In VivoActivation by Ethanol of Plasma Membrane ATPase of ...

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Oct 1, 1990 - Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991,

p.

Vol. 57, No. 3

830-835

0099-2240/91/030830-06$02.00/0 Copyright © 1991, American Society for Microbiology

In Vivo Activation by Ethanol of Plasma Membrane ATPase of Saccharomyces cerevisiae M. FERNANDA ROSA' AND ISABEL SA CORREIA2* Departamento de Energias Renovdveis, LNETI, 1699 Lisbon Codex,1 and Laborat6rio de Engenharia Bioquimica, Instituto Superior Tecnico, Av. Rovisco Pais, 1096 Lisbon Codex,' Portugal Received 1 October 1990/Accepted 14 December 1990

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed. brane ATPase during fermentation did not appear reasonable to us. Moreover, we recently proved that the activity of plasma ATPase of S. cerevisiae growing in the presence of another lipophilic agent, octanoic acid, increased 1.5-fold compared with the value in cells grown in its absence (24). The present work was undertaken to clarify the effect of ethanol on the activity of S. cerevisiae plasma membrane ATPase.

The activity of the proton-pumping ATPase in the plasma membrane of fungi generates a proton gradient that couples ATP hydrolysis to the extrusion of protons across the membrane, resulting in the establishment of a transmembrane proton electrochemical gradient which drives the secondary transport (3, 19, 20, 22). Considering the high energy investment resulting from the proton pump working close to its maximum capacity, this is observed only under special circumstances, and usually ATPase activity is maintained at much lower values (19, 20). For example, when cells of Saccharomyces cerevisiae were incubated in vivo with glucose, the plasma ATPase activity increased as much as 10-fold (18). This proton-pumping ATPase was also activated in vivo by acid pH, and this activation appeared to constitute a mechanism to regulate internal pH (6). Ethanol interacts with membranes by insertion into the hydrophobic interior, increasing the polarity of this region, weakening the hydrophobic barrier to the free exchange of polar molecules and hydrophobic interactions, and affecting the positioning of proteins within the membranes (8). Therefore, the structural alteration of plasma membrane should be among the mechanisms underlying ethanol toxicity, and the plasma membrane ATPase might be a critical target. The transmembrane proton flow was found to be sensitive to the range of ethanol concentrations that significantly reduced the fermentation rate (2, 3, 13). The dissipation of the proton gradient induced by ethanol may involve the inhibition of plasma membrane ATPase activity as proposed by Cartwright et al. (3), the stimulation of passive proton influx due to the nonspecific increase of plasma membrane permeability (11, 17, 22), or a combination of both. Considering the observations of Dombek and Ingram (4) that internal pH remained near neutrality through batch fermentation, instead of decreasing as expected, and considering the significant increase of ethanol-induced stimulation of the passive proton influx (11), the possible inhibition of plasma mem-

MATERIALS AND METHODS

Microorganism. The respiratory mutant S. cerevisiae IGC 3507 III (11, 16, 22-24) was used. Growth media and conditions. Cells of S. cerevisiae were batch cultured in liquid medium (pH 5.4) containing 30 g of glucose, 5 g of yeast extract (Difco Laboratories), 1.7 g of yeast nitrogen base (Difco), 5 g of ammonium sulfate, each per liter, and increasing concentrations of added ethanol. Growth was carried out with orbital agitation at 30°C, and media were inoculated with cells pregrown in an identical medium with 100 g of glucose per liter (initial biomass, 0.14 g [dry weight] per liter). Analysis of ethanol, biomass, and glucose during growth. At suitable times, samples were taken, centrifuged, and analyzed for glucose (dinitrosalicilic acid method [12]) and ethanol by gas chromatography (15). Growth was followed by optical density at 640 nm (OD640), and, at the stationary phase, biomass concentration was determined by cell dry weight. Activity of plasma membrane ATPase. (i) Sampling of yeast cells. To study the effect of ethanol on the plasma membrane ATPase of S. cerevisiae, the activity of the ATPase was determined in the total membrane fraction prepared from cells grown in the presence of increasing concentrations of ethanol as described above and harvested under standardized conditions (in the mid-exponential phase when the biomass concentration reached about 0.5 mg [dry weight] per ml [OD64O = 1.75]). After centrifugation at 4°C, the cell pellets were resuspended in their appropriate supernatants

