Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain1
Author for correspondence: Alonso Rodríguez-Navarro. Tel: +34 91 3365751. Fax: +34 91 3365757. e-mail: arodrignavar{at}bit.etsia.upm.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: cation, pump, Schizosaccharomyces pombe, Leishmania, Trypanosoma
The GenBank accession numbers for the sequences reported in this paper are: Pleurotus ostreatus ENA1, AJ420741; Phycomyces blakesleeanus ENA1, AJ420742; Ph. blakesleeanus PCA1, AJ420743; Blakeslea trispora ENA1, AJ420744; B. trispora BCA1, AJ420745; B. trispora BCA2, AJ420746.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast with Na+ and Ca2+ effluxes, research on K+ efflux has been scarce. This can be explained because K+ is the most abundant cation in the cells, and in most natural environments the external K+ concentration is much lower than the internal concentration. In these conditions, the K+ diffusion potential is not very different from the membrane potential, with the consequence that non-specific loss is insignificant. Exceptions to this rule are the cellular milieu encountered by intracellular parasites and that found by organisms growing on plant debris, whose composition is dominated by the cellular components of plant cells. Furthermore, in the latter case, the effect of drying may increase the concentration of K+ well over the normal cytoplasmic concentration. In these two cases, an effective K+ efflux system is required. The most representative example of intracellular life is the mitochondrion, and also it is the best example of the indispensability of K+ efflux (Nicholls & Ferguson, 1997 ). This is mediated by an electroneutral K+/H+ antiporter (Garlid, 1996
), whose existence was predicted by Mitchell 40 years ago (Mitchell, 1961
).
Fungusplant associations conquered the lands that emerged from the sea some 450 million years ago (Hass et al., 1994 ; Redecker et al., 2000
; Simon et al., 1993
; Taylor et al., 1994
, 1999
; Wilkinson, 2001
). These associations include fungal growth on plant debris and fungalplant symbioses, either mutualistic or parasitic. In mycorrhizal mutualistic associations, the membrane of the fungal cell does not contact the cytoplasm of the plant cell from which it is separated by the interface matrix (Harrison, 1999
), but even in this case the fungal cells may be exposed to high K+ if the plant cells lyse. Therefore, when associated with plants, the fungal cell is circumstantially or permanently exposed to high K+. This suggests that, as in the case of the mitochondrion, fungi need an effective K+-efflux system.
Biochemical and functional analyses of several fungal Na+ ATPases have identified some of these ATPases as bifunctional enzymes for Na+ and K+ (Bañuelos & Rodríguez-Navarro, 1998 ; Benito et al., 1997
; Haro et al., 1991
), suggesting that these ATPases may be the predicted K+ efflux system of fungi. These ATPases form a phylogenetic cluster, named IID (Axelsen & Palmgren, 1998
) or ENA (Benito et al., 2000
), which surprisingly includes the CTA3 ATPase of Schizosaccharomyces pombe, originally described as a Ca2+-ATPase (Ghislain et al., 1990
; Halachmi et al., 1992
). The function of CTA3 as a Ca2+-ATPase and not as a Na+-ATPase was supported by the conservation in CTA3 of several amino acids typical of Ca2+-ATPases and was consistent with the functional absence of a Na+-ATPase in Schiz. pombe, where Na+ tolerance and Na+ efflux is dominated by the Na+/H+ antiporter SOD2 (Jia et al., 1992
; Hahnenberger et al., 1996
). However, with the increasing number of P-ATPases isolated and studied, the phylogenetic isolation of CTA3 from other Ca2+-ATPases also increased, and therefore so did the doubts about the real function of CTA3.
We here report that the Schiz. pombe CTA3 ATPase is a K+-efflux ATPase and that the capacity of ENA ATPases to pump Na+ has evolved recently as an adaptation of fungi to salinity. We also discuss the existence and function of ENA ATPases in the parasites Leishmania and Trypanosoma, which in some cases have to overcome a problem of K+ stress similar to that existing in fungi.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The coding region of the Schiz. pombe cta3 gene was cloned by PCR and inserted into plasmid pDR195. Fragments of the Ph. blakesleeanus and B. trispora ENA genes were amplified from genomic DNA using the degenerate primers described elsewhere (Benito et al., 2000 ). The PCR products were cloned into the PCR2.1-Topo vector using the TOPO TA cloning kit (Invitrogen). Plasmid pDR195 with the cta3 insert is toxic in bacteria and was constructed directly in yeast. The correct sequence of the construct transformed into the yeast strain was checked by sequencing PCR amplifications of two fragments. Sequencing was done with an automated ABI PRISM 377 DNA sequencer (Perkin-Elmer). For Southern blot hybridizations, genomic DNA was digested with restriction enzymes. Then, after electrophoresis and blotting to a nylon membrane, it was hybridized in the presence of 50% formamide. For Northern blots, total RNA was extracted (Carlson & Botstein, 1982
), fractionated through formaldehyde-agarose gels and blotted onto a nylon membrane. Membranes were hybridized with DNA probes that were labelled with [
-32P]dATP by the random priming method (Feinberg & Vogelstein, 1983
).
