Keywords

8.1 Introduction

Ion homeostasis is a fundamental requirement for all organisms. Many different minerals are required for essential biochemical processes, but accumulation of these elements is toxic. As these elements are present as charged molecules in aqueous cellular environments, they are not able to freely diffuse across cell membranes. Thus, all living organisms have developed efficient systems to acquire and store these elements and robust mechanisms to maintain homeostatic concentrations to avoid toxicity.

Saccharomyces cerevisiae has been developed into a productive model to study many aspects of ion homeostasis based on its advantages as an experimental system and the high level of conservation throughout evolution of many proteins that transport ions (Saier 2000; Wolfe and Pearce 2006; Botstein and Fink 2011). Moreover, this model system is amenable to genome-level approaches, which have extensively characterized the yeast ‘ionome’ and defined genes and gene networks that contribute to its maintenance (Eide et al. 2005; Yu et al. 2012). Remarkably, in these studies, a relatively low number of genetic alterations were shown to have large effects on the mineral composition of yeast cells: approximately 5 % of the strains analyzed in rich media (212 of 4,358 knock-outs) and 9 % of the strains analyzed in minimal media (1065 of 11,890 haploid and diploid knock-outs and overexpression strains) showed significant differences in the relative concentrations of the 13–17 cations tested. These studies have revealed an important role for mitochondrial and vacuolar function and the ESCRT pathway (involved in vesicle trafficking) in the regulation of yeast ion homeostasis. Additionally, many of the strains identified displayed alterations in the accumulation of multiple elements. Only a scarce number of mutants were shown to be defective in only one element. These results indicate that the mechanisms that have evolved to maintain ion homeostasis are robust and in many cases act in a coordinated manner.

Potassium is a key monovalent cation necessary for multiple aspects of cell growth and survival, for example compensation of negative charges of macromolecules to maintain electroneutrality, cell turgor and volume, enzyme activity, protein synthesis, and maintenance of proper membrane potential and intracellular pH. In most cell types, potassium is accumulated against its concentration gradient to relatively high amounts, whereas sodium accumulation is actively avoided because of its toxicity. In many mammalian cell types, this low sodium/potassium ratio at the cellular level is actively maintained by P type Na+, K+ ATPases, which drive sodium out of the cell in exchange for potassium (Skou and Esmann 1992). The resulting sodium gradient is used for the coupled uptake of many ions and nutrients via secondary, sodium-coupled carriers. Essentially, yeast cells maintain low sodium/potassium ratios through efficient and selective potassium uptake (and not sodium), efficient efflux of excess sodium and efficient sequestration of sodium in the vacuole. In the majority of these transport processes, a proton motive force created by H+-ATPases is required.

This review will focus on our current knowledge regarding potassium and sodium transport and how homeostasis of these ions is achieved and maintained in baker’s yeast. As mentioned above, although not directly involved in transporting potassium or sodium themselves, H+-ATPases are key regulators of these transport processes and so will be discussed first. General aspects of potassium and sodium uptake and efflux will be considered and our current knowledge regarding the structure and function of the implicated transporters will be presented. Our understanding of how potassium homeostasis is regulated and how yeast cells respond to both excess extracellular sodium and potassium and potassium starvation will be discussed. Finally, some applications of this knowledge to other fields will also be presented.

8.2 The Role of H+-ATPases in Potassium and Sodium Transport

8.2.1 Pma1

In S. cerevisiae, the plasma membrane H+-ATPase encoded by the PMA1 gene is largely responsible for creating the proton motive force across the plasma membrane. This proton gradient drives nutrient uptake by secondary, proton-coupled carriers (Barnett 2008). The PMA1 gene is essential and it encodes a 100 kDa P2-type ATPase that is highly stable and abundant in the yeast plasma membrane and has been estimated to consume at least 20 % of cellular ATP (Benito et al. 1991; Morsomme et al. 2000). The enzyme is activated by glucose and acidic internal pH and, not surprisingly, alterations in its activity have an important impact on intracellular pH and ion homeostasis (Serrano 1983; Perlin et al. 1988; Goossens et al. 2000). Mutants with partial loss of function of the PMA1 gene are unable to grow at low external pH and display tolerance to cations due to alterations in the membrane potential that lead to a decrease in the uptake of positively charged molecules, such as Hygromycin B (McCusker et al. 1987; Perlin et al. 1988). The S. cerevisiae genome contains a second gene, PMA2 which is approximately 90 % identical to PMA1 (Schlesser et al. 1988). Although the Pma2 protein can pump protons and can substitute for Pma1 when expressed under the control of a strong promoter, in standard growth conditions, this gene is expressed at very low levels and therefore does not have an important impact on ion homeostasis (Supply et al. 1993).

Transcriptional regulation of PMA1 (and in some cases PMA2) has been described in response to carbon source (mediated by the Rap1 and Gcr1 transcription factors), during the diauxic shift, entry into stationary phase and stress conditions (Rao et al. 1993; Portillo 2000; Fernandes and Sá-Correia 2003). As mentioned, on the protein level, decreased intracellular pH activates the enzyme, as does glucose addition. The mechanism of activation by acidic pH is not clear, but it may reflect the pH optimum of the enzyme that has been observed in reconstituted systems or post-translational modifications yet to be defined on the molecular level. Glucose activation of Pma1 rapidly results in an increase in the Vmax and a decrease in the affinity for ATP and is mediated, at least in part, by phosphorylation of the autoinhibitory C-terminal domain. Although the exact molecular mechanism has yet to be fully elucidated, several Pma1 phosphorylation sites have been implicated. Specifically, the phosphorylation of threonine 912 is required for glucose activation, but appears to be constitutive, while phosphorylation of serine 911 is induced by glucose addition and is also necessary for full Pma1 activation (Lecchi et al. 2007). The NPR family kinases Ptk2 and Hrk1 have been shown to positively regulate Pma1 activity (Goossens et al. 2000). Evidence has been presented suggesting that Ptk2 phosphorylates serine 899 of Pma1 (Eraso et al. 2006). Moreover, a role for the PP1-type phosphatase, Glc7 in the regulation of Pma1 activity has been proposed (Williams-Hart et al. 2002). In addition, the Yck1 and Yck2 casein kinases have been reported to negatively regulate Pma1 activity (Estrada et al. 1996). Other studies have suggested a role for calcium-dependent signaling in glucose-mediated Pma1 activation, although the mechanism is still unknown (Trópia et al. 2006; Pereira et al. 2008; Bouillet et al. 2012).

8.2.2 V-ATPase

The vacuolar H+-ATPase (V-ATPase) is also involved in determining the electrical potential across membranes of intracellular compartments and accordingly, it plays a crucial role in several physiological processes, including ion homeostasis (Kane 2007). The V-ATPase is a protein complex composed of a soluble, multi-subunit V1 catalytic region and a membrane-embedded, multi-subunit VO region, whose structural organization is similar to the F1Fo-ATPase (Nishi and Forgac 2002; Zhang et al. 2008). Two V-ATPase complexes have been identified. The first complex, which is present in vacuolar membranes contains the Vph1 subunit in the Vo complex and is responsible for acidifying the vacuole. In the second complex, Stv1 substitutes Vph1 and this complex is responsible for the acidification of the Golgi apparatus/endosomes, where it is targeted (Tarsio et al. 2011). The V-ATPase is regulated on the level of complex formation/dissociation. This regulation seems to be conserved evolutionarily and is complex. For example, glucose starvation, decreasing intracellular pH, and poor nutrient conditions favor the dissociation and concomitant reduction in the activation of the V-ATPase, whereas glucose re-addition and increasing intracellular pH have the opposite effect (Kane 2012).

In S. cerevisiae, experimental evidence has been reported that shows that the Pma1 plasma membrane and the V-ATPases act coordinately to control cytosolic pH homeostasis (Martínez-Muñoz and Kane 2008). The electrogenic nature of their combined activities is a major determinate in the generation of not only plasma membrane, but also organellar membrane potential. As mentioned, this electrochemical gradient is used for the uptake of nutrients from the cell environment by proton-coupled carriers (Barnett 2008). It also thought to play an important role in the ability of yeast cells to accumulate potassium against a steep concentration gradient and to enable the extrusion and organellar distribution of potassium and sodium via proton-coupled antiporters (Gaber 1992; Rodríguez-Navarro 2000; Arino et al. 2010).

8.3 Potassium Uptake and Efflux

Since as early as the 1940s, researchers proposed a relationship between potassium and proton transport in yeast and during the following years many aspects of these transport processes were characterized (Borst-Pauwels 1981). The steady state intracellular potassium concentration in yeast cells is maintained between 200 and 300 mM depending on the strain and growth conditions and is thought to depend on continuous uptake and efflux processes (Lapathitis and Kotyk 1998; Arino et al. 2010). As mentioned, the membrane potential generated by the plasma membrane H+-ATPase is vital for potassium uptake in yeast. However, the coordination of potassium fluxes across the plasma membrane is also crucial to maintain proper membrane potential, as demonstrated by the hyperpolarization of mutants defective in high affinity uptake and the depolarization observed in mutants lacking potassium efflux systems (Madrid et al. 1998; Maresova and Sychrova 2005; Kinclova-Zimmermannova et al. 2006; Maresova et al. 2006). Thus, it is clear that the coordination of these processes is crucial for yeast cell growth and survival. In the next sections, the proteins responsible for mediating the uptake and efflux of potassium across the plasma membrane will be discussed.

