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Review

Intracellular pH is a tightly controlled signal in yeast☆

Rick Orij, Stanley Brul, Gertien J. Smits ⁎Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands

a b s t r a c ta r t i c l e i n f o

Article history:Received 30 September 2010Received in revised form 15 March 2011Accepted 15 March 2011Available online 21 March 2011

Keywords:Cytosolic pHSecond messengerHomeostasisDynamic regulationNutrient signalling

Background: Nearly all processes in living cells are pH dependent, which is why intracellular pH (pHi) is atightly regulated physiological parameter in all cellular systems. However, in microbes such as yeast, pHi

responds to extracellular conditions such as the availability of nutrients. This raises the question of how pHi

dynamics affect cellular function.Scope of review: We discuss the control of pHi, and the regulation of processes by pHi, focusing on the modelorganism Saccharomyces cerevisiae. We aim to dissect the effects of pHi on various aspects of cell physiology,which are often intertwined. Our goal is to provide a broad overview of how pHi is controlled in yeast, andhow pHi in turn controls physiology, in the context of both general cellular functioning as well as of cellulardecision making upon changes in the cell's environment.Major conclusions: Besides a better understanding of the regulation of pHi, evidence for a signaling role of pHi

is accumulating.We conclude that pHi responds to nutritional cues and relays this information to alter cellularmake-up and physiology. The physicochemical properties of pH allow the signal to be fast, and affect multipleregulatory levels simultaneously.General significance: The mechanisms for regulation of processes by pHi are tightly linked to the moleculesthat are part of all living cells, and the biophysical properties of the signal are universal amongst all livingorganisms, and similar types of regulation are suggested in mammals. Therefore, dynamic control of cellulardecision making by pHi is therefore likely a general trait. This article is part of a Special Issue entitled: SystemsBiology of Microorganisms.

© 2011 Elsevier B.V. All rights reserved.

1. pH in biological systems

1.1. Definition of biological pH

The pH of a solution is defined as the negative logarithm of thehydrogen ion activity in water. If we assume the inside of a yeast cellto be a watery solution, we can estimate the number of free protons inthe cytosol of a single cell (48 μm3 [1,2] at a pH of 7 [3]) at no morethan ~3000. In contrast, global analysis of protein expression in yeasttells us that the number of protein molecules in a cell is in the order ofmillions [4]. Each of these proteins has multiple protonatable groupswhich can either donate or take up a proton. In addition, acidicmetabolites are also in excess compared to free protons. For instance,the concentration of inorganic phosphate (Pi) in yeast cells isestimated at around 50 mM [5], five orders of magnitude higherthan that of protons. This difference in the numbers of free protonsand potential buffer molecules is important for our perception of pHi.The classical view of the pH of a solution as the concentration of freeprotons in a bulk of water does not seem to apply. Because of this free

proton-to-buffer component ratio, cellular pH will be intrinsicallybuffered by all weakly acidic and basic components of the cell(Table 1). To understand biological function, it is more useful to knowthe relative protonation of all weakly acidic or basic molecules in therelevant cellular compartment. This requires knowledge of the in vivopKa values of acidic and basic groups in their actual cellular context,currently unavailable. Estimating the pKa value of amino acid sidechains, for instance, is far from trivial because of the tertiary structureof proteins. Residues on the protein surface, which are largelysurrounded by water, have an actual pKa that will be different fromthe same residues localized in the protein interior. This can beparticularly relevant when these residues are close to, or part of, anactive site [6,7].

1.2. Determination of pHi in yeast cells

To study the effects of pHi in vivo the need for accurate pHi

determinations is paramount. Methods for determination of pHi inyeast cells usually rely on the determination of the relativeprotonation state of a particular molecule. They include

31P nuclear

magnetic resonance (NMR) [8–12], equilibrium distribution of (radiolabeled) weak acids [13–15] or probing cells with pH-sensitive dyeslike C.SNARF-1 [16], 9-aminoacridine [17] and fluorescein [16,18,19].A disadvantage of some of these techniques is that they require

Biochimica et Biophysica Acta 1810 (2011) 933–944

☆ This article is part of a Special Issue entitled: Systems Biology of Microorganisms.⁎ Corresponding author. Tel.: +31 20 525 5143; fax: +31 20 525 7934.

E-mail addresses: p.j.orij@uva.nl (R. Orij), s.brul@uva.nl (S. Brul), g.j.smits@uva.nl(G.J. Smits).

0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.bbagen.2011.03.011

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extensive manipulation of cells. This manipulation can have significanteffect on the pHi [20]. In addition,many of these techniques are not ableto spatially resolve pH differences within cells. Clearly such spatialresolution is relevant asdifferent organelleswithin eukaryotic cells havetheir own specific pH value [21,22]. Recently, the use of pH-sensitivefluorescent proteins has made it possible to determine pHi in living,unperturbed cells, in a time-resolved and organelle specific manner[23]. One such probe, ratiometric pHluorin [24] was adapted by us andothers for in vivo pHi measurements in Saccharomyces cerevisiae [3,25–29].While thismethod provides a reliable and convenient experimentalsetup it does have its limitations. The pKa of the fluorophore is around7.2 [30] which results in a quick deterioration of measurementresolution at pH values above 8.0 or below 5.5 [3]. In addition,expressionof high levels of pHluorin protein could influence aparticularluminal pH by buffering, especially if the expected pH of the organelle isnear the pKa of the fluorophore, due to the low number free protonscompared to the number of pHluorin molecules.

1.3. pH buffering and homeostasis

As discussed above (Section 1.1), almost all proteins in living cellscontain weakly acidic and basic amino acid residues that can act asbuffers. In addition to proteins, metabolites can also act as buffers. Pi,

for example, has been implicated as an important cellular buffer [31].In the cell, all weakly acidic and basic molecules together constitutethe complex buffer that is the cytosol. The buffer capacity (β) of thisbuffer is defined as the amount of strong base or strong acid thatwould have to be added to increase or increase pH by 1 unit. β isdependent on pH and is largest around the pKa of the buffer molecule.Because the cellular molecules have many different pKa values, β willchange with pHi. Table 1, adapted from Ref. [32], lists the molecules orgroups present in the cell that contribute to β. Because of thiscomplexity, β has not been well defined in yeast. One possiblecontribution to the complexity of the cellular buffer is the volatilecarbon dioxide (CO2). CO2 is continuously produced by fermentingyeast cells and, being apolar, can diffuse freely over membranes. In afully aerated system where CO2 can escape from the culture thedissolved intracellular CO2 concentration can be regarded as constant.However, in cells with pHi ~7.0, it will to some extent react with waterforming HCO3

−. When the cell is faced with a sudden lowering of pH,protons are buffered by this HCO3

− to form H2CO3 (with a pKa = 3.6,but a net pK of the total reaction around 6.4) which in turn isconverted to CO2 and H2O. Mammalian cells are exposed to bloodwhich typically contains about 25 mM of HCO3

− [21]. This type ofbuffering accounts for a significant amount of mammalian cellular β(50–66%) [33]. However, in yeast it appears to constitute no more

Table 1Common pH dependent molecules in biology. Common weak acids and weak-bases in a cell are listed. Note that for amino acid side chains or termini the pKa values given are those offree amino acids. These values can change drastically when incorporated into a protein depending on the structure of the protein and its surroundings.

protein residues

metabolites

amino-terminus

carboxyl-terminus

aspartic/glutamic acid

arginine

cysteine

histidine

lysine

tyrosine

ammonium

phosphate

pKa

8.0

3.1

4.4

12.0

8.5

6.5

10.0

10.0

9.2

2.1, 7.2, 12.7

H++N+ H

H

H

N H

H

H++N+ H

H

H

N H

H

O

C OH C O

O

- H++

C OH

O

C O

O

H++

H++N+ C

H

H N H

H

N H

H

N C

H

H N H

N H

H

S H H++S -

H++C

HN+ CH

CH NH

C

N CH

CH NH

OH H++-O

H++ N+ H

H

H

N H

H

H H

OH

P

OH

OHO

OH

P

O

OHO + H + + 2H+ + 3H+

-

OH

P

O

OO

-

-

O

P

O

OO

-

-

-

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than 15–40% of β [34]. A consequence of this difference between yeastand mammalian cells is the fact that mammalian cells have manydifferent forms of carbonic anhydrases that can accelerate theconversion of CO2 and H2O to HCO3

− and H+, or vice versa, while inyeast only one such protein has been identified [35].

2. Effect of pH on the molecules of life

2.1. pHi effects on metabolites

At neutral pHi nearly all water-soluble biosynthetic intermediatesare ionized because they are either phosphorylated or carboxylated,with the exception of excretory and fermentation products such as CO2

[36]. This prevents the metabolites from leaking out of the cell. Anexample of this is found in glycolysis, where the uncharged glucose isphosphorylated by hexokinase upon transport into the cell by thehexose transporter and all consecutive metabolites are charged (Fig. 1).

As discussed above (Section 1.3), Pi makes a significant contributionto cellular buffer capacity. Its protonation state can, however, also havefunctional consequences. The phosphate ion has 3 pKa values (2.1, 7.2,and 12.7) and can exist in 4 different states in aqueous solution(PO4

3−, HPO42−, H2PO4

− and H3PO4). At neutral pH, only HPO42−

(62%) and H2PO4− (38%) are present in significant amounts. The

functional difference of the 2 forms of the ion can be crucial. In musclecells it was shown thatHPO4

2− andH2PO4− bind to the Ca2+-ATPasewith

equal affinity, but only binding of H2PO4− leads to phosphorylation [37].

Coupled phosphates, whether to metabolites, lipids, or proteins, haveonly two sites remaining for protonation, with one very high pKa and asecond pKa value around neutrality. Phosphorylation of proteins andmetabolites has important regulatory roles in the cell and will affectmolecular behavior with respect to pHi (see Section 2.3).

2.2. Protein folding and function are pH dependent

Every biochemistry textbook teaches about the pH dependence ofenzyme activity. A single protonation event of a basic group in the

active site of an enzyme can change the interaction with aneighboring residue in this site, change the affinity for the substrate,and influence the reaction the enzyme catalyzes. Changes in pHinfluence charged residues of amino acid side chains which can lead tochanges in protein folding, causing reduced activity. Conformationalstability of proteins has been shown to be pH dependent [38], andextreme pH can lead to protein denaturation. In addition, electrostaticinteractions between charged surface residues can have significanteffects on protein stability and activity [39]. pH also influences theinteractions of proteins with other proteins, lipids and metabolites.The classical example of the influence of pH on the interactionbetween a protein and a metabolite is the Bohr effect which describeshow an increasing concentration of protons will reduce the affinity ofhemoglobin for oxygen [40].

Another example of how pHi can influence a specific reaction is thepH dependence of the oxidation–reduction potential of specificreductases and dehydrogenases. In glutathione redox reactions,glutathione reductase catalyzes the transfer of two electrons fromNADPH to glutathione disulfide yielding two molecules of glutathioneand NAD+. Lipoamide dehydrogenase catalyzes the reverse reactionwhere the dithiol is oxidated and the pyridine reduced. By measuringthe potential at different pH values the oxidation–reduction poten-tials were shown to be dependent on protonation state in both thedehydrogenase [41] and the reductase [42].