* Corresponding author. 830

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ETHANOL AND S. CEREVISIAE PLASMA MEMBRANE ATPase

to obtain a cell density of 15 mg (dry weight) per ml. These

-o

cell suspensions (total volume, 4 ml) were quickly frozen

In

(-70°C) until use, after the addition of concentrated solu-

tions of N-[tris(hydroxymethyl)methylglycine], EDTA, and dithiothreitol (final concentrations of 100, 5, and 2 mM, respectively) (5). (ii) Preparation of the total membrane fraction. Frozen samples were thawed, and the cell suspensions, in aliquots of 2 ml, were disintegrated with 1.5 ml of glass beads (Sigma; 0.5-mm diameter) by mixing with a vortex mixer for 1 min intercalated by 1 min on ice (repeated eight times). The homogenates were diluted with 5 ml of a medium containing 0.33 M sucrose, 0.1 M Tris (adjusted to pH 8 with HCl), 5 mM EDTA, and 2 mM dithiothreitol. After centrifugation for 3 min at 900 x g, the supernatants were decanted and centrifuged for 45 min at 40,000 x g (Beckman JA-20.1 rotor) at 4°C. The total membrane fraction was resuspended in a medium containing 20% glycerol, 10 mM Tris (adjusted to pH 7.5 with NaOH), 0.1 mM EDTA, and 0.1 mM dithiothreitol. Yields ranging from 35 to 60 ,ug of protein per mg of dry biomass were obtained. Protein concentrations obtained in the total membrane fractions ranged from 5 to 8.5 mg/ml. Protein concentrations were determined by the method of Bradford (1) by using bovine serum albumin (Sigma) as the standard. For comparison, the membrane fractions were also extracted by cell disintegration for a total of 2 min by vortexing with the glass beads. (iii) ATPase assay. The ATPase activity of the total membrane fraction (50 to 85 ,ug of protein per 500 RI of the assay mixture) was determined in the assay medium containing 50 mM 2-(N-morpholino)ethanesulfonic acid (pH 5.7, adjusted with Tris), 10 mM MgSO4 7H20, 50 mM KCI, 5 mM sodium azide (to inhibit mitochondrial ATPase), 0.2 mM ammonium molybdate (to inhibit acid phosphatases), and 100 mM KNO3 (to inhibit vacuolar ATPase) (18). Under these conditions, ATPase activity could be attributed predominantly to plasma membrane ATPase, since orthovanadate (100 ,uM), a specific inhibitor of plasma membrane ATPase (18, 20), was found to inhibit 85 to 90% of the ATPase activity determined in its absence. After 5 min of incubation of the assay mixture at 30°C for thermostabilization, the enzyme assay was started by the addition of a concentrated solution of ATP (Sigma) (final concentration, 2 mM). After 2, 4, 6, and 8 min, the reaction was stopped by the addition of 500 ,I of trichloroacetic acid (10% [wt/vol]), at 4°C, to 500 ,1u of the reaction mixture. The membranes were then separated by centrifugation, and 900 ,ul of 0.8 N HCl with 0.5% (vol/vol) ammonium molybdate was added to the supematant. The concentration of Pi liberated was determined as described by Fiske (7), using Na2HPO4 H20 as the standard. The ATPase activity was measured in nanomoles of mol Pi released min-' mg of protein-1. Characterization of basal and activated ATPase. The kinetic properties of the plasma membrane ATPase in the total membrane fraction prepared from cells grown in the absence (presenting a basal value) or with 6% (vollvol) added ethanol (presenting a higher specific activity) were compared (Km for ATP, pH profiles, and sensitivity to orthovanadate). The Km for ATP was calculated by a least-squares fitting to the Lineweaver-Burk plot of ATPase activity versus ATP concentration for concentrations of ATP in the range of 0.4 to 2.0 mM. The pH profile was drawn in the range of 4.5 to 7.5 (by the addition of 2 mM Tris). To evaluate the inhibition of plasma ATPase by orthovanadate, the crude membrane fraction was preincubated for 10 min in an assay medium containing increasing concentrations of Na3VO4- 14H20 -

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pH 5.4, 3% glucose) in the presence of increasing concentrations of ethanol. Results are representative of the many growth experiments

performed.

