MICROBIOLOGY LETTERS. Alkali^metal^cation in ux and e ux systems in nonconventional yeast species. K + uptake systems MINIREVIEW

Transcription

1 MICROBIOLOGY LETTERS MINIREVIEW Alkali^metal^cation in ux and e ux systems in nonconventional yeast species José Ramos 1, Joaquín Ariño 2 & Hana Sychrová 3 1 Departamento de Microbiología, Universidad de Córdoba, Córdoba, Spain; 2 Departament de Bioquimica i Biologia Molecular, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain; and 3 Department of Membrane Transport, Institute of Physiology Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic Correspondence: Hana Sychrova, Department of Membrane Transport, Institute of Physiology AS CR, Videnska 1083, Prague 4, Czech Republic. Tel.: ; fax: ; sychrova@biomed.cas.cz Received 8 November 2010; revised 2 January 2011; accepted 5 January Final version published online 1 February DOI: /j x Editor: Derek Sullivan Keywords Ena ATPase; Nha antiporter; Trk transporter; Hak transporter; Acu ATPase; Tok channel. Abstract To maintain optimal intracellular concentrations of alkali metal cations, yeast cells use a series of influx and efflux systems. Nonconventional yeast species have at least three different types of efficient transporters that ensure potassium uptake and accumulation in cells. Most of them have Trk uniporters and Hak K 1 H 1 symporters and a few yeast species also have the rare K 1 (Na 1 )-uptake ATPase Acu. To eliminate surplus potassium or toxic sodium cations, various yeast species use highly conserved Nha Na 1 (K 1 )/H 1 antiporters and Na 1 (K 1 )-efflux Ena ATPases. The potassium-specific yeast Tok1 channel is also highly conserved among various yeast species and its activity is important for the regulation of plasma membrane potential. All yeast species need to regulate their intracellular concentrations of alkali metal cations, i.e. maintain rather high and stable potassium content and eliminate surplus toxic sodium cations. For this purpose, yeast cells possess a broad variety of plasma-membrane and organellar transporters that mediate the fluxes of cations with differing mechanisms and affinities. According to the analyses of the sequenced genomes, all yeasts probably possess conserved and efficient potassium uptake systems in their plasma membranes, two types of alkali metal cation efflux systems (antiporters and ATPases), and most of them also possess cation channels (Fig. 1). The alkali metal cation transport systems of the most-studied (and model) yeast species Saccharomyces cerevisiae have been recently reviewed elsewhere (Arino et al., 2010), so this minireview will try to summarize current knowledge on the plasma-membrane transport systems of nonconventional yeasts. Besides the second most widely used yeast model, Schizosaccharomyces pombe, alkali metal cation transporters have been recently characterized in many osmotolerant yeast species, i.e. species that can grow in the presence of very high salt or sugar concentrations, and in pathogenic Candida species that are also rather osmotolerant and for whom potassium and sodium homeostasis maintenance is thought to contribute to their pathogenicity. K + uptake systems Three types of efficient potassium uptake systems, differing in their transport mechanism and primary protein structure, have been identified so far in nonconventional and pathogenic yeast species. The high relevance of the potassium uptake process is highlighted by the fact that, with a single exception (Zygosaccharomyces rouxii), all yeasts whose genomes have been sequenced are probably endowed with more than one potassium uptake system. TRK The TRK (Transport of K + ) family of transporters seems to be the most widely distributed in yeasts, although in only three species is their presence not accompanied by the existence of another system with a different mechanism (Table 1). Recently, the Trk family of transporters in nonanimal cells has been reviewed (Corratgé-Faillie et al., 2010). In S. cerevisiae, transport depends mainly on TRK1,

2 2 J. Ramos et al. Table 1. An overview of genes encoding active potassium influx systems and outward rectifying potassium channel in yeasts Systems for K 1 influx the role of the Trk2 protein in potassium supply is marginal and its transport activity is undetectable in the presence of TRK1 (Arino et al., 2010). In S. pombe, two Trk proteins have also been found and characterized (Soldatenkov et al., 1995; Calero et al., 2000). Sptrk1 1 and Sptrk2 1 are, in contrast to S. cerevisiae, equally important for the cell when growing under standard conditions and the presence of any of them is enough to enable growth at very low potassium concentrations. Schizosaccharomyces pombe cells lacking both trk genes can still