The effect of pH on enzyme activity becomesmore unpredictable ifwe consider the effect on metabolic pathways. In enzymology, the pHdependence of the enzymes in the glycolytic pathway is extensivelystudied. Efforts have been made to optimize experimental conditionsto measure the highest activity of glycolytic enzymes [43] as well asfinding conditions that approximate the cell's interior [5]. Mostenzymes were shown to have different pH optimums and enzymesare differently affected in their activity by changes in pH. However,the effect of pH on total flux through any pathway is difficult toexplain only by pH effects on its enzymatic components because otherfactors, such as substrate and product concentrations, also play amajor role [44].

glucose glucose-6-phosphate frucose 6-phosphate

frucose 1,6-bisphosphate

dihydroxyacetonephosphate

glyceraldehyde3-phosphate

1,3-bisphospho-glycerate

3-phospho-glycerate

2-phospho-glycerate

phosphoenolpyruvate pyruvate

ATP ADP

ADP

ATP

ADP ATP

ADPATP ADPATP

H O

H O

hexose transporter

hexokinase phosphoglucoseisomerase

phosphofructokinase

aldolase

triosephosphateisomerase

glyceraldehyde phosphate dehydrogenase

phosphoglyceratekinase

phosphoglyceratemutase

enolase

pyruvate kinase

glucose H+H

H+H

NAD+NADH

PiH+

NADNADH

PiPH+H

H+H

Fig. 1. Charged glycolytic intermediates and acidification by glycolysis. To prevent spontaneous leakage from the cells interior all glycolytic intermediates are charged. Green dotsrepresent carbon atoms, yellow dots phosphate groups and red dots carboxyl groups. Upon entering the cell through the hexose transporter, glucose is immediately phosphorylated(yellow dots). Note that prior to cleavage of the glucose molecule by aldolase, it is phosphorylated again by phosphofructokinase so the reaction by aldolase results in 2 chargedproducts. The reaction catalyzed by phosphoglycerate kinase results in the formation of ATP and leaves a carboxyl group on 3-phospho-glycerate. In the final reaction pyruvate is leftwith only the carboxyl group. The pKa of pyruvic acid is 2.49, which means that at physiological pH nearly all of it exists in the dissociated, charged form. In addition, note thatglycolysis yields 2 protons per molecule glucose. The actual contribution of these protons to cellular acidification also depends on the redox state and the ATP–ADP cycle.

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2.3. Phospholipids and pHi

Some lipids in yeast, such as triacylglycerols and steryl esters, arenon-polar. These lipids function mainly as a chemical depot which canbemobilizedupon requirementandare stored in specific compartmentsnamed lipid particles [45]. Other lipids, such as phospholipids, sterols,sphingolipids and glycolipids, form the basis of biological membranes.These all have polar head groups with a charge that is pH dependent.Phospholipids are a major component of biological membranes andtheir polar headgroups contributemajorly to the surface charge of thesemembranes. Neutral or zwitterionic phospholipids like phosphatidyl-choline (PC) and phosphatidylethanolamine (PE) reside mostly in thenon-cytosolic layer while negatively charged phospholipids such asphosphatidylserine (PS) and phosphatidylinositol (PI) predominatelyface the cytosol [46]. Phosphatidic acid (PA) is theprecursor for allmajorphospholipids in yeast [47], although it represents only aminor fractionof the lipids in membranes. Like the other phospholipids, PA has a polarheadgroup that canbeprotonated or deprotonated (Fig. 2).Otherminorphospholipids, such as phosphatidylinositol-3-monophosphate (PI(3)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphati-dylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) are also enriched on thecytosolic side of the membrane. The asymmetrical distribution ofcharged lipids creates a negative inside surface that has been shown todetermine the targeting of proteins containing polycationic motifs inhigher eukaryotes [48]. In HeLa cells for example, PIP2 and PIP3 wereshown to be required to target and retain proteins such as K-Ras, thatcontain clusters of positively charged amino acids, to the plasmamembrane (PM) [49]. In addition, inmacrophages the PM inner surfacecharge was shown to be regulated by internalization of PS andsimultaneous hydrolysis of PIP2 upon calcium influx to regulate proteinlocalization [50].

An example of pHi influence on the interaction of lipids and proteinsin yeast is the pH dependence of the interaction of the transcriptional

regulator Opi1p and PA [51]. At neutral pHi, Opi1p binds PA directly atthe endoplasmic reticulum (ER) [52] and INO1, encoding the rate-limiting enzyme in inositol biosynthesis, is freely transcribed. Upon adecrease in pHi of less than 0.5 unit by a depletion of glucose PA isprotonated and as a result, Opi1p loses its affinity for PA and istranslocated to the nucleus to inhibit transcription of INO1 [51]. In thisexample PA ‘senses’ the change in pH and the change in its protonationstate affects the localization of the transcriptional repressor.

Other likely lipid candidates for pH sensing would be thephosphatidylinositol phosphates (PIPs) mentioned above. In yeast,PIPs have been shown to be involved in vesicular trafficking andrecruitment of specific proteins to the cytoplasmic face of intracellularmembranes [53]. Phosphoinositides form a complex intra- and inter-molecular hydrogen bond network that leads to an intra- andintermolecular dissipation of the charges. Minor pH changes or changesin the overall environment of the lipidswill have a pronounced effect onthe hydrogen bond pattern and, therefore, the charge of the respectivephosphoinositide head groups [54]. The pH-dependent complexionization behavior of PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3 was shownto depend on the phosphate substitution pattern at the inositol ring, butchanges are always affected in physiologically relevant pH values [54].Hydrolysis of PI(4,5)P2 by phospholipase C leads to the formation ofinositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which areboth second-messenger molecules [55,56].

3. Regulation of pHi

3.1. pH compartmentalization

The compartmentalized nature of eukaryotic cells allows them tophysically separate metabolites, proteins, and as a consequencebiochemical processes. Most organelles have their own specific pHvalue, associated with processes that take place in those compartments

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

-

-

phophatidic acid (PA)

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

CH2

CH2

NCH3

CH3

CH3

+

phophatidylcholine (PC)

-

phophatidylethanolamine (PE)

CH2

O

P

O

OO

CHCH2

OO

CC O O

R R

CH2

CH2

NH3+

-

OH

H

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

-OH OH

HH

H HO

OH H

H

phophatidylinositol (PI)

CH2

O

P

O

OO

CHCH2

OO

CC O O

R R

C

CH2

NH3+

-

COOH -

phophatidylserine (PS)

OH

H

CH2

O

P

O

OO

HC HC2

OO

C

R R

-

HH

H HO

OH H

H

phophatidylinositol-3-phosphate (PI(3)P)

P

O

OO --

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

-

-

phophatidic acid (PA)

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

--

--

phophatidic acid (PA)

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

CH2

CH2

NCH3

CH3

CH3

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

CH2

CH2

NCH3

CH3

CH3

phophatidylcholine (PC)

--

phophatidylethanolamine (PE)

CH2

O

P

O

OO

CHCH2

OO

CC O O

R R

CH2

CH2

NH3

-

CH2

O

P

O

OO

CHCH2

OO

CC O O

R R

CH2

CH2

NH3

-

OH

H

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

-OH OH

HH

H HO

OH H

H

phophatidylinositol (PI)

OH

H

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

-OH OH

HH

H HO

OH H

H

phophatidylinositol (PI)

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

CH2

O

P

O

OO

HC HC2

OO

CC O O

R R

--OH OH

HH

H HO

OH H

H

phophatidylinositol (PI)

CH2

O

P

O

OO

CHCH2

OO

CC O O

R R

C

CH2

NH3

-

COOH -

CH2

O

P

O

OO

CHCH2

OO

CC O O

R R

C

CH2

-

COOH -

phophatidylserine (PS)

OH

H

CH2

O

P

OO

OO

HC HC2

OO

C

R R

-OH

HH

H HO

OH H

H

phophatidylinositol-3-phosphate (PI(3)P)

P

O

OO --

P

O

OO ----

OC O

Fig. 2. Charged head groups of phospholipids. Polar head groups of phospholipids commonly found in biological membranes are listed. PA is the precursor for all other lipids. Notethat PA, PI, PI(3)P and PS are negatively charged while PE and PC are neutral due to their zwitterionic nature. Because of their complex ionization and dependence on environmentalinteractions, data on pKa values of the various charged groups are scarce. However, we can hypothesize on the charges that are likely to have pKa values near neutral that could bephysiologically relevant by influencing their interaction with other molecules in case of a change in protonation state. The last charge on phosphate groups is likely to always staycharged in the cell as the highest pKa for Pi is 12.7. The same goes for the positive charge on the amines of PS and PE where the pKa of free ammonium is 9.2. The pKa values of thesecond negative charge of the phosphate group as seen in PA and PI(3)P are most likely to be near neutral and potentially change their protonation state upon a fluctuation in pHi.

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[22,57]. Almost all ions vary in concentration over the PM but also overmembranes of the internal organelles. The ion concentration differencescreate gradients, which are essential for cell functioning as they containenergy used to drivemany cellular processes. H+ gradients over variousyeast membranes have been shown to be involved in the transport ofnutrients by either symport or antiport [58–60], and the ΔpH over themitochondrial inner membrane, created by activity of the electrontransport chain, is used for ATP synthesis [61].

All ionic gradients over a membrane together constitute themembrane potential (Δψ). PMΔψ is considered to be themain drivingforce for ion and nutrient translocation, but it has also been shown tobe involved in lateral segregation of proteins [62]. In mammalian cellsthe Δψ over the PM is primarily maintained by the Na+/K+ ATPase.The energy stored in the Na+-electrochemical gradient generated bythe Na+/K+ ATPase provides the driving force for the Na+/H+

exchangers that predominantly regulate cytosolic pH in mostmammalian cells [21]. In yeast however, the PM Δψ has beenshown to be mainly controlled by the H+-electrochemical gradient[63], although the K+-electrochemical gradient seems to contribute aswell [64,65]. The 2 main proton ATPases in yeast membranes (P-typeand V-type, discussed in the following paragraphs) are thought tocontribute to building Δψ (Fig. 3) [66]. While H+ is important forgenerating Δψ, the membrane potential in turn is shown to affect theactivity of the pumps that contribute to this gradient [67]. A change inthe gradients of one of the ionic componentswill have an effect onΔψ,and therefore influence the other ions. In this way, Δψ couples variousion gradients, such that the transporters of ions other than H+

contribute to pHi regulation, while the H+ gradient in turn affects thegradients of other ions. Indeed, a recent study into the ‘ionome’ of thecomplete yeast knock-out collection revealed that in most mutantsdeviations in concentration of ions seldom affected only one, andspecific ions displayed correlated behavior [68].

The proteins residing in the different organelles can also play animportant role in organellar ion homeostasis; studies on the isoelectric

points of proteins from different organelles show a correlation oflocalization and predicted charge in these compartments, suggesting aco-evolution of isoelectric points and subcellular pH environments[69,70]. The pH of any organelle is a result of a complex interplaybetween buffering of the luminal molecules, passive proton leaks andthe activity of pumps and channels. Dissecting which factors contributemost to the pH of a particular organelle is challenging. To this extent theuse of mathematical models has been shown to be very useful inmammalian systems [71]. The maintenance of proper organelle pHhomeostasis does not only have a local effect but also influences cellularspatial dynamics, such as vesicle trafficking between organelles in thesecretory and endosomal pathways.