Cation losses.
Yeast cells with different K+ and Na+ contents were obtained by growing and preincubating the cells in the arginine phosphate medium supplemented with KCl or NaCl as described in each case. To perform the experiments, the cells were suspended in 10 mM Ca2+-MES buffer, pH 6·0, 2% glucose, supplemented with RbCl as recorded in each case. At intervals, the cells were collected on Millipore membrane filters, rapidly washed with 20 mM MgCl2, acid-extracted, and analysed by atomic emission spectrophotometry (Rodríguez-Navarro & Ramos, 1984 ). Flux experiments were repeated three times. The initial values of the cation contents varied slightly among experiments (SD<10% of the mean), but the rates of the losses were almost identical.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To obtain a definitive answer on the function of CTA3, we completed a previous study on the expression of the cta3 transcript (Nishikawa et al., 1999 ), and cloned and expressed the cta3 gene in Sacch. cerevisiae mutants that are sensitive to high K+ or Na+, ena1-4 nha1, or to low Ca2+, pmr1. In the report of Nishikawa et al. (1999)
the expression of cta3 was enhanced by Na+, which was very surprising, because, as we have mentioned, it is very unlikely that CTA3 pumps Na+. A likely possibility was that the Na+ added induced a rapid Na+ uptake and an increase in the cytoplasmic pH. Consistent with this possibility we found that a high external pH and especially the presence of
, which alkalinizes the cytoplasm, strongly enhanced the expression of cta3. We also found that its expression was insensitive to changes in Ca2+ concentration (Fig. 1
). Simultaneously, we found that cta3 suppressed the sensitivity of the ena1-4 nha1 mutant to high K+ (Fig. 2
), but slightly increased its Na+ tolerance, and that it had no effect at all on the sensitivity of the pmr1 mutant to low Ca2+ concentrations (not shown). Consistent with its capacity to increase the K+ tolerance of the ena1-4 nha1 mutant, CTA3 also induced a rapid K+ efflux (Fig. 3
). Taken together, these results demonstrate that CTA3 is a K+-ATPase.
|
|
|
|
In phylogenetic trees, ENA ATPases form a cluster independent from Ca2+-ATPases (Axelsen & Palmgren, 1998 ; Bañuelos & Rodríguez-Navarro, 1998
; Benito et al., 2000
) and ENA sequences can be easily identified by BLAST search in databases using the ScENA1 sequence. We carried out this search in several databases and found that ENA genes existed in many different fungi (Table 2
), to be added to those described in Sacch. cerevisiae (Haro et al., 1991
), Schwanniomyces occidentalis (Bañuelos & Rodríguez-Navarro, 1998
), N. crassa (Benito et al., 2000
), D. hansenii (Almagro et al., 2001
) and Schiz. pombe (present results). Interestingly, the genome sequencing project of N. crassa has produced the sequence of a third ENA ATPase in this species, which we named NcENA3 (Table 2
). The ATPases encoded in the four repeats of the gene tandem in Sacch. cerevisiae, are either identical (ENA2 and ENA3), or almost identical (ENA1, ENA2 and ENA4). Also the pairs of ATPases in Schw. occidentalis, D. hansenii and Candida albicans are highly related. In contrast, the three ENA ATPases in N. crassa are very different (Fig. 5
). The search in databases also identified two more ENA sequences in the parasites Leishmania donovani (GenBank accession no. AF067495) and Trypanosoma brucei (GenBank accession no. AF145723). The Trypanosoma sequence is a shorter sequence that shows 60% identity with the corresponding fragment of the Leishmania sequence. In both cases, the authors reported the sequences as encoding Ca2+-ATPases.