grow at a similar rate to the wild type when the external concentration of K 1 is above 20 mm, and they are able to transport Rb 1 (K 1 analogue) with a low affinity. Therefore, the existence of a third, less efficient, K 1 transporter cannot be ruled out. However, it is also possible that the K 1 influx in the mutant is due to an ectopic process similar to the one described for S. cerevisiae (Madrid et al., 1998). Fig. 1. Transport systems for potassium and/or sodium influx and efflux in plasma membranes of nonconventional yeast species. TRK1 TRK2 HAK1 ACU1 TOK1 S. cerevisiae S. pombe 1 1 K. lactis 1 1 D. hansenii 1 1 D. occidentalis 1 1 Z. rouxii 1 1 C. albicans 1 1 Pseudogene 1 P. stipitis Y. lipolytica K 1 channel Kluyveromyces lactis is endowed with a TRK homologous gene whose product works as a low-affinity K 1 transporter (Miranda et al., 2002). Trk transporters have been studied in two Debaryomyces species (Debaryomyces occidentalis, former Schwanniomyces occidentalis, and Debaryomyces hansenii), and DoTrk1 was found to be involved in the potassium uptake and in the control of the membrane potential (Banuelos et al., 2000). Debaryomyces hansenii TRK1 was expressed in an S. cerevisiae mutant lacking its endogenous potassium transporters. This expression resulted in partial recovery of growth and ability to retain K 1 at low concentrations (Prista et al., 2007). Recently, DhTrk1 has been proposed to work as a uniporter under nonlimiting K 1 conditions (Martínez et al., 2011). The Candida albicans Trk1 transporter has been functionally compared with the Trk systems of S. cerevisiae. It has been proposed that its main function, potassium transport,

3 Potassium and sodium transporters in yeasts 3 is quantitatively conserved while its secondary function, chloride efflux, exhibits important differences from ScTrk1 (Miranda et al., 2009). Interestingly, CaTrk1 and CaTok1 have been proposed to be the effectors in killing C. albicans with the cationic protein Histatin 5, with Trk1 providing the essential pathway for the ATP loss observed during treatment with this toxic protein (Baev et al., 2004). HAK Many nonconventional yeasts (Hansenula polymorpha, Debaryomyces, C. albicans) as well as mycelial fungi (Neurospora crassa; Haro et al., 1999) contain, besides Trk, a second type of K 1 transporter coded by HAK genes (High Affinity K + transporter) (Table 1; Rodriguez-Navarro, 2000; Arino et al., 2010). Yeast HAK transporters are homologous to the Kup system of Escherichia coli. The role of Hak1 in potassium transport has been studied in two Debaryomyces species, D. occidentalis (Banuelos et al., 1995, 2000) and D. hansenii (Prista et al., 2007), containing both the TRK1 and HAK1 genes. Heterologous expression of DoHAK1 in S. cerevisiae mutants with defective K 1 uptake improved both their growth at low K 1 - and potassium-transport capacity. It has been proposed that HAK transporters work as K 1 H 1 symporters with a high concentrative capacity and that they are expressed under K 1 starvation. Under certain conditions, Na 1 can substitute for H 1 in D. hansenii and in this case, K 1 Na 1 symport would be the operating mechanism of transport. The expression of DhHAK1 requires not only low external K 1 but also low Na 1, because in the absence of K 1, the presence of Na 1 prevents the expression of the gene. Further, the addition of millimolar concentrations of either K 1 or Na 1 to D. hansenii cells provokes a fast decrease in HAK1 gene expression (Martínez et al., 2011). ACU The existence of a third type of K 1 uptake system, ACU ATPases (Alkali Cation Uptake), has been reported recently. This system is not widely distributed in nonconventional yeasts, but is present in some of them, such as Ustilago maydis or Pichia sorbitophila (Benito et al., 2004). The ACU ATPases form a novel subfamily of P-type ATPases involved in high-affinity K 1 or Na 1 uptake. In U. maydis, twoacu genes have been identified and studied (Umacu1 and Umacu2). Deletion of the acu1 and acu2 genes and subsequent transport studies showed that they encode transporters mediating a high-affinity K 1 and Na 1 uptake. This finding was also confirmed by the heterologous expression of UmAcu2 ATPase in S. cerevisiae mutants. Besides P. sorbitophila, other yeasts have genes or pseudogenes whose translated sequences show high similarity to the Acu proteins of U. maydis (Benito et al., 2004), for example Pichia stipitis (Jeffries et al., 2007). Potassium TOK channels Whereas a database search indicates that the genomes of most Candida, Zygosaccharomyces, Yarrowia or Pichia yeast species contain a gene orthologous to the S. cerevisiae TOK1 (coding for the only known yeast outward K 1 rectifier), the best-known nonconventional