8.3.1 Trk1 and Trk2

In 1984, Rodriguez-Navarro and Ramos proposed a dual mode of potassium transport by showing that yeast displayed both high and low affinity potassium uptake depending on the growth history of the cells (Rodríguez-Navarro and Ramos 1984). In 1988, the first potassium transporter gene, TRK1 was cloned on the basis of its ability to complement a yeast mutant defective in potassium uptake (Gaber et al. 1988). TRK1 is a non-essential gene that encodes an integral membrane protein of 1235 amino acids (Fig. 8.1). Based on the structure of the KcsA K+ channel from Streptomyces lividans, Trk1 has been proposed to be composed of four repetitions of an M1PM2 motif (Durell and Guy 1999). M1 and M2 are transmembrane segments that are connected by the P helix (Fig. 8.1). An extensive mutagenesis analysis has identified residues in the second transmembrane helix (M2) of the fourth M1PM2 repetition (M2D) of Trk1 as being crucial for potassium transport (Haro and Rodríguez-Navarro 2003). It has been proposed that the four M1PM2 repetitions of the Trk1 monomer fold into a symmetric array and that four Trk1 monomers form a tetramer in the plasma membrane (Durell and Guy 1999). Although initial reports suggested that Trk1 is localized in plasma membrane lipid “rafts”, further characterization of the protein distribution in the yeast plasma membrane shows that essentially all integral membrane proteins are found in two classes of microdomains that share biochemical properties with mammalian “rafts”, but the overall organization and function of these microdomains appears to be quite different (Yenush et al. 2005; Malinsky et al. 2013).

Fig. 8.1
figure 1

Saccharomyces cerevisiae plasma membrane potassium and sodium transport proteins. For each protein the standard name, systematic name, Yeast transporter information code based on the Transport classification system (YETI), transporter type, proposed topology and substrate specificity are shown (cations in bold are preferred substrates). Numbers at the end of each sequence represent the length of the protein. See text for more details and references

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Whereas wild type strains are able to grow in low micromolar potassium concentrations and exhibit high affinity and high velocity potassium uptake (V max 30 nmol/mg cells/min and K m of 0.024 mM), strains lacking TRK1 are unable to grow in 0.1 mM KCl and show a marked reduction in potassium uptake kinetics, demonstrating that Trk1 is a major contributor to high affinity potassium uptake (Rodríguez-Navarro and Ramos 1984; Gaber et al. 1988). Each transporter has two cation binding sites and normally functions as a K+ co-transporter, thought to be driven by the membrane potential created by the Pma1 H+-ATPase. However, this affirmation assumes a plasma membrane potential of −300 mV, which has not be confirmed experimentally in S. cerevisiae. Thus, other scenarios, such as Trk1 acting as a K+-Na+ symporter cannot be ruled out (reviewed in: (Arino et al. 2010)).

TRK1 orthologues have been identified in other yeast, fungi and higher plants (Rodríguez-Navarro 2000). In fact, S. cerevisiae contains a second gene, TRK2 which encodes a protein that is 55 % identical to Trk1 (Ko and Gaber 1991). The proposed topology is the same for Trk2, with the main structural difference residing in the length of the second cytosolic segment (Trk1 642 aa; Trk2 326 aa) (Fig. 8.1). Deletion of the TRK2 gene has little effect on yeast growth, although the potassium requirements of the double trk1 trk2 mutant increase 10-fold, as compared to the trk1 simple mutant (Ko et al. 1990). Trk2 was initially proposed to mediate low affinity transport. However, later studies showed that Trk2, when expressed from a strong promoter, can mediate high/moderate affinity potassium uptake (Ramos et al. 1994; Michel et al. 2006). Thus, although Trk2 participates in potassium uptake, Trk1 is the dominant transporter, likely due to the higher expression of the TRK1 gene. Interestingly, the Trk transporters have also been shown to mediate the efflux of anions such as Cl−, I− and Br− and SCN− and NO3 −, presumably through the pore created by the formation of the Trk1 or Trk2 tetramers (Kuroda et al. 2004; Rivetta et al. 2011). Although the physiological significance of this activity detected in electrophysiology experiments is not clear, it has been proposed to balance charges generated by Pma1 proton pumping activity (Rivetta et al. 2011).

As mentioned, Trk1 is the transporter responsible for potassium uptake and as such plays an important role in yeast physiology. Although there is no evidence for transcriptional regulation of either TRK1 or TRK2 in response to cation-related stresses, many proteins have been identified that affect the activity of this transporter, presumably at the post-translational level (Fig. 8.2). For example, the functionally redundant protein kinases encoded by the HAL4 (SAT4) and HAL5 genes were identified as positive regulators of Trk1 (Mulet et al. 1999). Overexpression of these genes confers tolerance to toxic concentrations of NaCl or LiCl and this phenotype requires the presence of the TRK1 and TRK2 genes. Moreover, the double hal4 hal5 mutant presents defects in Rb+ uptake and a slow growth phenotype in minimal media that can be ameliorated with increased external potassium. Evidence for direct phosphorylation of Trk1 by these kinases is lacking. However, it has been shown that the Hal4 and Hal5 kinases are required for Trk1 plasma membrane accumulation (Perez-Valle et al. 2007). The deletion of the last 35 amino acids of the Trk1 protein stabilizes the transporter in the plasma membrane, suggesting that this region is implicated in plasma membrane delivery and/or maintenance. Interestingly, several other nutrient transporters, in addition to Trk1, also fail to accumulate at the plasma membrane in hal4 hal5 mutants leading to defects in both carbon and nitrogen metabolism, suggesting a more general role for the Hal4 and Hal5 kinases (Perez-Valle et al. 2010).

Fig. 8.2
figure 2

Schematic representation of transporters and regulators controlling potassium and sodium transport in Saccharomyces cerevisiae. The major contributors to the known signal transduction pathways involved in potassium and sodium homeostasis are depicted. Discontinuous lines represent interactions that have not been fully documented. See text for details and references. (LE late endosome)

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The Arl1 protein, which encodes a G protein of the Ras superfamily involved in protein trafficking, has been suggested to modulate Trk1 activity, as toxic cation sensitivity and a reduction in Rb+ uptake has been documented in the mutant strain (Munson et al. 2004). Moreover, both HAL4 and HAL5 act as multi-copy suppressors of the arl1 mutant strain. However, in this report no defect in Trk1 protein levels or trafficking was observed in arl1 mutants, so the mechanism through which Arl1 regulates potassium transport has yet to be elucidated. Other protein kinases such as Sky1 and Snf1 have also been implicated in the regulation of Trk1. Mutants lacking the SR protein kinase SKY1 show alterations in Rb+ uptake and membrane potential, suggested to be mediated by alterations in Trk1 activity, although the mechanism is unknown and other researchers have described a Trk1-independent role for Sky1 in the regulation of ion homeostasis (Erez and Kahana 2002; Forment et al. 2002). In the case of the AMP kinase homologue Snf1, mutant strains are unable to fully activate potassium uptake. Moreover, it was shown that the residual kinase activity of a non-phosphorylated Snf1 isoform can activate high affinity potassium uptake, but again, the molecular basis is unknown (Portillo et al. 2005). Interestingly, two Snf1 phosphorylation sites are listed in the Phosphogrid database for Trk1, although they have not be confirmed directly (www.phosphogrid.org). The gene encoding the trehalose-6-phosphate synthase gene (TPS1) has been shown to activate Trk1 (Mulet et al. 2004). Several lines of evidence suggest that the mechanism involves the direct or indirect activation of Trk1 by glucose phosphates (Glc-1-P and Glc-6-P), which would be in agreement with earlier studies showing that potassium uptake is activated by increased levels of phosphorylated sugars (Alijo and Ramos 1993).

Protein phosphatases have also been reported to modulate Trk1 activity. First, early reports suggested that the Ca2+/calmodulin-dependent calcineurin phosphatase is required for Trk1 to properly discriminate between potassium and sodium under conditions of salt stress (Mendoza et al. 1994). More recently, it was shown that the absence of calcineurin also affects high affinity potassium uptake in the absence of salt stress (Casado et al. 2010). The mechanism of this regulation is thought to involve the calcineurin-dependent regulation of the HAL5 gene. Several lines of evidence suggest that a second protein phosphatase, Ppz1 is an important regulator of Trk1 activity. Strains lacking PPZ1 and the related PPZ2 gene are tolerant to toxic cations, as are strains that overexpress the Ppz1 regulatory subunit HAL3 (Ferrando et al. 1995; Posas et al. 1995; de Nadal et al. 1998). In addition, strains lacking the PPZ1 and PPZ2 genes display increased turgor pressure and increased pH, due to excess potassium accumulation (Yenush et al. 2002). These phenotypes require the presence of the TRK1 and TRK2 genes. Furthermore, Ppz1 was shown to co-localize and physically interact with Trk1 and in ppz1 ppz2 mutants an increase in Trk1 phosphorylation levels are observed (Yenush et al. 2005). Taken together, these data suggest that Ppz1 is a negative regulator of Trk1. Moreover, the interaction between Ppz1 and Hal3 is pH-dependent, leading to a model in which the Hal3-Ppz1 complex participates in the maintenance of internal potassium concentrations by responding to changes in internal pH. The kinase(s) responsible for Trk1 phosphorylation and the mechanism by which this class of post-translational modification alters the properties of the transporter still need to be defined.

An alternative approach that has been taken to identify regulators of Trk1 is high-throughput screening of the yeast mutant collection looking for genes whose disruption leads to increased or decreased tolerance to toxic cations, such as hygromycin B (Barreto et al. 2011; Fell et al. 2011). In these screens, 150–200 mutants encoding genes belonging to several functional groups, including protein kinases and phosphatases, transcription, cell cycle, and DNA processing were enriched. Some of the regulators identified in both screens have been mentioned above, such as Arl1, Sky1, Hal4, and Hal5. Interestingly, both screens also identified many mutants related to various aspects of vesicle trafficking, such as SNARE proteins and components of the CORVET and HOPS complexes. However, many of these mutants are not defective in Trk1 plasma membrane accumulation as might be expected, thus their participation in the regulation of potassium uptake remains to be defined.