|
|
Using all known fungal Ca2+- and Na+-ATPases, for which we had sequences long enough for a phylogenetic study, along with several plant Ca2+-ATPases, we constructed a phylogenetic tree. Fig. 5(a) shows the tree constructed using sequences that cover almost the whole large cytoplasmic loop connecting the fourth and fifth transmembrane fragments, but the same result is obtained using the complete sequences (Benito et al., 2000
). This tree produced the same clusters as previous studies (Axelsen & Palmgren, 1998
; Benito et al., 2000
), leaving the Leishmania sequence outside the group of Ca2+-ATPases and included in the group of ENA ATPases. However, the Leishmania sequence diverged substantially from the fungal sequences. This was expected from the studies with other proteins (Baldauf & Palmer, 1993
), but made it difficult to predict its phylogenetic position. To further investigate if the Leishmania ATPase belonged to the ENA cluster, we constructed a bootstrap tree using this ATPase, some selected ENA ATPases, fungal and plant Ca2+-ATPases, and the sequence of the PMA1 H+-pump as the outgroup. The branching order supporting the notion that the Leishmania ATPase belongs to the ENA cluster is robust, supported by 97·4% of bootstraps (Fig. 5b
). In the ENA-type fungal sequences, there is a hemiascomycete clade, which is consistent with evolutionary studies of fungi (Auwera & Wachter, 1996
; Berbee & Taylor, 1993
). The two ATPases of the two Zygomycetes Ph. blakesleeanus and B. trispora are also related each other. In contrast, the three ATPases of N. crassa are very divergent among themselves, and also with the hemiascomicete clade.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The first conclusion of our results is that now ENA ATPases can be perceived not as a phylogenetic group of enzymes of variable activity, but as a group of enzymes with a common activity, the mediation of K+ or Na+ efflux. The biochemical behaviour of ScENA1 (Benito et al., 1997 ) and the present results (Table 1
) indicate that many of these ATPases exhibit a low specificity for K+ or Na+, whereas some are more specific for K+ or Na+. All the ENA ATPases studied up to now are plasma membrane enzymes, but further studies are needed to rule out their presence in other membranes. The second conclusion is the universal presence of ENA ATPases in fungi. ENA genes have been identified in 20 species among Basidiomycetes, Ascomycetes and Zygomycetes, and they have been found in all fungi in which their presence has been investigated, even in Na+-sensitive strains.
In contrast to the universal presence of ENA ATPases in fungi, they are absent in plants (Garciadeblas et al., 2001 ). The strong contrast between plants and fungi regarding ENA ATPases can be understood if the original function of these ATPases were K+ extrusion. Life originated in the sea, and it is evident that early living cells needed an efficient Na+ efflux system. This requirement has been permanent for animal cells, because they have been permanently exposed to sea water, or similar fluid, and an ancestor P-ATPase evolved to the present Na+,K+-ATPase. This is not the case for fungi and plants, which left the sea probably during the Precambrian era. They last shared a common ancestor about a billion years ago (Doolittle et al., 1996
; Lee, 1999
), but later evolved together in an oligotrophic medium with a low Na+ content (Retallack & Germán-Heins, 1994
; Retallack, 1997
). Fungi and plants adapted to these conditions using the same H+-pump ATPase (Serrano, 1988
) and similar K+ and Na+ transporters (Rodríguez-Navarro, 2000
), but only plant-associated fungi were exposed, at least circumstantially, to the high K+ concentrations of the plant cells.
In cells with a very negative membrane potential, as is the case for fungal cells (Rodríguez-Navarro et al., 1986 ; Slayman, 1965a
, b
), exposure to a high K+ environment brings about an almost inevitable K+ influx which must be balanced with K+ efflux. In mitochondria, an electroneutral K+/H+ antiporter mediates K+ efflux and volume control (Garlid, 1996
), because the mitochondrion external medium is under a homeostatic control and the mitochondrial matrix pH can be maintained without exception at a pH slightly higher than cytoplasmic pH. Fungal cells are also furnished with a K+/H+ antiporter (Bañuelos et al., 1998
; Camarasa et al., 1996
; Ramírez et al., 1998
; Sychrová et al., 1999
), but its function cannot be permanent, because of the variability of the environmental pH. Therefore, the most reasonable explanation for the existence of the fungal K+-efflux ATPase is the adaptation of a P-type ATPase to mediate K+ efflux when the external pH is high.