yeasts S. pombe and D. hansenii seem to lack a similar system. Electrophysiological experiments have shown that the primary function of this channel in S. cerevisiae is K 1 efflux contributing to the maintenance of a stable plasma membrane potential (Arino et al., 2010). Information on the activity of Tok channels in yeasts is scarce, but in C. albicans the gene has been identified, the function of the protein studied and deletion mutants characterized (Baev et al., 2003). Homozygous deletion of CaTOK1 completely abolishes the currents and gating events characteristic of the Tok1 channel. The same study also reported that mutants lacking this gene showed an increased viability after treatment with the potent salivary toxin Histatin 5, which induces the efflux of cellular ATP, potassium and magnesium (Baev et al., 2003). More recently, it has been shown that K 1 efflux via CaTok1 is required for the progression of an apoptosis-like process in Candida cells. Because K 1 efflux is one of the earliest events of the apoptotic process in metazoan cells and is presumed to be necessary for activating biochemical apoptotic pathways, the authors propose that the effect of channelmediated K 1 efflux on apoptosis has been evolutionary conserved among species ranging from yeasts to humans (Andres et al., 2008). Alkali--metal--cation/H 1 antiporters Transport systems mediating the exchange of alkali metal cations for protons exist in the plasma membranes of probably all organisms, and in the membranes of most eukaryotic organelles (Arino et al., 2010). Genes homologous to S. cerevisiae NHA1 (Na/H Antiport) have been found in all sequenced yeast genomes and members of the plasma-membrane NHA family have been so far characterized in 10 nonconventional yeast species, c.f. below. However, in six of them, the characterization of their transport capacity and substrate specificity is purely based on data obtained upon their heterologous expression in S. cerevisiae, and only for four species (S. pombe, Z. rouxii, C. albicans and C. glabrata) this information has been complemented with phenotype and transport studies in deletion/overexpression mutants. The main substrates of the yeast antiporters are sodium and/or potassium cations, together with their analogues lithium and rubidium. Members of the NHA family differ in their length (from 468 for SpSod2 to 985 amino acid residues in S. cerevisiae and C. parapsilosis antiporters) and this difference is related to the length of their C-termini. The N-termini predicted 12

4 4 J. Ramos et al. transmembrane segments and connecting hydrophilic loops are highly conserved (Pribylova et al., 2006; Krauke & Sychrova, 2008). According to the number of NHA proteins in the plasma membrane and to their functional specialization, the 10 yeast species can be divided in two subgroups, one containing three members (S. pombe, Z. rouxii, Yarrowia lipolytica) in which the original NHA1 gene has been probably duplicated and the two antiporters gained differing functions (sodium detoxification and maintenance of potassium homeostasis), whereas in the larger subgroup, only one plasma-membrane antiporter with multiple functions exists. In S. pombe, one of the two antiporters, Spsod2 was the very first characterized yeast transport system with an Na 1 / H 1 antiport mechanism (Jia et al., 1992). Its identification was based on a selection for increased tolerance to Li 1 in salt-sensitive S. pombe cells. Deletion of the gene led to diminished NaCl tolerance, its overexpression resulted in an increased Na 1 export from cells and the heterologous expression in S. cerevisiae cells confirmed the antiporter s role in sodium efflux and tolerance, as well as its inability to transport potassium cations (Jia et al., 1992). Much later, the second S. pombe antiporter (SpSod22) was identified, and its characterization, though only via expression in S. cerevisiae, showed that it efficiently transports K 1 and is thus probably the main system for the maintenance of potassium homeostasis in S. pombe cells exposed to surplus K 1 cations (Papouskova & Sychrova, 2007). The Y. lipolytica genome also contains two genes encoding members of the NHA family (Papouskova & Sychrova, 2006). Their heterologous expression in S. cerevisiae cells revealed that while the YlNha1 protein mainly increased cell tolerance to potassium and contributed to potassium homeostasis in the presence of very high concentrations of extracellular KCl, YlNha2p displayed a remarkable transport capacity for sodium, in fact, the highest so far measured for any yeast Na 1 /H 1 antiporter. The two plasma-membrane antiporters of the osmotolerant yeast Z. rouxii (ZrSod2-22 and ZrNha1) have been characterized in detail both in