Living cells accumulate potassium (K+) to fulfil multiple functions. It is well documented that the model yeast Saccharomyces cerevisiae grows at very different concentrations of external alkali cations and keeps high and low intracellular concentrations of K+ and sodium (Na+) respectively. However less attention has been paid to the study of the intracellular distribution of these cations. The most widely used experimental approach, plasma membrane permeabilization, produces incomplete results, since it usually considers only cytoplasm and vacuoles as compartments where the cations are present in significant amounts. By isolating and analysing the main yeast organelles, we have determined the subcellular location of K+ and Na+ in S. cerevisiae. We show that while vacuoles accumulate most of the intracellular K+ and Na+, the cytosol contains relatively low amounts, which is especially relevant in the case of Na+. However K+ concentrations in the cytosol are kept rather constant during the K+-starvation process and we conclude that, for that purpose, vacuolar K+ has to be rapidly mobilized. We also show that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that both in wild-type and vacuolar mutants, nuclei contain and keep a relatively constant and important percentage of total intracellular K+ and Na+, which most probably is involved in the neutralization of negative charges.

INTRODUCTION

Potassium (K+) is a fundamental element for living cells. It is usually scarce in the majority of ecosystems and it is well known that it is required to fulfil multiple cellular functions, such as regulation of intracellular pH, compensation of negative charges in many macromolecules, or maintenance of cell volume, turgor or plasma membrane electrochemical potential [13]. In contrast, sodium (Na+) is the most abundant cation in natural environments, but its accumulation is toxic for most organisms. It has been reported by several groups that the model yeast Saccharomyces cerevisiae is able to grow in a broad range of external [K+], that the intracellular concentration usually ranges from 200 to 300 mM under normal growth conditions and that it decreases at limiting K+ or in the presence of high Na+ [2,3]. Therefore S. cerevisiae cells tend to accumulate K+ against high concentration gradients, and the main plasma membrane potassium transporter Trk1 plays a crucial role in the process [2,3]. Two recent genome-wide studies identified multiple genes from different functional categories involved in K+ homoeostasis highlighting the complexity of the process [4,5]. In addition, little attention has been paid to the detailed analysis of K+ and Na+ intracellular distribution in yeast. The subcellular localization of cations under different external conditions has not been definitively clarified, despite the fact that subcellular compartmentalization is critical for eukaryotic cellular physiology and, for example, it is generally accepted that vacuolar Na+ sequestration plays an important role in both in yeast and plant cells [6,7]. The existence of K+/Na+ transporters in organelles such as vacuoles and endosome/prevacuolar compartments [810] or mitochondria [11] reinforces the possible physiological importance of cation compartmentalization.

By using energy-dispersive X-ray microanalysis, it was reported more than three decades ago that the intracellular distributions of K+, rubidium (Rb+) and caesium (Cs+) were very similar, and that the concentrations of these ions in the cytoplasm were approximately equal to those in the nucleus and twice those found in the vacuole [12]. However, possibly due to the fact that the technique was not readily available for many groups or maybe because those experiments were performed under non-physiological conditions (prior to analysis, yeast cells were suspended in water for 1 day), the fact is that this work did not have the impact and significance that it may have had. The only relatively widely used approach to investigate the question of subcellular cation localization in yeast is the use of substances such as cytochrome c [1315], DEAE dextran [16] or digitonin [17] to permeabilize the plasma membrane. This is a convenient and rapid method, but the main limitation of the procedure is that the results are too simplistic, since it usually considers only two important compartments where the cations are present in significant amounts; K+ extracted after permeabilization is considered the cytoplasmic fraction, whereas the remaining amount is ascribed to the vacuole. After using these procedures, most of the investigations conclude that cations accumulate in the vacuolar fraction to significantly higher amounts than in the cytoplasmic fraction [13,15,16], which is in contradiction with what was previously published by Roomans and Sevéus [12].

On the basis of the isolation of the main yeast organelles, we have optimized a procedure to determine the subcellular location of K+ and Na+ in S. cerevisiae wild-type cells harvested under several conditions. Moreover, we have applied the same procedure to four different vacuolar mutants. We conclude that while vacuoles accumulate most intracellular K+ and Na+, the cytosol contains relatively low amounts of theses cations. However, the concentrations of K+ in the cytosol are kept rather constant during the K+-starvation process and for that purpose vacuolar K+ has to be rapidly mobilized. We also found that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that in all strains we have studied, the nuclei have and maintain a relatively constant and important percentage of the total intracellular K+ and Na+.

EXPERIMENTAL

Yeast strains and culture conditions

The S. cerevisiae wild-type and isogenic mutant strains used in the present study and their genotypes are listed in Table 1. Yeast cells were cultured with shaking at 28°C. ‘Normal K+ cells’ were grown in YPD medium [1% (w/v) yeast extract and 2% (w/v) glucose], ‘Na+-grown cells’ were grown in YPD supplemented with 0.8 M NaCl, and ‘K+-starved cells’ were obtained by growing cells in synthetic K+-free medium [YNB-F (0.17% yeast nitrogen base without amino acids, ammonium sulfate and K+ (pH 5.8), Formedium™] supplemented with 50 mM KCl and incubating them for an additional 4 h in the same medium lacking added K+ [18]. Cells were generally harvested during the exponential growth phase at D600 values close to 1.0. In the case of nhx1 mutant grown in Na+, cells were collected at D600 values of 0.2–0.3.

Table 1
Strains used in the present study
Strain Genotype Gene function 
BY4741 MATa his3Δ1 leuΔ0 met15Δ0 ura3Δ0  
Δptc1 ptc1::NAT1 PP2C type protein phosphatase 
Δvam6 vam6:: kan MX4 Vacuolar morphogenesis protein 
Δvnx1 vnx1::LEU2 Vacuolar Na+ (K+)/H+exchanger 
Δnhx1 nhx1::LEU2 Prevacuolar–vacuolar Na+(K+)/H+exchanger 
Strain Genotype Gene function 
BY4741 MATa his3Δ1 leuΔ0 met15Δ0 ura3Δ0  
Δptc1 ptc1::NAT1 PP2C type protein phosphatase 
Δvam6 vam6:: kan MX4 Vacuolar morphogenesis protein 
Δvnx1 vnx1::LEU2 Vacuolar Na+ (K+)/H+exchanger 
Δnhx1 nhx1::LEU2 Prevacuolar–vacuolar Na+(K+)/H+exchanger 

Growth curves

To estimate the growth of the different strains, liquid YPD, supplemented or not with 0.8 M NaCl, was inoculated with yeast cells (D600=0.05) and growth was monitored for 25 h.

Isolation of main organelles from yeast

All centrifugation steps were carried out at 4°C in plastic tubes and all buffers were kept on ice. Protocols were adapted in order to use K+- and Na+-free buffers. Figure 1 shows the general workflow followed in these experiments.

Steps in the workflow for the determination of intracellular cation distribution

Figure 1
Steps in the workflow for the determination of intracellular cation distribution

Time taken for the preparation was less than 4 h.

Figure 1
Steps in the workflow for the determination of intracellular cation distribution

Time taken for the preparation was less than 4 h.

Isolation of vacuoles

Vacuoles were isolated as described previously [19,20] with minor modifications. Briefly, cells were grown, washed and treated with lyticase-100KU (2 mg/g of cells for 55 min). The spheroplasts were resuspended in 10 mM Tris/Mes (pH 6.9), 0.1 mM MgCl2 and 12% Ficoll, homogenized using a Dounce homogenizer and centrifuged at 26600 g for 60 min. The white layer at the top of the tubes, which contained most of the vacuoles, was collected and resuspended in the same buffer.

The vacuole protein concentration was determined using the Bradford protein assay [21]. For measuring V-ATPase (vacuolar ATPase) activity, we followed the procedure described by Cagnac et al. [9] with minor modifications. Vacuole protein (25 μg) was added to a buffer containing 50 mM tetramethyl ammonium chloride, 5 μM Acridine Orange, 5 mM Tris/Mes (pH 7.5), 3.125 mM MgSO4, 1 mM sodium azide (to inhibit F1F0-ATPase) and 1 mM sodium vanadate (to inhibit P-type ATPases) [22]. The V-ATPase activity was activated by the addition of 5 mM Tris-ATP, and time-dependent fluorescence changes were monitored using a fluorometer (SPECTRAFluor Plus, Tecan), with excitation and emission wavelengths at 485 and 535 nm respectively. The vacuolar activity was inhibited with the addition of 1 μM bafilomycin A. The V-ATPase activity was defined as the difference between the activities obtained with and without 1 μM bafilomycin A in the total homogenate and in the vacuole fraction.

Isolation of nuclei

The isolation of nuclei from yeasts was performed following the procedure described by Zhang and Reese [23]. Basically, the cells were grown, washed and treated with lyticase-100KU. The spheroplasts obtained were resuspended in sorbitol digestion buffer [1.4 M sorbitol, 40 mM Hepes-(NH4)2SO4 (pH 7.5) and 0.5 mM MgCl2], supplemented with 1 mM PMSF and 2 mM 2-mercaptoethanol, homogenized using a Dounce homogenizer and centrifugated at 21500 g for 30 min. The pellet was resuspended in digestion buffer.

To estimate the proportion of nuclei recovered, 100 μl of the nuclei suspension was diluted in 900 μl of digestion buffer and D600 was measured. The reading should be approximately 0.2 for 1 g of cells [23]. In addition, the number of nuclei was determined in spheroplasts and the nuclear fraction using minor modifications of the Prescott–Breed method [24]. Basically, the spheroplasts and the nuclei fraction was thinly smeared on microscope slides and equilibrated with PBS. DAPI staining solution was added and the slides were incubated for 5 min at room temperature (25°C). Background fluorescence was eliminated adding antifade reagent (Slowfade® Gold antifade reagent, Life Technologies). Samples were observed and nuclei counted using a fluorescence microscope with a Mercury-arc lamp (the excitation maximum of DAPI bound to dsDNA is 358 nm and the emission maximum is 461 nm).

Isolation of mitochondria

Isolation of mitochondria was performed following the procedure described by Zotova et al. [25] with some modifications. After lyticase-100KU treatment, the spheroplasts were centrifuged, washed and resuspended in 2.4 M sorbitol and 0.2 M NH4PO3 (pH 7.4). The spheroplasts were homogenized in a Dounce homogenizer. Mitochondria were recovered by centrifugation at 11000 g and the pellet was resuspended in the same buffer.

Mitocondrial protein concentration was determinated by the Bradford protein assay [21]. Cytochrome c oxidase activity was measured as described previously [26,27]. The oxidation of cytochrome c by cytochrome c oxidase was followed spectrophotometrically at 550 nm for 30 s, assuming ϵ1 cm=19600 per mol for horse heart cytochrome c [28]. The mitochondrial fraction cytochrome c oxidase activity was expressed as a percentage of the total homogenate activity.

Isolation of ER (endoplasmic reticulum)

ER was obtained by using the Endoplasmic Reticulum Isolation kit (Sigma) with some minor modifications. Spheroplasts were prepared and homogenized as described above [19,20]. The homogenate was centrifuged at 1000 g for 10 min. Then, the corresponding supernatant was centrifuged at 15000 g for 15 min. The resulting supernatant was the so-called post-mitochondrial fraction. This fraction was then precipitated with 7 volumes of 8 mM CaCl2 and centrifuged at 8000 g for 10 min. The ER partitioned to the insoluble fraction, which was resuspended in 10 mM Tris/Mes (pH 6.9), 0.1 mM MgCl2 and 12% Ficoll buffer.

The ER protein concentration was determined using the Bradford protein assay [21]. The ER NADPH-cytochrome c activity was determined using the Cytochrome c Reductase (NADPH) Assay kit (Sigma) and was expressed as a percentage of the total homogenate activity.

Isolation of the Golgi apparatus

The Golgi apparatus was isolated as described previously [29] with some minor modifications. Spheroplasts were homogenized and centrifuged, and the post-mitochondrial fraction was obtained as described for ER. A 1 ml aliquot of that fraction was placed on the top of a gradient containing 1 ml steps of 22, 26, 30, 34, 38, 42, 46, 50 and 54% (w/w) sucrose in 10 mM Hepes (pH 7.5) and 1 mM MgCl2. Gradients were centrifuged at 30000 rev./min for 150 min in a Beckman SW41 rotor. Fractions of 660 μl (12-drop) were collected from the top to the bottom. The sucrose density was measured with a refractometer (Atago). Protease inhibitors were added to the fractions.

Step 26 was enriched with Golgi apparatus protein. The Golgi GDPase activity was measured in vesicle fractions of the different steps in buffer containing 0.2 M imidazole (pH 7.5), 10 mM CaCl2, 0.1% Triton X-100 and 2 mM GDP; 100 μl of this solution, containing 20 μg of sample protein, was incubated at 30°C for 30 min. The reaction was stopped by transferring the tubes to ice and adding 10 μl of 10% SDS. To determine the amount of phosphate released during hydrolysis, 200 μl of ultra-pure water and 700 μl of Ames reagent (1:6 mixture of 10% ascorbic acid and 0.42% ammonium molybdate in 0.5 M sulfuric acid) were added to each tube; following incubation at 45°C for 20 min, absorbance was measured at 660 nm [30]. The Golgi apparatus fraction GDPase activity was expressed as a percentage of the total homogenate activity.

Cytosolic fraction

In order to obtain the cytosol, spheroplasts were homogenized and centrifuged, and the post-mitochondrial fraction was isolated as described for the ER. That fraction was centrifuged at 35000 rev./min for 60 min and then the supernatant was again centrifuged at 45000 rev./min for 150 min (in a Beckman 50.2 TI rotor). The resulting supernatant corresponded to the soluble fraction of the cytoplasm [31].

Cross-contamination activity

The vacuolar cross-contamination in the nuclei, and mitochondrial and ER fractions was determined fluorimetrically by measuring the V-ATPase activity [19,20]. The nuclei cross-contamination was estimated by using a fluorescence microscope to observe nuclei [24,32] and spectrophotometrically to determine the presence of nucleic acids [23] in the vacuolar, ER and mitochondrial fractions. The degree of mitochondrial cross-contamination in the vacuolar, ER and nuclear fractions was determined spectrophotometrically measuring cytochrome c oxidase activity [26,27]. The Golgi apparatus cross-contamination was determined spectrophotometrically measuring Golgi GDPase activity in vacuolar, mitochondrial, nuclei and ER fractions.

Plasma membrane permeabilization procedure

In order to permeabilize yeast plasma membranes, the procedure described by Okorokov et al. [13] was slightly modified. Cells were washed with 0.9 M mannitol, 1 mM Hepes, 5 mM MgSO4 and 10 mM CaCl2. To make the plasmalemma readily permeable to cations, the cells were treated for 15 min with 2% cytochrome c solution (0.9 M mannitol, 2% cytochrome c, 18 μg/ml antimycin, 1 mM Hepes, 5 mM MgSO4, 10 mM CaCl2 and 5 mM 2-deoxy-D-glucose). Permeabilized cells were recovered by centrifugation and the supernatant was kept (the ‘cytoplasmic fraction’). Then vacuoles in the pellet were lysed by osmotic shock by adding 5 ml of water (the‘vacuolar fraction’).

K+ and Na+ measurements

To estimate internal K+ and Na+ content, cell samples were collected on Millipore filters, which were rapidly washed with 20 mM MgCl2. Whole cells [33] and subcellular fractions were then extracted with acid and K+ and Na+ were analysed by atomic emission spectrophotometry. Cation content in the different organelles was calculated taking into consideration the percentage of nuclei, vacuoles, mitochondria, Golgi apparatus and ER isolated. In all cases values are expressed as nmol of K+/Na+ per mg of dry weight cells.

Statistical analyses of the results

In all cases three to four independent experiments were used for every condition. Values shown are means±S.E.M. In some experiments, the significance of differences between mean values was determined by ANOVA using Tukey's test. A P value<0.05 was considered significant. Statistical processing was carried out using SPSS software.

RESULTS

Efficiency of the isolation procedure

Yeast cells obtained under several conditions were treated as described in the Experimental section in order to determine the intracellular cation distribution, efficiency of the isolation procedures and cross contaminations. As a first step, we checked that intracellular K+ was not lost during the spheroplasting procedure both in wild-type and vacuolar mutants (less than 5% of total K+ was lost in all cases). After the different organelle isolation steps, 91% (±8%), 40% (±8%), 27% (±3%), 71% (±6%) and 87% (±3%) of the nuclei, vacuoles, mitochondria, ER and Golgi apparatus were isolated and used to determine their cation content. Those percentages are comparable with what has been previously described in the literature when these organelles are isolated for different purposes [3441]. Moreover our control tests to determine possible cross-contaminations showed that the mitochondria, vacuoles, Golgi apparatus or ER contaminating other fractions were usually below 5%. Owing to the isolation procedure, the Golgi apparatus fraction is practically free of other organelles [29]. Vacuolar, ER and mitochondrial fractions were virtually free from nuclei contamination since on the one hand, no nuclei were detected by microscopy and, on the other hand, spectrophotometric reading of nucleic acids gave values of approximately 0.001 for 1 g of cells compared with 0.2 in the control [23].

Taken together, these results indicate a low degree of cross-contamination in the different fractionation procedures.

Subcellular K+ distribution

Intracellular cation distribution was studied in wild-type yeast and vacuolar mutants. Our objective was to analyse a set of different mutants affected in vacuolar physiology. For that purpose, we selected two mutants lacking a cation transporter (vnx1 and nhx1) and, to determine whether possible changes observed were due to the ion transport activity of these proteins or to general defects in vacuolar morphology, we included two other mutants for comparison: ptc1 and vam6. These mutants also display alterations in vacuolar morphology, although these mutants are not expected to affect ion transport, the fact is that they are Na+/lithium (Li+)-sensitive [4244]. All strains grew similarly in rich medium without potassium limitation (normal K+ cells) (Figure 2A) and although nhx1 mutants, lacking a K+(Na+)/H+ antiporter located in prevacuolar and vacuolar membranes [4547], showed a slight delay in growth, both the growth rate constant and maximum biomass yield were similar to those obtained for the wild-type, vam6, ptc1 and the vnx1 mutant, lacking the K+(Na+)/H+ transporter in the tonoplast [9]. Under these growth conditions, total intracellular K+ content was determined in all of the strains. It is worth highlighting that the vam6 strain accumulated approximately 20% more K+ than the wild-type with highly significant differences (Tukey’s test, P<0.001) (Table 2). Intracellular distribution of the cation in wild-type cells showed that the vacuole was the intracellular compartment containing more K+ (approximately 45% of the total). It is relevant that nuclei contained 29% of the total K+ and only a minor amount of this cation was present in the mitochondria, Golgi apparatus and ER. (Figure 2B). Finally, it is important to mention that when complex YPD or synthetic YNB-F plus 50 mM KCl were used, growth rates were comparable (0.27 and 0.24 h−1 respectively) (Figure 2A and [18]), as were intracellular K+ concentrations (571±38 and 564±47 nmol/mg respectively) and K+ distribution (results not shown).

Subcellular K+ localization in wild-type and vacuolar mutants

Figure 2
Subcellular K+ localization in wild-type and vacuolar mutants

(A) Growth curves of normal K+ cells. Yeast cells were inoculated in YPD medium at D600=0.01 and a representative curve is shown. (B and C) Percentage relative to total intracellular K+ located in vacuole, nucleus, mitochondria, Golgi apparatus, ER and cytosol in normal K+ cells (B) and K+-starved cells (C). Data are means±S.E.M. for four independent experiments. (D) Radar graphs of quantitative K+ changes (nmol/mg) after starvation in wild-type and vacuolar mutants.

Figure 2
Subcellular K+ localization in wild-type and vacuolar mutants

(A) Growth curves of normal K+ cells. Yeast cells were inoculated in YPD medium at D600=0.01 and a representative curve is shown. (B and C) Percentage relative to total intracellular K+ located in vacuole, nucleus, mitochondria, Golgi apparatus, ER and cytosol in normal K+ cells (B) and K+-starved cells (C). Data are means±S.E.M. for four independent experiments. (D) Radar graphs of quantitative K+ changes (nmol/mg) after starvation in wild-type and vacuolar mutants.

Table 2
Total K+ content in normal K+ cells and in K+-starved cells

Cells were grown in YPD (normal K+ cells) or in YNB-F supplemented with 50 mM KCl and then starved for 4 h (K+-starved cells). Data are means±S.E.M.. Different letters represent statistically significant differences among the strains under the same conditions (Tukey’s test, P<0.05). * denotes a highly significant difference by Tukey’s test (P<0.001).

 Total intracellular K+ (nmol/mg of cells) 
Strain Normal K+ cells K+-starved cells 
BY4741 564±47b 265±14c 
Δptc1 558±14b 237±14b 
Δvam6 680±40c281±29c 
Δvnx1 593±40b 209±23a 
Δnhx1 508±25a 202±18a 
 Total intracellular K+ (nmol/mg of cells) 
Strain Normal K+ cells K+-starved cells 
BY4741 564±47b 265±14c 
Δptc1 558±14b 237±14b 
Δvam6 680±40c281±29c 
Δvnx1 593±40b 209±23a 
Δnhx1 508±25a 202±18a 

We applied the same isolation procedure in four different vacuolar mutants. We first checked that the alterations in vacuoles of the mutants did not lead to different organellar yields. In general, the total protein yield was 38–42 mg/g of cells in all strains, and in all cases 15–17% corresponded to vacuolar proteins. The fact that, for example, ER proteins were 11–13% of total protein in all strains reinforced the idea that cell organelle isolation outcomes in all of the mutants was sufficiently similar to wild-type to ensure that the comparisons were sufficiently robust. Although affected in very different processes, all of them showed altered distribution of K+ with decreased amounts of this cation in the vacuole and increased amounts in the cytosol. This redistribution was more relevant in the case of the two mutants affected in transport in which the cytosol accumulated even more K+ than the vacuole. For example, in the vnx1 mutant, we measured 37% of internal K+ in the cytosol and only 21% in the vacuole. Moreover, K+ in the nuclei, mitochondria, Golgi apparatus and ER was not greatly affected by these mutations, although we observed an increase in K+ in the mitochondrial fraction of the ptc1 mutant (Figure 2B). Importantly, we did not observe increased amounts of vacuolar cross-contamination of other fractions in these mutants, suggesting that vacuolar integrity was not compromised during the extraction process. Moreover, we examined the isolated vacuoles from each of these strains microscopically and did not observe significant differences in the morphology of the purified vacuoles, thus discarding an artefact or vacuolar fragility.

In a subsequent experiment, we prepared K+-starved cells. Cells were grown in YNB-F supplemented with 50 mM KCl. After 4 h of incubation in the same medium without added K+, all strains lost more than 50% of their intracellular K+ content (Table 2). Determination of the intracellular distribution of K+ revealed that vacuolar K+ spectacularly decreased in wild-type cells and that the percentage in the cytosol was 3.3-fold higher than in normal K+ cells, which means higher cytosolic K+ amounts under starvation conditions. Therefore it is conceivable that K+ was transported from the vacuole to the cytosol in order to fulfil cytosolic requirements. Finally, it is worth mentioning that the nuclei kept a similar percentage of K+ after starvation (Figure 2C).

The general behaviour described above was also observed in all of the mutant strains. However, the changes in the vacuolar and cytosolic distribution of K+ during starvation were much less relevant in the mutants, probably because of the impaired vacuolar physiology which probably leads to the observed decreased vacuolar and increased cytosolic K+ concentrations before the starvation process (Figure 2C). Figure 2(D) shows radar graphs illustrating the quantitative changes in K+ distribution after the K+-starvation process in the wild-type and the vacuolar mutants.

We next decided to use the wild-type strain to compare our results with those obtained with the most widely used procedure to determine intracellular cation distribution: plasma membrane permeabilization. This procedure is straightforward and less time consuming, but too simplistic since it focuses almost exclusively on vacuolar and cytoplasmic compartmentalization and tends to ignore the nuclear K+ content contribution to total intracellular K+. Our parallel experiments, in which the plasma membrane was permeabilized with cytochrome c, on the one hand fit with previous observations showing the higher content of alkali cations in vacuoles of cells grown without K+ limitation [13,16], since we measured 55/45% in ‘vacuolar’ and ‘cytoplasmic’ fractions of normal K+ cells and, on the other hand, confirmed the drastic mobilization of vacuolar K+ to the cytoplasm during the starvation process (28/72%) (results not shown).

Subcellular Na+ distribution

When the YPD medium was supplemented with 0.8 M NaCl (‘Na+ grown cells’) we observed that while the wild-type and vnx1 mutant grew similarly, vam6 and ptc1 were slightly more affected and nhx1 mutants lacking the vacuolar K+(Na+)/H+ antiporter presented a severe growth impairment (Figure 3A). Under these conditions, all strains accumulated significant amounts of Na+, close to the K+ values (Table 3). It is worth highlighting that the nhx1 mutant, the strain showing poorer growth in the presence of Na+, was the only one in which total intracellular amounts of Na+ were higher than those of K+. The next step was to analyse the intracellular cation distribution. Results in Figures 3(B) and 3(C) show that, also under these growth conditions, wild-type cells accumulated more cations in the vacuole than in the cytosol, and that this was much more significant in the case of Na+, which was kept at very low levels in the cytosol, suggesting a Na+-sequestering function for the vacuole. This function was also confirmed by parallel plasma membrane permeabilization experiments which indicated that approximately 60% of the total Na+ was kept in the ‘vacuolar fraction’ (results not shown). Very interestingly, nuclei contained both Na+ and K+ in not very different amounts, which was not the case for wild-type mitochondria, Golgi apparatus or ER, which contained lower percentages of Na+ than of K+. Once again, the four vacuolar mutants showed altered distribution of cations, containing less K+ and Na+ in the vacuoles and higher amounts of both cations in the cytosol. The ratio of vacuolar compared with cytosolic Na+ clearly illustrates the defective cation distribution in these mutants: while this ratio was 13.7 for the wild-type, in the different mutants it reached values from 2.2 (ptc1) to 0.7 (vnx1). Radar graphs of the Na+ content in the wild-type and in the vacuolar mutants are shown in Figure 3(D). These results show that while the wild-type maintains a virtually Na+-free cytosol, all of the mutants contain higher amounts of Na+ in the cytosol, being especially relevant in the case of the two mutants affected in transport (vnx1 and nhx1).

Subcellular K+ and Na+ localization in wild-type and vacuolar mutants

Figure 3
Subcellular K+ and Na+ localization in wild-type and vacuolar mutants

(A) Growth curves of Na+-grown cells. Yeast cells were inoculated in YPD medium supplemented with 0.8 M NaCl at D600=0.01 and a representative curve is shown. (B and C) Percentage relative to total intracellular K+ (B) and Na+ (C) located in vacuole, nucleus, mitochondria, Golgi apparatus, ER and cytosol. Data are means±S.E.M. for four independent experiments. (D) Radar graphs of quantitative Na+ distribution (nmol/mg) in wild-type and vacuolar mutants.

Figure 3
Subcellular K+ and Na+ localization in wild-type and vacuolar mutants

(A) Growth curves of Na+-grown cells. Yeast cells were inoculated in YPD medium supplemented with 0.8 M NaCl at D600=0.01 and a representative curve is shown. (B and C) Percentage relative to total intracellular K+ (B) and Na+ (C) located in vacuole, nucleus, mitochondria, Golgi apparatus, ER and cytosol. Data are means±S.E.M. for four independent experiments. (D) Radar graphs of quantitative Na+ distribution (nmol/mg) in wild-type and vacuolar mutants.

Table 3
Total K+ and Na+ content in Na+-grown cells

Cells were grown in YPD supplemented with 0.8 M NaCl. Data are means±S.E.M. Different letters represent statistically significant differences among the strains (Tukey’s test, P<0.05).

 Total intracellular K+ and Na+ in Na+-grown cells 
Strain K+ (nmol/mg of cells) Na+ (nmol/mg of cells) 
BY4741 260±20a 228±19a 
Δptc1 364±28c 328±47b 
Δvam6 275±12a 245±22a 
Δvnx1 322±31b 255±19a 
Δnhx1 282±46a 334±20b 
 Total intracellular K+ and Na+ in Na+-grown cells 
Strain K+ (nmol/mg of cells) Na+ (nmol/mg of cells) 
BY4741 260±20a 228±19a 
Δptc1 364±28c 328±47b 
Δvam6 275±12a 245±22a 
Δvnx1 322±31b 255±19a 
Δnhx1 282±46a 334±20b 

DISCUSSION

K+ and Na+ homoeostasis are well-studied processes in yeast and intracellular values of these cations in S. cerevisiae wild-type and mutants grown under different conditions have been frequently reported [3]. However, less attention has been paid to the subcellular localization of these ions. The most widely used approach to this subject is the use of several substances that specifically permeabilize the plasma membrane. As mentioned above, although this procedure seems to be too simplistic, several groups have reported higher concentrations of K+ in the vacuole than in the cytoplasm [1315]. The results of the present study with wild-type cells grown under non-limiting K+ conditions fit with this idea. Moreover, our results show that cytoplasmic values obtained by using cytochrome c were significantly higher than cytosolic values obtained by isolating organelles in both normal K+ cells and K+-starved cells. These results suggest much higher contamination of the cytoplasmic fraction coming from other organelles when the cytochorome c permeabilization process was used. We also demonstrate that the presence of significant amounts of K+ in the nucleus cannot be ignored, as reported by Roomans and Sevéus [12]. Vacuoles are the organelles which accumulate higher amounts of K+ and, in contrast, cytosolic K+ is relatively low. However, the amounts in the cytosol seem to be very important for cell physiology, i.e. regulation of enzymatic activities [48], as suggested by the extraordinary mobilization of vacuolar K+ to the cytosol during the starvation process in order to keep the levels constant. An open question that remains is the substrate accumulated by the vacuole in K+-starved cells in order to keep osmotic potential since, in addition to the decrease in K+ content, cells lose high amounts of polyphosphates during the K+-starvation process (D. Canadell and J. Ariño, personal communication).

In the case of Na+-grown cells, the subcellular distribution of this cation was similar to what was observed for K+, that is, higher amounts in the vacuole than in the cytosolic fraction. However, in this case, Na+ levels in the cytosol were kept especially low, reinforcing the idea of Na+ as a toxic element.

The four mutants used in the present study lack very different genes, but all of them have impaired vacuolar physiology. It is of note that all of them showed altered intracellular cation distribution. The four vacuolar mutants contained higher amounts of K+ in the cytosol and lower amounts in the vacuole. Specifically, both nhx1 and vnx1 were less effective in accumulating K+ in the vacuole, and ptc1 and vam6 showed a less-severe phenotype, supporting a specific role for the cation transport activity of Nhx1 and Vnx1 in intracellular cation distribution and validating our experimental procedure. These results indicate defective vacuolar accumulation and the absence of a possible sensing mechanism which would regulate K+ transport from the external medium to the cytoplasm. In addition, the four mutant strains showed a different response to K+ starvation than the wild-type strain. Moreover, none of these strains were able to maintain the very low cytosolic amounts of Na+ observed in the wild-type, although their Na+ sensitivity greatly varied from one mutant to another. This behaviour illustrates the complexity of the targets of Na+ sensitivity. In our hands, the vnx1 mutant was not Na+-sensitive, which is in agreement with previous reports [9], and the ptc1 and vam6 showed moderate sensitivity. In this respect, it is worth mentioning that in two genome-wide studies, the growth of vam6 mutants was proposed to be unaffected [49] or to be very slightly inhibited [50] by NaCl. Finally, the most susceptible strain, the nhx1 mutant, accumulated more intracellular Na+ than the wild-type, the K+/Na+ ratio was the lowest and, in addition, an important percentage of this Na+ was in the cytosol, which most probably is linked to its sensitive character. At this point it is worth commenting that Nhx1 regulates membrane fusion of endosomal vesicles and acidification of cytosol and vacuole lumen [46]. In summary, although the underlying reason may be different depending on the mutation, the fact is that all of the mutants showed altered vacuolar cation content and problems to adapt to changes in external medium.

From a general point of view, the cation content in other organelles such as mitochondria, Golgi apparatus and ER was relatively low and constant under the different conditions used in the present study. We cannot completely rule out the possibility of some K+ loss during the cell homogenization in the case of the ER or Golgi; however, it seems to be reasonable to expect low amounts of K+ in these organelles and the results of the present study appear coherent with this idea and with the whole set of data obtained. The possibility of significant amounts of K+ in yeast nuclei has usually been overlooked, although Roomans and Sevéus [12] did mentioned it in their pioneering paper in 1976. We now show that nuclei from wild-type cells contain approximately one-third of the intracellular K+ and Na+ and that this amount is kept quite constant in all of the vacuolar mutants studied and is independent of the external conditions (K+ starvation or the presence of Na+). Apparently, it does not matter if the cell uses K+ or Na+, since in Na+-grown cells, both K+ and Na+ are present. Nuclear pores are big enough to allow K+ and Na+ leak; however, our measurements suggest that important amounts of these cations are found in nuclear fractions and it is tempting to propose that they can be used to neutralize part of the negative charges in this organelle, as has been previously proposed in mammalian cells where Na+ and K+ have been directly implicated in chromosome structure through electrostatic neutralization and a functional interaction with non-histone proteins [51].

Finally we would like to highlight that we have established a robust and optimized procedure to determine subcellular cation localization in yeast that can be used to analyse wild-type cells under several external conditions and many different mutants affected in cation homoeostasis that are not sufficiently understood at present.

Abbreviations

     
  • ER

    endoplasmic reticulum

  •  
  • V-ATPase

    vacuolar ATPase

AUTHOR CONTRIBUTION

Rito Herrera contributed to the experimental design, carried out the experiments and analysed the results. María Álvarez contributed to the design of the research, helped with the organelle isolation procedure and analysed the results. Samuel Gelis helped with the interpretation of the data, scientific discussion and writing the paper. José Ramos conceived and designed the study, supervised the project and wrote the paper with input from all of the authors.

We thank L. Yenush and J. Ariño for critical comments, D. Canadell and J. Ariño for sharing information on intracellular polyphosphates, and the Translucent Consortium for many useful discussions. We also thank S. Demyda, F. Calahorro, and the groups of M.M. Malagón and J.M. Villalba for technical advice.

FUNDING

This work is a part of TRANSLUCENT-2, a SysMo ERA-NET funded Research project and was supported by the Ministry of Science and Innovation, Spain [grant numbers BFU2008-04188-C03-03 and EUI 2009-04153 (to J.R.)]. R.H. is a recipient of a Panama Government fellowship (SENACYT-IFARHU).

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