3.2. Control of cytosolic pH, proton translocating ATPases

Although pHi is highly dependent on nutrient availability andother environmental conditions such as the metabolic acidification ofthe medium during batch cultivation (see Section 4), cytosolic pH(pHc) in yeast cells growing on glucose is maintained aroundneutrality [3]. The master regulator of pHc in baker's yeast is the P2-type H+-ATPase Pma1p [72,73]. P-type ATPases are membraneproteins that couple transport of cations to ATP hydrolysis. Yeastpossesses sixteen P-type ATPases, two of which transport protons[74]. Pma1p, a single 100-kDa polypeptide, is one of the mostabundant proteins in the PM and pumps protons out of the cell [75] ata stoichiometry of 1 H+ translocated per ATP hydrolyzed, as foundfrom studies in Neurospora crassa and physiological analysis ofmaltose transport in brewer's yeast [76]. PMA1 is an essential gene,which makes it difficult to study since at least 30% of the wild-typeATPase activity is required for growth [77]. Using a range of Pma1point mutants with varying ATPase activity revealed that the ATPasewas rate limiting for growth and that ATPase activity correlated withpHc [78]. Different studies with Pma1 mutants showed that many ofthese mutations resulted in an inability of strains to grow at low pH or

Fig. 3. Contributors to pH regulation. The pH values of the different compartment were taken from various sources in the literature as cited in the main text. The two maincontributors to the building of Δψ by export of protons against the gradient at the cost of ATP are the P-Type ATPase Pma1p in the PM and the V-type ATPase (Vma1p) in variousorganellar membranes. The alkali cation/H+ exchangers in the PM (Nha1p) and various organellar membranes (Vnx1p, Vcxp1, Nhx1p, Kha1p) as well as the K+ transporters Trk1,2puse the energy stored in the Δψ to symport or transport ions and contribute to pHi homeostasis. In the mitochondrial inner membrane protons are pumped out of the mitochondrialmatrix by activity of the electron transport chain (ETC). The ΔpH and the Δψ are utilized by the F1F0-ATPase to synthesize ATP from ADP and Pi. The phosphate carrier (PiC) is alsodependent on ΔpH and symports Pi together with protons. The ADP/ATP carrier (AAC) exchanges ADP for ATP over the membrane and is also dependent on Δψ.

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in the presence of weak acids, suggesting a reduced ability to extrudeprotons from the cell [79]. Transcriptionally, PMA1 was shown to beexpressed under the control of the transcription factor RAP1/TUF [80],and its expression is influenced by carbon source availability [81].However, because of its abundance in the PM [78] and the relativelylong half-life of the protein [82] it is believed that Pma1 regulation ismainly post-translational. Glucose has been shown to inducephosphorylation of Pma1p [83], which causes a increased affinity forATP and an increased Vmax [84]. Mass spectrometry studies haveidentified Ser911 and Thr912 in the C-terminal tail of the protein asthe phosphorylation sites required for activation [85]. Additional datashow involvement of glucose sensing by Snf3p, as well as dependenceon G protein Gap2p, protein kinase C, the ubiquitin–proteasomeproteolytic pathway and interactionwith tubulin [86–88]. The activityof the pump is itself also pH-sensitive, with the activity increasingupon cytosolic acidification [89], causing it to be automaticallyactivated when needed.

S. cerevisiae possesses a second P2-type H+-ATPase, Pma2p, whichcan functionally replace the activity of Pma1p when under control ofthe PMA1 promoter [90]. However, PMA2 is not essential, and undernormal growth conditions it is expressed at a much lower level thanPMA1. The absolute levels of Pma2p in the PM and the absence of aneffect of a deletion of PMA2 on viability or stress tolerance suggest avery minor role for Pma2p in pHi regulation.

While Pma1p in the PM is the primary contributor to pHc

regulation, the activity of other pumps, such as the ATPase in thevacuole, is also important [25]. Vacuolar-type H+-ATPases (V-ATPases) are responsible for acidification of a variety of organellesin the secretory pathway, including the vacuole, endosomes, and lateGolgi apparatus [91]. In contrast to P-type H+-ATPases, V-ATPases inyeast do not reside in the PM but in the membranes of variousorganelles. In contrast to the single-subunit P-type H+-ATPases, V-ATPases consist of approximately fourteen subunits arranged in twosubcomplexes. The peripheral subcomplex V1 harbors the sites forATP hydrolysis, while the V0 subcomplex is embedded in themembrane and contains the proton pore. V-ATPases were shown tobe involved in pHc regulation by pumping protons out of the cytosol[25]. Interestingly, Pma1p is partially mislocalized in mutants lackingV-ATPase subunits (vma mutants) [25,92]. Vma mutants were shownto have 25–40% less Pma1p in the PM [25], suggesting a complexinteraction of different pHc controlling proteins. However, thecontribution of V-ATPases to pHc regulation does not solely consistof the localization of Pma1p. Specific inhibition of V-ATPase activitywith concanamycin A has an immediate effect on pHc withoutaffecting Pma1p localization [25]. Pumping excess protons to thevacuole is a very efficient way of maintaining pHc, as it also acidifiesthe vacuole and contributes to the H+ electrochemical gradient that isneeded for the Δψ across the vacuolar membrane. V-ATPases are notessential for growth at optimal growth conditions, but mutants areunable to grow at a pH below 3 and above 7, and show otherphenotypes such as sensitivity to elevated Ca2+ levels [91]. Thisnarrowed pH range for growth of the vmamutantsmight be explainedby the reduced amount of Pma1p in the PM. Like P-type ATPases, V-ATPases are also activated by glucose by a mechanism that regulatesthe extent of association between the V1 and V0 domains in responseto glucose concentration [93,94]. Together, the P- and V-type ATPasesare responsible for transporting protons out of the cytosol at the costof ATP, generating Δψ that can be used for transport of metabolites orother ions.

3.3. Control of cytosolic pH; cation transporters

One class of transporters that have been shown to have asignificant role in pHc homeostasis comprises alkali cation/H+

exchangers. Most alkali cation/H+ transporters exchange protonsand monovalent cations such as Na+ or K+ over membranes. The

main source of energy of these secondary transporters is the protongradients that are created by the ATPases [95]. The extent to whichthey contribute to pHc homeostasis is not well quantified. Asmentioned before (Section 3.1), the H+ and K+ gradients are themain contributors to Δψ. While Pma1p is considered to be the majorgenerator of Δψ, the K+ transporters Trk1p and Trk2p are thought tobe themajor consumers [66]. These potassium transporters have beenimplicated to be involved in pHc homeostasis. In a double mutant ofthe PPZ1 and PPZ2 genes, which encode two protein phosphatases, anincrease in pHc and a decrease in Δψ were observed that were shownto be a result of an increase in Trk activation rather than a Pma1pinhibition [96]. Furthermore, it was shown that Ppz1p physicallyinteracts with Trk1p and the level of Trk1p phosphorylation isincreased in ppz1,2 mutants [97].

The PM Na+/H+ antiporter Nha1p has been shown to be involvedin pHc homeostasis and has a minor but significant role. Deletion ofNHA1was shown to result in an increased pHc [26,98]. A second Na+/H+ antiporter that has been shown to be involved in pHc homeostasisis Kha1p, which resides in the membrane of the Golgi apparatus [99].KHA1 mutants produced a greater acidification of the mediumcoincident with a pHi alkalinization [100]. Another Na+/H+ exchang-er, Nhx1p, was identified in the membrane of the late endosome.Deletion of its gene resulted in a decreased pHc, and an increased saltsensitivity [26]. In addition, mutational studies on NHX1 revealed thatthese mutations result in trafficking defects that were linked with pHhomeostasis [101]. Lastly, two exchangers were identified in thevacuolar membrane. Vnx1p was shown to exchange both sodium andpotassium cations for protons in the vacuole [102] and Vcx1p, a Ca2+/H+ exchanger [103] that was recently revealed to also have K+/H+

activity [104]. Yeasts possess many more transporters which are pHdependent, such as the putative Cl−/H+ antiporter Gef1p [105] andvarious nutrient/H+ symporters [106]; their role in pHc regulationremains unclear.

3.4. Regulation of organellar pH

As discussed above (Section 3.2), V-ATPases are the main protonpumps in the vacuolar membrane and are responsible for acidificationof this organelle and generation of the Δψ over the membranes of thevacuole, Golgi apparatus and (late) endosome. The Na+(K+)/H+

exchangers Nhx1p, in the endocytic pathway [26], together withVnx1p and Vcx1p in the vacuolar membrane [104], depend on theactivity of V-ATPases but also contribute to vacuolar pH. Vacuolar pH innon-growing yeast has been reported to be ~6.2 [25,107]. Addition ofglucose, which activates V-ATPases and resembles the growth situation,resulted in a decrease of vacuolar pH to around 5.6 [25]. Theoretically,vmamutants may still be able to (partly) acidify their vacuoles throughendocytosis of acidic medium at low pH; whether this actuallycontributes to vacuolar acidification in the wild-type is still underdebate [108,109]. Another protein involved in vacuolar pH regulation isYhc3p. Yhc3p, also known as Btn1p, is a vacuolar membrane proteinand the yeast homolog of human CLN3 involved in Batten disease, a rareneurodegenerative disorder. Deletion of BTN1 resulted in aberrantvacuolar pH and altered V-ATPase activity. Furthermore, Btn1p localizesto the vacuole and is also required for vacuolar arginine transport. Thespecific functional role of Btn1p remains unclear but functions as a H+

uncoupler protein or a H+-antiporter are hypothesized [110].In contrast to the considerable data on cytosolic and vacuolar pH,

surprisingly little data on the pH of other organelles are available inyeast. In mammalian cells the ER was shown to be highly permeableto protons and its pH is therefore near neutral, identical to that of thecytosol [111]. Vesicular compartments of the pathway acidifyprogressing through the Golgi, resulting in a pH of 5.2 for secretorygranules [112]. Correct pH maintenance throughout the pathway isvital for enzyme function in the respective compartments and hasbeen shown to be crucial for post-translational modifications [113]

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and targeting [114] of proteins. The membrane of the nucleus haspores open to small molecules. Therefore, it is logical to assume thatnuclear pH is uniform with pHc. However, a nuclear pH gradient hasbeen reported in mammalian cells [115]. Also, data on the peroxi-somal pH of yeast cells are ambiguous. Yeast peroxisomes arereported to have an alkaline pH, with measurements as high as 8.2in cells grown on oleate [116], while other studies report peroxisomalpH to be acidic [29]. Even the existence of a proton gradient over theperoxisomal membrane remains a matter of debate [117].