S. cerevisiae and in Z. rouxii cells. First, the sodium-specific Sod2 (and its silent copy Sod22) from a highly salt-tolerant strain (ATCC 4298) and later the Sod2-22 from the less halotolerant CBS732 strain were characterized upon expression in S. cerevisiae (Iwaki et al., 1998; Kinclova et al., 2001b). Both ZrSod2 and ZrSod22 were shown to enhance the NaCl tolerance of a salt-sensitive S. cerevisiae strain, but only ZrSOD2 was confirmed to be transcribed in Z. rouxii cells (Watanabe et al., 1995; Iwaki et al., 1998), so the high salt tolerance of the Z. rouxii ATCC 4298 strain was not based on the presence and expression of two copies of genes encoding a sodium-specific Na 1 /H 1 antiporter. Only one copy of the SOD2 gene has been found in the Z. rouxii CBS732 genome. As it was not only highly identical to ZrSOD2 but also contained a sequence unique to ZrSOD22, it was named ZrSOD2-22 (Kinclova et al., 2001b). Heterologous expression in an S. cerevisiae strain lacking its own alkali metal cation exporters revealed that all three Z. rouxii SOD antiporters transport only sodium and lithium (Iwaki et al., 1998; Kinclova et al., 2001b, 2002), similar to the S. pombe sod2 antiporter. A later search for a putative potassium proton antiporter in Z. rouxii led to the identification of the ZrNHA1 gene, and characterization of its product in S. cerevisiae confirmed the predicted ability of ZrNha1 to mediate the efflux of potassium (Pribylova et al., 2008). The difference between both ZrSod2-22 and ZrNha1 transporters in their substrate preferences (sodium vs. potassium) and physiological functions (sodium detoxification vs. maintenance of potassium homeostasis) has been demonstrated directly in Z. rouxii cells lacking or overexpressing the two antiporters (Pribylova et al., 2008). In general, the three sodium-specific antiporters (SpSod2, YlNha2 and ZrSod2-22) possess shorter C-terminal hydrophilic parts than their potassium-transporting paralogues, and YlNha2 and ZrSod2-22 antiporters have an extremely high capacity to export sodium cations (Kinclova et al., 2001b; Papouskova & Sychrova, 2006), much higher than ScNha1 or other yeast antiporters with broad substrate specificities described below. One plasma-membrane antiporter with a broad substrate specificity for at least four alkali cations (K 1,Na 1,Li 1, Rb 1 ) has been characterized in two osmotolerant yeast species, D. hansenii (Velkova & Sychrova, 2006) and P. sorbitophila (Banuelos et al., 2002) and in five members of the Candida genus C. albicans, C. dubliniensis, C. parapsilosis, C. glabrata and C. tropicalis (Kinclova et al., 2001a; Kamauchi et al., 2002; Krauke & Sychrova, 2008, 2011). All of these transporters have been characterized upon heterologous expression in S. cerevisiae. Phenotypes of increased salt tolerance as well as direct measurements of cation efflux showed that the individual transporters, though having the same large substrate specificity, differ in their capacity to transport cations, for example C. parapsilosis and C. albicans antiporters being the most and those of C. dubliniensis and C. glabrata being the least efficient (Krauke & Sychrova, 2008, 2011). Candida albicans and C. glabrata deletion mutants lacking the genes encoding Na 1 /H 1 antiporters have been constructed (Soong et al., 2000; Kinclova-Zimmermannova & Sychrova, 2007; Krauke & Sychrova, 2011) and characterization of their phenotype and transport capacity revealed that though these two antiporters are able to transport both potassium and sodium cations when expressed in S. cerevisiae, their absence in Candida cells only results in an increased sensitivity to high external potassium concentrations and did not alter their tolerance to NaCl. Detailed measurements of

5 Potassium and sodium transporters in yeasts 5 alkali metal cation efflux in wild-type cells, deletion and reintegration mutants confirmed that the two transporters play only a marginal role in sodium detoxification, but are highly important for cell survival in the presence of high external potassium concentrations. Thus these antiporters of C. albicans and C. glabrata are the very first known examples of the plasma-membrane Na 1 /H 1 antiporter family from prokaryotes and lower eukaryotes, whose primary function is not the elimination of toxic sodium cations, but contribution to the optimal intracellular potassium concentration, and thereby to cell volume, turgor and membrane potential. ENA ATPases The ENA (Efflux of Natrium) family belongs to the category of type-iid ATPases, and its members are found exclusively in fungi, bryophyta and protozoa (see Rodriguez-Navarro & Benito, 2010, for a recent review). ENA homologues exist in all so-far sequenced yeast genomes. Mainly from studies