8.3.2 Proteins Involved in Low Affinity Potassium Uptake

As mentioned, S. cerevisiae cells display high and low affinity potassium uptake depending on the growth history of the cells and the media employed. Under normal growth conditions, where the potassium concentration is not limiting, Trk1 would mediate the majority of the so-called low affinity potassium uptake. When the extracellular potassium concentration decreases, Trk1 switches to a high affinity mode to mediate growth in the presence of as little as 10 μM K+. Importantly, deletion of both TRK1 and TRK2 in S. cerevisiae is not lethal. These mutant strains display ectopic low affinity potassium uptake, indicating that additional mechanisms of potassium uptake must exist (Madrid et al. 1998). Electrophysiology studies revealed inward potassium currents in trk1 trk2 mutants, whose activity is inhibited by calcium (Bihler et al. 1998, 2002). A putative channel was proposed to be responsible for these currents and named NSC1 (non-specific cation channel), but the protein responsible was not identified. It has been proposed that the “very low affinity” potassium uptake observed in trk1 trk2 strains is mediated by multiple transport processes (reviewed in: (Arino et al. 2010)). Recently, two putative low affinity potassium transporter proteins were identified that may account for some of these currents. Kch1 and Kch2 (Prm6) were identified as necessary components of the pheromone-induced activation of the high affinity Ca2+ influx system (HACS) (Stefan et al. 2013). These fungal-specific proteins are predicted to have several transmembrane segments and have been shown to localize to the yeast plasma membrane. The inward rectifying currents are notably reduced in strains lacking both KCH1 and KCH2 and overexpression of either gene improves the growth of trk1 trk2 strains in low potassium medium, supporting a role for these proteins as potassium transporters or channels. However, under normal growth conditions, their activity appears to be eclipsed by much higher Trk1 activity. The fact that inward rectifying currents are still observed in strains lacking trk1 trk2 kch1 and kch2 indicate that additional mechanisms of potassium uptake are present. Candidates for these uptake systems include non-specific uptake by the Qdr2 drug/H+ antiporter and sugar and amino acid permeases (Ko et al. 1993; Wright et al. 1997; Vargas et al. 2007). Finally, deletion of the gene encoding a small hydrophobic protein called Pmp3, which is highly conserved in yeast and plants, has been proposed to facilitate cation uptake in a Trk1,2-independent manner, via an unknown mechanism (Navarre and Goffeau 2000).

8.3.3 Tok1

At least three different transporters contribute to potassium efflux in S. cerevisiae. Although both Ena1 and Nha1 can transport potassium, they were first identified based on their capacity for sodium efflux, and so will be discussed below. The third protein, Tok1, is an outwardly rectifying plasma membrane potassium channel and it is the only potassium-specific efflux system described in yeast (Gustin et al. 1986; Bertl et al. 1993; Ketchum et al. 1995; Zhou et al. 1995; Reid et al. 1996). The TOK1 gene encodes a protein of 691 amino acids that contains eight transmembrane segments, the last four of which participate in the formation of two pore-forming P domains responsible for K+ conductance (Fig. 8.1) (Ketchum et al. 1995; Martinac et al. 2008). The activity of the channel is regulated by both membrane potential and external potassium (Bertl et al. 1993; Vergani et al. 1997; Fairman et al. 1999). Accordingly, depolarization of the membrane leads to channel opening and potassium efflux, presumably to restore proper membrane potential. Gating of the channel is regulated by the carboxy terminal cytosolic segment, which prevents channel closure (Loukin and Saimi 2002). Although the electrophysiological data generated both in yeast and Xenopus oocytes clearly define the activity of the Tok1 channel, the physiological role of this potassium efflux activity remains unclear, as no growth-related phenotypes have been detected for the tok1 mutant strain (Gustin et al. 1986; Ketchum et al. 1995; Zhou et al. 1995; Lesage et al. 1996; Reid et al. 1996; Loukin et al. 1997; Bertl et al. 1993, 1998, 2003). The function of Tok1 may involve plasma membrane potential maintenance as it has been shown that tok1 mutant strains are depolarized, while strains overexpressing TOK1 are hyperpolarized (Maresova et al. 2006). Tok1 has also been reported to be phosphorylated almost immediately upon sodium chloride treatment by the Hog1 MAP kinase (Proft and Struhl 2004). The HOG signaling pathway is a conserved Mitogen Activated Protein Kinase (MAPK) pathway, which in conditions of hyperosmotic stress leads to the activation of the Hog1 MAP kinase (reviewed in (de Nadal et al. 2002)). Although the functional consequences of this phosphorylation were not examined in detail in this study, mathematical modeling predicts that Hog1-mediated phosphorylation of Tok1 reduces Na+ influx under NaCl stress (Ke et al. 2013).

8.4 Sodium Uptake and Efflux

As discussed earlier, due to its toxicity, sodium accumulation is actively avoided by yeast cells. Under normal laboratory growth conditions, the amount of intracellular sodium is very low. In the presence of high external concentrations, sodium is thought to enter the cell in various ways, principally by displacing potassium. For example, Trk1 and Trk2 can transport sodium, although the affinity is much lower than for potassium (Haro and Rodríguez-Navarro 2002). In fact, in the presence of high sodium, Trk1 is thought to undergo an undefined modification which improves its capability to discriminate between the two cations and thus favor potassium uptake (Mendoza et al. 1994). Sodium also enters through other non-specific, low-affinity potassium transporters, such as NSC1. These transporters do not appear to discriminate between these two cations, as trk1 trk2 mutant strains, which depend on these low-affinity transport mechanisms, accumulate more sodium than the wild type strain (Gómez et al. 1996).

8.4.1 Pho89

Interestingly, in S. cerevisiae one sodium-dependent nutrient transporter has been described. The PHO89 gene encodes a sodium-phosphate co-transporter protein of 574 amino acids with twelve predicted membrane-spanning domains (Persson et al. 1999) (Fig. 8.1). Expression of the PHO89 gene is induced by both phosphate limitation and alkaline pH (Martinez and Persson 1998; Serrano et al. 2002). The transporter, whose Km value for inorganic phosphate is 0.5 μM, is highly specific for sodium and maximum phosphate uptake is observed at 25 mM NaCl and pH 9.5 (Martinez and Persson 1998). Another related gene, PHO84 encodes a proton-coupled phosphate transporter, which is responsible for phosphate uptake at acidic pH (Persson et al. 1999). Pho89 is the only known sodium-dependent secondary nutrient transporter in S. cerevisiae. Recent work shows the detectable accumulation of intracellular Na+ as a result of Pho89 activity only in the absence of Ena1 (Serra-Cardona et al. 2014). Moreover, in this same study it was shown that the transcription of both PHO89 and ENA1 are coordinately regulated during alkaline stress. Thus, it appears that Ena1 activity is likely to suffice to avoid accumulation of toxic levels of intracellular sodium introduced via Pho89.

8.4.2 Nha1

Two classes of transport proteins have been shown to be important for sodium efflux in S. cerevisiae, Nha1 and the Ena family of ATPases. A role for Nha1 in tolerance to toxic sodium concentrations was initially shown by its recovery in a screen to identify genes improving the growth of a salt sensitive strain (Prior et al. 1996). The NHA1 gene encodes a protein of 985 amino acids, which is predicted to contain twelve transmembrane segments and a large cytosolic carboxy terminal domain (550 amino acids) (Fig. 8.1). The overall structure and transporter activity is conserved in all kingdoms of life, although diversity exists in the physiological function of this family of transporters. In the case of S. cerevisiae, Nha1 is localized to the plasma membrane and acts as a dimeric, electrogenic proton antiporter with similar affinity for both K+ and Na+ that is also capable of transporting Rb+ and Li+ (Bañuelos et al. 1998; Mitsui et al. 2005; Ohgaki et al. 2005). Thus, under acidic pH conditions, Nha1 is able to transport sodium out of the cell, although this is unlikely to be its most important physiological function. Accordingly, loss or increase of Nha1 function has been shown to influence cytosolic pH, membrane potential, Trk1-dependent potassium uptake and to be involved in the initial adaptation to both osmotic and alkaline pH stress (Prior et al. 1996; Sychrová et al. 1999; Bañuelos et al. 2002; Proft and Struhl 2004; Kinclova-Zimmermannova et al. 2006; Kinclova-Zimmermannova and Sychrova 2006).

The expression of the NHA1 gene has not been found to be regulated under osmotic or pH stress conditions and thus is thought to represent a constitutively expressed housekeeping gene (Bañuelos et al. 1998). Extensive mutagenesis studies in several yeast species have identified many amino acids required for activity and substrate specificity (reviewed in (Arino et al. 2010)). Several functions have also been ascribed to the large carboxy terminal tail. For example, a short 16 amino acid sequence predicted start at the end of the last transmembrane segment and continue into the beginning of the large cytosolic domain is required for proper function and targeting to the plasma membrane, while amino acids 920–930 have been implicated in Li+ transport (Kinclová et al. 2001; Mitsui et al. 2004a). In addition, regions of the Nha1 carboxy terminus have also been defined which are responsible for the ability of NHA1 overexpression to rescue the synthetic lethality of a mutant strain lacking both the SIT4 phosphatase gene and the HAL3 gene encoding the regulatory subunit of the Ppz1 phosphatase (Simón et al. 2003). The sit4 hal3 double mutant has been reported to have a defect in the G1/S transition of the cell cycle and the identification of NHA1 as a multi-copy suppressor has led to the suggestion that Nha1 plays a role in cell cycle progression, although the mechanism of the cell cycle arrest of this mutant and the basis of the NHA1-mediated rescue are not known (Simón et al. 2001).

