Correspondence: Hana Sychrová, Department of Membrane Transport, Institute of Physiology AS CR, Videnska 1083, 142 20 Prague 4, Czech Republic. Tel.: +420 241 062 667; fax: +420 241 062 488; e-mail: email@example.com
Saccharomyces cerevisiae extrudes K+ cations even when potassium is only present in scarce amounts in the environment. Lost potassium is taken up by the Trk1 and Trk2 uptake systems. If the Trk transporters are absent or nonfunctional, the efflux of potassium is significantly diminished. A series of experiments with strains lacking various combinations of potassium efflux and uptake systems revealed that all three potassium-exporting systems the Nha1 antiporter, Ena ATPase and Tok1 channel contribute to potassium homeostasis and are active upon potassium limitation in wild-type cells. In trk1Δ trk2Δ mutants, the potassium efflux via potassium exporters Nha1 and Ena1 is diminished and can be restored either by the expression of TRK1 or deletion of TOK1. In both cases, the relative hyperpolarization of trk1Δ trk2Δ cells is decreased. Thus, it is the plasma-membrane potential which serves as the common mechanism regulating the activity of K+ exporting systems. There is a continuous uptake and efflux of potassium in yeast cells to regulate their membrane potential and thereby other physiological parameters, and the cells are able to quickly and efficiently compensate for a malfunction of potassium transport in one direction by diminishing the transport in the other direction.
The maintenance of intracellular alkali-metal-cation homeostasis is a complex process which is important for the survival of all organisms. Yeast cells usually spend more energy to accumulate and maintain the high intracellular concentration of potassium that is required for many physiological processes (e.g. protein synthesis, enzyme activation, cell volume, membrane potential and intracellular pH regulation) (Rodriguez-Navarro, 2000), and to maintain low cytosolic concentrations of toxic cations such as sodium or lithium (Arino et al., 2010). The tolerance of yeast cells to high external concentrations of alkali-metal-cation salts is determined by several factors, the most important being the presence and activity of plasma-membrane and organellar cation transporters. Yeast species vary significantly in their ability to grow in either the presence of high concentration of salts or in external potassium-limiting conditions. This difference is based on the presence/absence of distinct transporters and on the specific ways they are regulated [for a review, see (Arino et al., 2010; Ramos et al., 2011)]. Significant differences in salt sensitivity/tolerance and cation homeostasis were not only found among different yeast species but were even observed between two laboratory strains of Saccharomyces cerevisiae [BY4741 and W303 (Petrezselyova et al., 2010)].
In the S. cerevisiae plasma membrane, two transport systems are employed to accumulate the necessary amount of potassium and three transporters serve to eliminate surplus intracellular alkali metal cations and maintain the optimum concentration of potassium. The uptake and accumulation of K+ is mediated by the Trk1 (Gaber et al., 1988) and Trk2 (Ko & Gaber, 1991; Ramos et al., 1994) uniporters whose activity is driven by the membrane potential generated by the Pma1 H+-ATPase (Arino et al., 2010). The TRK1 gene encodes the main potassium uptake system, a high-affinity transporter whose activity influences K+ and pH homeostases, cell turgor and plasma-membrane potential [for a review, see (Arino et al., 2010)]. The transporter encoded by the TRK2 gene is also involved in K+ accumulation and in the regulation of membrane potential; however, its expression level is rather low and the effect of its absence is much less pronounced (Navarrete et al., 2010; Petrezselyova et al., 2011). The presence of the TRK1 and TRK2 genes enables S. cerevisiae cells to grow even in very limited (micromolar) concentrations of potassium, and their absence (trk1Δ trk2Δ strains) strongly reduces the ability of cells to grow if the external potassium concentration is not at least 50 mM (Navarrete et al., 2010).
Three systems differing in their transport mechanisms and activity regulation mechanism were found to mediate the export of K+ from S. cerevisiae cells. Tok1 is a voltage-gated outward rectifying K+-specific channel (Gustin et al., 1986) that opens upon plasma-membrane depolarization (Bertl et al., 2003) and serves for fine tuning plasma-membrane potential (Bertl et al., 2003; Maresova et al., 2006). The other two K+ exporters are active, have broader substrate specificity and serve to export surplus potassium and eliminate toxic sodium and lithium cations from the cytosol. The Nha1 Na+(K+)/H+ antiporter (Prior et al., 1996) is a constitutively expressed housekeeping protein that uses the inward gradient of H+ (created by the Pma1 H+-ATPase) as a driving force to export Na+, K+, Li+ and Rb+ (Banuelos et al., 1998; Kinclova et al., 2001). The activity of Nha1 also plays a role in the maintenance of plasma-membrane potential and regulation of cell volume and internal pH (Sychrova et al., 1999; Kinclova-Zimmermannova et al., 2006; Arino et al., 2010). The Tok1 and Nha1 proteins are expressed at rather low levels and their activity is regulated at the post-translational level, for example by Hog1-mediated phosphorylation upon osmotic stress (Proft & Struhl, 2004).
On the other hand, the third system exporting alkali metal cations, Ena Na+(K+)-ATPase (Haro et al., 1991), is mainly regulated at the expression level. It is strongly induced by osmotic stress or alkaline external pH (Ruiz & Arino, 2007; Arino et al., 2010). Ena ATPase is the main sodium and lithium detoxifying system in S. cerevisiae, but it also contributes significantly to high potassium tolerance (Banuelos et al., 1998). The number of ENA gene copies varies among the different S. cerevisiae strains (e.g. ENA1, ENA2 and ENA5 in BY4741), but the dominant role in Na+ and K+ export is played by ENA1 (Wieland et al., 1995; Arino et al., 2010).
Surprisingly, S. cerevisiae cells can survive without the five plasma-membrane potassium transporters. The BY4741-derived quintuple mutant strain (BYT12345, trk1∆ trk2∆ tok1∆ nha1∆ ena1-5∆) is viable and grows well if it is supplemented with an appropriate (not too low and not too high) external potassium concentration (Navarrete et al., 2010). In fact, these cells survive potassium-limiting concentrations better than double mutants lacking only the Trk1 and Trk2 K+ uptake systems (Navarrete et al., 2010). A detailed study of the phenotypes and physiological parameters of wild-type BY4741 and Trk-deficient strains showed several surprising results, not only under potassium-limiting, but also under potassium-sufficient conditions. Even in the presence of sufficient amounts of potassium, the deletion of TRK1 and TRK2 resulted in a significant plasma-membrane hyperpolarization, a decrease in intracellular pH and in a diminished ability to acidify external media. Furthermore, trk1∆ trk2∆ cells were more sensitive to high concentrations of NaCl, LiCl and cationic drugs such as spermine, hygromycin B and tetramethylammonium (Navarrete et al., 2010). Additional phenotypes of trk1∆ trk2∆ mutants were observed during K+ starvation. In media with micromolar potassium concentrations, trk1∆ trk2∆ cells did not divide and their relative hyperpolarization became more evident. Interestingly, at nonlimiting concentrations of KCl (above 50 mM), the wild-type and trk1∆ trk2∆ strains had a comparable cell size and potassium content, whereas upon transfer to potassium-limiting conditions, the wild-type cells released more potassium and diminished their size to a higher extent than the trk1∆ trk2∆ cells (Navarrete et al., 2010). This data indicated that a net potassium efflux occurs during potassium starvation in wild-type cells but not in cells lacking the TRK1 and TRK2 genes.
In this study, we examined the contribution of individual K+ export systems (Tok1, Nha1, Ena1) to the observed K+ loss during potassium starvation of wild-type cells, and we analysed the physiological basis of their relative inactivity in the trk1∆ trk2∆ strain.
Materials and methods
Strains, media and plasmids
The S. cerevisiae BY4741 (MATa his3∆1 leu2∆ met15∆ ura3∆; EUROSCARF) strain and its derivatives lacking one or various combinations of the TRK1, TRK2, TOK1, NHA1 and ENA1-5 genes were used in this study. All mutant strains were prepared by homologous recombination using the Cre-loxP system (Gueldener et al., 2002) and their genotypes are listed in Table 1.
Yeast strains were grown either in standard YPD, in standard YNB or in K+-free YNB-F [0.175% YNB-F w/o amino acids, ammonium sulphate and potassium (Formedium) adjusted to pH 5.8 and supplemented with 0.4% ammonium sulphate and various concentrations of KCl (Navarrete et al., 2010)] at 30 °C. All media contained 2% glucose, and both YNB and YNB-F were supplemented either with Brand Supplement Mix (BSM) or BSM w/o uracil. Solid media were supplemented with 2% agar.
YEp352-based plasmid pTRK1 was used for TRK1 expression under the control of its own promoter (≈ 500 bp). It was constructed and kindly provided by S. Petrezselyova.
Potassium tolerance tests
To compare the growth of yeast cells at various concentrations of KCl, drop tests on solid YNB-F media were performed according to (Krauke & Sychrova, 2011). Growth was tested either at limited potassium concentrations (approximately 15 μM to 50 mM), at standard potassium concentration (200 mM) or in the presence of high KCl concentrations (up to 2.0 M KCl). Plates were incubated at 30°°C and the growth of cells was recorded for 3–5 days.
Estimation of K+ content changes upon potassium limitation
Cells were grown overnight in YNB-F + 200 mM KCl to OD600 ≈ 0.5, washed, resuspended (t = 0 min) in K+-free YNB-F and incubated for 60 min. Samples were withdrawn at t = 0, 30 and 60 min, washed, resuspended in the incubation buffer [10 mM Tris, 0.1 mM MgCl2, pH adjusted to 4.4 with citric acid and then increased to 4.5 with Ca(OH)2] and rapidly collected by filtration using Millipore filters. Cells on filters were washed immediately with 20 mM MgCl2, acid-extracted and the intracellular K+ concentration was estimated by atomic absorbtion spectrophotometry (Kinclova et al., 2001). Samples were taken in triplicate for each time point and the average results of three independent experiments are presented.
Measurement of K+ efflux rate
Cells grown overnight in YNB-F + 200 mM KCl (OD600 ≈ 0.5) were harvested, washed, resuspended (t = 0 min) in the incubation buffer [10 mM Tris, 0.1 mM MgCl2, 10 mM RbCl, 2% glucose, pH adjusted to 4.4 with citric acid and then increased to 4.5 with Ca(OH)2] and incubated for 60 min. Samples were withdrawn at 1, 5, 10, 25, 30, 35, 45 and 60 min, collected by filtration using Millipore filters and processed as described above. The experiment was repeated three times and a representative result is shown.
Estimation of relative plasma-membrane potential
Relative changes in plasma-membrane potential (∆ψ) were measured using the fluorescent dye diS-C3(3) (3,3′-dipropylthiacarbocyanide iodide; 0.1 mM stock solution in ethanol). Cells were grown overnight in YNB-F + 200 mM KCl to OD600 ≈ 0.5, washed, resuspended (t = 0 min) in K+-free YNB-F and incubated for 60 min. Cells were harvested, washed twice with 10 mM MES (pH 6.0, adjusted with triethanolamin), resuspended in the same buffer to a final OD600 of 0.1 and the potentiometric probe was added to a final concentration of 0.2 μM. The ratio of 560/580 nm emission intensities was immediately measured with an ISS PC1 spectrofluorometer (excitation: 531 nm) and processed as described previously (Maresova et al., 2009; Navarrete et al., 2010).
Estimation of cell size
Cell diameter was estimated for cells growing in YNB-F + 200 mM KCl and for cells incubated in K+-free YNB-F for 60 min. A cell counter (CASY™ model TT; Innovatis) with a 60 μm capillary was used. The experiment was repeated twice, each time 2 × 104 cells were analysed for each strain and each set of conditions. Intervals containing most typical 60% of the cell population were visualized using a box plot diagram with the mean diameter from the observed interval (3–9 μm) inside the box.
Results and discussion
K+ exporters are not functional in a trk1∆ trk2∆ strain in potassium-limiting conditions
When S. cerevisiae cells were grown in standard YNB or YPD media (containing approximately 20 mM K+), their intracellular potassium concentration was approximately 600 nmol per mg dry wt, that is roughly 0.3 M, depending on the strain and media pH (data not shown). When the growth medium contained a higher potassium level, for example as in our standardly used YNB-F supplemented with 200 millimoles KCl/l, the intracellular concentration of potassium in cells having all plasma-membrane potassium transporters was around 650 nmol per mg dry wt, and those of cells lacking all five K+ transporters (BYT12345; trk1∆ trk2∆ tok1∆ nha1∆ ena1-5∆) was slightly higher (700 nmol per mg dry wt; Fig. 1). The small increase in potassium content (approximately 10%) when the cells grew in the presence of tenfold higher concentration of KCl (20 vs. 200 mM) suggested a very efficient regulation of potassium influx and efflux. If the cells were grown in the presence of 50 or 200 mM KCl and subsequently incubated in YNB-F without the addition of KCl (i.e. in the presence of only 15 μM K+), their intracellular concentration of potassium decreased [Fig. 1 and (Navarrete et al., 2010)]. Surprisingly, the decrease in intracellular potassium content was more pronounced in the wild-type cells (BY4741; about 50% of intracellular K+ lost in 60 min) than in cells without potassium uptake systems (BYT12, trk1∆ trk2∆; 15% lost in 60 min). This result suggested that the potassium efflux via export system(s) was prominent in the BY4741 wild type but not in the mutant lacking the Trk1 and Trk2 uptake systems. Comparison of the data obtained for the BYT12 (trk1∆ trk2∆) and BYT12345 (trk1∆ trk2∆ tok1∆ nha1∆ ena1-5∆) cells showed the same level of K+ loss and confirmed the hypothesis that the three systems exporting potassium are not functional in BYT12 (trk1∆ trk2∆) cells upon potassium starvation (Fig. 1). This observation led to two main questions: Which of the three potassium exporters was responsible for the potassium efflux in BY4741 cells, and what mechanism was responsible for the reduced potassium efflux in trk1∆ trk2∆ cells?
All three potassium-exporting systems participate in K+ loss observed under potassium-limiting conditions
To elucidate which of the three K+ export systems is responsible for the observed efflux of K+ from BY4741 cells during potassium starvation, the changes in intracellular K+ content were followed in a series of mutants lacking one or two or all three K+ exporter genes (TOK1, NHA1 and ENA) (Fig. 2). Surprisingly, values obtained for single mutants were similar to those obtained for the wild-type BY4741 cells. Within one hour under potassium-limiting conditions, the intracellular content of potassium in these strains dropped from the initial concentration of about 700 nmol K+ per mg dry weight to approximately 350 nmol K+ per mg dry weight. A similar loss of K+ in all single deletion mutants and the wild type indicated that the remaining two exporters were able to substitute for the activity of the missing exporter. The initial K+ concentration in BYT45 strain, lacking both active export systems (nha1∆ ena1-5∆), was slightly higher than in the wild type, but the loss of K+ during starvation was strongly reduced (Fig. 2), which corresponded to the stable intracellular potassium concentration observed when the rate of potassium efflux from cells was measured (cf. Fig. ;5a; cells BYT45 [YEp352]). This result suggested that the Nha1 and Ena systems are the two main systems involved in potassium efflux. The observed significant role of Ena ATPase was surprising, as under our experimental conditions (pH 5.8, 200 mM KCl) the expression of Ena1 should not be very high (Ruiz & Arino, 2007; Arino et al., 2010).
The role of the Tok1 channel became evident when potassium content and efflux were measured in the BYT345 (tok1∆ nha1∆ ena1-5∆) strain. The potassium concentration in BYT345 grown in YNB-F + 200 mM KCl was significantly higher (850 nmol K+ per mg dry weight; Fig. 2) than in BYT45 (nha1∆ ena1-5∆) or the wild type (BY4741), but the loss of K+ from BYT345 cells was slightly smaller than from BYT45 cells (approximately 9.1 ± 1.7% vs. 12.2 ± 2.2% of internal K+ lost in 60 min; Fig. 2). Our results clearly showed that the activity of the Tok1 channel significantly influences the potassium content in cells grown at standard potassium levels and that its contribution to potassium export in potassium-limiting conditions is lower than those of the active exporters Nha1 and Ena1.
Deletion of TOK1 in BYT12 (trk1∆ trk2∆) strain results in hypersensitivity to low K+ concentrations
To study the contribution of the activity of K+ efflux system to cell growth under potassium-limiting conditions, we used strains derived from BYT12 (trk1∆ trk2∆) bearing additional deletions of TOK1 (BYT123), NHA1 (BYT124), ENA1-5 (BYT125) or the double deletion of NHA1 and ENA1-5 (BYT1245). First, the growth of these strains was characterized and compared with the wild type (BY4741) and BYT12345 (trk1∆ trk2∆ tok1∆ nha1∆ ena1∆) in either high or limiting concentrations of KCl (Fig. 3). Similar growth of all tested strains was only observed on plates containing 0.2 M KCl, that is under conditions closest to the optimal intracellular concentration of potassium. The tested strains also grew similarly well on 50 mM KCl, with the exception of BYT123 (trk1∆ trk2∆ tok1∆), which grew slightly slower (Fig. 3). As expected, the growth of strains lacking more than one potassium exporter (BYT1245 and BYT12345) was inhibited at high external potassium concentrations. Surprisingly, the inability of the BYT124 strain (trk1∆ trk2∆ nha1∆) to grow on high KCl (e.g. 2 M, Fig. 3) indicated a major role of Nha1p in eliminating surplus K+. Although the ENA1 gene is highly expressed under these high-osmolarity conditions (Ruiz & Arino, 2007; Arino et al., 2010), it is the antiporter and not the ATPase that is crucial for maintaining the optimum intracellular concentration of potassium.
The growth of all tested strains lacking TRK1 and TRK2 genes was strongly reduced under limiting potassium conditions. No significant difference in growth was observed between BYT12 (trk1∆ trk2∆), BYT124 (trk1∆ trk2∆ nha1∆), BYT125 (trk1∆ trk2∆ ena1-5∆) and BYT1245 (trk1∆ trk2∆ nha1∆ ena1-5∆) in 20 and 30 mM KCl. In agreement with previously published results (Navarrete et al., 2010), we observed a slightly increased growth ability of strains lacking the three K+ exporters (BYT12345) at limiting external potassium concentrations (Fig. 3). This advantage is most probably due to the fact that these cells do not lose internal K+ during cell growth and thus need less time to accumulate the necessary amount of potassium to establish cell turgor and accomplish cell division. The phenotype of reduced growth at suboptimal potassium concentrations observed for BYT123 at 50 mM KCl became even more evident at lower KCl concentrations (Fig. 3). This observation suggested that the efflux activity of the Nha1 antiporter and Ena ATPases was restored in the strain lacking Trk uptake systems and the Tok1 channel. As the absence of Trk systems leads to a hyperpolarization (Madrid et al., 1998; Navarrete et al., 2010) and deletion of TOK1 to a depolarization (Maresova et al., 2006) of the plasma membrane, we hypothesized that the observed phenotype of TOK1 deletion (reduced growth of cells with tok1∆ in the trk1∆ trk2∆ background at low KCl) might result from a plasma-membrane depolarization.
Efflux of K+ during potassium starvation is regulated by membrane potential
To verify this hypothesis, we estimated three basic physiological parameters (potassium content, relative membrane potential and cell size) of BYT123 (trk1∆ trk2∆ tok1∆) cells under standard (i.e. in YNB-F with 200 mM KCl) or limiting (60 min in YNB-F without added KCl) potassium conditions and compared them with those of the BY4741, BYT12 (trk1∆ trk2∆) and BYT12345 (trk1∆ trk2∆ tok1∆ nha1∆ ena1∆) strains. While almost 90% of intracellular K+ remained in BYT12 cells after 60 min in YNB-F (similarly as in BYT12345, Fig. 4a), 25% of K+ was exported from BYT123 and 50% from the wild type (Fig. 4a). The results showed that deletion of the TOK1 gene restored the efflux of potassium from cells lacking Trk1 and Trk2 systems and this efflux depended on the presence of the Nha1 and Ena1-5 efflux systems (Fig. 4a). The deletion of TOK1 also counteracted the relative hyperpolarization of BYT12 cells (Fig. 4b) and resulted in a ‘wild-type' size of BYT123 cells (Fig. 4c). During 60 min of K+ starvation, the mean diameter of cells dropped by about 11%, similar to wild-type and BYT123 cells, but the mean diameter of BYT12 and BYT12345 decreased only slightly (approximately 5%; Fig. 4c). From all these results, we concluded that the phenotypes observed upon deletion of TOK1 in the BYT12 (trk1∆ trk2∆) background resulted from the changed membrane potential (relative depolarization of BYT123 compared to BYT12) and that it was the relative hyperpolarization that affected the efflux of potassium via exporters when cells were exposed to low potassium concentrations and simultaneously could not exploit the high-affinity potassium uptake systems. Due to this hyperpolarization, cells contained more potassium cations and were consequently bigger.
Our data (very low growth rate of trk1∆ trk2∆ tok1∆ cells in media with low KCl) revealed the importance of the Tok1 channel for the growth of cells in media with potassium-limiting concentrations. The positive role of overexpressed Tok1 in the growth of trk1 trk2 mutants under potassium-limiting conditions was observed earlier (Fairman et al., 1999) and the uptake of K+ via this channel was suggested but not experimentally proved. Based on our data, we believe that the TOK1 overexpression results in a hyperpolarization of the cell membrane [as shown in (Maresova et al., 2006)] which increases the ectopic potassium uptake (Madrid et al., 1998).
Expression of Trk1 restores efflux of potassium via Nha1 and Ena1-5 exporters
To confirm our hypothesis, we tried to depolarize cells by the overexpression of TRK1. When the potassium efflux rate was measured (Fig. 5a), it was evident that the efflux of potassium depended on the presence of both potassium uptake (Trk1) and potassium efflux (Nha1, Ena1-5) systems. Cells lacking Trk systems did not release potassium similarly to cells lacking the Nha1 antiporter and Ena ATPases (Fig. 5a; BYT12[YEp352] and BYT45[YEp352]). A K+ efflux comparable to that observed for the wild-type (BY4741[YEp352]) cells was restored by overexpressing of TRK1 (using pTRK1) in the BYT12 (trk1∆ trk2∆) strain (Fig. 5a). That the observed efflux was not mediated by the Trk1 transporter itself was evident from the absence of potassium efflux in cells expressing Trk1 but lacking the Nha1 and Ena1-5 systems (BYT 45[YEp352]; Fig. 5a). The estimation of relative membrane potential in cells transformed either with an empty vector (YEp352) or with pTRK1 (Fig. 5b) confirmed that the overexpression of the TRK1 gene resulted in a significant depolarization of the cell membrane.
Taken together, our results showed that, under potassium-limiting conditions, the three K+ export systems (Nha1, Ena1 and Tok1) do not mediate potassium efflux in the trk1∆ trk2∆ mutant but all of them are active in the wild type and participate in continuous potassium efflux. As the three export systems use distinct transport mechanisms and are regulated differently, the absence of potassium efflux in the trk1∆ trk2∆ mutant must be caused by a general mechanism. We show that it is the plasma-membrane potential that regulates not only the activity of the voltage-gated Tok1 channel [as has been shown earlier (Bertl et al., 2003)] but surprisingly, the hyperpolarization also diminishes the potassium efflux via Nha1 cation/H+ antiporter and Ena ATPases. The interconnection of potassium uptake and efflux systems was already shown in a study in which the absence of a potassium-exporting system (Nha1) was compensated for by a decrease in Trk1 affinity for potassium (Banuelos et al., 2002). Here we show the opposite effect: the absence of K+ uptake systems leads to the changes of potassium efflux rate of the active exporters. Our results confirm that S. cerevisiae cells need a continuous uptake and efflux of potassium to regulate their membrane potential and thereby other physiological parameters such as cell size and that they are able to quickly and efficiently compensate for an insufficiency in potassium transport in one direction by diminishing the transport in the other direction.
We are grateful to Pavla Herynkova for excellent technical assistance and Olga Zimmermannova for helpful discussions and critical reading of the manuscript. This work is a part of TRANSLUCENT-2, a SysMo ERA-NET funded Research Project, and was supported by grants MSMT LC531, Institutional Concept AV0Z50110509 and GA AS CR IAA 500110801. J.Z. thanks MSMT for a stipend, and to Specific University Research Project No. 33779266 awarded by Charles University Prague for additional financial support.