in the model yeast S. cerevisiae, it is commonly accepted that the role of the Ena ATPase is crucial for sodium detoxification at high external ph values, where the antiporter system cannot effectively exchange Na 1 for protons. However, ENA ATPases are not specific for sodium (or lithium) extrusion, but they also transport K 1, as it was initially deduced from the characterization of the Ena1 ATPase activity in S. cerevisiae (Benito et al., 1997). Further support for this notion came from the discovery of two ATPases (encoded by ENA1 and ENA2 genes) with different functions in D. occidentalis (Banuelos & Rodriguez-Navarro, 1998). These two genes complement the Na 1 sensitivity of an S. cerevisiae ena mutant strain. The expression of DoENA2 was increased by high ph, but both high ph and high sodium were required for the DoENA1 expression. Remarkably, whereas D. occidentalis mutants lacking ENA1 were less sodium tolerant, the mutation of ENA2 did not alter sodium tolerance, but resulted in sensitivity to high ph and decreased potassium efflux. From these results, it was concluded that both genes exhibit different cation specificities and that ENA ATPases can mediate the efflux of potassium (Banuelos & Rodriguez-Navarro, 1998). Besides D. occidentalis, the ENA ATPases have been characterized in several other halotolerant yeast species. Two ENA genes have been identified so far in D. hansenii. DhENA1 was expressed in the presence of high Na 1 concentrations, while the expression of DhENA2 also required high ph. Heterologous expression of the DhENA genes in an S. cerevisiae mutant indicated their function in sodium detoxification and extrusion (Almagro et al., 2001). Similarly, a gene encoding the Ena ATPase from Z. rouxii (ZrENA1) was isolated and characterized (Watanabe et al., 1999, 2002). Remarkably, although the expression of ZrENA1 was observed, it was not upregulated by NaCl stress. However, the protein was efficient at extruding sodium cations, because upon overexpression in a salt-sensitive S. cerevisiae strain, its presence increased NaCl tolerance. Nevertheless, it appears that in Z. rouxii cells, the extrusion of Na 1 might be carried out mainly via the Na 1 /H 1 antiporter. The extremely halotolerant black yeast Hortaea werneckii appears to contain two ATPases, HwEna1 and HwEna2, that are important for maintaining low intracellular Na 1 and K 1 content in this organism (Gorjan & Plemenitas, 2006). Although both genes are responsive to salt, the expression of HwENA1 is higher shortly after salt stress, whereas the expression of HwENA2 appears more prominent in adapted cells. The presence of ENA ATPases has also been investigated in another stress-tolerant fungus, Torulaspora delbrueckii. The isolated TdENA1 gene was able to increase NaCl tolerance when overexpressed in S. cerevisiae (Hernandez-Lopez et al., 2006), and its expression in T. delbrueckii was induced when cells were exposed to NaCl or LiCl. However, in contrast to what is found in S. cerevisiae, this response was not dependent on the presence of TdCrz1, encoding the homologue of the calcineurin-activated transcription factor ScCrz1. The authors postulated that T. delbrueckii and S. cerevisiae differ in the regulatory circuits and mechanisms that drive their adaptive response to salt stress. The genome of the salt-sensitive fission yeast S. pombe encodes a single ENA-related gene, denoted cta3 1. The cta3 1 gene product was initially proposed to work as an ATP-dependent calcium pump and not as a Na 1 -ATPase (Halachmi et al., 1992), but further work demonstrated that Cta3 preferentially mediates the efflux of potassium and not sodium (Benito et al., 2002). It has been shown that the increased cta3 1 expression in response to salt stress (both sodium and potassium) is mediated in S. pombe by the Wis1-Sty1 MAP kinase cascade and the Atf1 transcription factor (Nishikawa et al., 1999) and is also controlled by the transcriptional repressors Tup11 and Tup12 (Greenall et al., 2002). Interestingly, cation stress selectively causes chromatin structure alterations around CRE-like sequences in cta3 1, and this selectivity is lost in a tup11 tup12 doubledeletion mutant, suggesting that these Tup1-like repressors regulate the chromatin structure to ensure the specificity of gene activation (Hirota et al., 2004). As for pathogenic fungi, genes encoding Ena ATPases have been cloned and partially characterized in several Candida species and in Cryptococcus neoformans. It is worth noting that the absence of ENA-type ATPases in animal cells makes this protein a possible antifungal drug target. ENA21 and ENA22 have been identified in both C. albicans and C. dublinensis (Enjalbert et al., 2009). The basal expression of ENA21 was lower in C. dublinensis than in C. albicans and, in contrast to the latter, in which a fivefold induction was observed, the CdENA21 gene was not induced when