In yeast mitochondria, the proton motive force is crucial in bothfermenting and respiring cells. The lack of data on mitochondrial pHcontrol is therefore remarkable. Measurements in mammalian cellshave reported the mitochondrial matrix pH to be around 8 [57,118].Our own measurements in yeast have always been significantly loweraround 7.5 [3]. An explanation for the apparent difference betweenyeast and higher eukaryotes could be that yeasts have the ability toferment glucose, so that they do not require the use of mitochondria forenergy generation. However, growth on non-fermentable carbonsources such as ethanol and glycerol did not lead to highermitochondrial pH [3]. The outermitochondrial membrane is consideredto be permeable to protons. It contains the voltage dependent anionchannel (VDAC), also known as mitochondrial porin, that is permeableto molecules and ions up to 5 kDa [119]. The subject of voltage gatingproperty of VDAC is one of controversy [120]. The ability to change thesize of porin depends on a Δψ over the mitochondrial outer membrane,but how could a membrane with such large pores maintain a Δψ?Evidence for a Δψ over the outer mitochondrial membrane has beenprovided by pH-sensitive GFP measurements of the pH of themitochondrial intermembrane space in human cell lines, which wasshown to be significantly lower (~0.7 pH unit) than the pH of thecytosol [121]. While the difference between the pH of the cytosol andthe mitochondrial matrix in this study was no more than 0.2 pH units,the ΔpH over the inner mitochondrial membrane was measured to be0.9 pH units [121]. The activity of the electron transport chain in theinner mitochondrial membrane which extrudes protons out of thematrix generates this ΔpH and is considered to be themain contributorto the slightly alkaline pH of themitochondrial matrix [21]. This protongradient together with the Δψ is the driving force for ATP synthesis bythe F1F0-ATPase. However, not all of the energy stored in theelectrochemical gradient is used for ATP synthesis. The adeninenucleotide carrier that exchanges ADP and ATP across the innermembrane is also dependent on this gradient, as is the phosphatetranslocase that symports H+ and Pi needed for ATP generation [122].In addition, mitochondrial protein import is also dependent onΔψ overthe mitochondrial inner membrane [123]. Knowledge of alkali cation/H+ exchangers in the inner mitochondrial membranes that couldcontribute to pH regulation is limited. Mdm38p, Mrs7p and Ydl183cphave been shown to be required for mitochondrial K+/H+ exchange,but the pump itself still remains to be identified [124]. A genome-widescreen of simultaneous measurements of Ca2+ and H+ concentrationsin mitochondria of Drosophila recently identified a new Ca2+/H+

antiporter and confirmed the interdependence of cation gradients overthe mitochondrial membrane [125]. The lack of knowledge on howyeast regulates its mitochondrial pH with respect to the differentmembranes and the implication for Δψ, ΔpH, and ATP generationindicates the need for further study on this subject.

4. pHi responds to environmental changes

Although pHi is tightly controlled, pH values in living cells are notstatic but highly dynamic [3,25,93]. Yeast cells are able to withstandsubstantial shifts in pHi without loss of viability. Stress due to, forinstance, absence of nutrients or induced by weak acids can result inpHi shifts in the order of 3 pH units which can be fully counteracted[3]. In the next part of this review we will describe the regulation ofpHi in the face of environmental perturbations.

4.1. pHi and acid stress

Wild-type yeast can grow at medium pH values ranging from 2.5to 8.5, while growth and fermentation kinetics are unaffectedbetween 3.5 and 6.0 [126]. In addition, pHc remains stable aroundneutrality in response to shifts in external pH between 3.0 and 7.5 [3].The ability to withstand these substantial shifts is due to the combinedefforts of Pma1p [12] and V-ATPases [127] at low pH. Because theuptake of various nutrients in yeast depends on the proton gradientover the PM, this organism prefers an acidic external pH and growspoorly at neutral pH or above. Indeed, a number of signaling pathwayshave been identified to be involved in the response of yeast to alkalinepH, among which are the Slt2 MAP kinase, the calcineurin and theRim101 pathways, reviewed in Ref. [128]. Rim101p is a transcriptionalrepressor, which targets a.o. the genes encoding the transcriptionalrepressors Nrg1p and Smp1p. One of the targets of Nrg1p is the Na+

pump gene ENA1, which is induced at alkaline pH and required for iontolerance [129]. One possible mechanism by which the Rim101pathway has been suggested to affect the response to alkaline pH isreassembly of the cell wall. The Rim101 pathway is involved inremodeling the cell wall, in tandem with the Slt2 pathway [130]. TheSlt2 MAP kinase pathway, which is activated by protein kinase C, isinvolved in cell wall remodeling and is activated by alkaline externalpH. Deletion of WSC1 resulted in a considerable reduction of alkali-induced Slt2 activation which led to the hypothesis that thetransmembrane protein Wsc1p could act as a sensor for membranestress caused by high external pH [131]. Alkaline pH also induces atransient increase in intracellular calcium, which activates thecalcineurin pathway [132]. The calcium was shown to be taken upfrom the medium through the Cch1–Mid1 calcium channel. Uponcalcineurin activation, the Crz1p transcription factor enters thenucleus and binds to the promoters of a specific set of alkali-inducedgenes [133]. The many pathways involved in the response to alkalineexternal pH stress its importance in yeast physiology. However, howexternal alkaline pH and the cellular response to this stress affectintracellular pH is not yet understood.

In nature, yeast cells often encounter organic weak acids. Asdiscussed previously, a change in the external proton concentrationby the addition of strong acids or bases has limited effect on pHi. Thesefully ionized compounds cannot diffuse over biological membranesbecause of their charge. Weak acids, on the other hand, always exist inequilibrium between their protonated and deprotonated states. Atlow pH a substantial fraction of weak acid will be protonated, and thisprotonated form can easily pass the PM due to its ability to dissolve inthe membrane. In the near neutral cytosol most of the weak acid willdissociate, leading to intracellular proton and anion release. Both theproton and the anion can subsequently be exported from the cell atthe cost of ATP. However, once outside, the anions will re-associatewith protons due to the lowmedium pH and can diffuse back over themembrane constituting a cycle that could provide a constant drain onthe cells energy reserves. Thismechanism is the basis of classical weakacid theory reviewed in Ref. [134]. There have been many studies onthe effects of different weak acids on pHc of yeast [3,12,26,135–139].The ability of these weak acids to acidify the cytosol was originallythought to be the main contributor to growth inhibition [14]. This isstill believed to be the case for acetic acid [134], but studies on stressinduced by the lipophilic sorbic and benzoic acid indicated thatgrowth inhibition by these acids is mainly due to additional factors[136]. When lipophilic weak acids enter the cell, they activate thetranscription factor War1p which induces transcription of PDR12[140] encoding a PM ATP-binding cassette transporter weak acid thatlowers the intracellular levels of sorbate or benzoate by catalyzing theactive efflux of the preservative anion from the cell [134].The induction of Pdr12p is very fast, leading to high levels of proteinin the membrane within minutes after weak acid stress [140,141]. Onthe other hand, growth and pHc recovery are not observed until after

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1 hour [3]. These results may indicate a buffering capacity of the weakacid anion [130]. Proton extrusion during benzoic acid stress was alsoshown to be accompanied by K+ influx [142]. Yeast cells showsignificant accumulation of K+ in response to long-term exposure tobenzoic acid and deletion of TRK1 confers sensitivity to thispreservative. The combined efforts of Pma1p, Pdr12p and Trk1pseem vital for restoration of homeostasis leading to growth recovery.

4.2. pHi in response to nutrient availability

Yeast cells faced with a depletion of glucose arrest growth, are nolonger able to maintain pHc around neutrality and show a steadydecline in pHc which finally settles around external pH [3]. Culturesbuffered at slightly alkaline pH do not show this same decrease in pHc

upon glucose depletion [93]. Re-addition of glucose to starved cellsleads to a very rapid acidification of the cytosol in approximately30 seconds followed by an alkalinization to neutral pH before growthrecommences [3,143]. The rapid acidification is thought to be due tothe initiation of glycolysis [144]. Indeed, the total proton yield inglycolysis is 2 protons for every molecule of glucose (Fig. 1). Thesubsequent alkalinization is believed to be due to activation of Pma1p,which is activated upon glucose addition [145] and highly sensitive toATP levels which are raised by initiation of glycolysis [66]. These

fluctuations in pHc levels as a result of a change in nutrient availabilityare thought to have important signaling functions.

4.3. pHi as a signal

The physiological relevance of changes in pHi is apparent but ill-understood. Physiologically relevant pHi changes can be very smallones that are difficult to monitor in single cells (Section 2.3). Thequestion remains if these changes in pHi are an actual signal thattriggers a response or create the proper physiological environment forcertain processes to occur. In higher eukaryotes, increases in pHc havebeen shown to be correlated with cell proliferation [146], differen-tiation [147] and cell cycle progression [148], while a decrease in pHc

was shown to promote apoptosis [149]. The intracellular pH also playsan important role in cancer. The ability of tumor cells to metastasize isdependent on intracellular and extracellular pH, reviewed in Ref.[150]. The migration of cells might depend on a local pH gradient thatis induced by Na+/H+ exchangers on one side of the cell. The export ofprotons and resulting local pH alkalinization at this side has beensuggested to induce cytoskeletal rearrangement [151].

In terms of correlations between pHi fluctuations and possiblephysiological responses some data can also be found in yeast. Severalstudies have attempted to describe a direct role for pHc in cell cycle

nucleus7.0

vacuole

PKA

V-ATPase nucleusVo

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[H+]

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[cAMP]

[cAMP](1)

(2)

(3)

(4)(5)+++

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Opi1+++

Opi1+++

Opi1

Fig. 4. pH signaling of glucose availability. In the presence of glucose (A) pHc is maintained around neutral by the combined actions of Pma1p and V-ATPase (see main text for details).This neutral pHhas 2 direct effects: (1) the assembly of V-ATPase, which has been shown to be essential for protein kinase A (PKA) activation and (2) the transition of the protonated formof phosphatidic acid (PAH) in the ER membrane to the deprotonated form of phosphatidic acid (PA−). PA− has a high affinity for the transcriptional repressor Opi1 and causes it to beretained on the ERmembrane. Upon glucose starvation (B) the cytosol acidifies due to the inactivation of Pma1p and V-ATPase. This acidification causes (3) disassembly of V-ATPases and(4) protonation of phosphatidic acid. PAH has low affinity for Opi1 which enables it to move to the nucleus and repress key components of the lipid biosynthesis pathway.When glucoseis re-administered (C) a fast, transient acidification occurs (about 30 seconds, seemain text for details). This causes (5) a transient peak in the cAMP concentration,which has been shownto activate PKA. The activity of Pma1 and V-ATPase restores pHc to neutral.

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progression. Analysis of pHi with31P NMR with respect to cell cycle

progression showed a transient increase in pHi in synchronouscultures upon glucose refeeding that accompanied the progressionthrough the cell cycle checkpoint START, which could not be observedin asynchronous cultures [10]. Further studies using steady statedistribution of propionic acid to measure pHi in cell cycle mutantsconfirmed this observation [152]. However, later studies on cell cycleprogression in the fission yeast Schizosaccharomyces pombe correlat-ing cell length with pHi did not reveal cell cycle related changes in pHi

[20]. Besides the control of growth, or proliferation, other develop-mental decisions may also be regulated by pHc. In the pathogenicyeast Candida albicans, pHc alkalinization was shown to accompanygerm tube formation [153,154]. In addition, it has been suggested thatfungal hyphae contain a pH gradient that sustains polarized growth ofthese cellular structures [155,156]. However, in a recent study ofliving hyphae of the filamentous fungus Aspergillus niger usingmicroscopy and a ratiometric pHluorin derivative, longitudinalcytoplasmic pH gradients were not observed [157]. A final exampleis provided by several studies that report on the involvement of pHi inapoptosis in yeast. It was shown that the pro-apoptotic protein Baxinduces increased mitochondrial matrix pH and decreased pHc.Changes in pHi were followed by classic apoptotic phenomena suchas cytC release, caspase activation and mitochondrial swelling anddepolarization [28]. In addition, pHi regulation by the yeast vacuolewas shown to have an important role in apoptosis induced by aceticacid stress. Yeast strains devoid of a detectable vacuole were shown tohave a more severe pHi decrease, leading to necrosis instead ofapoptosis [158]. In yeast, glucose can induce apoptosis in the absenceof other nutrients [159]. It was recently discovered that mutants withdecreased Pma1p activity are more tolerant to apoptosis induced byglucose [160], suggesting a role of pHi involvement in apoptosis.