As mentioned above, Nha1 has been implicated in the initial adaptation to hyperosmotic stress. In addition to Tok1 (see above), upon salt stress, the Hog1 MAP kinase also very rapidly phosphorylates Nha1 on T765 and T876 (Proft and Struhl 2004). Experimental data presented by these authors show that, under certain conditions, this post-translational modification increases its ability to confer tolerance to NaCl and so was interpreted as activating Nha1 sodium extrusion activity. Subsequently, Kinclova-Zimmermannova and Sychrova showed that sorbitol treatment decreases Nha1 K+ efflux activity in a Hog1-dependent manner (Kinclova-Zimmermannova and Sychrova 2006). Further experiments are required to definitively determine the function and molecular mechanism of this post-translational modification. In agreement with a role for multiple phosphorylation in the regulation of Nha1, the phospho-binding 14-3-3 protein, Bmh1, was found to interact with Nha1 and to influence toxic cation tolerance (Zahrádka et al. 2012). However, the Nha1-Bmh1 interaction does not require the presence of the Hog1 kinase and the mechanism by which this interaction may affect Nha1 activity is as yet undefined. An additional 12 phosphorylation sites are listed in the Phosphogrid database in the carboxy terminus of Nha1, some of which are suggested to respond to salt stress and may represent candidates for 14-3-3 protein interaction sites (www.phosphogrid.org). Another protein, named Cos3 has also been described to interact with Nha1 (Mitsui et al. 2004b). Gain or loss of function of this gene has been shown to alter salt resistance in a Nha1-dependent manner, although the mechanism by which it may regulate the antiporter is unclear, especially considering that it is localized mostly to the vacuolar membrane.

8.4.3 Ena1

As mentioned, at acidic intracellular pH the Nha1 antiporter can extrude sodium, whereas at higher pH, the Ena1 transporter is principally responsible for sodium extrusion (Bañuelos et al. 1998). Chromosome IV of most yeast genomes contains 3–5 tandem copies encoding ENA P-type ATPases, which are classified in the fungal-specific IID subfamily (for reviews, see (Benito et al. 2002; Arino et al. 2010; Palmgren and Nissen 2011)). One exception is the CEN.PK strain and its derivatives that encode only one divergent ENA gene called ENA6 (Daran-Lapujade et al. 2009). In the rest of the strains analyzed, the ENA genes encode identical or nearly identical proteins that are 1091 amino acids long and are predicted to contain ten transmembrane segments and a larger cytosolic nucleotide-binding domain between the fourth and fifth membrane helices (Fig. 8.1). ENA transporters are localized to the plasma membrane and form a typical phospho-enzyme intermediate, using the energy generated from ATP hydrolysis to transport K+, Na+ or Li+ (with varying affinities) against their concentration gradient (Haro et al. 1991; Wieland et al. 1995; Benito et al. 1997). Lack of the ENA genes, either in the CEN.PK strains or by genetic manipulation deleting the complete cluster, leads to marked salt and alkaline pH sensitivity, confirming the role of these genes as important participants in sodium (and lithium) extrusion (Haro et al. 1991; Daran-Lapujade et al. 2009). At the post-translational level, very little is known regarding possible regulation of ENA proteins. Strains lacking the SRO7 gene, which encodes a protein involved in exocytosis homologous to the Drosophila Lgl tumor suppressor gene, were shown to be salt sensitive and to display defects in the proper accumulation of Ena1 at the plasma membrane, although no further progress has been made (Larsson et al. 1998; Wadskog et al. 2006). By contrast, a considerable amount of information is available regarding the transcriptional regulation of the key component of this gene cluster, ENA1. Here, the major contributors will be discussed, but for more details, excellent reviews are available (Ruiz and Arino 2007; Arino et al. 2010).

Under standard growth conditions, the expression of the ENA genes is low, as observed for the rest of the transport proteins discussed above. However, in contrast to other transporters whose mRNA levels are generally unaltered by environmental conditions, expression of the ENA1 gene is specifically and markedly increased in response to osmotic, saline and alkaline pH stress via the action of several signaling pathways (Mendoza et al. 1994; Márquez and Serrano 1996; Lamb et al. 2001) (Fig. 8.2). Under conditions of mild saline (0.3–0.4 M NaCl) and osmotic stress, the HOG pathway plays a dominant role in ENA1 induction (Marquez and Serrano 1996). As mentioned above, among the first regulatory events to occur upon Hog1 activation is the phosphorylation of Nha1 and Tok1 (Proft and Struhl 2004). However, activated Hog1 quickly accumulates in the nucleus and mediates the induction of ENA1 (and many other target genes) via several mechanisms (Ferrigno et al. 1998; Posas et al. 2000; Rep et al. 2000). First, Hog1 phosphorylates the bZip transcription factor Sko1 and converts the Sko1-Ssn6-Tup1 complex from a transcriptional repressor to an activator (Proft and Struhl 2002). In addition, the histone deacetylase complex Rpd3-Sin3 is recruited to the ENA1 promoter in a Hog1-dependent manner, facilitating the association of RNA polymerase II and transcriptional activation (De Nadal et al. 2004). Finally, Hog1, like other MAP kinases, has also been shown to be involved in transcriptional elongation of many of its target genes under stress conditions, but whether this activity of Hog1 is involved in ENA1 induction has not been reported (reviewed in (de Nadal and Posas 2011)).

Another important pathway regulating the induction of the ENA1 gene under stress conditions is mediated by the protein phosphatase, calcineurin (Mendoza et al. 1994). Calcineurin is a calcium/calmodulin-dependent, PP2B-type heterodimeric phosphatase composed of one of two redundant catalytic subunits (Cna1 or Cna2) and the regulatory subunit encoded by the CNB1 gene (Klee et al. 1988). Osmotic stress has been proposed to provoke a calcium burst responsible for the activation of the calcineurin pathway (Matsumoto et al. 2002). Induction of the expression of the gene encoding the Na+-ATPase by calcineurin occurs mainly through the dephosphorylation of the transcription factor Crz1 which has been shown to bind to two calcineurin-dependent response elements (CDRE) in the ENA1 promoter and activate transcription (Mendizabal et al. 2001). Mutations in genes encoding another protein phosphatase, Ppz1 and its regulatory subunit, Hal3, have also been shown to affect ENA1 expression (Ferrando et al. 1995; Posas et al. 1995). As discussed above, Ppz1 is a negative regulator of Trk1. In the ppz1 mutant, an increase in basal ENA1 transcription is observed and it has been shown to be fully dependent on the calcineurin/Crz1 pathway, suggesting that Ppz1 is a negative regulator of calcineurin (Ruiz et al. 2003). Mutants lacking both the PPZ1 and PPZ2 genes display an increase in internal K+ and a more alkaline cytosolic pH, which contribute to even higher basal levels of ENA1 (Yenush et al. 2002). In this case, both the calcineurin/Crz1 pathway and a second alkaline responsive element in the ENA1 promoter contribute to the higher mRNA levels (Ruiz et al. 2003).

Although it has been shown that the Hog1 and calcineurin pathways account for the vast majority of ENA1 induction in response to saline and osmotic stress, other pathways have also been identified that contribute to the regulation of ENA1 expression in response to different stresses (Marquez and Serrano 1996). For example, several studies have shown that the C2H2 family zinc finger transcription factor, Rim101 is important for ENA1 induction in conditions of alkaline stress, in cooperation with the AMP kinase homologue, Snf1 (see below) and the calcineurin pathway (Lamb et al. 2001; Serrano et al. 2002; Platara et al. 2006). Rim101 acts as a negative regulator of the Ngr1 repressor. Thus, upon activation of Rim101, Ngr1-mediated repression is released, leading to transcriptional activation of ENA1 (Lamb and Mitchell 2003). Mutants lacking RIM101 are sensitive to toxic cations and this phenotype was initially attributed to defects in the induction of ENA1 transcription. However, in response to moderate saline stress, ENA1 induction is not affected in rim101 mutants, likely due to the dominant role played by the Hog1 pathway. In this case, the Rim101 pathway is required for proper accumulation of the Ena1 protein (Marqués et al. 2015).

ENA1 expression has also been shown to respond to nutrient availability. For example, ENA1 expression is under glucose repression: expression is higher in media containing galactose, instead of glucose as the carbon source (Alepuz et al. 1997). This induction has been shown to require ENA1 promoter sequences that are bound by the Mig1 and Mig2 transcriptional repressors and to be mediated by the Snf1 kinase (Alepuz et al. 1997; Proft and Serrano 1999). Mutants lacking the snf1 gene are sensitive to toxic cation concentrations (Alepuz et al. 1997). In addition to its role as a regulator of Trk1, mentioned above, defects in ENA1 induction have also been postulated to contribute to this snf1 phenotype. However, in the case of salt stress, it appears that the Ngr1 repressor, and not Mig1, are involved in Snf1-mediated ENA1 induction (Ye et al.2008). Interestingly, in the case of alkaline stress, both MIG and Ngr1 promoter elements have been implicated in Snf1-dependent ENA1 induction (Platara et al. 2006). Signal transduction routes responding to nitrogen source quality can also influence ENA1 expression. More specifically, treatment with rapamycin, which inhibits the TORC1 signaling pathway, has been shown to lead to an increase in ENA1 mRNA levels (Crespo et al. 2001). The salt sensitivity of mutants in two TOR-regulated GATA transcription factors, Gln3 and Gat1, and the presence of GATA motifs in the ENA1 promoter suggest that these proteins mediate rapamycin-dependent ENA1 induction. However, additional studies showing the absence of ENA1 regulation by the Sit4 phosphatase, a regulator of Gln3, and the cytoplasmic localization of Gln3 under salt stress conditions have called into question the validity of this straightforward model (Masuda et al. 2000; Tate and Cooper 2007). Finally, the Protein kinase A (PKA) pathway has been implicated in the inhibition of ENA1 induction by controlling the subcellular localization and increasing the repressor activity of the Sko1 transcription factor and by antagonizing the calcineurin pathway, through the phosphorylation of Crz1 (Nakamura et al. 1993; Pascual-Ahuir et al. 2001; Proft et al. 2001; Kafadar and Cyert 2004).