Adaptation of plants to saline media occurred recently, when present plant families had already differentiated (Rozema, 1996 ), producing halophytes that always have close glycophyte relatives. The close evolution of plants and fungi makes it possible to predict the same recent adaptation of fungi to Na+. However, unlike plant cells, fungi had a K+-efflux ATPase with a high potential of adaptation for Na+ extrusion. To maintain the K+ efflux capacity of the cells, as well as a better adaptation to Na+, the ENA genes duplicated and generated pumps that were more active with Na+ (Table 1
). So far, the only identified fungus in which this duplication did not occur and which lacks a significant Na+-ATPase activity is Schiz. pombe. Although this fungus might be a singular case because it has a rather independent phylogenetic position among Ascomycetes (Gehrig et al., 1996
; Keogh et al., 1998
), further research may identify more cases.
The ENA gene duplication occurred very recently in the hemiascomycetes yeast Sacch. cerevisiae (97% identity between ScEna1p and ScEna4p), and earlier in C. albicans, Schw. occidentalis and D. hansenii (84, 73 and 68% identity, respectively, between the Ena1p and Ena2p in the same species). The case of Neurospora, with three rather divergent ENA ATPases (4248% identities among NcEna1p, NcEna2p and NcEna3p), may be a more refined model of adaptation to Na+. NcENA1 is rather Na+ specific (Table 1), and if either NcENA2 or NcENA3 is the K+-ATPase homologue to SpCTA3, there is still a third ATPase whose function is not yet clear.
The function of the Leishmania LdCA1 needs to be demonstrated, but it is possible that it fulfils the function of the fungal ENA ATPases, from which its divergence (Fig. 5) can be expected from evolutionary considerations (Baldauf & Palmer, 1993
). The existence of K+-efflux ATPases in Leishmania and Trypanosoma is not surprising. Some of them (not T. brucei), after a lysosomal recruitment, escape from the highly acidic lysosomal vacuole into the cytoplasm, facing a neutral pH and a high K+ content. Assuming that the plasma membrane of the parasitic cells is hyperpolarized (negative inside) (Glaser et al., 1992
), and that the cytoplasm is not at a higher pH than that of the host cells (Heyden & Docampo, 2000
), the existence of K+-efflux ATPases in the plasma membrane can be explained as discussed for fungi.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Auwera, G. V. d. & Wachter, R. D. (1996). Large-subunit rRNA sequence of the chytridiomycete Blastocladiella emersonii and implications for the evolution of zoosporic fungi. J Mol Evol 43, 476-483.[Medline]
Axelsen, K. B. & Palmgren, M. G. (1998). Evolution of substrate specificities in the P-type ATPase superfamily. J Mol Evol 46, 84-101.[Medline]
Baldauf, S. L. & Palmer, J. D. (1993). Animals and fungi are each others closest relatives, congruent evidence from multiple proteins. Proc Natl Acad Sci USA 90, 11558-11562.[Abstract]
Bañuelos, M. A. & Rodríguez-Navarro, A. (1998). P-type ATPases mediate sodium and potassium effluxes in Schwanniomyces occidentalis. J Biol Chem 273, 1640-1646.
Bañuelos, M. A., Quintero, F. J. & Rodríguez-Navarro, A. (1995). Functional expression of the ENA1 (PMR2)-ATPase of Saccharomyces cerevisiae in Schizosaccharomyces pombe. Biochim Biophys Acta 1229, 233-238.[Medline]
Bañuelos, M. A., Synchrová, H., Bleykasten-Grosshans, C., Souciet, J.-L. & Potier, S. (1998). The Nha1 antiporter of Saccharomyces cerevisiae mediates sodium and potassium efflux. Microbiology 144, 2749-2758.[Abstract]
Benito, B., Quintero, F. J. & Rodríguez-Navarro, A. (1997). Overexpression of the sodium ATPase of Saccharomyces cerevisiae. Conditions for phosphorylation from ATP and Pi. Biochim Biophys Acta 1328, 214-225.[Medline]
Benito, B., Garciadeblas, B. & Rodríguez-Navarro, A. (2000). Molecular cloning of the calcium and sodium ATPases in Neurospora crassa. Mol Microbiol 35, 1079-1088.[Medline]
Berbee, M. L. & Taylor, J. W. (1993). Dating the evolutionary radiations of the true fungi. Can J Bot 71, 1114-1127.