All these examples suggest that (transient) changes in pHi areimportant for cell functioning. The effect of pHi could be mediatedeither by the combined partial inhibition of multiple enzymaticreactions in the metabolic network, or a controlled response to aspecific pH related signal. However, in view of recent data, it hasbecome attractive to hypothesize that pHc itself could be an actualsignal or second messenger. Evidence of pHc as a genuine secondmessenger for nutrient availability in yeast is summarized in Fig. 4.First, the protein kinase A (PKA) pathway is regulated by pHc in twodifferent ways. The synthesis of cAMP, the compound required for thefinal activation of PKA, is controlled by pHc [161]. The concentration ofcAMP steeply increases upon glucose addition to non-fermentingcells, and this increase is required for the initial activation of PKA tostart growth [162]. Two independent signals, namely glucose itselfthrough the trimeric G-protein alpha subunit Gpa2, and the glucoseinduced pHc decrease through Ras, lead to the activation of theadenylate cyclase Cyr1 [163]. Additionally, the affinity of Cyr1 for itssubstrate, ATP, is strongly pH dependent, increasing upon decreasingpH [164]. Intracellular acidificationwill thus initiate PKA activity, evenindependent from the glucose signal. However, high glucose itselfleads to intracellular acidification in all likelihood because of theproton release during the initial steps of glycolysis [144], and thus thetwo are tightly linked. Recently, V-ATPase assembly was shown todepend on high pHc, and a rapid disassembly occurred upon glucosestarvation [93]. Correct assembly of the V-ATPases is also required forthe initiation of PKA signaling, as V-ATPase mutants were attenuatedfor glucose addition induced cAMP accumulation. Prolonged exposureto glucose leads to a PKA mediated feedback inhibition of cAMPsynthesis [165], although continuous PKA activity is required duringgrowth on glucose. Additionally, glucose is required for the mainte-nance of a high pHc during growth. How the continuedmild activity ofPKA during growth on glucose is maintained and how this links tocAMP concentrations are not yet fully understood. It is clear, however,that pHc is a crucial signal for the transitions between glucosestarvation, exposure, and depletion.

Cytosolic pH also functions as a signal for glucose availability toprevent unnecessary investments. The interaction of the transcrip-tional repressor Opi1 and the membrane phospholipid PA is releasedby a low pHc, resulting in the inactivation of the inositol biosyntheticpathway [51]. Thus, no transcription for micronutrient synthesis isinitiated in the absence of glucose. These examples raise the questionif other transcriptional regulators could be controlled in a similarfashion, as a plethora of processes associated with growth will have tobe arrested in case of glucose depletion.

4.4. pHi sensors

In order to specifically respond to changes in pHi the cell musthave ways to sense pHi. A pHi sensor should be able to sense a changein pHi by a change in protonation state and be able to functionallytransmit this change as a signal. Given the tight regulation of pHi

discussed above, putative pHi sensors must be able to sense small(~0.3 pH unit) changes in pHi and are therefore likely to containtitratable residues that have a pKa around neutrality. There have beenseveral reports of ion exchangers that have a pH sensing domain thatregulates the activity of these proteins [32]. In mammalian cells, theNHE1 PMNa+/H+ exchanger has an intracellular H+-binding site thatsenses pHc [166]. Detailed studies on the protonation states ofresidues, the NhaA Na+/H+ antiporter in Escherichia coli, revealed apH sensor that can only undergo protonation or deprotonationsubsequent to conformation changes [167].

The main pH regulators in yeast, Pma1p and V-ATPase, have beenshown to have pH dependent activity [89,110,168] but whether theseproteins are able to sense pHi remains incompletely understood. V-ATPase has been implicated as a pH sensor [93,169] and potentialresidues that can act as a sensor have been suggested in mammaliancells [170]. However, these hypotheses are still preliminary andrequire further experimentation. If the activity of a pH regulatingprotein is regulated by post-translational modifications such asphosphorylation and not directly by pHi itself, it is reasonable tohypothesize other factors in the cell sense pHi and transmit this signalto the regulating pumps. One such sensor has been suggested to bethe Ppz1p–Hal3p interaction [97]. An increase in pHc was shown todestabilize the interaction between the phosphatase Ppz1p and itsinhibitory subunit Hal3p. Ppz1p is proposed to subsequently inhibitthe activity of the Trk1p by dephosphorylation. This mechanism haltsalkalinization by the Trk1p K+ transporter to not overshoot thedesired neutral pH of the cytosol. The role for the PA–Opi1 interactionas pH sensor is a final example of a novel mechanism of cell regulation[51]. These types of interactions are very poorly studied in yeast butare likely to bemanifold. The important implications of this novel fieldof research implore further investigation.

5. Perspective

This review summarizes themany aspects involved in pH regulationin yeast. The waywe perceive this regulation has changed considerablyin the past couple of years. The pHi is not a static value but a highlydynamic property of cells that is sensitive to changes in environmentalconditions. Also, pHi proved to be not simply a permissive condition forall biochemical reactions in the cell to occur but was shown to be anactual part of signal transduction as a response to changing environ-mental conditions.

The nature of regulation of cellular processes by pHi is so simple andseems so universal it is hard to imagine that it would not be a conservedbiological phenomenon across all cellular organisms. The utilization ofinorganic phosphate as a cellular buffer and phosphorylation as a wayto activate proteins, which both are sensitive to pH changes in aphysiologically relevant pH range in all cellular systems, is a strongindication of this hypothesis. We believe that we need to be moreaware of these basic mechanisms in our attempt to understand cellular

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functioning. The uncovering of additional mechanisms under control ofintracellular pH promises to be an important and exciting topic forfuture research.

References

[1] F. Sherman, Getting started with yeast, Methods Enzymol. 350 (2002) 3–41.[2] A. Perktold, B. Zechmann, G. Daum, G. Zellnig, Organelle association visualized by

three-dimensional ultrastructural imaging of the yeast cell, FEMS Yeast Res. 7(2007) 629–638.

[3] R. Orij, J. Postmus, A. Ter Beek, S. Brul, G.J. Smits, In vivo measurement ofcytosolic and mitochondrial pH using a pH-sensitive GFP derivative inSaccharomyces cerevisiae reveals a relation between intracellular pH and growth,Microbiology 155 (2009) 268–278.

[4] S. Ghaemmaghami, W.K. Huh, K. Bower, R.W. Howson, A. Belle, N. Dephoure, E.K.O'Shea, J.S. Weissman, Global analysis of protein expression in yeast, Nature 425(2003) 737–741.

[5] K. van Eunen, J. Bouwman, P. Daran-Lapujade, J. Postmus, A.B. Canelas, F.I.Mensonides, R. Orij, I. Tuzun, J. van den Brink, G.J. Smits,W.M. van Gulik, S. Brul, J.J.Heijnen, J.H. de Winde, M.J. de Mattos, C. Kettner, J. Nielsen, H.V. Westerhoff, B.M.Bakker,Measuring enzyme activities under standardized in vivo-like conditions forsystems biology, FEBS J. 277 (2010) 749–760.

[6] F. Milletti, L. Storchi, G. Cruciani, Predicting protein pK(a) by environmentsimilarity, Proteins 76 (2009) 484–495.

[7] J. Mongan, D.A. Case, Biomolecular simulations at constant pH, Curr. Opin. Struct.Biol. 15 (2005) 157–163.

[8] R.B. Moon, J.H. Richards, Determination of intracellular pH by 31P magneticresonance, J. Biol. Chem. 248 (1973) 7276–7278.

[9] G. Navon, R.G. Shulman, T. Yamane, T.R. Eccleshall, K.B. Lam, J.J. Baronofsky, J.Marmur, Phosphorus-31 nuclear magnetic resonance studies of wild-type andglycolytic pathway mutants of Saccharomyces cerevisiae, Biochemistry 18 (1979)4487–4499.

[10] R.J. Gillies, K. Ugurbil, J.A. den Hollander, R.G. Shulman, 31P NMR studies ofintracellular pH and phosphate metabolism during cell division cycle ofSaccharomyces cerevisiae, Proc. Natl Acad. Sci. U.S.A. 78 (1981) 2125–2129.

[11] R.K. Sharma, V. Jain, Effects of 2-deoxy-D-glucose on the photosensitisation-induced bioenergetic changes in Saccharomyces cerevisiae as observed by in vivoNMR spectroscopy, Indian J. Biochem. Biophys. 31 (1994) 36–42.

[12] V. Carmelo, H. Santos, I. Sa-Correia, Effect of extracellular acidification on theactivity of plasma membrane ATPase and on the cytosolic and vacuolar pH ofSaccharomyces cerevisiae, Biochim. Biophys. Acta 1325 (1997) 63–70.

[13] M.T. Kresnowati, C. Suarez-Mendez, M.K. Groothuizen, W.A. van Winden, J.J.Heijnen, Measurement of fast dynamic intracellular pH in Saccharomycescerevisiae using benzoic acid pulse, Biotechnol. Bioeng. 97 (2007) 86–98.

[14] H.A. Krebs, D.Wiggins, M. Stubbs, A. Sols, F. Bedoya, Studies on themechanism ofthe antifungal action of benzoate, Biochem. J. 214 (1983) 657–663.

[15] S. Ramos, M. Balbin, M. Raposo, E. Valle, L.A. Pardo, The mechanism ofintracellular acidification induced by glucose in Saccharomyces cerevisiae, J.Gen. Microbiol. 135 (1989) 2413–2422.

[16] R.S. Haworth, B.D. Lemire, D. Crandall, E.J. Cragoe Jr., L. Fliegel, Characterisation ofproton fluxes across the cytoplasmic membrane of the yeast Saccharomycescerevisiae, Biochim. Biophys. Acta 1098 (1991) 79–89.

[17] G.K. Sureshkumar, R. Mutharasan, Intracellular pH responses of hybridoma and yeastto substrate addition and acid challenge, Ann. NY Acad. Sci. 745 (1994) 106–121.

[18] M. Halm, T. Hornbaek, N. Arneborg, S. Sefa-Dedeh, L. Jespersen, Lactic acidtolerance determined by measurement of intracellular pH of single cells ofCandida krusei and Saccharomyces cerevisiae isolated from fermented maizedough, Int. J. Food Microbiol. 94 (2004) 97–103.

[19] L.U. Guldfeldt, N. Arneborg, Measurement of the effects of acetic acid andextracellular pH on intracellular pH of nonfermenting, individual Saccharomycescerevisiae cells by fluorescence microscopy, Appl. Environ. Microbiol. 64 (1998)530–534.

[20] J. Karagiannis, P.G. Young, Intracellular pH homeostasis during cell-cycleprogression and growth state transition in Schizosaccharomyces pombe, J. CellSci. 114 (2001) 2929–2941.

[21] J.R. Casey, S. Grinstein, J. Orlowski, Sensors and regulators of intracellular pH,Nat. Rev. Mol. Cell Biol. 11 (2010) 50–61.