8.5 Intracellular K+/Na+ Transport Proteins

One shortcoming of many of the approximations routinely used to study ion homeostasis in yeast is that the intracellular distribution of the different elements is not always considered. It has long been known that yeast cells accumulate many solutes, including cations, in the vacuole and this sequestration has been proposed to be important for both proper homeostasis and survival in response to ionic stress conditions (Okorokov et al. 1980; Perkins and Gadd 1993; Nass et al. 1997). The presence of ion transporters in the membranes of organelles indicates that subcellular compartmentalization and distribution of ions is actively maintained by the cell. The first attempts to measure the distribution of potassium in yeast was carried out in 1976 using energy-dispersive X-ray microanalysis (Roomans and Sevéus 1976). These authors concluded that the amount of potassium was similar in the cytoplasm and nucleus and that vacuoles contained half the amount of potassium found in the cytosol. For these experiments, the cells were incubated overnight in water, a treatment that is likely to distort the cation distribution as compared to cells that are actively growing. Several studies reported data estimating the cytosolic vs. vacuolar distribution in different yeast species by using treatments that specifically permeabilize the plasma membrane (Okorokov et al. 1980; Perkins and Gadd 1993; de Nadal et al. 1999; Montiel and Ramos 2007). Although informative, these approaches do not account for ion content in other compartments, as all of the non-cytoplasmic ion content is generally attributed to the vacuole. More recently, Herrera and co-workers used subcellular fractionation protocols and atomic emission spectrophotometry to better define the distribution of both potassium and sodium under different growth conditions (Herrera et al. 2013). While their results confirm the accumulation of potassium and sodium in the vacuole relative to the cytosol, they also show that the nucleus contains an important percentage of the total intracellular potassium (and sodium, if present) which is maintained constant under different growth conditions, consistent with the results reported by Roomans and Sevéus (Roomans and Sevéus 1976). The authors propose that potassium and sodium enter non-specifically through nuclear pores and act to neutralize the negative charges found in this organelle, analogous to that reported in mammalian cells (Strick et al. 2001). On the other hand, they show that the amount of potassium (and especially sodium when added to the medium) is relatively low in the cytosol and find that the amount of cytosolic potassium does not markedly change during potassium starvation, indicating mobilization from the vacuole under these conditions. The main characteristics of the transporters that contribute to this subcellular distribution of potassium and sodium will be presented below and are shown schematically in Fig. 8.2.

8.5.1 Vacuole

8.5.1.1 Vnx1

The main proton-coupled antiporter mediating potassium or sodium transport across the vacuolar membrane is encoded by the VNX1 gene (Cagnac et al. 2007). The protein encoded by this gene is 908 amino acids long and predicted to contain 13 transmembrane segments and a 242 amino acid amino terminal cytosolic domain. Vnx1 was identified in a functional screen of all antiporter mutants predicted to be localized to the vacuolar membrane or endosomes looking for alterations in Na+/H+ or K+/H+ exchange activity in purified vacuoles (Cagnac et al. 2007). Protein sequence alignments place Vnx1 in the CAX (calcium exchanger) family, but this protein shows no calcium transport activity. Instead, this transporter exchanges protons for potassium or sodium ions, having a higher affinity for the latter. Thus, Vnx1 uses the proton gradient generated by the Vma1 H+-ATPase (see above) to mediate the transport of potassium (or sodium, if present) into the vacuole.

8.5.1.2 Vcx1 and Vch1

A second transporter, encoded by the VCX1 gene was subsequently shown to be responsible for the residual potassium/H+ exchange activity remaining in vacuoles purified from vnx1 mutants (Cagnac et al. 2010). This transporter, which is 411 amino acids long with 11 transmembrane helices, was first characterized as a vacuolar Ca2+/H+ exchanger and this likely represents its main activity, although as stated, Vcx1 can also transport K+ (Cunningham and Fink 1996; Pozos et al. 1996). The crystal structure of this protein was recently solved, which may aid in defining the molecular determinants of substrate specificity (Waight et al. 2013). A recent study has provided evidence that another transporter, encoded by the VCH1 gene functions as a vacuolar K+/Cl− co-transporter (Petrezselyova et al. 2013). Vch1 contains 1120 amino acids and 12 putative transmembrane segments. Although its transport activity has not been directly tested, based on sequence homology to other members of the cation-Cl− co-transporter (CCC) family, the subcellular localization and phenotypic data demonstrating a role in the proper maintenance of intracellular potassium and vacuolar morphology, Vch1 very likely mediates electroneutral symport of potassium and chloride ions into the vacuole (André and Scherens 1995; Petrezselyova et al. 2013).

8.5.2 Endosomes/Golgi

8.5.2.1 Nhx1

Among the organellar monovalent cation transport proteins, the endosomal Na+/H+ antiporter encoded by the NHX1 gene was the first identified in yeast and may be the most extensively characterized (Nass et al. 1997). The Nhx1 antiporter has 12 predicted hydrophobic domains distributed over a total length of 633 amino acids. Not all of the reports in the literature are consistent regarding Nhx1 topology, but the observation that the carboxy terminal sequence of Nhx1 has been shown to interact with at least one regulatory protein (see below) suggests that it is likely that this region of the protein is cytosolic (Wells and Rao 2001; Ali et al. 2004). Several reports have established that this transporter localizes to the membrane of late endosomes (the pre-vacuolar compartment), as well as recycling endosomes and the trans-Golgi network, where it contributes to pH maintenance within vesicles by mediating potassium (or sodium, if present) sequestration in these compartments in exchange for protons (Nass and Rao 1998; Brett et al. 2005; Kojima et al. 2012). Disruption of the gene leads to several phenotypes, including sensitivity to low pH and high salt, a decrease in cytosolic pH and vesicle trafficking defects, a function shown to require the ion transporter capacity (Bowers et al. 2000; Brett et al. 2005; Mukherjee et al. 2006). Accordingly, a role for Nhx1 in osmotic shock adaptation and sequestration of toxic cations and surplus potassium has been documented (Nass and Rao 1999; Quintero et al. 2000). Nhx1 was also described to be necessary for the recruitment of the ESCRT-0 component Vps27 to endosomes necessary for multi-vesicular body (MVB) formation in a cell-free assay, although a second report, using a genetic approach, suggests a role for Nxh1 downstream of MVB formation (Kallay et al. 2011; Mitsui et al. 2011). Finally, a link between Nhx1 and vesicle fusion and a physical interaction between the carboxy terminus of Nhx1 and a Rab family GTPase- activating protein (Gyp6) has been reported (Ali et al. 2004; Qiu and Fratti 2010). Therefore, it appears that Nhx1 may be involved in several aspects of vesicle trafficking in yeast.

8.5.2.2 Kha1

Kha1 is the sodium or potassium-proton antiporter that shares the highest level of homology to bacterial antiporters (Ramírez et al. 1998). The KHA1 gene encodes an 873 amino acid protein predicted to have 12 transmembrane segments, which, although initially thought to be a plasma membrane transporter, has been shown to localize to the membrane of the Golgi apparatus (Ramírez et al. 1998; Flis et al. 2005; Maresova and Sychrova 2005). The phenotypic characterization of the kha1 mutant alone or in combination with other mutants suggests that it acts as a proton-coupled antiporter facilitating the accumulation of potassium in this organelle (Maresova and Sychrova 2005). Specifically, the alkaline pH sensitivity of this mutant can be ameliorated by high external potassium. Additional studies have provided evidence for a broad substrate specificity by showing that in strains lacking the Arl1 GTPase, Kha1 increases potassium, sodium and lithium tolerance (Marešová and Sychrová 2010). Although the transporter activity has not yet been tested directly, Kha1 is thought to participate in the regulation of potassium and pH homeostasis in the Golgi apparatus, likely in coordination with the Gef1 chloride channel (Flis et al. 2005).