6 6 J. Ramos et al. C. dublinensis was exposed to 1 M NaCl. The expression of ENA22 was much lower than that of ENA21 in both species. The introduction of a single copy of CaENA21 into C. dubliniensis was subsequently shown to be sufficient to confer a high salt tolerance. These and others experiments supported the notion that differential ENA21 expression levels in C. dubliniensis and C. albicans contribute to the differing salt tolerances of these pathogens. Recently, the ENA1 gene from C. glabrata was isolated and characterized in comparison with the CgNha1 antiporter (Krauke & Sychrova, 2010). The major role of CgEna1 is the detoxification of sodium and lithium, and it has a very little potassium efflux capacity. A screen for possible candidates for virulence in the human pathogenic fungus C. neoformans identified a mutant strain carrying an insertion in a gene with the structural characteristics of fungal ENA ATPases (Idnurm et al., 2009). Interestingly, ena1 mutant strains were sensitive to alkaline ph conditions, but not to high salt concentrations. The expression of ENA1 was induced by high ph, irrespective of the presence of the calcineurin phosphatase (cna1 mutant), and the sensitivity to high ph of both mutations was additive, suggesting two independent pathways for survival under alkaline conditions. Deletion and complementation experiments confirmed the relevance of ENA1 for virulence in a mouse model. Six genes encoding type II P-type ATPases have been identified in N. crassa (Benito et al., 2000). However, only one of them fully complemented the Na 1 sensitivity of the S. cerevisiae ena mutant. Expression of this gene, termed NcENA1, was upregulated by Na 1 and high ph. Interestingly, in N. crassa, Ena1 seems to be highly specific for sodium transport and does not mediate potassium efflux (Benito et al., 2000; Rodriguez-Navarro & Benito, 2010). NcENA2 was able to only partly suppress the Na 1 sensitivity of an S. cerevisiae mutant (Benito et al., 2009). ENA ATPases have also been characterized in other species, for example plant pathogens Fusarium oxysporum (Caracuel et al., 2003) and U. maydis (Benito et al., 2009; Rodriguez-Navarro & Benito, 2010). The general trait is that at least two ENA genes are present, weakly expressed at low ph and in the absence of high K 1 and Na 1 levels, but are commonly induced at high salt and/or ph conditions. While in some yeasts these proteins are able to extrude both sodium and potassium, in other cases they are rather specific. In general, little is known about the regulation of the expression of ENA genes in yeasts other than S. cerevisiae and even less about the biochemistry of the encoded proteins. Further work will be needed in this direction, particularly if this ATPase is confirmed as a possible antifungal drug target. In general, yeast ENA ATPases and NHA antiporters are highly conserved and used jointly as systems ensuring extrusion of surplus alkali metal cations. Besides sodium, most of these yeast systems evolved the ability to export effectively potassium (together with the yeast TOK channels). On the other hand, potassium influx in yeast cells is mediated by at least three types of systems unevenly spread among the yeast species. The existence of TRK, HAK and ACU transporters in various combinations reflects phylogeny and original niches of the yeast species. Acknowledgements The authors collaborate within the context of TRANSLU- CENT, a SysMo ERA-NET-funded Research Consortium, and wish to express their gratitude to all members of the Consortium for many hours of fruitful and exciting scientific interaction. Work in J.R. s laboratory was supported by grants GEN C2-2-E/SYS, EUI and BFU C03-03 (MICINN, Spain). Work in J.A. s laboratory was supported by grants GEN C2-1- E/SYS, EUI and BFU C03-01 (MI CINN, Spain). J.A. is the recipient of an Ajut de Suport a les Activitats dels Grups de Recerca (Grant 2009SGR-1091) and an ICREA Academia award from the Generalitat de Catalunya. Work in H.S. s laboratory was supported by grants MSMT LC531 and COST OC10012, GA AS CR IAA , GA CR P503/10/0307 and AV0Z References Almagro A, Prista C, Benito B, Loureiro-Dias MC & Ramos J (2001) Cloning and expression of two genes coding for sodium pumps in the salt-tolerant yeast Debaryomyces hansenii. J Bacteriol 183: Andres MT, Viejo-Diaz M & Fierro JF (2008) Human lactoferrin induces apoptosis-like cell death in Candida albicans: critical role of K 1 -channel-mediated K 1 efflux. Antimicrob Agents Ch 52: Arino J, Ramos J & Sychrova H (2010) Alkali metal cation transport and