[22] A. Roos, W.F. Boron, Intracellular pH, Physiol. Rev. 61 (1981) 296–434.[23] R. Bizzarri, M. Serresi, S. Luin, F. Beltram, Green fluorescent protein based pH

indicators for in vivo use: a review, Anal. Bioanal. Chem. 393 (2009) 1107–1122.[24] G. Miesenbock, D.A. De Angelis, J.E. Rothman, Visualizing secretion and synaptic

transmission with pH-sensitive green fluorescent proteins, Nature 394 (1998)192–195.

[25] G.A. Martinez-Munoz, P. Kane, Vacuolar and plasma membrane proton pumpscollaborate to achieve cytosolic pH homeostasis in yeast, J. Biol. Chem. 283(2008) 20309–20319.

[26] C.L. Brett, D.N. Tukaye, S. Mukherjee, R. Rao, The yeast endosomal Na+K+/H+exchanger Nhx1 regulates cellular pH to control vesicle trafficking, Mol. Biol. Cell16 (2005) 1396–1405.

[27] L. Maresova, B. Hoskova, E. Urbankova, R. Chaloupka, H. Sychrova, Newapplications of pHluorin-measuring intracellular pH of prototrophic yeasts anddetermining changes in the buffering capacity of strains with affected potassiumhomeostasis, Yeast (2010).

[28] S. Matsuyama, J. Llopis, Q.L. Deveraux, R.Y. Tsien, J.C. Reed, Changes inintramitochondrial and cytosolic pH: early events that modulate caspaseactivation during apoptosis, Nat. Cell Biol. 2 (2000) 318–325.

[29] F.M. Lasorsa, P. Scarcia, R. Erdmann, F. Palmieri, H. Rottensteiner, L. Palmieri, Theyeast peroxisomal adenine nucleotide transporter: characterization of twotransport modes and involvement in deltapH formation across peroxisomalmembranes, Biochem. J. 381 (2004) 581–585.

[30] K. Nehrke, Intracellular pH measurements in vivo using green fluorescentprotein variants, Methods Mol. Biol. 351 (2006) 223–239.

[31] U. Pick, M. Bental, E. Chitlaru, M. Weiss, Polyphosphate-hydrolysis—a protectivemechanism against alkaline stress? FEBS Lett. 274 (1990) 15–18.

[32] J. Srivastava, D.L. Barber, M.P. Jacobson, Intracellular pH sensors: designprinciples and functional significance, Physiology (Bethesda) 22 (2007) 30–39.

[33] W.F. Boron, Regulation of intracellular pH, Adv. Physiol. Educ. 28 (2004) 160–179.[34] K. Sigler, A. Kotyk, A. Knotkova, M. Opekarova, Processes involved in the creation

of buffering capacity and in substrate-induced proton extrusion in the yeastSaccharomyces cerevisiae, Biochim. Biophys. Acta 643 (1981) 583–592.

[35] R. Gotz, A. Gnann, F.K. Zimmermann, Deletion of the carbonic anhydrase-likegene NCE103 of the yeast Saccharomyces cerevisiae causes an oxygen-sensitivegrowth defect, Yeast 15 (1999) 855–864.

[36] B.D. Davis, On the importance of being ionized, Arch. Biochem. Biophys. 78(1958) 497–509.

[37] Y.M. Khan, J.M. East, A.G. Lee, Effects of pH on phosphorylation of the Ca2+-ATPase of sarcoplasmic reticulum by inorganic phosphate, Biochem. J. 321 (Pt 3)(1997) 671–676.

[38] C.N. Pace, D.V. Laurents, J.A. Thomson, pH dependence of the urea and guanidinehydrochloride denaturation of ribonuclease A and ribonuclease T1, Biochemistry29 (1990) 2564–2572.

[39] M. Akke, S. Forsen, Protein stability and electrostatic interactions betweensolvent exposed charged side chains, Proteins 8 (1990) 23–29.

[40] C. Bohr, K.A. Hasselbalch, A. Krogh, Über einen in biologischer Beziehungwichtigen Einfluss, den die Kohlensäurespannung des Blutes auf dessenSauerstoffbindung übt, Skand. Arch. Physiol. 16 (1904) 401–412.

[41] R.G. Matthews, C.H. Williams Jr., Measurement of the oxidation–reductionpotentials for two-electron and four-electron reduction of lipoamide dehydro-genase from pig heart, J. Biol. Chem. 251 (1976) 3956–3964.

[42] D.M. Veine, L.D. Arscott, C.H. Williams Jr., Redox potentials for yeast, Escherichiacoli and human glutathione reductase relative to the NAD+/NADH redox couple:enzyme forms active in catalysis, Biochemistry 37 (1998) 15575–15582.

[43] P. VanHoek, J.P. VanDijken, J.T. Pronk, Effect of specific growth rate on fermentativecapacity of baker's yeast, Appl. Environ. Microbiol. 64 (1998) 4226–4233.

[44] V. Vojinovic, U. von Stockar, Influence of uncertainties in pH, pMg, activitycoefficients, metabolite concentrations, and other factors on the analysis of thethermodynamic feasibility of metabolic pathways, Biotechnol. Bioeng. 103(2009) 780–795.

[45] S. Rajakumari, K. Grillitsch, G. Daum, Synthesis and turnover of non-polar lipidsin yeast, Prog. Lipid Res. 47 (2008) 157–171.

[46] J.A. Op den Kamp, Lipid asymmetry in membranes, Annu. Rev. Biochem. 48(1979) 47–71.

[47] G.M. Carman, G.S. Han, Regulation of phospholipid synthesis in yeast, J. Lipid Res.50 (Suppl) (2009) S69–S73.

[48] S. McLaughlin, D. Murray, Plasma membrane phosphoinositide organization byprotein electrostatics, Nature 438 (2005) 605–611.

[49] W.D. Heo, T. Inoue, W.S. Park, M.L. Kim, B.O. Park, T.J. Wandless, T. Meyer, PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to theplasma membrane, Science 314 (2006) 1458–1461.

[50] T. Yeung, G.E. Gilbert, J. Shi, J. Silvius, A. Kapus, S. Grinstein, Membranephosphatidylserine regulates surface charge and protein localization, Science319 (2008) 210–213.

[51] B.P. Young, J.J. Shin, R. Orij, J.T. Chao, S.C. Li, X.L. Guan, A. Khong, E. Jan, M.R.Wenk,W.A. Prinz, G.J. Smits, C.J. Loewen, Phosphatidic acid is a pH biosensor thatlinks membrane biogenesis to metabolism, Science 329 (2010) 1085–1088.

[52] C.J. Loewen, M.L. Gaspar, S.A. Jesch, C. Delon, N.T. Ktistakis, S.A. Henry, T.P. Levine,Phospholipid metabolism regulated by a transcription factor sensing phospha-tidic acid, Science 304 (2004) 1644–1647.

[53] T. Strahl, J. Thorner, Synthesis and function of membrane phosphoinositides inbudding yeast, Saccharomyces cerevisiae, Biochim. Biophys. Acta 1771 (2007)353–404.

[54] E.E. Kooijman, K.E. King, M. Gangoda, A. Gericke, Ionization properties ofphosphatidylinositol polyphosphates in mixed model membranes, Biochemistry48 (2009) 9360–9371.

[55] R. Irvine, Cell signaling. The art of the soluble, Science 316 (2007) 845–846.[56] P.J. Belde, J.H. Vossen, G.W. Borst-Pauwels, A.P. Theuvenet, Inositol 1,4,5-

trisphosphate releases Ca2+ from vacuolar membrane vesicles of Saccharomycescerevisiae, FEBS Lett. 323 (1993) 113–118.

[57] J. Llopis, J.M. McCaffery, A. Miyawaki, M.G. Farquhar, R.Y. Tsien, Measurement ofcytosolic, mitochondrial, and Golgi pH in single living cells with greenfluorescent proteins, Proc. Natl Acad. Sci. U.S.A. 95 (1998) 6803–6808.

[58] B. Andre, An overview of membrane transport proteins in Saccharomycescerevisiae, Yeast 11 (1995) 1575–1611.

[59] D. Wipf, U. Ludewig, M. Tegeder, D. Rentsch, W. Koch, W.B. Frommer,Conservation of amino acid transporters in fungi, plants and animals, TrendsBiochem. Sci. 27 (2002) 139–147.

[60] K. Nishimura, K. Igarashi, Y. Kakinuma, Proton gradient-driven nickel uptake byvacuolar membrane vesicles of Saccharomyces cerevisiae, J. Bacteriol. 180 (1998)1962–1964.

942 R. Orij et al. / Biochimica et Biophysica Acta 1810 (2011) 933–944

Author's personal copy

[61] P. Mitchell, Coupling of phosphorylation to electron and hydrogen transfer by achemi-osmotic type of mechanism, Nature 191 (1961) 144–148.

[62] G. Grossmann, M. Opekarova, J. Malinsky, I. Weig-Meckl, W. Tanner, Membranepotential governs lateral segregation of plasma membrane proteins and lipids inyeast, EMBO J. 26 (2007) 1–8.

[63] D.S. Perlin, C.L. Brown, J.E. Haber, Membrane potential defect in hygromycin B-resistant pma1 mutants of Saccharomyces cerevisiae, J. Biol. Chem. 263 (1988)18118–18122.

[64] L. Maresova, E. Urbankova, D. Gaskova, H. Sychrova, Measurements of plasmamembrane potential changes in Saccharomyces cerevisiae cells reveal theimportance of the Tok1 channel in membrane potential maintenance, FEMSYeast Res. 6 (2006) 1039–1046.

[65] R. Madrid, M.J. Gomez, J. Ramos, A. Rodriguez-Navarro, Ectopic potassium uptakein trk1 trk2 mutants of Saccharomyces cerevisiae correlates with a highlyhyperpolarized membrane potential, J. Biol. Chem. 273 (1998) 14838–14844.

[66] A. Goossens, N. de La Fuente, J. Forment, R. Serrano, F. Portillo, Regulation of yeastH(+)-ATPase by protein kinases belonging to a family dedicated to activation ofplasma membrane transporters, Mol. Cell. Biol. 20 (2000) 7654–7661.

[67] D. Seto-Young, D.S. Perlin, Effect of membrane voltage on the plasmamembrane H(+)-ATPase of Saccharomyces cerevisiae, J. Biol. Chem. 266(1991) 1383–1389.

[68] D.J. Eide, S. Clark, T.M. Nair, M. Gehl, M. Gribskov, M.L. Guerinot, J.F. Harper,Characterization of the yeast ionome: a genome-wide analysis of nutrientmineral and trace element homeostasis in Saccharomyces cerevisiae, GenomeBiol. 6 (2005) R77.

[69] C.L. Brett, M. Donowitz, R. Rao, Does the proteome encode organellar pH? FEBSLett. 580 (2006) 717–719.

[70] P. Chan, J. Lovric, J. Warwicker, Subcellular pH and predicted pH-dependentfeatures of proteins, Proteomics 6 (2006) 3494–3501.

[71] M. Grabe, G. Oster, Regulation of organelle acidity, J. Gen. Physiol. 117 (2001)329–344.

[72] T. Ferreira, A.B. Mason, C.W. Slayman, The yeast Pma1 proton pump: a model forunderstanding the biogenesis of plasma membrane proteins, J. Biol. Chem. 276(2001) 29613–29616.

[73] R. Serrano, M.C. Kielland-Brandt, G.R. Fink, Yeast plasma membrane ATPase isessential for growth and has homology with (Na++K+), K+- and Ca2+-ATPases, Nature 319 (1986) 689–693.

[74] P. Catty, A. de Kerchove d'Exaerde, A. Goffeau, The complete inventory of theyeast Saccharomyces cerevisiae P-type transport ATPases, FEBS Lett. 409 (1997)325–332.

[75] P. Morsomme, C.W. Slayman, A. Goffeau, Mutagenic study of the structure,function and biogenesis of the yeast plasma membrane H(+)-ATPase, Biochim.Biophys. Acta 1469 (2000) 133–157.

[76] D.S. Perlin, M.J. San Francisco, C.W. Slayman, B.P. Rosen, H+/ATP stoichiometryof proton pumps from Neurospora crassa and Escherichia coli, Arch. Biochem.Biophys. 248 (1986) 53–61.

[77] A. Ambesi, M. Miranda, V.V. Petrov, C.W. Slayman, Biogenesis and function of theyeast plasma-membrane H(+)-ATPase, J. Exp. Biol. 203 (2000) 155–160.

[78] F. Portillo, R. Serrano, Growth control strength and active site of yeast plasmamembrane ATPase studied by site-directed mutagenesis, Eur. J. Biochem. 186(1989) 501–507.

[79] J.H. McCusker, D.S. Perlin, J.E. Haber, Pleiotropic plasma membrane ATPasemutations of Saccharomyces cerevisiae, Mol. Cell. Biol. 7 (1987) 4082–4088.

[80] E. Capieaux, M.L. Vignais, A. Sentenac, A. Goffeau, The yeast H+-ATPase gene iscontrolledby the promoter binding factor TUF, J. Biol. Chem. 264 (1989) 7437–7446.

[81] R. Rao, D. Drummond-Barbosa, C.W. Slayman, Transcriptional regulation byglucose of the yeast PMA1 gene encoding the plasma membrane H(+)-ATPase,Yeast 9 (1993) 1075–1084.

[82] B. Benito, E. Moreno, R. Lagunas, Half-life of the plasmamembrane ATPase and itsactivating system in resting yeast cells, Biochim. Biophys. Acta 1063 (1991)265–268.

[83] A. Chang, C.W. Slayman, Maturation of the yeast plasma membrane [H+]ATPaseinvolves phosphorylation during intracellular transport, J. Cell Biol. 115 (1991)289–295.

[84] F. Portillo, P. Eraso, R. Serrano, Analysis of the regulatory domain of yeast plasmamembrane H+-ATPase by directed mutagenesis and intragenic suppression,FEBS Lett. 287 (1991) 71–74.

[85] S. Lecchi, C.J. Nelson, K.E. Allen, D.L. Swaney, K.L. Thompson, J.J. Coon, M.R.Sussman, C.W. Slayman, Tandem phosphorylation of Ser-911 and Thr-912 at theC terminus of yeast plasma membrane H+-ATPase leads to glucose-dependentactivation, J. Biol. Chem. 282 (2007) 35471–35481.

[86] M.A. Souza, M.J. Tropia, R.L. Brandao, New aspects of the glucose activation of theH(+)-ATPase in the yeast Saccharomyces cerevisiae, Microbiology 147 (2001)2849–2855.

[87] N. de la Fuente, A.M. Maldonado, F. Portillo, Glucose activation of the yeastplasma membrane H+-ATPase requires the ubiquitin–proteasome proteolyticpathway, FEBS Lett. 411 (1997) 308–312.

[88] A.N. Campetelli, G. Previtali, C.A. Arce, H.S. Barra, C.H. Casale, Activation of theplasma membrane H-ATPase of Saccharomyces cerevisiae by glucose is mediatedby dissociation of the H(+)-ATPase-acetylated tubulin complex, FEBS J. 272(2005) 5742–5752.

[89] P. Eraso, C. Gancedo, Activation of yeast plasma membrane ATPase by acid pHduring growth, FEBS Lett. 224 (1987) 187–192.

[90] P. Supply, A. Wach, D. Thines-Sempoux, A. Goffeau, Proliferation of intracellularstructures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisiae,J. Biol. Chem. 268 (1993) 19744–19752.

[91] P.M. Kane, The where, when, and how of organelle acidification by the yeastvacuolar H+-ATPase, Microbiol. Mol. Biol. Rev. 70 (2006) 177–191.

[92] N. Perzov, H. Nelson, N. Nelson, Altered distribution of the yeast plasmamembrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants, J. Biol.Chem. 275 (2000) 40088–40095.

[93] R. Dechant, M. Binda, S.S. Lee, S. Pelet, J. Winderickx, M. Peter, Cytosolic pH is asecondmessenger for glucose and regulates the PKA pathway through V-ATPase,EMBO J. (2010).

[94] K.J. Parra, P.M. Kane, Reversible association between the V1 and V0 domains ofyeast vacuolar H+-ATPase is an unconventional glucose-induced effect, Mol.Cell. Biol. 18 (1998) 7064–7074.

[95] J. Arino, J. Ramos, H. Sychrova, Alkali metal cation transport and homeostasis inyeasts, Microbiol. Mol. Biol. Rev. 74 (2010) 95–120.

[96] L. Yenush, J.M. Mulet, J. Arino, R. Serrano, The Ppz protein phosphatases are keyregulators of K+ and pH homeostasis: implications for salt tolerance, cell wallintegrity and cell cycle progression, EMBO J. 21 (2002) 920–929.

[97] L. Yenush, S. Merchan, J. Holmes, R. Serrano, pH-Responsive, posttranslationalregulation of the Trk1 potassium transporter by the type 1-related Ppz1phosphatase, Mol. Cell. Biol. 25 (2005) 8683–8692.

[98] H. Sychrova, J. Ramirez, A. Pena, Involvement of Nha1 antiporter in regulation ofintracellular pH in Saccharomyces cerevisiae, FEMS Microbiol. Lett. 171 (1999)167–172.

[99] L. Maresova, H. Sychrova, Physiological characterization of Saccharomycescerevisiae kha1 deletion mutants, Mol. Microbiol. 55 (2005) 588–600.

[100] J. Ramirez, O. Ramirez, C. Saldana, R. Coria, A. Pena, A Saccharomyces cerevisiaemutant lacking a K+/H+ exchanger, J. Bacteriol. 180 (1998) 5860–5865.

[101] S. Mukherjee, L. Kallay, C.L. Brett, R. Rao, Mutational analysis of theintramembranous H10 loop of yeast Nhx1 reveals a critical role in ionhomoeostasis and vesicle trafficking, Biochem. J. 398 (2006) 97–105.

[102] O. Cagnac, M. Leterrier, M. Yeager, E. Blumwald, Identification and character-ization of Vnx1p, a novel type of vacuolar monovalent cation/H+ antiporter ofSaccharomyces cerevisiae, J. Biol. Chem. 282 (2007) 24284–24293.

[103] T.C. Pozos, I. Sekler, M.S. Cyert, The product of HUM1, a novel yeast gene, isrequired for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers, Mol. Cell. Biol. 16 (1996) 3730–3741.

[104] O. Cagnac, M.N. Aranda-Sicilia, M. Leterrier, M.P. Rodriguez-Rosales, K. Venema,Vacuolar cation/H+ antiporters of Saccharomyces cerevisiae, J. Biol. Chem. 285(2010) 33914–33922.

[105] N.A. Braun, B. Morgan, T.P. Dick, B. Schwappach, The yeast CLC proteincounteracts vesicular acidification during iron starvation, J. Cell Sci. 123(2010) 2342–2350.

[106] J. Horak, Yeast nutrient transporters, Biochim. Biophys. Acta 1331 (1997) 41–79.[107] R.A. Preston, R.F. Murphy, E.W. Jones, Assay of vacuolar pH in yeast and

identification of acidification-defective mutants, Proc. Natl Acad. Sci. U.S.A. 86(1989) 7027–7031.

[108] H. Nelson, N. Nelson, Disruption of genes encoding subunits of yeast vacuolar H(+)-ATPase causes conditional lethality, Proc. Natl Acad. Sci. U.S.A. 87 (1990)3503–3507.

[109] A.L. Munn, H. Riezman, Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast: identification of six new END genes, J. Cell Biol. 127(1994) 373–386.

[110] S. Padilla-Lopez, D.A. Pearce, Saccharomyces cerevisiae lacking Btn1p modulatevacuolar ATPase activity to regulate pH imbalance in the vacuole, J. Biol. Chem.281 (2006) 10273–10280.

[111] J.H. Kim, L. Johannes, B. Goud, C. Antony, C.A. Lingwood, R. Daneman, S. Grinstein,Noninvasive measurement of the pH of the endoplasmic reticulum at rest andduring calcium release, Proc. Natl Acad. Sci. U.S.A. 95 (1998) 2997–3002.

[112] P. Paroutis, N. Touret, S. Grinstein, The pH of the secretory pathway:measurement, determinants, and regulation, Physiology (Bethesda) 19 (2004)207–215.

[113] L. Carnell, H.P. Moore, Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport andsorting of secretogranin II, J. Cell Biol. 127 (1994) 693–705.

[114] E. Chanat, W.B. Huttner, Milieu-induced, selective aggregation of regulatedsecretory proteins in the trans-Golgi network, J. Cell Biol. 115 (1991)1505–1519.

[115] O. Seksek, J. Bolard, Nuclear pH gradient in mammalian cells revealed by lasermicrospectrofluorimetry, J. Cell Sci. 109 (Pt 1) (1996) 257–262.

[116] C.W. van Roermund, M. de Jong, I.J. L, J. van Marle, T.B. Dansen, R.J. Wanders, H.R.Waterham, The peroxisomal lumen in Saccharomyces cerevisiae is alkaline, J. CellSci. 117 (2004) 4231–4237.

[117] F.M. Lasorsa, P. Pinton, L. Palmieri, P. Scarcia, H. Rottensteiner, R. Rizzuto, F.Palmieri, Peroxisomes as novel players in cell calcium homeostasis, J. Biol. Chem.283 (2008) 15300–15308.

[118] M.F. Abad, G. Di Benedetto, P.J. Magalhaes, L. Filippin, T. Pozzan, MitochondrialpH monitored by a new engineered green fluorescent protein mutant, J. Biol.Chem. 279 (2004) 11521–11529.

[119] M. Colombini, A candidate for the permeability pathway of the outermitochondrial membrane, Nature 279 (1979) 643–645.

[120] M. Colombini, VDAC: the channel at the interface between mitochondria and thecytosol, Mol. Cell. Biochem. 256–257 (2004) 107–115.

[121] A.M. Porcelli, A. Ghelli, C. Zanna, P. Pinton, R. Rizzuto, M. Rugolo, pH differenceacross the outer mitochondrial membrane measured with a green fluorescentprotein mutant, Biochem. Biophys. Res. Commun. 326 (2005) 799–804.

[122] M. Klingenberg, The ADP–ATP translocation in mitochondria, a membranepotential controlled transport, J. Membr. Biol. 56 (1980) 97–105.

943R. Orij et al. / Biochimica et Biophysica Acta 1810 (2011) 933–944

Author's personal copy

[123] N. Pfanner, K.N. Truscott, Powering mitochondrial protein import, Nat. Struct.Biol. 9 (2002) 234–236.

[124] L. Zotova, M. Aleschko, G. Sponder, R. Baumgartner, S. Reipert, M. Prinz, R.J.Schweyen, K. Nowikovsky, Novel components of an active mitochondrial K(+)/H(+) exchange, J. Biol. Chem. 285 (2010) 14399–14414.

[125] D. Jiang, L. Zhao, D.E. Clapham, Genome-wide RNAi screen identifies Letm1 as amitochondrial Ca2+/H+ antiporter, Science 326 (2009) 144–147.

[126] T.M. Matthews, C. Webb, Culture systems, in: M.F. Tuite, S.G. Oliver (Eds.),Saccharomyces (Biotechnology Handbooks), Plenum Press, New York, 1991,pp. 249–282.

[127] T.T. Diakov, P.M. Kane, Regulation of V-ATPase activity and assembly byextracellular pH, J. Biol. Chem. (2010).

[128] J. Arino, Integrative responses to high pH stress in S. cerevisiae, OMICS 14 (2010)517–523.

[129] T.M. Lamb, A.P. Mitchell, The transcription factor Rim101p governs ion toleranceand cell differentiation by direct repression of the regulatory genes NRG1 andSMP1 in Saccharomyces cerevisiae, Mol. Cell. Biol. 23 (2003) 677–686.

[130] F. Castrejon, A. Gomez, M. Sanz, A. Duran, C. Roncero, The RIM101 pathwaycontributes to yeast cell wall assembly and its function becomes essential in theabsence of mitogen-activated protein kinase Slt2p, Eukaryot. Cell 5 (2006)507–517.

[131] R. Serrano, H. Martin, A. Casamayor, J. Arino, Signaling alkaline pH stress in theyeast Saccharomyces cerevisiae through theWsc1 cell surface sensor and the Slt2MAPK pathway, J. Biol. Chem. 281 (2006) 39785–39795.

[132] R. Serrano, A. Ruiz, D. Bernal, J.R. Chambers, J. Ariño, The transcriptional responseto alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediatedsignalling, Mol. Microbiol. 46 (2002) 1319–1333.

[133] L. Viladevall, R. Serrano, A. Ruiz, G. Domenech, J. Giraldo, A. Barceló, J. Ariño,Characterization of the calcium-mediated response to alkaline stress inSaccharomyces cerevisiae, J. Biol. Chem. 279 (2004) 43614–43624.

[134] M. Mollapour, A. Shepherd, P.W. Piper, Novel stress responses facilitateSaccharomyces cerevisiae growth in the presence of the monocarboxylatepreservatives, Yeast 25 (2008) 169–177.

[135] N. Arneborg, L. Jespersen, M. Jakobsen, Individual cells of Saccharomycescerevisiae and Zygosaccharomyces bailii exhibit different short-term intracellularpH responses to acetic acid, Arch. Microbiol. 174 (2000) 125–128.

[136] D. Bracey, C.D. Holyoak, P.J. Coote, Comparison of the inhibitory effect of sorbic acidand amphotericinBon Saccharomyces cerevisiae: is growth inhibitiondependent onreduced intracellular pH? J. Appl. Microbiol. 85 (1998) 1056–1066.

[137] A.D. Warth, Effect of benzoic acid on glycolytic metabolite levels andintracellular pH in Saccharomyces cerevisiae, Appl. Environ. Microbiol. 57(1991) 3415–3417.

[138] M.T. Kresnowati, W.A. van Winden, W.M. van Gulik, J.J. Heijnen, Energetic andmetabolic transient response of Saccharomyces cerevisiae to benzoic acid, FEBS J.275 (2008) 5527–5541.

[139] N.P. Mira, M. Palma, J.F. Guerreiro, I. Sa-Correia, Genome-wide identification ofSaccharomyces cerevisiae genes required for tolerance to acetic acid, Microb CellFact 9 (2010) 79.

[140] A. Kren, Y.M. Mamnun, B.E. Bauer, C. Schuller, H. Wolfger, K. Hatzixanthis, M.Mollapour, C. Gregori, P. Piper, K. Kuchler, War1p, a novel transcription factorcontrolling weak acid stress response in yeast, Mol. Cell. Biol. 23 (2003)1775–1785.

[141] M.N. Papadimitriou, C. Resende, K. Kuchler, S. Brul, High Pdr12 levels in spoilageyeast (Saccharomyces cerevisiae) correlate directly with sorbic acid levels in theculture medium but are not sufficient to provide cells with acquired resistance tothe food preservative, Int. J. Food Microbiol. 113 (2007) 173–179.

[142] N. Macpherson, L. Shabala, H. Rooney, M.G. Jarman, J.M. Davies, PlasmamembraneH+ and K+ transporters are involved in the weak-acid preservative response ofdisparate food spoilage yeasts, Microbiology 151 (2005) 1995–2003.

[143] J.A. den Hollander, K. Ugurbil, T.R. Brown, R.G. Shulman, Phosphorus-31 nuclearmagnetic resonance studies of the effect of oxygen upon glycolysis in yeast,Biochemistry 20 (1981) 5871–5880.

[144] F. Rolland, J.H. De Winde, K. Lemaire, E. Boles, J.M. Thevelein, J. Winderickx,Glucose-induced cAMP signalling in yeast requires both a G-protein coupledreceptor system for extracellular glucose detection and a separable hexosekinase-dependent sensing process, Mol. Microbiol. 38 (2000) 348–358.

[145] R. Serrano, In vivo glucose activation of the yeast plasma membrane ATPase,FEBS Lett. 156 (1983) 11–14.

[146] S.P. Denker, D.C. Huang, J. Orlowski, H. Furthmayr, D.L. Barber, Direct binding ofthe Na–H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton

and cell shape independently of H(+) translocation, Mol. Cell 6 (2000)1425–1436.

[147] A. Boussouf, S. Gaillard, Intracellular pH changes during oligodendrocytedifferentiation in primary culture, J. Neurosci. Res. 59 (2000) 731–739.

[148] L.K. Putney, D.L. Barber, Na–H exchange-dependent increase in intracellular pHtimes G2/M entry and transition, J. Biol. Chem. 278 (2003) 44645–44649.

[149] D. Lagadic-Gossmann, L. Huc, V. Lecureur, Alterations of intracellular pHhomeostasis in apoptosis: origins and roles, Cell Death Differ. 11 (2004)953–961.

[150] C. Stock, A. Schwab, Protons make tumor cells move like clockwork, PflugersArch. 458 (2009) 981–992.

[151] R.A. Gatenby, E.T. Gawlinski, A.F. Gmitro, B. Kaylor, R.J. Gillies, Acid-mediatedtumor invasion: a multidisciplinary study, Cancer Res. 66 (2006) 5216–5223.

[152] S. Anand, R. Prasad, Rise in intracellular pH is concurrent with ‘start’ progressionof Saccharomyces cerevisiae, J. Gen. Microbiol. 135 (1989) 2173–2179.

[153] S. Kaur, P. Mishra, R. Prasad, Dimorphism-associated changes in intracellular pHof Candida albicans, Biochim. Biophys. Acta 972 (1988) 277–282.

[154] E. Stewart, S. Hawser, N.A. Gow, Changes in internal and external pHaccompanying growth of Candida albicans: studies of non-dimorphic variants,Arch. Microbiol. 151 (1989) 149–153.

[155] G.D. Robson, E. Prebble, A. Rickers, S. Hosking, D.W. Denning, A.P.J. Trinci, W.Robertson, Polarized growth of fungal hyphae is defined by an alkaline pHgradient, Fungal Genet. Biol. 20 (1996) 289–298.

[156] F. Alcantara-Sanchez, C.G. Reynaga-Pena, R. Salcedo-Hernandez, J. Ruiz-Herrera,Possible role of ionic gradients in the apical growth of Neurospora crassa, AntonieLeeuwenhoek 86 (2004) 301–311.

[157] T. Bagar, K. Altenbach, N.D. Read, M. Bencina, Live-cell imaging and measure-ment of intracellular pH in filamentous fungi using a genetically encodedratiometric probe, Eukaryot. Cell 8 (2009) 703–712.

[158] A. Schauer, H. Knauer, C. Ruckenstuhl, H. Fussi, M. Durchschlag, U. Potocnik, K.U.Frohlich, Vacuolar functions determine the mode of cell death, Biochim. Biophys.Acta 1793 (2009) 540–545.

[159] D. Granot, A. Levine, E. Dor-Hefetz, Sugar-induced apoptosis in yeast cells, FEMSYeast Res. 4 (2003) 7–13.

[160] F.A. Hoeberichts, J. Perez-Valle, C. Montesinos, J.M. Mulet, M.D. Planes, G. Hueso,L. Yenush, S.C. Sharma, R. Serrano, The role of K(+) and H(+) transport systemsduring glucose- and H(2)O(2)-induced cell death in Saccharomyces cerevisiae,Yeast (2010).

[161] J.M. Thevelein, M. Beullens, F. Honshoven, G. Hoebeeck, K. Detremerie, J.A. denHollander, A.W. Jans, Regulation of the cAMP level in the yeast Saccharomycescerevisiae: intracellular pH and the effect of membrane depolarizing compounds,J. Gen. Microbiol. 133 (1987) 2191–2196.

[162] J.M. Thevelein, J.H. de Winde, Novel sensing mechanisms and targets for thecAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae, Mol.Microbiol. 33 (1999) 904–918.

[163] S. Colombo, P. Ma, L. Cauwenberg, J. Winderickx, M. Crauwels, A. Teunissen, D.Nauwelaers, J.H. deWinde, M.F. Gorwa, D. Colavizza, J.M. Thevelein, Involvementof distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae, EMBO J. 17(1998) 3326–3341.

[164] C. Purwin, K. Nicolay, W.A. Scheffers, H. Holzer, Mechanism of control ofadenylate cyclase activity in yeast by fermentable sugars and carbonyl cyanidem-chlorophenylhydrazone, J. Biol. Chem. 261 (1986) 8744–8749.

[165] K. Mbonyi, L. van Aelst, J.C. Argüelles, A.W. Jans, J.M. Thevelein, Glucose-inducedhyperaccumulation of cyclic AMP and defective glucose repression in yeaststrains with reduced activity of cyclic AMP-dependent protein kinase, Mol. Cell.Biol. 10 (1990) 4518–4523.

[166] P.S. Aronson, Kinetic properties of the plasma membrane Na+–H+ exchanger,Annu. Rev. Physiol. 47 (1985) 545–560.

[167] E. Olkhova, C. Hunte, E. Screpanti, E. Padan, H. Michel, Multiconformationcontinuum electrostatics analysis of the NhaA Na+/H+ antiporter of Escherichiacoli with functional implications, Proc. Natl Acad. Sci. U.S.A. 103 (2006)2629–2634.

[168] V. Carmelo, P. Bogaerts, I. Sa-Correia, Activity of plasma membrane H+-ATPaseand expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cellsgrown at optimal and low pH, Arch. Microbiol. 166 (1996) 315–320.

[169] C. Recchi, P. Chavrier, V-ATPase: a potential pH sensor, Nat. Cell Biol. 8 (2006)107–109.

[170] V. Marshansky, The V-ATPase a2-subunit as a putative endosomal pH-sensor,Biochem. Soc. Trans. 35 (2007) 1092–1099.

944 R. Orij et al. / Biochimica et Biophysica Acta 1810 (2011) 933–944