Department of Membrane Transport, Institute of Physiology CzAcadSci, 14220 Prague 4, Czech Republic1
Laboratory of Microbiology and Genetics, UPRES A-7010-CNRS, Université Louis Pasteur, 67083 Strasbourg, France2
Author for correspondence: Hana Sychrová. Tel: +420 2 475 26 67. Fax: +420 2 475 24 88. e-mail: sychrova{at}biomed.cas.cz
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ABSTRACT |
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Keywords: Na+ efflux, K+ efflux, salt tolerance, transport
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INTRODUCTION |
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The Na+/H+ antiporter responsible for sodium and lithium tolerance of the fission yeast Schizosaccharomyces pombe is encoded by the sod2 gene and is believed to be the sole sodium extrusion system in this yeast (Jia et al., 1992 ). Three genes encoding Na+/H+ antiporters were identified in two osmotolerant Zygosaccharomyces rouxii strains: ZSOD2 and ZSOD22 in ATCC 42981 (Iwaki et al., 1998
; Watanabe et al., 1995
), and ZrSOD2-22 in CBS 732 (Kinclová et al., 2001a
). Although the gene (ZENA1) encoding Na+-ATPase was also identified in Z. rouxii, it was shown that the tolerance of cells to high external concentrations of Na+ and Li+ depends mainly on the Na+/H+ antiporter activity (Watanabe et al., 1999
). On the other hand, in the model yeast Saccharomyces cerevisiae, the most efficient sodium-eliminating system is Na+-ATPase (encoded by the ENA1-4/PMR2AE genes: Haro et al., 1991
; Wieland et al., 1995
), while the Na+/H+ antiporter (encoded by the NHA1 gene: Prior et al., 1996
) plays a minor role in the salt tolerance of Sacch. cerevisiae cells (Bañuelos et al., 1998
). Finally, a homologous gene (CNH1) encoding the Na+/H+ antiporter was isolated from two Candida albicans strains, SC5314 (Soong et al., 2000
) and MEN (Kinclová et al., 2001b
). Deletion of CNH1 leads to retardation of growth and a highly elongated morphology of cells, but it has little effect on the sensitivity of C. albicans cells to high concentrations of sodium and lithium (Soong et al., 2000
).
The Sacch. cerevisiae Nha1p mediates the efflux not only of toxic Na+ and Li+, but also of K+ and Rb+ (Bañuelos et al., 1998 ; Kinclová et al., 2001c
) and its function in cells seems to be more general than elimination of toxic cations. The Nha1p is perhaps involved in the regulation of intracellular pH (Sychrová et al., 1999
) and intracellular potassium concentration (Bañuelos et al., 1998
; Kinclová et al., 2001c
). Recently, we have shown that C. albicans Cnh1p transports, besides toxic sodium and lithium, also potassium and rubidium (Kinclová et al., 2001b
). Both Schiz. pombe sod2p and Z. rouxii ZSod2 antiporters have been already expressed in a Sacch. cerevisiae strain, but their transport activity for alkali metal cations has never been directly measured. The Na+/H+ antiporter activity was estimated only as improved growth of Sacch. cerevisiae transformants (expressing either Schiz. pombe sod2p or Z. rouxii ZSod2 antiporters) in the presence of LiCl or NaCl (Hahnenberger et al., 1996
; Iwaki et al., 1998
). In the case of Schiz. pombe sod2p, its activity in Sacch. cerevisiae cells was observed also as a Na+-dependent proton uptake (Hahnenberger et al., 1996
). Different vectors and promoters were used to express Schiz. pombe and Z. rouxii antiporters in a Sacch. cerevisiae strain that, moreover, contained a functional NHA1 copy (Hahnenberger et al., 1996
; Iwaki et al., 1998
).
In order to compare directly the transport properties of all known yeast Na+/H+ antiporters, we expressed them under the same conditions (vector and promoter) in a Sacch. cerevisiae mutant lacking sodium-extrusion systems, Na+-ATPases and the Na+/H+ antiporter. Cells containing different antiporters were tested for their tolerance to Na+, Li+, K+ and Rb+, and the efflux activity of sod2p and ZrSod2-22p was measured in comparison with that of Nha1p. Our results showed that the expression of Z. rouxii and Schiz. pombe genes improved considerably the sodium and lithium tolerance of Sacch. cerevisiae cells, but neither sod2p nor ZrSod2-22p was involved in the transport of K+ or Rb+. The Z. rouxii antiporter seems to be the most efficient sodium- and lithium-extruding system of all yeast antiporters as its activity maintains the lowest Na+ concentration in cells growing in the presence of NaCl.
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METHODS |
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Plasmids.
Multi-copy plasmids used in this work for expression of genes encoding different yeast Na+/H+ antiporters were as follows: pNHA1-985 harbouring the Sacch. cerevisiae NHA1 gene (Kinclová et al., 2001c ), pZrSOD2-22 containing the ZrSOD2-22 gene from Z. rouxii CBS732 (Kinclová et al., 2001a
) and pCNH1-G23 harbouring the CNH1 gene from C. albicans MEN (Kinclová et al., 2001b
). For cloning the Schiz. pombe sod2 gene (which contains an intron: Jia et al., 1992
), the total RNA was isolated from Schiz. pombe cells (Carlson & Botstein, 1982
) and reverse-transcribed with the First Strand cDNA Synthesis Kit (Pharmacia Biotech). The reverse-transcription products were used for PCR amplification (Peltier Thermal Cycler, PTC-200 MJ Research; Taq DNA polymerase, Boehringer Mannheim) with the antisense-anchor-specific primer (5'-GAAGAATTCGCGGCCGCAGGAA-3': Bañuelos & Rodríguez-Navarro, 1998
) and a sense-sod2-gene-specific primer (5'-CGGGATCCATTACTATGGGCTGGAG-3'). This procedure yielded one DNA fragment (1·5 kb) corresponding to cDNA of the sod2 gene with BamHI and EcoRI restriction sites (underlined in the primer sequences) in the upstream and downstream ends of the open reading frame, respectively. The amplified fragment was first inserted into the pCRII-TOPO vector using the TOPO TA Cloning kit (Invitrogen). Subsequently, the 1·5 kb long BamHIEcoRI fragment was cloned into the multi-copy YEp352 vector (Hill et al., 1986
) behind the NHA1 promoter region (Bañuelos et al., 1998
), resulting in the plasmid pSpsod2.
Genetic and molecular methods; DNA sequencing and sequence analysis.
Standard protocols for nucleic acid manipulations, and yeast and E. coli transformations, were used (Bloch et al., 1992 ; Sambrook et al., 1989
). DNA sequencing was carried out using the Thermo Sequenase radiolabelled terminator cycle sequencing kit (Amersham Life Science). Both strands of the sod2 cDNA fragment were completely sequenced, and Lasergene99 (DNASTAR) was used for DNA and protein sequence analyses.
Salt tolerance determination.
The growth capacity of yeast cells in the presence of NaCl, LiCl, KCl and RbCl was tested as described earlier (Kinclová et al., 2001c ).
Cation contents and loss.
To estimate the intracellular concentration of alkali metal cations, aliquots of cells were withdrawn from the incubation mixture at various time intervals, cells were collected on Synpor membrane filters (0·85 µm pore diameter, Czech Republic), rapidly washed with 20 mM MgCl2, acid-extracted and analysed by atomic absorption spectrophotometry (Haro et al., 1991 ; Rodríguez-Navarro & Ramos, 1984
). The alkali metal cation efflux was estimated as described earlier (Kinclová et al., 2001c
). Lithium uptake measurements were done according to Camacho et al. (1981)
. Cells were grown in YNB media to OD600
0·3, harvested, washed and resuspended in the incubation buffer [20 mM MES containing 0·1 mM MgCl2, 2% glucose and adjusted to pH 5·5 with Ca(OH)2]. At time zero, LiCl was added to the suspension (final concentration 50 mM) and samples of cells were withdrawn at 510 min intervals during 60 min. For estimation of internal cation contents in growing cells, fresh YNB media (40 ml) without or supplemented with the indicated amounts of NaCl were inoculated to OD600
0·02 from cell cultures grown overnight in 20 ml standard YNB. Then growth was assessed by measuring the increase in OD600 of the cell suspension for 16 h. At OD600
0·2, 15 ml of the cultures was harvested, washed and resuspended in 15 ml MES buffer [20 mM MES, 0·1 mM MgCl2 adjusted to pH 5·5 with Ca(OH)2], and two aliquots of 5 ml were used for determination of Na+ and K+ contents. All measurements of cation transport and content were repeated at least three times; the deviation among parallel results was always <5%.
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RESULTS |
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Yeast Na+/H+ antiporters have different substrate specificity
To determine the substrate specificity of Schiz. pombe sod2 and Z. rouxii ZrSod2-22 antiporters, first the growth of S. cerevisiae B31 cells expressing heterologous sod2p and ZrSod2-22p was tested on plates containing increasing amounts of sodium, lithium, potassium and rubidium salts (Fig. 1). Cells transformed with an empty vector (negative control) were very sensitive to all these cations and only the presence of Nha1p (positive control) conferred high potassium and rubidium tolerance (Fig. 1
). Cells with the ZrSod2-22 and sod2 antiporters could not grow on plates supplemented with high concentrations of potassium or rubidium. They tolerated the same amounts of external KCl and RbCl as control cells without any antiporter (800 mM and 500 mM, respectively). These results indicated that neither potassium nor rubidium was a substrate for sod2p or ZrSod2-22p.
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Efflux of Na+, Li+ and K+ mediated by yeast alkali-metal-cation/H+ antiporters
To verify if the different cell tolerances to alkali metal cations observed in drop tests really resulted from the transport activity of Na+/H+ antiporters, the efflux of alkali metal cations from B31 cells harbouring either the heterologous sod2 or ZrSod2-22 antiporters or the Sacch. cerevisiae Nha1p was measured. In the control cells, lacking any antiporter, the internal concentration of all cations tested did not change significantly during the experiment; thus the observed effluxes of cations from cells were mediated exclusively by Na+/H+ antiporters (Fig. 2). In agreement with the results obtained in drop tests, no loss of potassium was observed in cells containing either ZrSod2-22p or sod2p. Only Nha1p mediated potassium efflux from cells (Fig. 2a
).
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Lithium uptake
The high lithium efflux activity of ZrSod2-22 antiporter was also confirmed when lithium uptake was measured. No difference in uptake of Li+ among cells containing empty vector and cells with Nha1p or the sod2p was observed (data not shown), but cells harbouring the ZrSod2-22 antiporter contained considerably lower amounts of Li+ than control cells after only 10 min incubation in the presence of 50 mM LiCl, and this difference increased during the experiment (Fig. 3). The apparently lower rate of lithium uptake was more likely due to the simultaneous ZrSod2-22 antiporter activity.
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The high efflux activity of ZrSod2-22p for sodium was reflected also in the highest concentration of potassium maintained in cells (Fig. 4d). The cells with Cnh1p and sod2p had almost the same internal amounts of sodium and potassium, although these two antiporters differ in substrate specificity. This result suggested that Cnh1p probably discriminated between Na+ and K+ and it transported sodium with higher affinity.
From all these results one can conclude that the functional expression of all four yeast Na+/H+ antiporters in Sacch. cerevisiae influenced the intracellular concentration of sodium in cells (and thus the intracellular K+/Na+ ratio), which in turn affected Na+ tolerance of cells.
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DISCUSSION |
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The Na+/H+ antiporter ZrSod2-22, isolated from the osmotolerant Z. rouxii, seems to be the most effective sodium and lithium antiporter system of the yeast family. It improved considerably the tolerance of Sacch. cerevisiae B31 cells to sodium and especially to lithium at all pH values tested. Sodium and lithium efflux measurements and NaCl growth experiments confirmed its high export capacity. We have already shown that the ZrSod2-22 antiporter could substitute very effectively for the low Na+-ATPase activity as its overexpression improved the salt tolerance of some Sacch. cerevisiae wild-type strains (Kinclová et al., 2001a ). Thus the ZrSod2-22 antiporter could be a good candidate for heterologous expression to improve the salt tolerance of plants.
To conclude, in spite of their high sequence homology, the family of yeast plasma membrane Na+/H+ antiporters can be divided, as regards substrate specificity and probably cell function, into two distinct subfamilies: (1) the subfamily with substrate specificity only for Na+ and Li+ (Schiz. pombe, Z. rouxii antiporters) and with a primary detoxication function in cells, and (2) the subfamily (Sacch. cerevisiae, C. albicans antiporters) mediating transport of all alkali metal cations that, besides elimination of toxic cations, probably have a role in other cell functions (regulation of intracellular K+ concentration, pH and cell volume). The distribution of yeast antiporters into subfamilies does not reflect the level of protein identity, as the closest by sequence comparison, Sacch. cerevisiae Nha1p and Z. rouxii ZrSod2-22p (Table 2), belong by their substrate specificity to different subfamilies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 19 June 2001;
revised 1 November 2001;
accepted 15 November 2001.
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