homeostasis in yeasts. Microbiol Mol Biol R 74: Baev D, Rivetta A, Li XS, Vylkova S, Bashi E, Slayman CL & Edgerton M (2003) Killing of Candida albicans by human salivary histatin 5 is modulated, but not determined, by the potassium channel Tok1. Infect Immun 71: Baev D, Rivetta A, Vylkova S, Sun JN, Zeng GF, Slayman CL & Edgerton M (2004) The Trk1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5. J Biol Chem 279: Banuelos MA & Rodriguez-Navarro A (1998) P-type ATPases mediate sodium and potassium effluxes in Schwanniomyces occidentalis. J Biol Chem 273: Banuelos MA, Klein RD, Alexander-Bowman SJ & Rodriguez- Navarro A (1995) A potassium transporter of the yeast Schwanniomyces occidentalis homologous to the Kup system of

7 Potassium and sodium transporters in yeasts 7 Escherichia coli has a high concentrative capacity. EMBO J 14: Banuelos MA, Madrid R & Rodriguez-Navarro A (2000) Individual functions of the HAK and TRK potassium transporters of Schwanniomyces occidentalis. Mol Microbiol 37: Banuelos MA, Ramos J, Calero F, Braun V & Potier S (2002) Cation/H 1 antiporters mediate potassium and sodium fluxes in Pichia sorbitophila. Cloning of the PsNHA1 and PsNHA2 genes and expression in Saccharomyces cerevisiae. Yeast 19: Benito B, Quintero FJ & Rodriguez-Navarro A (1997) Overexpression of the sodium ATPase of Saccharomyces cerevisiae: conditions for phosphorylation from ATP and P i. Biochim Biophys Acta 1328: Benito B, Garciadeblas B & Rodriguez-Navarro A (2000) Molecular cloning of the calcium and sodium ATPases in Neurospora crassa. Mol Microbiol 35: Benito B, Garciadeblas B & Rodriguez-Navarro A (2002) Potassium- or sodium-efflux ATPase, a key enzyme in the evolution of fungi. Microbiology 148: Benito B, Garciadeblas B, Schreier P & Rodriguez-Navarro A (2004) Novel p-type ATPases mediate high-affinity potassium or sodium uptake in fungi. Eukaryot Cell 3: Benito B, Garciadeblas B, Perez-Martin J & Rodriguez-Navarro A (2009) Growth at high ph and sodium and potassium tolerance in media above the cytoplasmic ph depend on ENA ATPases in Ustilago maydis. Eukaryot Cell 8: Calero F, Gomez N, Arino J & Ramos J (2000) Trk1 and Trk2 define the major K 1 transport system in fission yeast. J Bacteriol 182: Caracuel Z, Casanova C, Roncero MI, Di Pietro A & Ramos J (2003) ph response transcription factor PacC controls salt stress tolerance and expression of the P-Type Na 1 -ATPase Ena1 in Fusarium oxysporum. Eukaryot Cell 2: Corratgé-Faillie C, Jabnoune M, Zimmermann S, Very AA, Fizames C & Sentenac H (2010) Potassium and sodium transport in non-animal cells: the Trk/Ktr/HKT transporter family. Cell Mol Life Sci 67: Enjalbert B, Moran GP, Vaughan C, Yeomans T, Maccallum DM, Quinn J, Coleman DC, Brown AJ & Sullivan DJ (2009) Genome-wide gene expression profiling and a forward genetic screen show that differential expression of the sodium ion transporter Ena21 contributes to the differential tolerance of Candida albicans and Candida dubliniensis to osmotic stress. Mol Microbiol 72: Gorjan A & Plemenitas A (2006) Identification and characterization of ENA ATPases HwEna1 and HwEna2 from the halophilic black yeast Hortaea werneckii. FEMS Microbiol Lett 265: Greenall A, Hadcroft AP, Malakasi P, Jones N, Morgan BA, Hoffman CS & Whitehall SK (2002) Role of fission yeast Tup1- like repressors and Prr1 transcription factor in response to salt stress. Mol Biol Cell 13: Halachmi D, Ghislain M & Eilam Y (1992) An intracellular ATPdependent calcium pump within the yeast Schizosaccharomyces pombe, encoded by the gene cta3. Eur J Biochem 207: Haro R, Sainz L, Rubio F & Rodriguez-Navarro A (1999) Cloning of two genes encoding potassium transporters in Neurospora crassa and expression of the corresponding cdnas in Saccharomyces cerevisiae. Mol Microbiol 31: Hernandez-Lopez MJ, Panadero J, Prieto JA & Randez-Gil F (2006) Regulation of salt tolerance by Torulaspora delbrueckii calcineurin target Crz1p. Eukaryot Cell 5: Hirota K, Hasemi T, Yamada T, Mizuno KI, Hoffman CS, Shibata T & Ohta K (2004) Fission yeast global repressors regulate the specificity of chromatin alteration in response to distinct environmental stresses. Nucleic Acids Res 32: Idnurm A, Walton FJ, Floyd A, Reedy JL & Heitman J (2009) Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis. Eukaryot Cell 8: Iwaki T, Higashida Y, Tsuji H, Tamai Y & Watanabe Y (1998) Characterization of a second gene (ZSOD22) ofna 1 /H 1 antiporter from salt-tolerant yeast Zygosaccharomyces rouxii and functional expression of ZSOD2 and ZSOD22 in Saccharomyces cerevisiae. Yeast 14: Jeffries TW, Grigoriev IV, Grimwood J et al. (2007) Genome sequence of the lignocellulose-bioconverting and xylosefermenting yeast Pichia stipitis. Nat Biotechnol 25: Jia ZP, McCullough