Buckel, W. (2001). Sodium ion-translocating decarboxylases. Biochim Biophys Acta 1505, 15-27.[Medline]
Camarasa, C., Prieto, S., Ros, R., Salmon, J. M. & Barre, P. (1996). Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles. Yeast 12, 1301-1313.[Medline]
Carafoli, E. (1994). Biogenesis, plasma membrane calcium ATPase: 15 years of work on the purified enzyme. FASEB J 8, 993-1002.
Carlson, M. & Botstein, D. (1982). Two differentially regulated mRNAs with different 5' ends encode secreted and intracellular forms of yeast invertase. Cell 28, 145-154.[Medline]
Cunningham, K. W. & Fink, G. R. (1994). Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases. J Cell Biol 124, 351-363.[Abstract]
Dimroth, P., Jockel, P. & Schmid, M. (2001). Coupling mechanism of the oxalacetate decarboxylase Na+ pump. Biochim Biophys Acta 1505, 1-14.[Medline]
Doolittle, R. F., Feng, D.-F., Tsang, S., Cho, G. & Little, E. (1996). Determining divergence times of the major kingdoms of living organisms with a protein clock. Science 271, 470-477.[Abstract]
Feinberg, A. P. & Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13.[Medline]
Garciadeblas, B., Benito, B. & Rodríguez-Navarro, A. (2001). Plant cells express several stress calcium ATPases but apparently no sodium ATPase. Plant Soil 235, 181-192.
Garlid, K. D. (1996). Cation transport in mitochondria the potassium cycle. Biochim Biophys Acta 1275, 123-126.[Medline]
Gehrig, H., Schüßler, A. & Kluge, M. (1996). Geosiphon pyriforme, a fungus forming endocytobiosis with Nostoc (cyanobacteria), is an ancestral member of the Glomales, evidence by SSU rRNA analysis. J Mol Evol 43, 71-81.[Medline]
Geisler, M., Axelsen, K. B., Harper, J. F. & Palmgren, M. G. (2000). Molecular aspects of higher plant Ca2+-ATPases. Biochim Biophys Acta 1465, 52-78.[Medline]
Ghislain, M., Goffeau, A., Halachmi, D. & Eilan, Y. (1990). Calcium homeostasis and transport are affected by disruption of cta3, a novel gene encoding Ca2+-ATPase in Schizosaccharomyces pombe. J Biol Chem 265, 18400-18407.
Glaser, T. A., Utz, G. L. & Mukkada, A. J. (1992). The plasma membrane electrical gradient (membrane potential) in Leishmania donovani promastigotes and amastigotes. Mol Biochem Parasitol 51, 9-15.[Medline]
Hahnenberger, M. K., Jia, Z. & Young, P. G. (1996). Functional expression of the Schizosaccharomyces pombe Na+/H+ antiporter gene, sod2, in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 93, 5031-5036.
Halachmi, D., Ghislain, M. & Eilam, Y. (1992). An intracellular ATP-dependent calcium pump within the yeast Schizosaccharomyces pombe, encoded by the gene cta3. Eur J Biochem 207, 1003-1008.[Abstract]
Haro, R., Garciadeblas, B. & Rodríguez-Navarro, A. (1991). A novel P-type ATPase from yeast involved in sodium transport. FEBS Lett 291, 189-191.[Medline]
Harrison, M. J. (1999). Biotrophic interfaces and nutrient transport in plant/fungal symbioses. J Exp Bot 50, 1013-1022.[Abstract]
Hass, H., Taylor, T. N. & Remy, W. (1994). Fungi from the Lower Devonian Rhynie chert, mycoparasitism. Am J Bot 81, 29-37.
Heyden, N. v. d. & Docampo, R. (2000). Intracellular pH in mammalian stages of Trypanosoma cruzi is K+-dependent and regulated by H+-ATPases. Mol Biochem Parasitol 105, 237-251.[Medline]
Jia, Z. P., McCullough, N., Martel, R., Hemminngsens, S. & Young, P. G. (1992). Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast. EMBO J 11, 1631-1640.[Abstract]
Keogh, R. S., Seoighe, C. & Wolf, K. H. (1998). Evolution of gene order and chromosome number in Saccharomyces, Kluyveromyces and related fungi. Yeast 14, 443-457.[Medline]
Lee, M. S. Y. (1999). Molecular clock calibrations and metazoan divergence dates. J Mol Evol 49, 385-391.[Medline]
Mitchell, P. (1961). Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191, 144-148.