(O to 400 ,uM) at pH 5.7 and 30°C, and the enzyme reactions were started as described above. Cycloheximide and in vivo activation of ATPase. Cells grown in the absence of ethanol and harvested under standardized conditions were resuspended in the growth medium either without ethanol or with 6% (vol/vol) ethanol plus 0.1% (wt/vol) cycloheximide. After 0, 15, and 55 min of incubation at 30°C, cells were harvested and the total membrane fraction was extracted to determine the specific activity of plasma membrane ATPase. Reversibility of ATPase activation. Cells grown with 6% (vol/vol) added ethanol (30°C, pH 5.4) and presenting a more active plasma membrane ATPase were harvested by centrifugation when biomass reached the standardized value of 0.5 mg (dry weight) per ml (equivalent to an OD640 of 1.75). The pellet was resuspended in a volume of fresh growth medium without ethanol (0.5 mg [dry weight] per ml). Cells were incubated at 30°C with orbital agitation, and after 15, 45, and 90 min of incubation, samples were taken and the total membrane fraction was extracted to assay plasma ATPase activity. Effects of ethanol on the activity of plasma membrane ATPase in the membrane fraction. To examine the effect of ethanol on the in vitro activity of plasma membrane ATPase, the crude membrane fraction extracted from cells of S. cerevisiae grown in the absence of ethanol was incubated in the mixture for ATPase assay, at 30°C and pH 5.7, in the presence of increasing concentrations of ethanol (up to 12% [vol/vol]), for 3 or 60 min before starting the enzyme reaction by the addition of ATP.

RESULTS Effects of ethanol on the kinetics and energetics of growth with ethanol. The addition of ethanol in the range of 0 to 10% (vol/vol) (these concentrations allowed balanced growth until glucose exhaustion) to a growth medium with 30 g of glucose per liter depressed the specific growth rate of S. cerevisiae IGC 3507 III (Fig. 1). Growth and ethanol yields

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FIG. 2. Specific activity of the plasma membrane ATPase in the total membrane fraction extracted from cells of S. cerevisiae IGC 3507 III grown without or with increasing concentrations of ethanol and harvested under standardized conditions (a) and from cells grown with 6% (vol/vol) of ethanol and incubated in fresh medium without ethanol for 0 to 90 min (b). Bars represent the standard deviation from at least three independent experiments with two enzyme assays each. The results of the ATPase reversion experiment are the means of two independent experiments with two enzyme assays each.

(grams of dry biomass or grams of ethanol per gram of glucose consumed) also suffered a deep decrease for ethanol concentrations above 6% (vol/vol) (Fig. 1). However, for concentrations of added ethanol equal to or less than 6% (vol/vol), the much slighter decrease of the biomass yield of the respiratory mutant strain under study was associated with an increase in the ethanol yield (Fig. 1). In vivo activation by ethanol of plasma membrane ATPase. (i) Activity of plasma membrane ATPase in cells grown with or without ethanol. The specific activity of plasma membrane ATPase determined in the crude membrane fractions prepared from cells grown in the absence of ethanol was found to be significantly below the activity in cells grown with concentrations of ethanol up to 10% (vol/vol) (Fig. 2a). The maximal activity was detected in cells grown with ethanol in the concentration range of 6 to 8% (vol/vol) and reached a value three times higher than the activity in cells grown in its absence (Fig. 2a). Cells used for a comparison of the specific activities of plasma membrane ATPase were grown with or without increasing concentrations of ethanol and harvested in the mid-exponential phase of growth, when the OD640 reached 1.75. By that time, media pH as well as the concentrations of glucose consumed and ethanol produced presented similar values (media pH range, 4.95 to 5.04; residual glucose range, 26 to 29 g liter-1). Under these standardized conditions, and although the cells to be compared were batch cultured, the differences observed in the activity of plasma membrane ATPase might be attributable mainly to the presence of ethanol in the growth media. The values of the specific activity of plasma membrane ATPase determined during the course of this work were below the levels usually mentioned in the literature (18). According to our Materials and Methods, for the preparation of the crude membrane fraction, cells were disintegrated with glass beads by vortexing them for a total of 8 min instead of only 2 min as indicated by other authors (18). By using disintegration conditions identical to those more frequently reported in the literature, we were also able to calculate higher specific activities. This was closely associated with lower values of the total protein present in the crude membrane fraction that were obtained after the shorter grinding period (Table 1). The increase of the ATPase activity in cells grown with 6% (vol/vol) ethanol was also confirmed under these conditions, and the percentage of

