiometry of the H⁺/octanoic acid exchange.     

The pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast.

Exposure of Saccharomyces cerevisiae to sorbic acid strongly induces two plasma membrane proteins, one of which is identified in this study as the ATP-binding cassette (ABC) transporter Pdr12. In the absence of weak acid stress, yeast cells grown at pH 7.0 express extremely low Pdr12 levels. However, sorbate treatment causes a dramatic induction of Pdr12 in the plasma membrane. Pdr12 is essential for the adaptation of yeast to growth under weak acid stress, since Deltapdr12 mutants are hypersensitive at low pH to the food preservatives sorbic, benzoic and propionic acids, as well as high acetate levels. Moreover, active benzoate efflux is severely impaired in Deltapdr12 cells. Hence, Pdr12 confers weak acid resistance by mediating energy-dependent extrusion of water-soluble carboxylate anions. The normal physiological function of Pdr12 is perhaps to protect against the potential toxicity of weak organic acids secreted by competitor organisms, acids that will accumulate to inhibitory levels in cells at low pH. This is the first demonstration that regulated expression of a eukaryotic ABC transporter mediates weak organic acid resistance development, the cause of widespread food spoilage by yeasts. The data also have important biotechnological implications, as they suggest that the inhibition of this transporter could be a strategy for preventing food spoilage.     

Active extrusion of potassium in the yeast Saccharomyces cerevisiae induced by low concentrations of trifluoperazine

Trifluoperazine (TFP), the antipsychotic drug, induces substantial K+ efflux, membrane hyperpolarization and inhibition of H+-ATPase in the yeast Saccharomyces cerevisiae. Investigations on the mechanism of these effects revealed two different processes observed at different incubation conditions. At an acidic pH of 4.5 and an alkaline pH of 7.5, K+ efflux was accompanied by substantial proton influx which led to intracellular acidification and dissipation of delta psi formed by cation efflux. The results indicated nonspecific changes in membrane permeability. Similar results were also observed when cells were incubated at pH 5.5-6.0 with higher concentrations of TFP (above 75 microM). On the other hand, low concentrations of TFP (30-50 microM) at pH 5.5-6.0 caused marked membrane hyperpolarization and K+ efflux unaccompanied by the efflux of other cations and by H+ influx. Our experiments indicate that under these conditions K+ efflux was an active process. (1) K+ efflux proceeded only in the presence of a metabolic substrate and was inhibited by metabolic inhibitors. (2) When 0.3-0.9 mM-KCl was present in the medium at pH 6.0, the concentration of K+ within the cells (measured at the end of the incubation with TFP) was much lower than the theoretical concentration of Kin+ if the distribution of K+ between medium and cell water was at equilibrium (at zero electrochemical gradient). (3) Valinomycin decreased the net K+ efflux and decreased the membrane hyperpolarization induced by TFP, probably by increasing the flux of K+ into the cells along its electrochemical gradient. (4) Conditions which led to active K+ efflux also led to a marked decrease in cellular ATP level. The results indicate that under a specific set of conditions TFP induces translocation of K+ against its electrochemical gradient.     

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells cultivated aerobically or anaerobically

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells were examined on the basis of the cell growth rate, NAD(P)H concentration, reactive oxygen production, plasma membrane H⁺-ATPase activity, and ethanol production. In aerobically or anaerobically cultured yeast cells, NAD(P)H concentration decreased with increasing concentration of menadione, and the recovery of NAD(P)H concentration was proportional to the cell growth rate. However, there was no relationship among the inhibition of the cell growth and reactive oxygen production, plasma membrane H⁺-ATPase activity, and ethanol production. Among them, ethanol production showed resistance to the cytotoxicity of menadione, suggesting the resistance of glycolysis to menadione. The growth inhibitory effect of menadione depended on the rapid decrease and the recovery of NAD(P)H rather than production of reactive oxygen species regardless of aerobic culture or anaerobic culture and presence or absence of mitochondrial function. The recovery of NAD(P)H concentration after the addition of menadione might depend on menadione-resistant glycolytic enzymes.     

Yeast plasma membrane Ena1p ATPase alters alkali-cation homeostasis and confers increased salt tolerance in tobacco cultured cells

In plants, the plasma membrane Na(+)/H(+) antiporter is the only key enzyme that extrudes cytosolic Na(+) and contributes to salt tolerance. But in fungi, the plasma membrane Na(+)/H(+) antiporter and Na(+)-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na(+) and Li(+) efflux across the plasma membrane during salt stress and for K(+) efflux at high pH and high K(+). To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na(+) and Li(+) than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K(+) than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na(+), Li(+), and K(+)) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K(+)-ATPase activity, Na(+)-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress.     

Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis.

