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1 Centro de Botânica Aplicada à Agricultura, Instituto Superior de Agronomia, TU Lisbon, 1349-017 Lisboa, Portugal
2 Departamento Microbiología, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, E-14071 Córdoba, Spain
Correspondence
Catarina Prista
cprista{at}isa.utl.pt
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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trk2 mutant of Saccharomyces cerevisiae, unable to grow at low K+. This expression resulted in partial recovery of growth and ability to retain K+ at low concentrations. In liquid media, 0.5 M NaCl affected growth of these S. cerevisiae transformants as it does in D. hansenii, resulting in a much less deleterious effect than in wild-type S. cerevisiae. Kinetics of Rb+ uptake in the transformants suggest that DhTRK1p and DhHAK1p code for moderate-affinity K+ transporters exhibiting a sigmoid response against Rb+ concentration and presenting a deviation from classic Michaelis–Menten kinetics at low substrate concentrations. Rb+ uptake by the DhTRK1p transporter was stimulated by millimolar concentrations of Na+ at pH 4.5. The good performance of DhTRK1p in the presence of NaCl may be a key feature in the halotolerance of D. hansenii.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Rodriguez-Navarro, 2000). Because these interactions are not mimicked by Na+ or by any other alkaline cation, K+ has become absolutely necessary for living cells. Furthermore, the majority of eukaryotic cells, with the exception of a few yeasts, algae and halophytes, do not tolerate environments with high Na+ concentration and low water potential, and Na+ is toxic for most of them (Serrano 1996; Garciadeblas et al., 2003).
Although in most cells the intracellular K+ concentrations are quite similar under unstressed conditions, ranging between 100 and 500 mM, the external K+ concentrations to which cells are exposed can vary a lot, ranging from millimolar (in sea water and must) to micromolar in K+-depleted soils. In environments where K+ is scarce, cells need to accumulate K+ against a concentration gradient in order to maintain optimal intracellular concentrations. Several K+ transporters from the TRK-HKT and HAK-Kup families, which allow highly efficient K+ uptake, have been described in eukaryotic walled cells (Rodriguez-Navarro, 2000). Transporters of the TRK-HKT type seem to be present in all fungi and plants studied so far (Rodriguez-Navarro, 2000), and they probably evolved from K+ channels (Durell et al., 1999). This type of transporter is postulated to mediate the symport of two alkaline cations (Haro & Rodriguez-Navarro, 2002). In contrast, transporters of the HAK-Kup type have been identified in all plants but not in all fungi.
In fact, neither in Saccharomyces cerevisiae nor in Schizosaccharomyces pombe has the existence of HAK transporters been reported. In Debaryomyces occidentalis, a HAK-Kup transporter was identified and proposed to function as a K+/H+ symporter (Rodriguez-Navarro, 2000). Recently, a third type of K+ transporter, a K+-ATPase, was identified in the filamentous fungus Ustilago maydis and in the yeasts Pichia sorbitophyla and Candida albicans (Benito et al., 2004). Although both TRK and HAK transport K+ with high affinity (in the case of TRK when cells are grown in low K+), they are competitively inhibited by Na+ in all yeasts and plants tested so far (Haro et al., 1999; Haro & Rodriguez-Navarro, 2002). This inhibition is more evident when the Na+ : K+ ratio is high; this is probably due to a situation of K+-starvation even in the presence of K+ when the concentration of Na+ is high (Gomez et al., 1996).
Debaryomyces hansenii is a yeast that occurs in marine environments and is tolerant to salt and alkaline pH (Norkrans, 1966; Butinar et al., 2005); it can be also isolated from brines, cheese and other salty, low-water-activity environments (Mounier et al., 2005; Cocolin et al., 2006). Over the past 30 years, a few authors have described several features of this yeast related to its extreme halotolerance; these studies have focused on glycerol production and accumulation (Larsson et al., 1990; Lages et al., 1999), cation fluxes (Norkrans & Kylin, 1969; Prista et al., 1997; Gonzalez-Hernandez et al., 2004), and pH (Mortensen et al., 2006) and Na+ targets (Aggarwal et al., 2005). Some of these studies have considered D. hansenii as a halophilic yeast (Gonzalez-Hernandez et al., 2004) and used this organism as a model for the study of salt tolerance mechanisms in eukaryotic walled cells (Prista et al., 2002, 2005). The recent release of the complete sequence and annotation of the D. hansenii genome by the Génolevure consortium (Dujon et al., 2004) revealed that most known pathways involved in salt tolerance in S. cerevisiae are also present in D. hansenii (Prista et al., 2005) and also brought new perspectives to molecular studies in this yeast.
Norkrans & Kylin (1969) reported that higher NaCl concentrations were required to completely inhibit the uptake of potassium in D. hansenii than in S. cerevisiae. More recently, for D. hansenii, our group reported the non-inhibitory effect of Na+ on K+ uptake, when K+ is present at low levels, and the non-toxic effect of Na+ under these conditions, even when it is accumulated in relatively high intracellular concentrations (Prista et al., 1997). So far the molecular bases for these features have not been clearly established. As part of a study aimed at characterizing the major mechanisms involved in the extreme halotolerance of D. hansenii, we decided to clone and characterize the two major K+ uptake systems previously described in yeasts. The experiments described below analyse important aspects concerning the mechanisms of K+ uptake in D. hansenii and characterize the effects of Na+ on DhHAK1- and DhTRK1-mediated K+ transport. We propose that these two K+ uptake systems may play an important physiological role as part of the global strategy evolved in D. hansenii to achieve K+ nutrition while coping with high NaCl concentrations, thereby providing a competitive advantage in many salty environments such as the sea.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. All S. cerevisiae strains used in this work are derivatives of W303-1A (MATa). Type strain D. hansenii PYCC2968 (CBS 767) was used as the source of genomic DNA. S. cerevisiae W3 (W303-1A trk1 : : LEU2 trk2 : : HIS3) was used as recipient strain in complementation experiments with the plasmids listed in Table 1. Escherichia coli DH5
(Hanahan, 1985) was used for routine propagation of the plasmids.
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; Prista et al., 1997) with 2 % (w/v) glucose and distinct KCl and NaCl concentrations was used. These media were supplemented, when required, with the adequate requirements for prototrophic growth (Pronk, 2002). E. coli DH5 was routinely maintained in Luria–Bertani (LB) medium at 37 °C; ampicillin (100 µg ml–1) and X-Gal (4 µg ml–1) were added (Sambrook et al., 1989) when required.
Recombinant DNA techniques.
Plasmid isolation was performed by alkaline extraction as described by Birnboim & Doly (1979), and modified as in Sambrook et al. (1989). For plasmid isolation from yeasts, we used the procedure described by Hoffman & Winston (1987). Agarose gel electrophoresis and restriction site mapping were performed according to standard methods (Sambrook et al., 1989). Yeast genomic DNA from D. hansenii for PCR amplification was isolated as described by Cryer et al. (1975) after a previous treatment with lyticase (5 mg ml–1).
Total RNA was extracted from exponential-phase yeast cells after 5 h incubation under several conditions. Cells were then collected by membrane filtration as described by Belinchon et al. (2004), frozen using liquid nitrogen and kept at –80 °C until RNA extraction was performed. Total RNA was extracted by the hot phenol extraction protocol (Schmitt et al., 1990), modified by Daniela Castro (personal communication) as described below. Frozen cells were resuspended in 470 µl 100 mM sodium acetate (pH 5.0), 5 mM MgCl2 plus 0.1 vol. SDS 10 %, w/v, 5 µl DEPC and 500 µl glass beads, and vortexed for 1 min. After vortexing, cells were subjected to three hot phenol extractions (5 min at 65 °C) with 1 vol. phenol/chloroform/isoamyl alcohol (25 : 24 : 1) pH 5.0 and one extraction with 1 vol. chloroform/isoamyl alcohol at room temperature. RNA precipitation was performed as described by Schmitt et al. (1990). Total RNA was fractionated through formaldehyde-agarose gels and transferred to N+-Hybond membranes (GE Healthcare). Hybridization was performed with digoxigenin (DIG)-labelled probes prepared from internal fragments of DhTRK1 and DhHAK1. Fragments were labelled using the DIG system (Roche) by random priming, according to the manufacturer's instructions. Hybridizations were performed in DIG Easy Hyb (Roche) at 50 °C. Membranes were then washed under high-stringency conditions and exposed to X-ray films for a maximum of 72 h.
Cloning the DhTRK and the DhHAK genes.
Sequence data for D. hansenii were obtained from the Génolevures Consortium Website at http://cbi.labri.u-bordeaux.fr/Genolevures/ by performing TBLASTN search with Trk1p and Hak1p sequences from D. occidentalis against the D. hansenii genomic sequence. Using the BLASTP 2.2.14 program (NCBI) (Altschul et al., 1997), ORFs showing homology to the Trk1p protein of S. cerevisiae and D. occidentalis and to the Hak1p proteins of D. occidentalis and C. albicans were identified. To express both DhTrk1p and DhHak1p from D. hansenii, YEp352 vector was used. Based on the nucleotide sequences of these ORFs together with the contiguous upstream and downstream regions, primers were designed in order to amplify a region from approximately 1000 bp upstream of the ATG start codon to 500 bp after the TAA stop codon for each gene. Forward f1TRK (5'-CCGCTCGAGCGGACATACGGCTGGTCTGTGGAAAAA-3') and f1HAK (5'-CCGCTCGAGCGGACCAAGACCGCAAGAACA-3') primers, modified to incorporate a restriction site for XhoI (underlined), and reverse r1TRK (5'-CGCGGATCCTGTCAAGCTCTTGCAATTTTC-3') and r1HAK (5'-CGCGGATCCGCGCAAGAATGGATTGACCTGAAC-3') primers, modified to incorporate a restriction site for BamHI (underlined), were used to amplify DNA fragments containing full-length ORFs encoding DhTRK1 (f1TRK and r1TRK) and DhHAK1 (f1HAK and r1HAK), using genomic DNA from D. hansenii CBS 767 as a template for PCR. PCR amplification was carried out in an Eppendorf thermocycler with DNA polymerase from BIOTOOLS, for 35 cycles, at 68 °C (annealing temperature chosen according to the primer characteristics). The amplified products were digested with XhoI and BamHI, purified using the ‘GFX PCR DNA and Gel Band Purification’ kit (GE Healthcare) and cloned into YEp352 vector previously digested with BamHI and SalI and purified with the same kit. Cloning was performed according to standard protocols described by Sambrook et al. (1989). Constructs were named as CPpTRK, for plasmids containing the DhTRK1 gene, and CPpHAK, for plasmids containing the DhHAK1 gene (Table 1
). The plasmids were cloned into E. coli DH5, amplified, subjected to extraction and restriction analysis, and finally sequenced using an ABI Prism automated DNA sequencer (Perkin–Elmer). Transformation of S. cerevisiae W3 was performed by the lithium acetate method described by Geitz & Schiestl (1995). Transformants were selected on minimal medium without uracil, leucine and histidine. Fifty-three transformants containing CPpTRK and 159 transformants containing CPpHAK were obtained. One representative clone from each transformant (CPTRK and CPHAK) was used for heterologous expression studies.
Nucleotide sequence and analysis.
DNA and protein sequences for comparative analysis were obtained from BLASTP 2.2.14 (Altschul et al., 1997). Multiple protein alignments were performed by using CLUSTAL W version 1.83 (Thompson et al., 1994) and phylogenetic trees were obtained by using TreeView X (Page, 1996).
Growth assays.
The capability of yeast strains to grow in the presence of low KCl with or without NaCl was assessed on solid YNB or in minimal liquid medium (Rodriguez-Navarro & Ramos, 1984; Prista et al., 1997), supplemented with KCl and NaCl to the desired final concentrations. For solid media assays, transformants were grown for 24 h in 5 ml YNB liquid medium without amino acids and with the required auxotrophic supplements to a final density of approximately 3x107 cells ml–1. Plates were inoculated with 10 µl drops of serial 10-fold dilutions of saturated cultures and incubated at 28 °C. Growth was recorded after 1 and 2 weeks. For liquid media assays, transformants were grown to mid-exponential phase, for 24 h in 5 ml YNB liquid medium with 50 mM KCl without amino acids and with the auxotrophic requirements. After being harvested and washed twice with cold ultrapure water, cells were inoculated in modified arginine phosphate medium (Rodriguez-Navarro & Ramos, 1984) supplemented with KCl and NaCl as indicated, and the pH was adjusted to 5.6. The inoculum was calculated in order to obtain an initial OD640 of 0.1 in the arginine phosphate medium. Cells were grown at 28 °C. Growth rates were determined from the absorbance vs time curves measured at 640 nm (Spectronic 20D, Milton Roy).
K+ content and K+ and Rb+ uptake assays.
K+ content assays were performed essentially as described by Ramos et al. (1990). Cells were grown in minimal medium without uracil, leucine and histidine, with 500 µM KCl, to an OD640 of 0.3. The culture was split into two portions. One portion was immediately collected on Millipore filters (0.45 µm), rapidly washed with 20 mM MgCl2 and treated with HCl for a minimum of 24 h (Rodriguez-Navarro & Ramos, 1984). The other portion was harvested by centrifugation, washed twice with cold ultrapure water, resuspended in 10 mM MES (pH 5.6, adjusted with Ca(OH)2, 2 %, w/v, glucose) to the original OD640 and incubated at 28 °C in an orbital shaker for K+ starvation. After 5 h incubation, cells were collected and treated by the methods described above for K+ non-starved cells.
The intracellular K+ content was analysed by atomic emission spectrophotometry (Rodriguez-Navarro & Ramos, 1984).
For Rb+ uptake, influx assays in K+-starved cells were performed as described previously (Ramos et al., 1994). Cells grown in minimal medium (pH 4.5) with 10 mM KCl to mid-exponential phase were harvested, washed twice with cold ultrapure water and potassium-starved in the same medium without KCl. After a 5 h starvation period, cells were harvested by centrifugation, washed twice with cold ultrapure water, and resuspended at OD640 0.5–0.6 in Tris/citrate buffer (20 mM citric acid adjusted to pH 4.5 with Tris/HCl, 2 %, w/v, glucose) with or without NaCl. At time zero, RbCl was added at the required concentration. Samples were taken periodically and treated as previously described. In short-term experiments, uptake was linear with time and initial uptake rates were obtained from the slope of this line.
Data analysis.
All the experiments were repeated at least twice. The agreement among repetitions was high and typically the standard deviations were lower than 10 % of the mean. In order to study the complex kinetics of Rb+ influx, we used the equation from Garciadeblas et al. (2003):
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) and the second term to the rate through a transporter with two binding sites for Rb+ (Haro & Rodriguez-Navarro, 2002). In this case, the kinetic parameters presented in Table 3 were obtained fitting the experimental data points to the equation by non-linear regression analysis using GraphPad Prism version 4.00 for Mac OS X (Graphpad Software, www.graphpad.com). Several fittings were performed for the whole range and partial ranges of concentrations. Tentative parameters were always obtained by graphic approaches (Eddie–Hofstee or double-reciprocal plots).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The phylogenetic analysis of DhTRK1p and other fungal transporters of the same family revealed that DhTRK1p is most closely related to the DoTRK1p transporter from D. occidentalis and the CaTRK1p transporter from C. albicans, and less related to Schiz. pombe and S. cerevisiae TRK transporters (Fig. 1a).
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; Durell et al., 1999) were found, with the sequence of four M1PM2 segments consisting of an N-terminal outer-membrane helix (M1), a P-loop region (P) and a C-terminal inner transmembrane helix (M2). The glycine residues conserved in the four P fragments, in M2b, and immediately after Pd were also present (Durell & Guy, 1999). We also observed a smaller region located between the third and fourth transmembrane regions, the absence of a highly hydrophobic region located at Q1040 and a reduced number of conserved potential N-linked glycosylation sites (data not shown), which is consistent with the high similarity between DhTRK1p and ScTRK2p (Ko & Gaber, 1991).
Cloning and sequence analysis of the DhHAK1 gene
Using the same approach that was applied for the TRK transporter, we found two almost consecutive ORFs in chromosome E (anti-sense strand) for the HAK-type transporter; these ORFs were separated by 39 bp and were annotated as pseudogenes in the Génolevure database. The complete region containing the two pseudogenes was amplified and sequenced, confirming the existence of the stop codon at the position +1287 of the putative ORF, the same position as described in the Génolevure database (Dujon et al., 2004). The translation of the amplified sequence revealed two in-frame and consecutive sequences, which corresponded to a hypothetical protein of 817 aa, divided by a stop codon located at the position of amino acid 430, and showing a strong homology to DoHAK1p and several other HAK-KUP transporters. Phylogenetic analysis of DhHAK1p (Fig. 1b) showed that the protein clustered within the group of yeast HAK transporters. As expected, the protein had both the 12 transmembrane regions and the conserved regions usually found in HAK transporters (Rodriguez-Navarro, 2000). This was not the case for the conserved amino acid sequence in the first transmembrane domain GXXXGDXGTSPLY, described by Senn et al. (2001) as characteristic of some high-affinity HAK transporters from plants like HvHAK1p.
Expression of DhTRK1 and DhHAK1 transcripts
We performed Northern blot analysis for DhTRK1 and DhHAK1 transcripts in D. hansenii using total RNA prepared from D. hansenii cells incubated for 5 h in minimal medium, under potassium starvation and non-starvation conditions, and in the absence or presence of 0.5 M NaCl. Under K+ starvation conditions, the expression of DhHAK1 was only observed in the absence of NaCl (results not shown). Under the same conditions, we did not observe expression of DhTRK1p. This negative result has been previously reported for Nctrk-1p, ScTRK1p and ScTRK2p (Haro et al., 1999), and DoTRK1p (Banuelos et al., 2000).
Functional characterization of DhTRK1p and DhHAK1p
To verify the K+ transport capability of the proteins encoded by DhTRK1 and DhHAK1 we amplified the chromosomal regions corresponding to DhTRK1 and DhHAK1 together with their own putative promoter and termination regions. The amplified fragments were cloned and used to transform a trk1trk2 S. cerevisiae mutant, W3, lacking the endogenous TRK transporters and incapable of growing under low K+ conditions. Characterization of all transformants harbouring DhTRK1 or DhHAK1 was performed. Two of each category were selected for phenotypic characterization.
DhTRK1p and DhHAK1p improve growth under low K+ conditions.
In comparative growth tests performed in solid minimal medium with different K+ concentrations (pH 4.5, 5.6 and 7.5), all the transformants bearing DhTRK1 or DhHAK1 behaved in a similar way. Fig. 2 shows the results in minimal medium supplemented with 10 µM and 50 mM KCl. Strain CPZERO, harbouring YEp352, was used as a control, showing that the plasmid had no effect on the phenotype of the trk1trk2 strain. The presence of either CPpTRK or CPpHAK led to partial recovery of the ability to grow under low K+ concentrations, which is a characteristic of S. cerevisiae W303 carrying its native K+ transporters.
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). In minimal medium with 500 µM KCl and 0.5 M NaCl, the behaviour of CPTRK and CPHAK was similar to that of D. hansenii. In D. hansenii and in the transformants the specific growth rate decreased by 30 %, while in S. cerevisiae W303, bearing the endogenous K+ transporters, 0.5 M NaCl had a strong inhibitory effect on growth.
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; Rodriguez-Navarro & Rubio, 2006) and in accordance with the data obtained by Northern blot analysis described above.
DhTRK1 and DhHAK1 encode two K+ transporters.
To investigate if DhTRK1 and DhHAK1 code for K+ uptake transporters, we used Rb+ as a K+ analogue to perform uptake assays in CPTRK and CPHAK K+-starved cells. pH 4.5 was chosen for detailed kinetic studies, since it was previously shown that, at this pH, Na+ stimulated Rb+ uptake and K+ accumulation in D. hansenii (Prista et al., 1998).
When we analysed the initial Rb+ uptake rates for CPTRK and CPHAK, the kinetics analysis of Rb+ uptake indicated the existence of two transport components, one corresponding to the ectopic low-affinity uptake presenting estimated kinetic parameters similar to those described for the trk1trk2 S. cerevisiae mutant (Table 3) (Ramos et al., 1994), and the other exhibiting a sigmoid response against Rb+ concentration and presenting a deviation from Michaelis–Menten kinetics which yielded a convex Eadie–Hofstee plot at low Rb+ concentrations (Fig. 3).
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, composed of two terms, a component for the low-affinity system and a quadratic rate equation similar to the one described for ScTRK1 (Haro & Rodriguez-Navarro, 2002) (see Methods). At very low Rb+ concentrations (below 50 µM) it was possible to detect the effect of the constant K2 (usually much lower than K1). The customary Km is really the K1 constant corresponding to the kinetic constant for the second binding site as was proposed by Haro & Rodriguez-Navarro (2002). The kinetic parameters estimated after fitting all the experimental data to the equation are summarized in Table 3. The values of K1 of 1.38 and 0.53 mM Rb+ for DhTRK1p and DhHAK1p, respectively, suggest that these genes encode moderate-affinity Rb+ transporters. As reported for other members of the HAK-Kup family (Banuelos et al., 1995; Haro et al., 1999), the affinity of DhHAK1p was higher than that of DhTRK1p.
Effect of Na+ on Rb+ transport by DhTRK1p and DhHAK1p
Several studies on K+ (Rb+) influx have shown that Na+ is taken up by S. cerevisiae and produces a competitive inhibition effect on K+ (Rb+) uptake (Ramos & Rodriguez-Navarro, 1986), probably leading to a situation of K+ deprivation deleterious to the cells. Previous studies on Rb+ uptake in D. hansenii have shown that Na+ concentrations up to 50 mM do not inhibit Rb+ uptake at pH 4.5 (Prista et al., 1997). The differences in K+ transport performance between D. hansenii and S. cerevisiae, together with the weaker growth inhibition observed for the CPTRK and CPHAK strains (see above), suggested that, if DhTRK1 and DhHAK1 mediate the main pathway of K+(Rb+) uptake in D. hansenii, as seems to be the case, they could show different behaviour in response to Na+, compared to S. cerevisiae. To address this possibility we tested the effect of NaCl on Rb+ influx in CPTRK and CPHAK. Fig. 4 shows the initial Rb+ uptake rates for CPTRK and CPHAK K+-starved cells in the absence and in the presence of 1, 5 and 10 mM NaCl. In CPHAK, increasing NaCl concentrations led to an increasingly higher inhibitory effect, with 10 mM NaCl resulting in a threefold decrease in Rb+ uptake rate. The results obtained indicate that the Ki of Na+ for DhHAK is in the millimolar range, much higher than for Rb+ (results not shown). In contrast, in the transformant bearing the DhTRK1p transporter no inhibition was observed up to 10 mM NaCl, at pH 4.5. At this pH, in CPTRK, we were even able to observe significant stimulation (40 %) at 10 mM NaCl relative to uptake in the absence of NaCl.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Bonetti et al., 1995) and C. albicans (Santos et al., 1993). This mechanism could lead to a non-standard translation process of termination codon readthrough in which the stop codon UGA, often described as a particularly leaky codon (Surguchov, 1988), would be avoided. The fact that CPHAK cells showed a clearly different phenotype with respect to growth, K+ content and Rb+ uptake, as compared with CPZERO, supported the idea that a functional DhHAK1p was being expressed in CPHAK clones, consistent with the hypothesis of an existing non-standard translation process of termination codon readthrough. If the stop codon was effective, this would imply that a truncated protein lacking 47 % of the C-terminal amino acid sequence (the last four transmembrane regions and the hydrophilic C terminus) was functional. This is very improbable since in the case of HAK-Kup transporters it has been shown that the hydrophilic C terminus has an essential role in HAK-Kup transporter functionality (Schleyer & Bakker, 1993).
Several studies have shown that high-affinity K+ uptake systems are induced upon K+ starvation and internal K+ depletion in yeast and plant cells (Haro et al., 1999; Martinez-Cordero et al., 2004). A difficult point to explain in our results is the observation that DhHAK was not detectably expressed in K+-starved cells when NaCl was present. It is conceivable that, rather than a low extracellular K+ concentration, intracellular K+ depletion is required for HAK expression. The presence of NaCl would activate DhTRKp. In this case starvation would be less efficient in promoting internal K+ depletion, and for this reason the expression of DhHAK would not be detectable.
In growth assays performed at 0.5 mM KCl, a concentration 100 times lower than that reported for optimal growth of the trk1trk2 strain (Gaber et al., 1988), the presence of 0.5 M NaCl affected growth parameters of CPTRK and CPHAK in a similar way to that observed for D. hansenii growth (Table 2), and in a way much less dramatic than for wild-type S. cerevisiae. In D. hansenii, even though a lower capacity of the K+ transport systems was observed, it was proposed that a higher Na+ intracellular content could help cells to overcome the lack of K+ when it is scarce (Gonzalez-Hernandez et al., 2004). This cannot be the case for the S. cerevisiae transformant strains, since Na+ should still be toxic when accumulated inside these cells.
In the transformants bearing D. hansenii K+ transporters, the growth phenotype of wild-type S. cerevisiae was not completely recovered (Fig. 2). This result can be explained by the lower K+ (Rb+) affinity in the transformant strains (Table 3) as compared to S. cerevisiae (Ramos et al., 1994). This may lead to a decrease in growth rate due to a lower intracellular K+ content, a consequence of the reduced ability to capture K+ from a medium where the availability of this cation is limited. The lower K+ (Rb+) affinity exhibited by DhTRK1p and DhHAK1p compared with the affinities described for the same type of transporters in other yeasts (Rodriguez-Navarro, 2000) is in accordance with the lower affinity for Rb+ observed in D. hansenii (Prista et al., 1997).
Both DhTRK1p and DhHAK1p showed similar Michaelis–Menten-deviated kinetic behaviours. For DhTRK1p, this type of deviation is in accordance with previously described kinetics for other known TRK-HKT transporters (Garciadeblas et al., 2003; Haro & Rodriguez-Navarro, 2003) and can be attributed to the existence of two Rb+ (K+) binding sites with different affinities, the first of them being occupied by the residual K+ (up to 1.5 µM) resulting from cell content loss in K+-free buffer, as suggested by Haro & Rodriguez-Navarro (2002). In the case of DhHAK1p, this kind of deviation from Michaelis–Menten kinetics has never been described for HAK transporters. Nevertheless, it agrees with the idea that some HAK transporters may be, in some conditions, K+/H+ symporters as was described for plant HAK transporters (Santa-Maria et al., 1997; Senn et al., 2001). At pH 4.5, the H+ concentration is sufficiently high to interfere with the binding of Rb+ to the first site especially when Rb+ is low, as was described for K+ transporters in the case of K+ and Rb+ (Borst-Pauwels, 1981; Haro & Rodriguez-Navarro, 2002). The fact that Rb+-uptake assays to estimate kinetic parameters are usually performed at pH 6.0, a proton concentration significantly lower than we used (1 µM H+ vs 32 µM H+ in our case) may explain the differences in the results. This inhibition is relieved upon increasing the Rb+ concentration.
The initial Rb+ influx rates obtained at pH 4.5 with increasing NaCl concentrations (Fig. 4) show that NaCl has different effects on each transporter. The effect of NaCl on the DhTRK1p-mediated Rb+ uptake at pH 4.5 was significantly different from that usually observed for transporters of this type such as ScTRK1p and trk-1 from Neurospora crassa. For ScTRK1p, 2 mM NaCl inhibited Rb+ influx by 35 % at the same Rb+ concentration (Haro & Rodriguez-Navarro, 2002) and for Nctrk-1 we estimated that 10 mM NaCl inhibited Rb+ influx by 30 % (Haro et al., 1999). Although these results were obtained for a different pH, the known independence of pH of ScTRK1 K+ influx (Rodriguez-Navarro & Ramos, 1984), together with consistent results presented by Haro & Rodriguez-Navarro (2002), led us to conclude that the effect of NaCl on K+ transport is quite peculiar in D. hansenii.
Taken together, these results suggest that DhTRK1p and DhHAK1p are the main K+ transporters in D. hansenii. They may have evolved to become rather low-affinity transporters, since potassium is relatively abundant in the marine habitats of this yeast. Potassium is an essential cation in all living systems. Since it is involved in several crucial cellular processes such as protein synthesis and the control of membrane potential and intracellular pH, cells need to maintain minimum intracellular levels of this cation in order to survive. Since NaCl is a competitive inhibitor of K+ uptake, under high NaCl concentrations most yeasts have problems in maintaining intracellular K+ content, a deleterious situation to the cell. Functional and robust K+ transport systems with the remarkable characteristic of not being, or at least being less, inhibited by NaCl may be critical features to guarantee survival and growth of D. hansenii in salty environments.
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ACKNOWLEDGEMENTS |
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Edited by: D. J. Jamieson
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Received 16 January 2007;
revised 5 May 2007;
accepted 30 May 2007.
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) and presence of 1 mM (
), 5 mM (
) and 10 mM (
) NaCl. Assays were performed in Tris/citrate buffer pH 4.5, as described in Methods.