activation was independent of the disintegration time (Table 1). (ii) In vivo reversion of ATPase activation. The in vivo activation of plasma membrane ATPase observed for yeast cells grown with ethanol was rapidly reversed, also in vivo, after ethanol removal (Fig. 2b). The level of the ATPase specific activity did not, however, reach values as low as the basal level (Fig. 2b), probably because incubation (30°C) in the growth medium without ethanol for 15 to 90 min led to cell growth (the OD640 of 1.6, observed when incubation in the fresh medium started, increased up to 1.8, 2.2, and 3.0 after 15, 45, and 90 min of incubation, respectively). In fact, we found that the specific activity of plasma ATPase increased when cells were harvested slightly later, during the exponential phase of growth (14). (iii) Characteristics of basal and activated plasma membrane ATPase. The comparison of several characteristics of the basal level and the ethanol-activated plasma ATPases suggested that no conformational change of the enzyme had occurred. In fact, although the Vmax values differed significantly, the sensitivities to orthovanadate (Fig. 3a), the Km values for ATP (0.56 and 0.49 mM for the basal and the activated enzymes, respectively [Fig. 3b]), and the pH profiles, with optimal values in the range of 5.5 to 6.0 (Fig. 3c), were virtually identical. These results, in addition to the fact that the activated state was preserved after the extraction of the total membrane fraction despite the decrease or the elimination of the ethanol incorporated in the plasma TABLE 1. Comparison of the specific activities of plasma membrane ATPase in cells grown without or with 6% (vol/vol) ethanol and of the protein concentrations in the crude membrane fractions obtained after cell disintegration' Sp act after disintegration for: Ethanol concn

(% [vol/vol])

0 6

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6.7 5.5

a Cell disintegration after a total of 2 or 8 min of grinding with glass beads (diameter, 0.5 mm).

VOL. 57, 1991

ETHANOL AND S. CEREVISIAE PLASMA MEMBRANE ATPase

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FIG. 3. Comparison of various characteristics of plasma membrane ATPase in the total membrane fraction prepared from cells of S. cerevisiae IGC 3507 III grown in the absence (O) or presence (A) of 6% (vol/vol) ethanol: sensitivity to orthovanadate (a), activity as a function of ATP concentration (Lineweaver-Burk plot) (b), and pH profile (c). Results are the means of at least two enzyme assays.

membrane, led us to hypothesize that these two forms might be essentially the same enzyme and to investigate the effect on the reported activation of an inhibitor of protein synthesis (cycloheximide). (iv) In vivo activation and protein synthesis. The incubation (30°C) of yeast cells, grown in the absence of ethanol, in the same growth medium plus 6% (vol/vol) ethanol led to the rapid increase of the plasma membrane ATPase activity (Fig. 4). This activation was not observed if incubation was carried out under identical conditions but in the absence of ethanol, although a slight increase in the ATPase activity was also observed, presumably due to the interference of cell growth (Fig. 4). The activation by ethanol appeared to be virtually dependent on protein synthesis on the basis of the lack of activation when cycloheximide (0.1% [wt/vol]) was added to the incubation medium with ethanol (Fig. 4). The results reported here are not fully conclusive considering that, even during the first 15 min of incubation, cell growth occurred and the specific growth rates were different under the various experimental conditions used. Therefore, after

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60 40 Time, (min) FIG. 4. Specific activity of the plasma membrane ATPase in the total membrane fraction extracted from cells of S. cerevisiae IGC 3507 III grown without ethanol and harvested as for the experiment in Fig. 2 and incubated up to 55 min, with agitation at 30°C, in growth media without ethanol (LI), with 6% (vol/vol) ethanol (A), or with 6% (vol/vol) ethanol plus cycloheximide (0.1%) (0). Results are representative of two different experiments with at least two enzyme

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assays

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15 min of incubation, cells were harvested at slightly different states of growth (the OD64o increased from 2.0 when incubation was started up to 2.45 when ethanol was absent and up to 2.2 or 2.1 when 6% [vol/vol] ethanol was added and cycloheximide was absent or present, respectively). After 55 min of incubation, the OD reached 3.1, 2.6, and 2.4 for these same growth conditions, respectively. In vitro inhibition of plasma membrane ATPase by ethanol. Although ethanol activated the plasma membrane ATPase of S. cerevisiae in vivo, it also inhibited this same enzyme in vitro (Fig. 5). Both the basal and the activated ATPase were similarly inhibited by ethanol (Fig. 5). When ethanol was present in concentrations above 7% (vol/vol), the specific activity of the activated plasma ATPase extracted from cells grown with ethanol decreased to values similar to those calculated for the nonactivated ATPase that was extracted from cells grown in its absence and incubated with identical concentrations on this inhibitor (Fig. 5).

DISCUSSION The proton-pumping ATPase from the plasma membrane commands vital physiological functions in the yeast cell. It is therefore expected that its activity might be tightly regulated. The dependence of the ATPase activity on the stage of cell growth, on the acidity, and on the glucose concentration in the growth medium are examples (5, 6, 21) of this tight regulation. The in vivo activation of the plasma membrane ATPase of S. cerevisiae IGC 3507 III by ethanol reported in this article shares various aspects with the in vivo activation by octanoic acid which has also been reported to occur in this strain (23). In fact, the activated state of the plasma membrane ATPase induced by either ethanol or octanoic acid exhibited the Km for ATP, the pH profile, and the sensitivity to orthovanadate identical to the characteristics of the basal ATPase present in cells grown in the absence of these two lipophilic molecules. However, while ethanol led to an increase of the maximal enzyme activity up to 3-fold the basal value, an increase of only 1.5-fold was observed for the activation by octanoic acid (24). Another difference between the effects of these two toxic compounds on plasma membrane ATPase is demonstrated by the fact that toxic concentrations of octanoic acid that induced ATPase activation in vivo were not found to inhibit this membrane enzyme in vitro (24), whereas ethanol, in the range of 0 to 10%

834

APPL. ENVIRON. MICROBIOL.

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FIG. 5. Effect on the specific activity of plasma membrane ATPase (absolute values [a and b] or percentage of inhibition [c and dl) of 3 (O, A) or 60 min (A, A) of incubation, with increasing concentrations of ethanol, of the crude membrane fraction extracted either from cells grown in the absence (a and c) or in the presence of 6% (vol/vol) ethanol (b and d). Results are the means of a least two enzyme assays.

(vol/vol), induced the activation of the ATPase in vivo but also inhibited this enzyme in vitro. These results are in agreement with those previously reported by Cartwright et al. (3) that demonstrated the inhibition of plasma membrane ATPase by ethanol. The mechanisms involved either on the in vivo activation by ethanol of the plasma membrane ATPase or the in vivo reversion of this activation after ethanol removal remain obscure. On the basis of our results, the possible stimulation of ATPase biosynthesis induced by these lipophilic molecules cannot be excluded. On the contrary, this appeared to be a reasonably adequate hypothesis since all of the characteristics that were tested for the activated or the basal ATPase were identical, suggesting that no conformational change had occurred and that these two forms could be the same enzyme rather than a modified protein. Moreover, the preservation of the activated state after the extraction of the total membrane fraction in addition to the effect of cycloheximide that appeared to prevent the in vivo ATPase activation are also in accordance with that hypothesis. However, it has been emphasized that, in S. cerevisiae, the overexpression of plasma membrane ATPase was detrimental for the cell and that the regulatory mechanisms should be based on the increase of the catalytic activity rather than on the amount of the enzyme (5, 6, 18, 19). Differences observed for several characteristics of basal and activated enzymes induced by acid (6) were, in fact, consistent with the last hypothesis. For Streptococcus faecalis, however, Kobayashi et al. (9, 10) proposed a model to examine whether the intracellular pH of cells grown in the presence of protonophores or in acid medium is regulated by changes in the amount and activity of the proton-pumping ATPase