Bacillus subtilis induced a set of general stress proteins in response to a salt or heat stress. Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g. 52 degrees C). In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations. A mild heat shock induced a cross-protection against lethal salt stress. The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress. Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli. The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.     

Cadmium-induced adaptive resistance and cross-resistance to zinc in Xanthomonas campestris

Cadmium (Cd) and zinc (Zn) are environmental pollutants affecting both soil and water. The toxicity resulting from the exposure of Xanthomonas campestris, a soil bacterium and plant pathogen, to these metals was investigated. Pretreatment of X. campestris with sub-lethal concentrations of Cd induced adaptive protection against subsequent exposure to lethal doses of Cd. Moreover, Cd-induced cells also showed cross-resistance to lethal concentrations of Zn. These induced protections required newly synthesized proteins. Unexpectedly, Zn-induced cells did not exhibit adaptive protection against lethal concentrations of Zn or Cd. These data suggested that the increased resistance to Cd and Zn killing probably involved other protective mechanisms in addition to ion efflux.     

Modulation of reactive oxygen species production during osmotic stress in Arabidopsis thaliana cultured cells: involvement of the plasma membrane Ca2+-ATPase and H+-ATPase

In Arabidopsis thaliana cells, hypoosmotic treatment initially stimulates Ca2+ influx and inhibits its efflux and, concurrently, promotes a large H2O2 accumulation in the external medium, representative of reactive oxygen species (ROS) production. After the first 10-15 min, Ca2+ influx rate is, however, lowered, and a large rise in Ca2+ efflux, concomitant with a rapid decline in H2O2 level, takes place. The drop of the H2O2 peak, as well as the efflux of Ca2+, are prevented by treatment with submicromolar concentrations of eosin yellow (EY), selectively inhibiting the Ca2+-ATPase of the plasma membrane (PM). Comparable changes of Ca2+ fluxes are also induced by hyperosmotic treatment. However, in this case, the H2O2 level does not rise, but declines below control levels when Ca2+ efflux is activated. Also K+ and H+ net fluxes across the PM and cytoplasmic pH (pH(cyt)) are very differently influenced by the two opposite stresses: strongly decreased by hypoosmotic stress and increased under hyperosmotic treatment. The H2O2 accumulation kinetics, followed as a function of the pH(cyt) changes imposed by modulation of the PM H+-ATPase activity or weak acid treatment, show a close correlation between pH(cyt) and H2O2 formed, a larger amount being produced for changes towards acidic pH values. Overall, these results confirm a relevant role for the PM Ca2+-ATPase in switching off the signal triggering ROS production, and propose a role for the PM H+-ATPase in modulating the development of the oxidative wave through the pH(cyt) changes following the changes of its activity induced by stress conditions.     

Specific protein phosphorylation occurs in molluscan red blood cell ghosts in response to hypoosmotic stress

Summary The regulation of cellular volume upon exposure to hypoosmotic stress is accomplished by specific plasma membrane permeability changes that allow the efflux of certain intracellular solutes (osmolytes). The mechanism of this membrane permeability regulation is not understood; however, previous data implicate Ca²⁺ as an important component in the response. The regulation of protein phosphorylation is a pervasive aspect of celllular physiology that is often Ca²⁺ dependent. Therefore, we tested for osmotically induced protein phosphorylation as a possible mechanism by which Ca²⁺ may mediate osmotically dependent osmolyte efflux. We have found a rapid increase in³²P_i incorporation into two proteins in clam blood cell ghosts after exposure of the intact cells to a hypoosmotic medium. The osmotic component of the stress, not the ionic dilution, was the stimulus for the phosphorylations. The osmotically induced phosphorylation of both proteins was significantly inhibited when Ca²⁺ was omitted from the medium, or by the calmodulin antagonist. chlorpromazine. These results correlate temporally with cell volume recovery and osmolyte (specifically free amino acid) efflux. The two proteins that become phosphorylated in response to hypoosmotic stress may be involved in the regulation of plasma membrane permeability to organic solutes, and thus. contribute to hypoosmotic cell volume regulation.     

Identification of a calmodulin-regulated soybean Ca(2+)-ATPase (SCA1) that is located in the plasma membrane

Ca(2)+-ATPases are key regulators of Ca(2+) ion efflux in all eukaryotes. Animal cells have two distinct families of Ca(2+) pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane. In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non-plasma membrane locations. Here, we identify a plant cDNA, designated SCA1 (for soybean Ca(2+)-ATPase 1), that encodes Ca(2+)-ATPase and is located at the plasma membrane. The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein. The Ca(2+)-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K(1/2) approximately 10 nM). Two calmodulin binding sequences were identified in the N-terminal domain. An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca(2+) pumps. Our results indicate that SCA1p is structurally distinct from the plasma membrane-localized Ca(2+) pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations. In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress.