Biochimica et Biophysica Acta, 1071 (1991) 375-391
375
© 1991 Elsevier Science Publishers B.V. All rights reserved 0304-4157/91/$03.50
B B A R E V 85390
Review
Mechanisms of acid extrusion in yeast Karel Sigler i and Milan H/Sfer 2 t Institute of Microbiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) and 2 Institute of Botany, University of Bonn, Bonn (Germany) (Received 30 May 1991)
Conte~ts i.
Int~roduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
375
II.
Su A. B. C. D E
~strate-induced acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of proton flux by external pH and buffering capacity . . . . . . . . . . . . . . . . . . . . . . Regulation by substrate uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation by cell concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation by metabolic state o f cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intracellular titratable acidity, during acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
376 376 377 378 376 378
!II.
Processes participating in acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Production o f CO2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Production o f organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Operation o f the plasma m e m b r a n e H ÷-translocating ATPase . . . . . . . . . . . . . . . . . . . . . . . 1. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Reaction mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Role of the ATPase in the build-up of A/.tH + and in p H regulation . . . . . . . . . . . . . . . . . 4. Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Interaction of H +-ATPase with ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. H + / K + exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
379 379 379 380 381 381 381 382 382 383
IV.
Acidification scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Signal transmission pathway as possible t r i g g e r / s o u r c e of acidity excretion . . . . . . . . . . . . . . B. M e a s u r e m e n t of m e m b r a n e potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Determination o f p H i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
384 384 386 386
V.
Acidification in the absence o f metabolic substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Spontaneous acidification caused by lifting of oxygen limitation . . . . . . . . . . . . . . . . . . . . . . . 1. Spontaneous and substrate-induced acidification in starved cells . . . . . . . . . . . . . . . . . . . . 2. Acidification induced by hydrogen peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
386 387 387 387
VI.
O t h e r types o f acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
388
VII. The role o f cellular polyelectrolytes in proton relations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
388
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
388
I. Introduction Correspondence: K. Sigler, Institute o f Microbiology, Czechoslovak A c a d e m y o f Sciences, Vfdefiskfi 1083, 142 20 Prague 4, Czechoslovakia.
Proton relations are known to play an important part in cell physiology. Contrary to early views [97,101], protons are not distributed passively across cell mem-
376 branes and this imposes a constant necessity on living cells to get rid of undesirable acidity. Metabolic and other processes may give rise to additional acid loads of shorter or longer duration. Cells have evolved mechanisms to cope with these loads and to make use of intracellular and transmembrane acidity changes and proton movements as energization devices or signals. The intracellular pH in many cell types is maintained around 7, although this does not hold absolutely [15]. Higher p H i values are found, e.g., in turnout cells; lower pH i values in dormant cell forms. Intracellular acidity affects, apart from metabolic rates, also permeability of membranes for ions [139], cell cycle [43], affinity of ATP to Mg 2+ [142], autoassociation of ATP molecules [158], cell proliferation and differentiation, formation of cytoskeleton components [149], joining of enzyme subunits into oligomers [77], or cellular cAMP level [173,174]. Both intra- and extracellular pH affect transport processes, irrespective of whether the driving force is the protonmotive force. These effects can be both catalytic and allosteric [12(~,121] and reflect also the nature of the transported ~olute [85]. Stabilization of intracellular acidity level is achieved by several basic mechanisms: (i) buffering systems (carbonate system, phosphate system, weak organic acids, cell polyelectrolytes)which serve for rapid pH i readjustment after a perturbation but are insufficient for preserving cell integrity upon a failure of membrane pH-regulating systems [71,139]; (ii) biochemical conversion of non-volatile acids and bases to neutral products [62,157]; (iii) transfer of acidity from cytosol to organeUes [3,34]; and (iv) expulsion of excess acidity from the cell via the plasma membrane. Passive proton flux due to intrinsic passive membrane permeability was estimated at about 0.1% of the maximum rate of H + recirculation in chloroplast and mitochondrial membranes [134]. The main tools of active H + pumping are H+-translocating ATPases of various types, [111]. In microbial cells they fulfill two mutually contrasting roles - pHi-homeostasis and a buildup of transmembrane proton gradients. A A~/'-induced recirculation of H + that would merely increase the passive backflow of protons into the cells and hence run counter the pHi-homeostasis, is prevented by ion fluxes electrically compensating the H + fluxes (CI- and K + channels in organelles, N a + / H + exchange in animal cells and bacteria, non-specific ionic leaks in fungi or H C O 3 - / C I - exchange in animal cells). In Neurospora crassa [144] an artificially imposed intraceUular acid load is alleviated by H + effiux from the cells via an electrogenic H+-pump, with a slower opening of a leak conductance pathway which causes eventually membrane depolarization, which in turn further stimulates the pump. The pH i oz ~east cells appears to be much more sensitive to perturbation than the membrane potential
[173,174]. lntlacellular acidification is usually accompanied by a rise in the cAMP level, but these two processes can be separated [173,174]. Internal acidification is also thought to be responsible for the effect of some antifungal agents (benzoic acid, sorbic acid) on various yeasts [25,70]. The processes responsible for and participating in both the pH i homeostasis and in the buildup and regulation of the transmembrane proton gradient in yeast cells are surveyed in this review. Excretion of acidity, which serves the yeast cells e.g. to ward off non-yeast microorganisms and to facilitate the H+-symports of nutrients, can be observed in a number of situations; it forms an essential part of cell response to diverse stimuli or interventions such as addition of a substrate, salts (KCI) or uncouplers in combination with oxygenation in the absence of substrate (see below), or addition of electron acceptors such as ferricyanide to the external medium. The contributory mechanisms are numerous and the overall process is the result of their orchestration. II. Substrate-induced acidification
A frequently encountered but still not completely understood situation in yeast cells that involves dramatic changes in metabolic state, acidity load on the cells, changes in the composition of the protonmotive force across the plasma membrane, and triggering of a multitude of ion transport processes, is the addition of a dissimilable substrate to (mostly resting) cells. Extrusion of acidity from th~ cells may bring the external pH as low as 1.5-1.7 [26]. The final pH in the suspension (or the transmembrane ApH) is characteristic for given yeast batch and ambient conditions and the acidification curve is typical for given yeast species and cell history. In energy-deprived cells the curve resolves into several segments which signify the start-up of various participato~,y processes (Fig. 1). For S. cerevisiae, an initial rapid segment, C l, with subsequent shoulder on the curve can also be found on the glucose uptake curve [159] and on intracellular inorganic phosphate level [135]. It was ascribed to a rapid osmotic equilibration of the substrate with subsequent slower onset of dissimilation [159]. The most suitable substrates for acidification in S. cerevisiae are glucose, fructose, mannose, sucrose, galactose (after induction), maltose (which, in contrast to the other sugars, is taken up by an H +-symport and may consequently cause an initial extracellular alkalinization; Ref. 30) and ethanol (in thoroughly aerated suspensions; see below).
II-A. Regulation of proton flux by external pH and buffering capacity On increasing the substrate and cell concentration both the rate and the overall extent of acid extrusion
377 7 --% ~
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10 mM) to a suspension of S. cerevisiae (Fig. 4) brings about a conspicuous oxygen evolution due to the action of cell catalase(s), followed by intensive respiration. The nicotinamide adenine nuc!eotide pool in the cells becomes oxidized and acidification of the medium takes place accompanied also by lowering of intracellular pH. The extracellular pH may drop as low as = 3.5. The process is likely to involve activation of the H ÷ATPase since external pH values below 4.5 cannot be reached by CO2, and no extracellular extrusion of organic acids takes place at the observed oxidized s~ate
388 of cellular NADH pool; also the acidification is associated with K + influx, although the latter lags somewhat behind H + extrusion. Moreover, both acidification and K + uptake can be completely prevented by diethylstilbestrol and dicyclohexylcarbodiimide. The difference compared with glucose-induced acidification is in that the H + efflux appears to be compensated solely by K +. Accordingly, when the extracellular K + level drops below a certain level, the peroxide-induced acid extrusion gives rise to membrane hyperpolarization which stops both further H + efflux and endogenous respiration, although the residual O 2 level in the suspension would easily support its continuation. The arrest of both respiration and acidification can be lifted by low concentrations of protonophores or by 100 mM KC! depolarizing the membrane [163]. It is not clear whether the intracellular acidification observed under these conditions can trigger the signal transmission pathway and give rise to the cAMP signal.
VI. Other types of acidification Some of the cellular buffering and H+-exchange systems are represented by polyelectrolytes localized on the cell surface. The cell wall of some yeasts, notably Candida albicans and Pichia humboldtii, has been reported to possess a high cation-exchanger capacity so that, on addition of a cation such as K +, Cs +, Na +, Li + or Ca 2+ a virtually instantaneous acidification ensues which can completely mask, e.g., pH 0 changes accompanying H+-symport of sugars or other nutrients into the cells [75], Acidification also accompanies the functioning of another system in the yeast plasma membrane coupled to transmembrane electron transport catalyzed by a membrane NADH-reductase similar to that found in erythrocytes or other cells [28]. The enzyme oxidizes NADH with a concomitant reduction of an extracellular electron acceptor such as ferricyanide [27,130]. Its activity decreases markedly on transition of the cells from the exponential to the stationary phase of growth. Inhibition of the process by pyrazol was taken as indicative of a requirement for alcohol dehydrogenase as a mediator of electron transfer from NADH to extracellular ferricyanide and attendant H + appearance in the medium. Opekarov~i and Sigler (unpublished data) failed to confirm this finding; however, the rate of both the ferricyanide reduction and the acidification was found to be strongly increased in the presence of glucose (after correction for glucose-induced acidification), indicating that glycolysis may serve as source of NADH. The rate of ferricyanide reduction and H + production was also strongly increased in the presence of vitamin K 3 that forms a redox intermediate between the cellular NADH reductase and ferricyanide. The
insensitivity of this type of acidification to DES points to its independence of the H+-ATPase [196].
VII. The role of cellular polyelectrolytes in proton relations The preceding sections concerned two main processes participating in buffering or pH-regulating phenomena in yeasts - acid-base equilibria of low-molecular buffers (CO2, organic acids) and the action of membrane transport systems ensuring H + (and K +) translocation between the cell and the medium. Another type of buffering substances is represented by extra- and intracellular polyelectrolytes whose protonation and deprotonation may evoke also mechanochemical phenomena. Chemical buffering and substrate-induced transmembrane H + movements usually mask the events occurring on the macromolecules (proteins, RNA) and special conditions are necessary to bring these events forth [188]. Titrations of suspension supernatants, whole suspensions and cell homogenates of C utilis showed that a fraction of the cell homogenate behaves as a weak acid with a 'dissociation constant' of about 4.6. THs dissociable or titratable fraction is directly proportional to the sum of cell proteins and nucleic acids which is proportional to the growth rate [7,189]. The electrical work necessary to dissociate 1 H + from the molecules of these biopolymers (0.28 eV) was found to be in the range of hydrogen bond energies (0.13-0.31 eV; Ref. 29). The number of dissociable hydrogen bonds per 1 g cell dry weight appears to correspond to approximately 25% H + in cell dry weight. This is in accord with the amount of water bound by globular prG~eins, about 20-30% of protein weight [191]. Hydration a n d / o r protonatio,n of biopolymers may thus play an important part iri intracellular pH control (cf. also Refs. 53, 54). I
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375
© 1991 Elsevier Science Publishers B.V. All rights reserved 0304-4157/91/$03.50
B B A R E V 85390
Review
Mechanisms of acid extrusion in yeast Karel Sigler i and Milan H/Sfer 2 t Institute of Microbiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) and 2 Institute of Botany, University of Bonn, Bonn (Germany) (Received 30 May 1991)
Conte~ts i.
Int~roduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
375
II.
Su A. B. C. D E
~strate-induced acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of proton flux by external pH and buffering capacity . . . . . . . . . . . . . . . . . . . . . . Regulation by substrate uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation by cell concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation by metabolic state o f cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intracellular titratable acidity, during acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
376 376 377 378 376 378
!II.
Processes participating in acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Production o f CO2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Production o f organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Operation o f the plasma m e m b r a n e H ÷-translocating ATPase . . . . . . . . . . . . . . . . . . . . . . . 1. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Reaction mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Role of the ATPase in the build-up of A/.tH + and in p H regulation . . . . . . . . . . . . . . . . . 4. Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Interaction of H +-ATPase with ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. H + / K + exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
379 379 379 380 381 381 381 382 382 383
IV.
Acidification scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Signal transmission pathway as possible t r i g g e r / s o u r c e of acidity excretion . . . . . . . . . . . . . . B. M e a s u r e m e n t of m e m b r a n e potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Determination o f p H i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
384 384 386 386
V.
Acidification in the absence o f metabolic substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Spontaneous acidification caused by lifting of oxygen limitation . . . . . . . . . . . . . . . . . . . . . . . 1. Spontaneous and substrate-induced acidification in starved cells . . . . . . . . . . . . . . . . . . . . 2. Acidification induced by hydrogen peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
386 387 387 387
VI.
O t h e r types o f acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
388
VII. The role o f cellular polyelectrolytes in proton relations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
388
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
388
I. Introduction Correspondence: K. Sigler, Institute o f Microbiology, Czechoslovak A c a d e m y o f Sciences, Vfdefiskfi 1083, 142 20 Prague 4, Czechoslovakia.
Proton relations are known to play an important part in cell physiology. Contrary to early views [97,101], protons are not distributed passively across cell mem-
376 branes and this imposes a constant necessity on living cells to get rid of undesirable acidity. Metabolic and other processes may give rise to additional acid loads of shorter or longer duration. Cells have evolved mechanisms to cope with these loads and to make use of intracellular and transmembrane acidity changes and proton movements as energization devices or signals. The intracellular pH in many cell types is maintained around 7, although this does not hold absolutely [15]. Higher p H i values are found, e.g., in turnout cells; lower pH i values in dormant cell forms. Intracellular acidity affects, apart from metabolic rates, also permeability of membranes for ions [139], cell cycle [43], affinity of ATP to Mg 2+ [142], autoassociation of ATP molecules [158], cell proliferation and differentiation, formation of cytoskeleton components [149], joining of enzyme subunits into oligomers [77], or cellular cAMP level [173,174]. Both intra- and extracellular pH affect transport processes, irrespective of whether the driving force is the protonmotive force. These effects can be both catalytic and allosteric [12(~,121] and reflect also the nature of the transported ~olute [85]. Stabilization of intracellular acidity level is achieved by several basic mechanisms: (i) buffering systems (carbonate system, phosphate system, weak organic acids, cell polyelectrolytes)which serve for rapid pH i readjustment after a perturbation but are insufficient for preserving cell integrity upon a failure of membrane pH-regulating systems [71,139]; (ii) biochemical conversion of non-volatile acids and bases to neutral products [62,157]; (iii) transfer of acidity from cytosol to organeUes [3,34]; and (iv) expulsion of excess acidity from the cell via the plasma membrane. Passive proton flux due to intrinsic passive membrane permeability was estimated at about 0.1% of the maximum rate of H + recirculation in chloroplast and mitochondrial membranes [134]. The main tools of active H + pumping are H+-translocating ATPases of various types, [111]. In microbial cells they fulfill two mutually contrasting roles - pHi-homeostasis and a buildup of transmembrane proton gradients. A A~/'-induced recirculation of H + that would merely increase the passive backflow of protons into the cells and hence run counter the pHi-homeostasis, is prevented by ion fluxes electrically compensating the H + fluxes (CI- and K + channels in organelles, N a + / H + exchange in animal cells and bacteria, non-specific ionic leaks in fungi or H C O 3 - / C I - exchange in animal cells). In Neurospora crassa [144] an artificially imposed intraceUular acid load is alleviated by H + effiux from the cells via an electrogenic H+-pump, with a slower opening of a leak conductance pathway which causes eventually membrane depolarization, which in turn further stimulates the pump. The pH i oz ~east cells appears to be much more sensitive to perturbation than the membrane potential
[173,174]. lntlacellular acidification is usually accompanied by a rise in the cAMP level, but these two processes can be separated [173,174]. Internal acidification is also thought to be responsible for the effect of some antifungal agents (benzoic acid, sorbic acid) on various yeasts [25,70]. The processes responsible for and participating in both the pH i homeostasis and in the buildup and regulation of the transmembrane proton gradient in yeast cells are surveyed in this review. Excretion of acidity, which serves the yeast cells e.g. to ward off non-yeast microorganisms and to facilitate the H+-symports of nutrients, can be observed in a number of situations; it forms an essential part of cell response to diverse stimuli or interventions such as addition of a substrate, salts (KCI) or uncouplers in combination with oxygenation in the absence of substrate (see below), or addition of electron acceptors such as ferricyanide to the external medium. The contributory mechanisms are numerous and the overall process is the result of their orchestration. II. Substrate-induced acidification
A frequently encountered but still not completely understood situation in yeast cells that involves dramatic changes in metabolic state, acidity load on the cells, changes in the composition of the protonmotive force across the plasma membrane, and triggering of a multitude of ion transport processes, is the addition of a dissimilable substrate to (mostly resting) cells. Extrusion of acidity from th~ cells may bring the external pH as low as 1.5-1.7 [26]. The final pH in the suspension (or the transmembrane ApH) is characteristic for given yeast batch and ambient conditions and the acidification curve is typical for given yeast species and cell history. In energy-deprived cells the curve resolves into several segments which signify the start-up of various participato~,y processes (Fig. 1). For S. cerevisiae, an initial rapid segment, C l, with subsequent shoulder on the curve can also be found on the glucose uptake curve [159] and on intracellular inorganic phosphate level [135]. It was ascribed to a rapid osmotic equilibration of the substrate with subsequent slower onset of dissimilation [159]. The most suitable substrates for acidification in S. cerevisiae are glucose, fructose, mannose, sucrose, galactose (after induction), maltose (which, in contrast to the other sugars, is taken up by an H +-symport and may consequently cause an initial extracellular alkalinization; Ref. 30) and ethanol (in thoroughly aerated suspensions; see below).
II-A. Regulation of proton flux by external pH and buffering capacity On increasing the substrate and cell concentration both the rate and the overall extent of acid extrusion
377 7 --% ~
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6
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~ o ~
w~ pH i
212°
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b "'"'~::::.":*,t.- . . . . . . .