which are dependent on intracellular pH. The same authors also put forward a hypothesis to explain the reversion of ATPase activation, i.e., when the low extracellular pH was increased, selective protein degradation occurred in cells grown at high pH. The rapid in vivo reversion of the activated state reported here when ethanol was removed from the growth medium also suggests that the higher enzyme activity observed in cells grown in the presence of 6% (vol/vol) ethanol cannot be attributed mainly to probable differences in the ATPase lipid environment occurring in the plasma membrane of cells grown in the presence of ethanol (25). The possible increase caused by ethanol in the permeability of the membrane vesicles to ATP was ruled out as an explanation for the in vivo increase of the activity of plasma membrane ATPase since this membrane enzyme was in fact inhibited in vitro when incubated with ethanol. It also remains to be seen how the in vivo activation and reversion reported here are regulated and whether we are facing a nonspecific activation involving other enzymes besides plasma membrane ATPase, as was reported to occur in S. faecalis growing in acid medium or in the presence of protonophores (9). On the basis of the literature, it is possible to think that the decrease of yeast intracellular pH induced by ethanol due either to the stimulation of H+ influx or to the in vitro inhibition of the plasma membrane ATPase (3, 11) (Fig. 5) could be associated with the signals for the in vivo activation of this membrane enzyme. On the other hand, activation might constitute a mechanism to regulate internal pH (5, 8-10). The role of the in vivo activation by octanoic acid of the plasma membrane ATPase of S. cerevisiae on the regulation of intracellular pH of cells grown with this lipophilic acid was, however, not proven (24).

VOL. 57, 1991

ETHANOL AND S. CEREVISIAE PLASMA MEMBRANE ATPase

The results reported here may also contribute to an understanding of the mechanisms underlying ethanol toxicity and tolerance in fermenting yeasts, namely, the plasma membrane proton-pumping ATPase as a target for ethanol. Although the mechanisms involved in the in vivo activation by ethanol of this enzyme remain obscure, this phenomenon appears to constitute a cell response towards the deleterious effects of ethanol. In fact, this activation counteracts the negative effects of ethanol, i.e., inhibiting the enzyme and increasing the plasma membrane permeability to protons. Interestingly, the increase of the ethanol yield and the simultaneous slight decrease of the biomass yield induced by concentrations of ethanol below 6% (vol/vol) were consistent with the increase of the ATPase activity in cells growing in the presence of this toxin as compared with cells grown in its absence, suggesting that they could be related. In fact, these results are consistent with the higher energy investment necessary for the ATPase to work with a higher activity (20) in the respiratory mutant under study that obtains energy only by fermentation. Our laboratory recently observed that the in vivo activation by octanoic acid of the plasma membrane ATPase of this yeast strain was also closely associated with the decrease of the biomass yield and the concomitant increase of the ethanol yield (24). For concentrations of ethanol above 7% (vol/vol), close to the maximal concentration for growth, significant decreases in both the growth and ethanol yields were, however, observed (Fig. 1). It was also within the range of ethanol concentrations of 7 to 10% (vol/vol) that the inhibition by ethanol of both the basal and the activated plasma membrane ATPase led to identical and very low values of activity (Fig. 5). This suggests that, for concentrations of ethanol above 7% (vol/vol), cells can no longer efficiently counteract its toxic effects. Studies continue to be done to elucidate and generalize the role of the in vivo activation of the proton-pumping ATPase in the development of ethanol tolerance by fermenting yeast strains.

6. 7. 8. 9.

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14. 15. 16.

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18. 19.

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4.

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Microbiol. 53:1286-1291. 5. Eraso, P., A. Cid, and R. Serrano. 1987. Tight control of the

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