8.5.3 Mitochondria

Ion fluxes are especially important in the mitochondria. The respiration-dependent negative membrane potential of mitochondria facilitates the entry of cations such as potassium, which, if not counter-acted, would result in excessive accumulation and osmotic swelling (reviewed in (Bernardi 1999)). Potassium-proton exchange (KHE) in the inner mitochondrial membrane is an essential element of Peter Mitchell’s chemiosmotic theory proposed in 1961 (Mitchell 1961). Although this activity has been well-documented in purified mitochondria from many different organisms (reviewed in (Bernardi 1999)), the identification of the protein(s) responsible for KHE has been elusive. In S. cerevisiae, three genes have been identified to play a role in KHE: MDM38, YLH47 (MRS7) and YDL183c (Nowikovsky et al. 2004; Froschauer et al. 2005; Zotova et al. 2010). MDM38, which was first identified in a comprehensive screen for searching for genes that affect mitochondrial function and morphology, appears to play to most important role in KHE (Dimmer et al. 2002; Nowikovsky et al. 2004, 2007; Zotova et al. 2010). Mdm38 is the orthologue of the human protein Leucine zipper–EF-hand–containing transmembrane 1 (LETM1), which is thought to be responsible for the seizures observed in patients with Wolf-Hirschhorn syndrome (Endele et al. 1999; Rauch et al. 2001; Schlickum et al. 2004). Expression of this gene in yeast can rescue the mitochondrial function and morphology phenotypes of mdm38 mutants (Nowikovsky et al. 2004). Although some authors suggest that LETM1 may be involved in mitochondrial Ca2+/H+ exchange, several lines of evidence suggest that the physiological function of Mdm38 and LETM1 is related to KHE (reviewed in (Nowikovsky and Bernardi 2014)). Ylh47 (Mrs7) is homologous to Mdm38, whereas the protein encoded by the YDL183c gene shares no sequence similarity. However overexpression of either YLH47 or YDL183c can suppress mdm38 mitochondrial dysfunction and the triple mdm38 ylh47 ydl183c mutant has more severe phenotypes than any of the single or double mutant combinations (Nowikovsky et al. 2004; Zotova et al. 2010). All three proteins are predicted to have a single membrane spanning domain and so are not likely to mediate KHE individually. However, all three proteins have been shown to be present in high molecular weight complexes and both Mdm38 and Ylh47 can oligomerize, leading to the hypothesis that these proteins are functionally redundant, necessary co-factors of an as yet unidentified KHE (Zotova et al. 2010) (Fig. 8.2).

8.6 Physiological Consequences and Cellular Responses to Alterations in Potassium and Sodium Concentrations

8.6.1 Saline Stress

Perturbations in the extracellular and/or intracellular concentrations of sodium and potassium lead to diverse cellular responses. As discussed above, sodium is actively extruded from yeast cells, so that a physiological response to low sodium (assuming sufficient potassium is present) is not expected. However, in the case of exposure to high concentrations of sodium, yeast cells respond on several levels and the response varies according to the severity and duration of the treatment. High sodium concentrations present a dual toxicity; ionic stress and hyperosmotic stress. One factor contributing to ionic toxicity is the capacity of sodium to displace potassium or in some cases magnesium in the active sites of some enzymes. For example, the HAL2 gene, which confers halotolerance upon overexpression, encodes for a nucleotidase that hydrolyses 3′-phosphoadenosine-5′-phosphate (PAP) to AMP which requires magnesium for catalysis (Murguía et al. 1996). Inhibition of this enzyme by low concentrations of lithium or sodium leads to the accumulation of toxic amounts of PAP and structural data suggests that lithium ions occupy a magnesium binding site necessary for proper catalysis (Albert et al. 2000).

To avoid sodium toxicity, yeast cells actively maintain a high K+/Na+ ratio. In response to saline stress, sodium extrusion, limitation of sodium entry and vacuolar sequestration are key processes, as discussed above. Ena1 and Nha1 are largely responsible for sodium extrusion under alkaline and acidic conditions, respectively and their activation represents one important physiological response to high sodium concentrations (Bañuelos et al. 1998). High salt concentrations also exert hyperosmotic shock and an essential component of the response to this class of stress is the metabolic adjustment toward production and accumulation of the compatible solute, glycerol to maintain water balance (for reviews see: (Blomberg 2000; Hohmann 2002)). Under these conditions, yeast cells also transiently arrest cell cycle progression and reduce both transcription and translation, presumably to provide time for adaptation, and Hog1 has been directly implicated in many of these processes (Teige et al. 2001; Proft and Struhl 2004; Clotet and Posas 2007; Melamed et al. 2008).

An important aspect of the salt stress response also involves remodeling of the gene expression profile. Several studies have examined the transcriptional response to high sodium concentrations and depending on the conditions employed, as many as 400 and 250 genes may be up-regulated or down-regulated, respectively (Posas et al. 2000; Rep et al. 2000; Causton et al. 2001; Yale and Bohnert 2001). Many of the genes whose mRNA levels are altered under saline stress are also regulated in a similar manner under a variety of stress conditions, and so represent a general stress response mediated in large part by Protein kinase A ((Hohmann et al. 2007) and references therein). The kinetics of the transcriptional regulation of individual genes during stress conditions varies widely, with many promoters responding quickly and transiently and others whose regulation is slower and in some cases prolonged, likely correlating with the function of the encoded protein in the acute response or long term adaptation, respectively. Hog1 is required for the regulation of a subset of genes in response to saline stress, including ENA1, as discussed above, and those necessary for glycerol production (Albertyn et al. 1994; Norbeck et al. 1996). Interestingly, the vast majority of the genes up-regulated in response to hyperosmotic stress are not required for cell survival under these conditions (Warringer et al. 2003). A recent report, using a novel signal rewiring approach, suggests that the Hog1-dependent induction of only the GPD1 (glycerol‐3‐phosphate dehydrogenase-1) and GPP2 (glycerol‐3‐phosphatase-2) genes, involved in glycerol biosynthesis, is necessary for osmoadaption (Babazadeh et al. 2014).

Analysis of gene expression has revealed many key features of stress responses. However, as mentioned, the alteration of the expression pattern of specific genes does not necessarily indicate an essential role for the encoded protein in stress adaptation. For example, even if an mRNA accumulates under certain stress conditions, the transcript must still be translated and the protein correctly processed, delivered and possibly activated in order to carry out its function. Several proteomics approaches have been undertaken to study changes in total protein accumulation under conditions of salt stress (reviewed in (Szopinska and Morsomme 2010)). Irrespective of the technique employed, all studies confirm the accumulation of key enzymes needed to shift metabolism towards glycerol production, underscoring the importance of this physiological response (Blomberg 1995; Norbeck and Blomberg 1996; Li et al. 2003; Soufi et al. 2009). A strong correlation between the subset of osmotic shock up-regulated proteins and their corresponding mRNA changes is observed in almost all cases, as would be expected. However, the overall relationship between the proteomic data and published mRNA changes are generally poor, indicating the complexity inherent in extrapolating from gene expression data, as mentioned above. One study analyzed specifically the plasma membrane proteins whose levels are affected during salt stress using a quantitative, gel-free iTRAQ labeling approach (Szopinska et al. 2011). Twelve plasma membrane proteins, including both eisosome components Lsp1 and Pil1, involved in endocytosis, were shown to accumulate, whereas 20 proteins, including Pma1 and ABC transporters, glucose and amino acid transporters, t-SNAREs, and proteins involved in cell wall biogenesis decreased during salt stress treatments. These data fit well with an increase in endocytosis of nutrient permeases in response to salt stress and are consistent with the decrease in amino acid uptake observed under these conditions (Norbeck and Blomberg 1998).

8.6.2 Increased Intracellular Potassium

Like sodium, addition of high extracellular concentrations of potassium (>1 M) also leads to hyperosmotic stress and so in this aspect the cellular response will be similar to that discussed above for sodium. Due to the efficacy of the Ena1 and Nha1 extrusion systems and the reduction in Trk1 activity, wild type cells do not accumulate high internal concentrations of potassium, even in the presence of very high extracellular potassium. However, mutants lacking the ENA gene cluster, NHA1 or both the PPZ1 and PPZ2 phosphatases are sensitive to high extracellular potassium, due to reduced extrusion or inability to inhibit uptake, respectively (Bañuelos et al. 1998; Yenush et al. 2002). The ppz1 ppz2 mutant has been used as a tool to study some aspects of the physiological consequences of steady state increases in intracellular potassium. These strains were shown to have an increase in cell size and intracellular pH and to display plasma membrane depolarization and constitutive activation of the Slt2/Mpk1 cell wall integrity pathway, suggesting that the cell wall is reinforced to counteract the tugor pressure resulting from increased intracellular potassium (Yenush et al. 2002; Merchan et al. 2004). Interestingly, resistance to DNA damaging agents is also reduced in ppz1 ppz2 mutants and these phenotypes are rescued by further disruption of the TRK1 and TRK2 genes or of the SLT2/MPK1 gene and are phenocopied by overexpression of a constitutively active version of the Slt2/Mpk1 MAP kinase kinase, MKK1 (Merchan et al. 2011). Thus, it appears that the constitutive activation of the MAP kinase pathway required for cell wall reinforcement in ppz1 ppz2 mutants is detrimental for some aspects of DNA integrity.

8.6.3 Potassium Starvation

Many studies have investigated various aspects of the physiological response to and consequences of lowering internal potassium concentrations, either by modifying the external media or by examining strains with genetic modifications that lead to reduced potassium uptake, namely the trk1 trk2 and hal4 hal5 mutants. Strains lacking the TRK1 and TRK2 genes are hyperpolarized and have a slightly decreased intracellular pH, even under non-limiting potassium conditions, despite the fact that the internal potassium concentration is not markedly different from the wild type control (Madrid et al. 1998; Navarrete et al. 2010). The hyperpolarization of the trk1 trk2 mutants explains the general sensitivity to toxic cations, whereas the reduced intracellular pH has been attributed to decreased Pma1 activity, which fits well with the reduction in amino acid uptake also observed in these mutants (Yenush et al. 2002; Navarrete et al. 2010). Mutants lacking the genes encoding for the HAL4 and HAL5 kinases share many of the same phenotypes with the trk1 trk2 mutants, such as acidic intracellular pH, decreased amino acid uptake and sensitivity to toxic cations, which is expected for strains lacking positive regulators of these potassium transporters (Perez-Valle et al. 2010). However, the hal4 hal5 mutants appear to have additional, Trk1-independent defects which lead to a decrease in the accumulation of many nutrient transporters at the plasma membrane, although the molecular mechanism underlying this defect has yet to be defined (Perez-Valle et al. 2007, 2010). Despite the fact that different conditions were used, analysis of the gene expression profiles of both hal4 hal5 and trk1 trk2 mutants shows a strong correlation among the genes that are up- or down-regulated (correlation coefficient = 0.77) and indicate cellular processes that are altered in both mutants, such as methionine biosynthesis (Perez-Valle et al. 2010; Barreto et al. 2012). Part of this phenotype may be explained by the marked reduction observed in the accumulation of the high affinity methionine permease, Mup1, which correlates with reduced methionine uptake (Perez-Valle et al. 2010). Studies of these mutants highlight aspects of cell function that are affected in strains where high affinity potassium uptake is permanently disabled. Another physiological situation is the adaptation process that takes place in response to a sudden drop in external potassium concentrations.

Several approaches have been taken to analyze wild type and mutant strains either grown in or shifted to media with limiting potassium concentrations in order to characterize the changes produced by the starvation and the cellular responses that lead to the re-establishment of potassium homeostasis. After several hours of potassium starvation, wild type strains lose 70 % of their internal potassium, the cell volume decreases by about 20 % and cells become hyperpolarized, but the internal pH remains essentially the same (Navarrete et al. 2010). The transcriptional response to potassium starvation has been studied in two ways. In the first approach, cells were grown in chemostat cultures in the presence of limiting concentrations of potassium (Hess et al. 2006). The transcriptional response was moderate, with a total of approximately 110 different genes up- or down-regulated more than threefold in the two lowest potassium concentrations tested (0.65 and 1.3 mM), as compared with the non-limiting potassium control. The majority of the affected transcripts encode proteins involved in nitrogen metabolism. Subsequent experiments revealed ammonium toxicity under limiting potassium conditions and suggest that yeast cells respond to this toxicity by secreting amino acids (Hess et al. 2006). Ammonium was suggested and later proven to enter through the Trk potassium transporters as part of a second study investigating the transcriptional response to short-term potassium starvation (Barreto et al. 2012). In this study, cells were grown in the presence of non-limiting potassium and then shifted to essentially potassium-free media (15 μM) and the transcriptional profile was determined at a series of time points using microarrays. More than 800 genes were shown to be up-regulated at least at one time point, whereas more than 900 genes were shown to be down-regulated. The bulk of the transcriptional response was not observed until 60 mins. Based on the transcriptional profile and further experiments, the shift to potassium-free media was shown to lead to a myriad of effects, including induction of oxidative stress, alterations in sulfur metabolism, phosphate starvation, pronounced reduction in genes necessary for ribosome biogenesis and translation, activation of the retrograde pathway, alteration of cell cycle-related gene and protein expression profiles and blockage of septin assembly. A similar study was also done using a different approach: Serial Analysis of Gene Expression (SAGE)-tag sequencing (Anemaet and van Heusden 2014). After 60 min of potassium starvation, mRNA levels of 105 and 172 genes were significantly up- or down-regulated, respectively. Although a lower number of genes were shown to be differentially expressed using this technique, there is a reasonable correlation between both studies, especially for genes related to the cell cycle and phosphate starvation. More recently, a detailed study confirmed and further characterized the phosphate deprivation response triggered by potassium starvation (Canadell et al. 2015). Proteomics approaches have also been employed to examine the changes at the level of protein accumulation in control and trk1 trk2 mutants and in both non-limiting potassium and in response to potassium starvation (Curto et al. 2010; Gelis et al. 2012). Whereas, in the trk1 trk2 mutants, no differentially expressed proteins were identified in non-limiting potassium medium, the studies using potassium-starved trk1 trk2 cells showed a marked decrease in the total amount of protein recovered after prolonged potassium starvation. However, as stated by the authors, in both studies the protein recovery was sub-optimal and so key changes in individual protein accumulation of proteins outside the pI and molecular weight range and/or below the abundance threshold may have gone undetected in these experimental approaches.

A mathematical model has helped to determine key events required for effective adaptation to potassium starvation (Kahm et al. 2012). This approach has revealed a complex interplay between biophysical forces and molecular regulation facilitating potassium homeostasis by predicting that proton extrusion and an increased rate of the bicarbonate reaction are vital for cells to maintain a minimal concentration of intracellular potassium in response to sudden starvation. Upon shifting cells to potassium-free media, potassium loss proceeds in two phases; an initial rapid loss, followed by a longer and slower decrease in internal potassium. In trk1 trk2 mutants, the second phase of potassium loss is much less pronounced than in the wild type cells, presumably due to the hyperpolarization of the membrane. This observation indicates that the lack of the high affinity transporters is not playing a pivotal role in net potassium loss during starvation. Using what is referred to as a reverse tracking algorithm, an initial burst of Pma1 activity and the bicarbonate reaction are predicted to be necessary to maintain the minimum amount of intracellular potassium required for viability. In both cases, this burst in activity will hyperpolarize the plasma membrane, but by two different mechanisms: Pma1 activation will lead to a decrease in the internal positive charge due to proton pumping outside the cell, whereas the bicarbonate reaction will lead to increased internal negative charge due to the accumulation of HCO3 − inside the cell. Importantly, the increase in Pma1 activity and transient activation of the bicarbonate reaction in response to potassium starvation predicted by the model were both confirmed experimentally. The mechanisms by which the cells sense and signal changes in the external potassium concentrations are still unknown, but this study highlights the usefulness of mathematical models to elucidate important aspects of cell physiology. These authors also present evidence showing that internal steady state potassium concentration is determined by the external concentration, thus indicating that potassium homeostasis is an example of non-perfect adaptation. A more recent study showed that the Trk1 and Trk2 transporters are required for the stabilization of intracellular potassium content by affecting the internal potassium concentrations attained at low extracellular potassium content (Herrera et al. 2014).

8.7 Extrapolations and Applications

As summarized above, a large number of laboratories have contributed to various aspects of the study of potassium and sodium transport in the model yeast S. cerevisiae. This information is important from a purely scientific point of view, but it also has many different applications, some of which will be mentioned here. For example, the experimental data generated has been used to construct mathematical models describing complex physiological processes, such as response to potassium starvation and to hyperosmotic shock (Klipp et al. 2005; Kahm et al. 2012; Ke et al. 2013). The predictive power of these models has confirmed the validity of these types of approaches and can serve as a framework for modeling processes in multi-cellular organisms.

On the other hand, the S. cerevisiae model system has been used as a point of reference to compare and contrast mechanisms of ion homeostasis in other yeast species, including those that cause disease in humans. Studies of the distribution and function of sodium and potassium transporters in non-conventional yeast species have been expertly reviewed (Ramos et al. 2011). Briefly, in most yeast species studied to date, surplus potassium and sodium are extruded via the joint participation of NHA antiporters, ENA ATPases and TOK potassium channels, whereas potassium uptake is mediated by various combinations of at least three types of systems unevenly spread among the yeast species: TRK and HAK (High Affinity K+) transporters and the ACU (Alkali Cation Uptake) ATPases. Yeast HAK transporters are homologous to the Kup system of Escherichia coli and have been proposed to work as K+–H+ symporters with a high concentrative capacity (Rodríguez-Navarro 2000). Whereas HAK transporters are found in many species, including higher plants, functional ACU ATPases have been described only in non-conventional yeast, such as Ustilago maydis, Pichia sorbitophila and the extremely halotolerant and adaptable fungus, Hortaea werneckii (Rodríguez-Navarro 2000; Benito et al. 2004; Plemenitaš et al. 2014). Thus, it appears that many of the general aspects of sodium and potassium transport described above are well-conserved, but depending on the niche, alternative strategies for acquiring and maintaining potassium and sodium homeostasis have evolved.

A large body of evidence indicates that excessive potassium efflux and intracellular potassium depletion are key early steps in apoptosis in mammalian cells (Yu 2003). Several studies suggest that these changes are also implicated in cell death in yeast. For example, prolonged potassium starvation has been shown to lead to cell death through a process in which many of the biochemical markers associated with apoptosis in metazoan cells are detected, such as phosphatidylserine externalization, changes in chromatin condensation, DNA and vacuole fragmentation, as well as enhanced accumulation of reactive oxygen species (ROS) (Lauff and Santa-María 2010). Moreover, both potassium and proton fluxes were shown to influence glucose-induced cell death (Hoeberichts et al. 2010). Using a series of mutants defective for Pma1 activity or potassium uptake or efflux, it was shown that cells that had either reduced Pma1 activity or maintained higher internal potassium concentrations were less sensitive to cell death produced by glucose addition to starved cells, whereas those with lower internal potassium were more sensitive. These effects were also correlated with ROS production and the authors suggest that this is a key event in inducing cell death under these conditions.

Thus, it appears that in yeast, as in mammalian cells, internal potassium homeostasis is vital for cell survival and conditions which alter this balance can lead to cell death. This notion is further supported by studies demonstrating a connection between the fungicidal activities of killer toxin K1, Histatin 5 (Hst 5) and lactoferrin with potassium homeostasis (Ahmed et al. 1999; Sesti et al. 2001; Baev et al. 2003, 2004; Andrés et al. 2008). Although not all the data reported are consistent with this hypothesis, Tok1 has been proposed to be the target of the yeast viral killer toxin K1, which has been shown to bind to and activate the channel from both sides of the plasma membrane (Ahmed et al. 1999; Sesti et al. 2001; Breinig et al. 2002). Hst5, a histidine-rich cationic protein produced in human saliva, is a key component of the non-immune defense system of the oral cavity that possesses both fungistatic and fungicidal activities against several potentially pathogenic fungi, such as Candida albicans, Candida glabrata, Candida krusei and Cryptococcus neoformans (Tsai and Bobek 1997a, b). This toxin induces non-cytolytic efflux of cellular ATP, potassium, and magnesium, implicating these ion movements in the mechanism of Hst5 toxicity. Genetic approaches suggest that Tok1 modulates Hst5-mediated toxicity, whereas Trk1 was shown to be a critical effector of its fungicidal activity in C. albicans (Baev et al. 2003, 2004). Similarly, lactoferrin, a protein present in all mammalian mucosal secretions, exhibits antifungal and antibacterial activities through a mechanism that is still being defined (Farnaud and Evans 2003). Lactoferrin causes a rapid release of potassium from C. albicans cells and cell death can be inhibited by high extracellular potassium or by treatment with chloride or potassium channel blockers, suggesting a role for potassium channels in the mechanism of action of this fungal toxin (Viejo-Díaz et al. 2004a, b; Andrés et al. 2008).

As alluded to above, alterations in potassium homeostasis also affect plasma membrane potential, nutrient uptake and survival at alkaline pH, which in turn have been linked to flocculation, invasiveness and virulence. For example, hyperactivation of Ppz phosphatases results in alteration of potassium transport leading to Protein kinase A activation and increased expression of the flocculin-encoding FLO11 gene, thus modulating flocculation and invasive growth in S. cerevisiae (González et al. 2013). Whether this mechanism is relevant in pathogenic fungi is still to be determined. A very relevant finding in this context is the identification of ENA1 as a virulence gene in Cryptococcus neoformans (Idnurm et al. 2009). A subset of a library of signature-tagged insertion mutants of this human pathogenic fungus was screened in a murine inhalation model to identify genes required for virulence. Inactivation of the ENA1 gene led to an avirulent phenotype, which was attributed to the reduced viability of this mutant under alkaline pH conditions. The ability of fungi to grow in slightly alkaline conditions is essential for pathogenesis because, in general, many human host environments have a relatively high pH (reviewed in: (Davis 2009)). This also explains the important role of the alkaline-responsive PacC/Rim101 pathway in pathogenic yeast virulence in found in several species (reviewed in (Cornet and Gaillardin 2014)).

Sensitivity to antifungal drugs is also affected in mutants with alterations in potassium homeostasis. Studies in S. cerevisiae have revealed that treatment with ketoconazole, miconazole or amiodarone leads to potassium efflux, similar to that observed for Hst5 and lactoferrin (Peña et al. 2009; Calahorra et al. 2011). Ketoconazole and miconazole are members of the azole class of antifungal drugs, whose main mechanism of toxicity is the inhibition of the biosynthesis of the fungal specific sterol, ergosterol by directly binding and inactivating cytochrome P-450, thus leading to alterations in the properties of the yeast plasma membrane (reviewed in: (Saag and Dismukes 1988)). Amiodarone is a cationic amphipathic drug that interacts preferentially with lipid membranes and has been used clinically as an anti-arrhythmic agent for many years (Mason 1987). It was shown to have broad fungicidal activity (Courchesne 2002). Studies aimed at determining the mechanism of action in yeast showed that mutation of PMA1, TOK1 or ENA1-4 protected against aminodarone toxicity, suggesting that initial drug-induced hyperpolarization is important in the mechanism of antifungal activity and this was confirmed by decreasing the membrane potential by glucose removal or addition of salts (Maresova et al. 2009). This transient hyperpolarization is followed by depolarization, Ca2+ and H+ influx and loss in cell viability (Courchesne and Ozturk 2003; Maresova et al. 2009; Peña et al. 2009). Other observations consistent with a role for the determinants of potassium homeostasis in fungal drug sensitivity of pathogenic yeast include studies showing that disruption of ENA1 and NHA1 in C. neoformans alter membrane potential and the sensitivity to several antifungal drugs (Jung et al. 2012). Finally, in the case of C. glabrata, treatment with another azole drug, fluconazole was shown to lead to membrane hyperpolarization and increased sensitivity to cationic drugs (Elicharova and Sychrova 2014). Moreover, in strains lacking ENA1 and/ or the CNH1 cation ATPase this combined treatment was even more effective.

Taken together, these observations clearly indicate that the proteins involved in determining and maintaining plasma membrane potential through the modulation of potassium homeostasis represent promising targets for complimentary antifungal treatments. The fact that, for example, the TRK1 gene, which has no homologues in mammalian cells, is present as a single copy in the C. albicans genome and the sequence of the MPM segments (see Sect. 8.3.1 on Trk1) is highly conserved among fungal species suggests that inhibitors of this protein have the potential to be broad-spectrum antifungal treatments with potentially low toxicity (Miranda et al. 2009).

S. cerevisiae is also used in a wide range of industrial processes. The impact of potassium homeostasis on nutrient uptake and cell survival will obviously affect many aspects of yeast performance in industrial fermentations. One poignant example was recently reported showing the direct effect of both external and internal potassium and pH on ethanol tolerance in conditions relevant for the industrial production of bioethanol (Lam et al. 2014). These authors showed that increasing the potassium concentration and the pH of the media leads to important improvements in both ethanol tolerance and production under high-glucose and high–cell-density conditions, essentially by boosting cell viability. These results were confirmed using yeast mutants that were engineered to have increased internal potassium and pH by increasing Trk1 activity (ppz1 ppz2 mutants) and ectopically overexpressing PMA1. The ethanol production of these modified laboratory strains was superior to industrial stains currently in use. The authors suggest that this protective effect of augmenting potassium and proton fluxes is due to the counteraction of the dissipation of the potassium and proton gradients caused by partial permeabilization of the plasma membrane in the presence of high ethanol concentrations. Their work shows that these complex, but genetically determined biophysical parameters may be key points of intervention for the development of yeast strains capable of higher bioethanol production in industrial settings.

The knowledge and reagents generated in S. cerevisiae have also served as a platform for discovery and characterization of ion transporters from both plants and mammals. For example, in the case of plant ion transporters, yeast mutants defective in high affinity potassium transport (trk1 trk2) were used to identify and determine structure/function relationships for the plant potassium channels KAT1 and AKT1 (Anderson et al. 1992, 1994; Schachtman et al. 1992; Sentenac et al. 1992; Uozumi et al. 1995; Nakamura et al. 1997). The Arabidopsis thaliana SOS (Salt Overly Sensitive) pathway, consisting of the SOS1 sodium ATPase, the SOS2 protein kinase and the SOS3 Ca2+ sensor was functionally reconstituted in yeast strains devoid of ENA1-4, NHA1 and NHX1 (Quintero et al. 2002). The Arabidopsis thaliana CHX17 gene was shown to complement the S. cerevisiae kha1 mutant phenotypes, suggesting that this transporter can function as a K+/H+ exchanger in the Golgi of yeast (Maresova and Sychrova 2006). Finally, several studies have used S. cerevisiae nhx1 or ena1-4 nha1 nhx1 mutants to study plant intracellular Na+/H+ exchangers from both rice and Arabidopsis thaliana (Gaxiola et al.1999; Quintero et al. 2000; Fukuda et al. 2004; Kinclová-Zimmermannová et al. 2004). These studies demonstrate the level of conservation that exists between yeast and plants and confirms the utility of yeast model systems for the study of higher organisms.

Not surprisingly, this same complementation approach has been used for the characterization of mammalian ion transporters as well. For example, several functional studies of inward rectifying potassium channels have been done using trk1 trk2 mutants (for examples see: (Tang et al. 1995; Minor et al. 1999; Hasenbrink et al. 2005; Haass et al. 2007; Schwarzer et al. 2008; D’Avanzo et al. 2010)). These heterologous expression systems have also been used for high-throughput screenings searching for small molecule modulators of potassium channels (Zaks-Makhina et al. 2004, 2009; Bagriantsev et al. 2013). The heterotrimeric ENaC sodium channel has also been functionally expressed in yeast and shown to increase salt sensitivity (Gupta and Canessa 2000). Mutations in this sodium channel have been linked to an inherited form of hypertension called Liddle’s Syndrome (Shimkets et al. 1994). Other transporters, such as the Na+,K+-ATPase, the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel and a Na+-phosphate co-transporter have also been studied by heterologous expression in yeast (for a review see: (Kolb et al. 2011)). Using ena1-4 nha1 nhx1 mutants, several mammalian Na+/H+ exchangers have also been characterized (Montero-Lomelí and Okorokova Façanha 1999; Flegelova et al. 2006; Xiang et al. 2007). One very interesting study used the yeast model system to characterize mutations in the human NHE9 Na+/H+ antiporter that have been associated with autism (Kondapalli et al. 2013). In this study, equivalent mutations found in autistic patients were introduced into the NHX1 gene and functional studies of these modified transporters showed that two of them led to a loss of Nhx1 function. Finally, as discussed earlier, another example of the utility of yeast to contribute to the definition of the function of the proteins encoded by disease related genes is the discovery that the LETM1 gene, responsible for seizures associated with some forms of Wolf-Hirschhorn Syndrome, can functionally complement the mdm38 yeast mutant mitochondrial KHE phenotypes (see above) (Nowikovsky et al. 2004).

In conclusion, our knowledge regarding potassium and sodium transport in yeast is quite extensive, but far from complete. Although most of the major transporters have been identified and extensively characterized, some fluxes, such as the low affinity potassium uptake NSC1 activity and the mitochondrial K+/H+ exchange across the inner membrane, await molecular characterization. Moreover, several ORFs with weak sequence homology to mammalian ion transporters still have unknown functions and may help to complete the picture, especially in the case of the intracellular distribution of potassium and sodium. Our understanding of the regulation of many of these transporters, especially on the post-translational level is also very limited. Integration of the signals leading to the establishment and maintenance of ion homeostasis in response to changing environments is an area where progress still needs to be made. Given the importance of this field to basic science and its applications ranging from industrial processes to plant salt and drought tolerance and mammalian physiology, disease states and drug discovery, new advances in the study of yeast potassium and sodium transport are likely to bring new insight with both expected and novel impacts in the future.