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10 mM) to a suspension of S. cerevisiae (Fig. 4) brings about a conspicuous oxygen evolution due to the action of cell catalase(s), followed by intensive respiration. The nicotinamide adenine nuc!eotide pool in the cells becomes oxidized and acidification of the medium takes place accompanied also by lowering of intracellular pH. The extracellular pH may drop as low as = 3.5. The process is likely to involve activation of the H ÷ATPase since external pH values below 4.5 cannot be reached by CO2, and no extracellular extrusion of organic acids takes place at the observed oxidized s~ate
388 of cellular NADH pool; also the acidification is associated with K + influx, although the latter lags somewhat behind H + extrusion. Moreover, both acidification and K + uptake can be completely prevented by diethylstilbestrol and dicyclohexylcarbodiimide. The difference compared with glucose-induced acidification is in that the H + efflux appears to be compensated solely by K +. Accordingly, when the extracellular K + level drops below a certain level, the peroxide-induced acid extrusion gives rise to membrane hyperpolarization which stops both further H + efflux and endogenous respiration, although the residual O 2 level in the suspension would easily support its continuation. The arrest of both respiration and acidification can be lifted by low concentrations of protonophores or by 100 mM KC! depolarizing the membrane [163]. It is not clear whether the intracellular acidification observed under these conditions can trigger the signal transmission pathway and give rise to the cAMP signal.
VI. Other types of acidification Some of the cellular buffering and H+-exchange systems are represented by polyelectrolytes localized on the cell surface. The cell wall of some yeasts, notably Candida albicans and Pichia humboldtii, has been reported to possess a high cation-exchanger capacity so that, on addition of a cation such as K +, Cs +, Na +, Li + or Ca 2+ a virtually instantaneous acidification ensues which can completely mask, e.g., pH 0 changes accompanying H+-symport of sugars or other nutrients into the cells [75], Acidification also accompanies the functioning of another system in the yeast plasma membrane coupled to transmembrane electron transport catalyzed by a membrane NADH-reductase similar to that found in erythrocytes or other cells [28]. The enzyme oxidizes NADH with a concomitant reduction of an extracellular electron acceptor such as ferricyanide [27,130]. Its activity decreases markedly on transition of the cells from the exponential to the stationary phase of growth. Inhibition of the process by pyrazol was taken as indicative of a requirement for alcohol dehydrogenase as a mediator of electron transfer from NADH to extracellular ferricyanide and attendant H + appearance in the medium. Opekarov~i and Sigler (unpublished data) failed to confirm this finding; however, the rate of both the ferricyanide reduction and the acidification was found to be strongly increased in the presence of glucose (after correction for glucose-induced acidification), indicating that glycolysis may serve as source of NADH. The rate of ferricyanide reduction and H + production was also strongly increased in the presence of vitamin K 3 that forms a redox intermediate between the cellular NADH reductase and ferricyanide. The
insensitivity of this type of acidification to DES points to its independence of the H+-ATPase [196].
VII. The role of cellular polyelectrolytes in proton relations The preceding sections concerned two main processes participating in buffering or pH-regulating phenomena in yeasts - acid-base equilibria of low-molecular buffers (CO2, organic acids) and the action of membrane transport systems ensuring H + (and K +) translocation between the cell and the medium. Another type of buffering substances is represented by extra- and intracellular polyelectrolytes whose protonation and deprotonation may evoke also mechanochemical phenomena. Chemical buffering and substrate-induced transmembrane H + movements usually mask the events occurring on the macromolecules (proteins, RNA) and special conditions are necessary to bring these events forth [188]. Titrations of suspension supernatants, whole suspensions and cell homogenates of C utilis showed that a fraction of the cell homogenate behaves as a weak acid with a 'dissociation constant' of about 4.6. THs dissociable or titratable fraction is directly proportional to the sum of cell proteins and nucleic acids which is proportional to the growth rate [7,189]. The electrical work necessary to dissociate 1 H + from the molecules of these biopolymers (0.28 eV) was found to be in the range of hydrogen bond energies (0.13-0.31 eV; Ref. 29). The number of dissociable hydrogen bonds per 1 g cell dry weight appears to correspond to approximately 25% H + in cell dry weight. This is in accord with the amount of water bound by globular prG~eins, about 20-30% of protein weight [191]. Hydration a n d / o r protonatio,n of biopolymers may thus play an important part iri intracellular pH control (cf. also Refs. 53, 54). I
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