Murata, T., Kawano, M., Irgarasi, K., Yamato, I. & Kakinuma, Y. (2001). Catalytic properties of Na+-translocating V-ATPase in Enterococcus hirae. Biochim Biophys Acta 1505, 75-81.[Medline]
Nicholls, D. G. & Ferguson, S. J. (1997). Bioenergetics 2. San Diego: Academic Press.
Nishikawa, T., Aiba, H. & Mizuno, T. (1999). The cta3+ 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, 183-187.[Medline]
Padan, E., Venturi, M., Gerchman, Y. & Dover, N. (2001). Na+/H+ antiporters. Biochim Biophys Acta 1505, 144-157.[Medline]
Ramírez, J., Ramírez, O., Saldaña, C., Coria, R. & Peña, A. (1998). A Saccharomyces cerevisiae mutant lacking a K+/H+ exchanger. J Bacteriol 180, 5860-5865.
Redecker, D., Kodner, R. & Graham, L. E. (2000). Glomalean fungi from the Ordovician. Science 289, 1920-1921.
Rentsch, D., Laloi, M., Rouhara, I., Schmelzer, E., Delrot, S. & Frommer, W. B. (1995). NTr1 encodes a high affinity oligopeptide transporter in Arabidopsis. FEBS Lett 370, 264-268.[Medline]
Retallack, G. J. (1997). Early forest soils and their role in Devonian global change. Science 276, 583-585.
Retallack, G. J. & Germán-Heins, J. (1994). Evidence from paleosols for the geological antiquity of rain forest. Science 265, 499-502.
Rodríguez-Navarro, A. (1971). Inhibition by sodium and lithium in osmophilic yeasts. Antonie Leeuwenhoek 37, 225-231.[Medline]
Rodríguez-Navarro, A. (2000). Potassium transport in fungi and plants. Biochim Biophys Acta 1469, 1-30.[Medline]
Rodríguez-Navarro, A. & Ramos, J. (1984). Dual system for potassium transport in Saccharomyces cerevisiae. J Bacteriol 159, 940-945.[Medline]
Rodríguez-Navarro, A., Blatt, M. R. & Slayman, C. L. (1986). A potassiumproton symport in Neurospora crassa. J Gen Physiol 87, 649-674.[Abstract]
Rozema, J. (1996). Biology of halophytes. In Halophytes and Biosaline Agriculture , pp. 17-30. Edited by R. Choukr-Allah, C. V. Malcolm & A. Hamdy. New York:Marcel Dekker.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual: Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sancho, E. D., Hernández, E. & Rodríguez-Navarro, A. (1986). Presumed sexual isolation in yeast populations during production of sherrylike wine. Appl Environ Microbiol 51, 395-397.
Serrano, R. (1988). Structure and function of proton translocating ATPase in plasma membranes of plants and fungi. Biochim Biophys Acta 947, 1-28.[Medline]
Shi, H., Ishitani, M., Kim, C. & Zhu, J.-K. (2000). The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter. Proc Natl Acad Sci USA 97, 6896-6901.
Simon, L., Bousquet, J., Lévesque, R. C. & Lalonde, M. (1993). Origin and diversification of endomycorrhizal fungi and coincidence with vascular land plants. Nature 363, 67-69.
Slayman, C. L. (1965a). Electrical properties of Neurospora crassa. Effects of external cations on the intracellular potential. J Gen Physiol 49, 69-92.
Slayman, C. L. (1965b). Electrical properties of Neurospora crassa. Respiration and the intracellular potential. J Gen Physiol 49, 93-116.
Stein, W. D. (1995). The sodium pump in the evolution of animal cells. Philos Trans R Soc Lond B 349, 263-269.[Medline]
Steuber, J. (2001). Na+ translocation by bacterial NADH:quinone oxidoreductase: an extension to the complex-I family of primary redox pumps. Biochim Biophys Acta 1505, 45-56.[Medline]
Sychrova, H., Ramírez, J. & Peña, A. (1999). Involvement of Nha1 antiporter in regulation of intracellular pH in Saccharomyces cerevisiae. FEMS Microbiol Lett 171, 167-172.[Medline]
Taylor, T. N., Remy, W. & Hass, H. (1994). Allomyces in the Devonian. Nature 367, 601.
Taylor, T. N., Hass, H. & Kerpt, H. (1999). The oldest fossil ascomycetes. Nature 399, 648.[Medline]
Wilkinson, D. M. (2001). Mycorrhizal evolution. Trends Ecol Evol 16, 64-65.[Medline]
Received 17 October 2001;
revised 29 November 2001;
accepted 7 December 2001.