N, Martel R, Hemminngsens S & Young PG (1992) Gene amplification at a locus encoding a putative Na 1 / H 1 antiporter confers sodium and lithium tolerance in fission yeast. EMBO J 11: Kamauchi S, Mitsui K, Ujike S, Haga M, Nakamura N, Inoue H, Sakajo S, Ueda M, Tanaka A & Kanazawa H (2002) Structurally and functionally conserved domains in the diverse hydrophilic carboxy-terminal halves of various yeast and fungal Na 1 /H 1 antiporters (Nhalp). J Biochem (Tokyo) 131: Kinclova O, Potier S & Sychrova H (2001a) The Candida albicans Na 1 /H 1 antiporter exports potassium and rubidium. FEBS Lett 504: Kinclova O, Potier S & Sychrova H (2001b) The Zygosaccharomyces rouxii strain CBS732 contains only one copy of the HOG1 and the SOD2 genes. J Biotechnol 88: Kinclova O, Potier S & Sychrova H (2002) Difference in substrate specificity divides the yeast alkali metal cation/h 1 antiporters into two subfamilies. Microbiology 148: Kinclova-Zimmermannova O & Sychrova H (2007) Plasmamembrane Cnh1 Na 1 /H 1 antiporter regulates potassium homeostasis in Candida albicans. Microbiology 153:

8 8 J. Ramos et al. Krauke Y & Sychrova H (2008) Functional comparison of plasma-membrane Na 1 /H 1 antiporters from two pathogenic Candida species. BMC Microbiol 8: 80. Krauke Y & Sychrova H (2011) Cnh1 Na 1 /H 1 antiporter and Ena1 Na 1 -ATPase play different roles in cation homeostasis and cell physiology of Candida glabrata. FEMS Yeast Res 11: Madrid R, Gomez MJ, Ramos J & Rodriguez-Navarro A (1998) Ectopic potassium uptake in trk1 trk2 mutants of Saccharomyces cerevisiae correlates with a highly hyperpolarized membrane potential. J Biol Chem 273: Martínez JL, Sychrova H & Ramos J (2011) Monovalent cations regulate expression and activity of the Hak1 potassium transporter in Debaryomyces hansenii. Fungal Genet Biol 48: Miranda M, Saldana C, Ramirez J, Codiz G, Brunner A, Ongay- Larios L, Coria R & Pena A (2002) The KlTrk1 gene encodes a low affinity transporter of the K 1 uptake system in the budding yeast Kluyveromyces lactis. Yeast 19: Miranda M, Bashi E, Vylkova S, Edgerton M, Slayman C & Rivetta A (2009) Conservation and dispersion of sequence and function in fungal TRK potassium transporters: focus on Candida albicans. FEMS Yeast Res 9: Nishikawa T, Aiba H & Mizuno T (1999) The cta31 gene that encodes a cation-transporting P-type ATPase is induced by salt stress under control of the Wis1-Sty1 MAPKK-MAPK cascade in fission yeast. FEBS Lett 455: Papouskova K & Sychrova H (2007) Schizosaccharomyces pombe possesses two plasma membrane alkali metal cation/h antiporters differing in their substrate specificity. FEMS Yeast Res 7: Papouskova M & Sychrova H (2006) Yarrowia lipolytica possesses two plasma membrane alkali metal cation/h 1 antiporters with different functions in cell physiology. FEBS Lett 580: Pribylova L, Papouskova K, Zavrel M, Souciet JL & Sychrova H (2006) Exploration of yeast alkali metal cation/h 1 antiporters: sequence and structure comparison. Folia Microbiol 51: Pribylova L, Papouskova M & Sychrova H (2008) The salt tolerant yeast Zygosaccharomyces rouxii possesses two plasmamembrane Na 1 /H 1 -antiporters (ZrNha1p and ZrSod2-22p) playing different roles in cation homeostasis and cell physiology. Fungal Genet Biol 45: Prista C, Gonzalez-Hernandez JC, Ramos J & Loureiro-Dias MC (2007) Cloning and characterization of two K 1 transporters of Debaryomyces hansenii. Microbiology 153: Rodriguez-Navarro A (2000) Potassium transport in fungi and plants. Biochim Biophys Acta 1469: Rodriguez-Navarro A & Benito B (2010) Sodium or potassium efflux ATPase a fungal, bryophyte, and protozoal ATPase. Biochim Biophys Acta 1798: Soldatenkov VA, Velasco JA, Avila MA, Dritschilo A & Notario V (1995) Isolation and characterization of SpTRK, a gene from Schizosaccharomyces pombe predicted to encode a K 1 transporter protein. Gene 161: Soong TW, Yong TF, Ramanan N & Wang Y (2000) The Candida albicans antiporter gene CNH1 has a role in Na 1 and H 1 transport, salt tolerance, and morphogenesis. Microbiology 146: Velkova K & Sychrova H (2006) The Debaryomyces hansenii NHA1 gene encodes a plasma membrane alkali metal cation antiporter with broad substrate specificity. Gene 369: Watanabe Y, Miwa S & Tamai Y (1995) Characterization of Na 1 /H 1 -antiporter gene closely related to the salttolerance of yeast Zygosaccharomyces rouxii. Yeast 11: Watanabe Y, Iwaki T, Shimono Y, Ichimiya A, Nagaoka Y & Tamai Y (1999) Characterization of the Na 1 -ATPase gene (ZENA1) from the salt-tolerant yeast Zygosaccharomyces rouxii. J Biosci Bioeng 88: Watanabe Y, Shimono Y, Tsuji H & Tamai Y (2002) Role of the glutamic and aspartic residues in Na 1 -ATPase function in the ZrENA1 gene of Zygosaccharomyces rouxii. FEMS Microbiol Lett 209: