The Na+,K+/H+ -antiporter Nha1 influences the plasma membrane potential of Saccharomyces cerevisiae

There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.     

Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions

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.     

Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants

Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. In Saccharomyces cerevisiae, the high K+ content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters formerly believed to extrude K+ from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p and presumed as a putative plasma membrane K+/H+ antiporter. We prepared kha1 and tok1-kha1 deletion strains in the B31 and MAB 2d background. Both the strains contain the ena1-4 and nha1 deletions; that means they lack the main active sodium and potassium efflux systems. MAB 2d has additional trk1 and trk2 deletions, i.e. is impaired in active K+ uptake as well. We performed a large physiological study with these strains to specify the phenotype of kha1 deletion. In our experiments, no difference in K+ content or efflux was observed in strains lacking the KHA1 gene compared with control strains. Two main phenotype manifestations of the kha1 deletion were growth defect on high external pH and hygromycin sensitivity. The correlation between these phenotypes and the kha1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of green fluorescent protein (GFP)-tagged Kha1p showed that this antiporter is localized preferentially intracellularly (in contrast to the plasma membrane Na+/H+ antiporter Nha1p). Based on these findings, Kha1p is probably not localized in plasma membrane and does not mediate efflux of alkali metal cations from cells, but is important for the regulation of intracellular cation homeostasis and optimal pH control, similarly as the Nhx1p.     

Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations

Saccharomyces cerevisiae cells express three defined potassium-specific transport systems en-coded by TRK1, TRK2 and TOK1. To gain a more complete understanding of the physiological function of these transport proteins, we have constructed a set of isogenic yeast strains carrying all combinations of trk1delta, trk2delta and tok1delta null mutations. The in vivo K+ transport characteristics of each strain have been documented using growth-based assays, and the in vitro biochemical and electrophysiological properties associated with K+ transport have been determined. As has been reported previously, Trk1p and Trk2p facilitate high-affinity potassium uptake and appear to be functionally redundant under a wide range of environmental conditions. In the absence of TRK1 and TRK2, strains lack the ability specifically to take up K+, and trk1deltatrk2delta double mutant cells depend upon poorly understood non-specific cation uptake mechanisms for growth. Under conditions that impair the activity of the non-specific uptake system, termed NSC1, we have found that the presence of functional Tok1p renders cells sensitive to Cs+. Based on this finding, we have established a growth-based assay that monitors the in vivo activity of Tok1p.     

A molecular target for viral killer toxin: TOK1 potassium channels.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes.     

Factors and processes involved in membrane potential build-up in yeast: diS-C3(3) assay.

No methods are currently available for fully reliable monitoring of membrane potential changes in suspensions of walled cells such as yeast. Our method using the Nernstian cyanine probe diS-C3(3) monitors even relatively fast changes in membrane potential delta psi by recording the shifts of probe fluorescence maximum lambda max consequent on delta psi-dependent probe uptake into, or exit from, the cells. Both increased [K+]out and decreased pHout, but not external NaCl or choline chloride depolarise the membrane. The major ion species contributing to the diS-C3(3)-reported membrane potential in S. cerevisiae are thus K+ and H+, whereas Na+ and Cl- do not perceptibly contribute to measured delta psi. The strongly pHout-dependent depolarisation caused by the protonophores CCCP and FCCP, lack of effect of the respiratory chain inhibitors rotenone and HQNO on the delta psi, as well as results obtained with a respiration-deficient rho- mutant show that the major component of the diS-C3(3)-reported membrane potential is the delta psi formed on the plasma membrane while mitochondrial potential forms a minor part of the delta psi. Its role may be reflected in the slight depolarisation caused by the F1F0-ATPase inhibitor azide in both rho- mutant and wildtype cells. Blocking the plasma membrane H(+)-ATPase with the DMM-11 inhibitor showed that the enzyme participates in delta psi build-up both in the absence and in the presence of added glucose. Pore-forming agents such as nystatin cause a fast probe entry into the cells signifying membrane damage and extensive binding of the probe to cell constituents reflecting obviously disruption of ionic balance in permeabilised cells. In damaged cells the probe therefore no longer reports on membrane potential but on loss of membrane integrity. The delta psi-independent probe entry signalling membrane damage can be distinguished from the potential-dependent diS-C3(3) uptake into intact cells by being insensitive to the depolarising action of CCCP.     

Potassium Uptake Through the TOK1 K+ Channel in the Budding Yeast

The current through TOK1 (YKC1), the outward-rectifying K⁺ channel in Saccharomyces cerevisiae, was amplified by expressing TOK1 from a plasmid driven by a strong constitutive promoter. TOK1 so hyper-expressed could overcome the K⁺ auxotrophy of a mutant missing the two K⁺ transporters, TRK1 and TRK2. This trk1Δtrk2Δ double mutant hyperexpressing the TOK1 transgene had a higher internal K⁺ content than one expressing the empty plasmid. We examined protoplasts of these TOK1-hyperexpressing cells under a patch clamp. Besides the expected K⁺ outward current activating at membrane potential (V _m ) above the K⁺ equilibrium potential (E _K+ ), a small inward current was consistently observed when the V _m was slightly below E _K+ . The inward and the outward currents are similar in their activation rates, deactivation rates, ion specificities and Ba²⁺ inhibition, indicating that they flow through the same channel. Thus, the yeast outwardly rectifying K⁺ channel can take up K⁺ into yeast cells, at least under certain conditions. Received: 1 October 1998/Revised: 9 December 1998     

Monitoring of real changes of plasma membrane potential by diS-C3(3) fluorescence in yeast cell suspensions

The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.     

Lymphocyte membrane potential assessed with fluorescent probes

The membrane potential of mouse spleen lymphocytes has been assessed with two fluorescent probes. 3,3'-Dipropylthiadicarbocyanine (diS-C3-(5)) was used for most of the experiments. Solutions with high K+ concentrations depolarised the cells. Valinomycin, an inophore which adds a highly K+-selective permeability membranes, slightly hyperpolarised cells in standard (6 mM K+) solution, and in 145 mM K+ solution produced a slight additional depolarisation. These findings indicate a membrane whose permeability is relatively selective for K+. Very small changes in potential were seen when choline replaced Na+, or gluconate replaced Cl-, supporting the idea of K+ selectivity. The resting potential could be estimated from the K+ concentration gradient at which valinomycin did not change the potential-the "valinomycin null point" - and under the conditions used the resting potential was approx.-60 mV. B cell-enriched suspensions were prepared either from the spleens of nu/nu mice or by selective destruction of T cells in mixed cell populations. The membrane potential of these cells was similar to that estimated for the mixed cells. In solution with no added K+, diS-C3-(5) itself appeared to depolarise the lymphocytes, in a concentration dependent manner. With the 100 nM dye normally used, the membrane potential in K+-free solution was around -45 mV, and 500 nM dye almost completely depolarised the cells. In standard solution quinine depolarised the cells. Valinomycin could still depolarise these cells indicating that depolarisation had not been due to dissipation of the K+ gradient. Since in K+-free solution diS-C3-(5) blocks the Ca2+-activated K+ channels in human red blood cell ghosts and quinine also blocks this K+ channel it is suggested that the resting lymphocyte membrane may have a similar Ca2+-activated K+ permeability channel. Because of the above mentioned effect of diS-C3-(5) and other biological side effects, such as inhibition of B cell capping, a chemically distinct fluorescent probe of membrane potential, bis(1,3-diethylthiobarbiturate)-trimethineoxonol was used to support the diS-C3-(5) data. This new probe proved satisfactory except that it formed complexes with valinomycin, ruling out the use of this ionophore. Results with the oxonol on both mixed lymphocytes and B cell-enriched suspensions gave confirmation of the conclusions from diS-C3-(5) experiments and indicated that despite its biological side effects, diS-C3-(5) could still give valid assessment of membrane potential.     

Deletions of SKY1 or PTK2 in the Saccharomyces cerevisiae trk1Deltatrk2Delta mutant cells exert dual effect on ion homeostasis

Sky1p and Ptk2p are protein kinases that regulate ion transport across the plasma membrane of Saccharomyces cerevisiae. We show here that deletion of SKY1 or PTK2 in trk1,2Delta cells increase spermine tolerance, implying Trk1,2p independent activity. Unexpectedly, trk1,2Deltasky1Delta and trk1,2Deltaptk2Delta cells display hypersensitivity to LiCl. These cells also show increased tolerance to low pH and improved growth in low K(+), as demonstrated for deletion of PMP3 in trk1,2Delta cells. We show that Sky1p and Pmp3p act in different pathways. Hypersensitivity to LiCl and improved growth in low K(+) are partly dependent on the Nha1p and Kha1p antiporters and on the Tok1p channel. Finally, Dhh1p, a RNA helicase was demonstrated to improve growth of trk1,2Deltasky1Delta cells in low K(+). Overexpression of Dhh1p improves the ability of trk1,2Delta cells to grow in low K(+) while dhh1Delta cells are sensitive to spermine and salt ions. A model that integrates these results to explain the mechanism of ion transport across the plasma membrane is proposed.     

TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae.

We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion.     

CgCYN1, a plasma membrane cystine-specific transporter of Candida glabrata with orthologues prevalent among pathogenic yeast and fungi

We describe a novel plasma membrane cystine transporter, CgCYN1, from Candida glabrata, the first such transporter to be described from yeast and fungi. C. glabrata met15Δ strains, organic sulfur auxotrophs, were observed to utilize cystine as a sulfur source, and this phenotype was exploited in the discovery of CgCYN1. Heterologous expression of CgCYN1 in Saccharomyces cerevisiae met15Δ strains conferred the ability of S. cerevisiae strains to grow on cystine. Deletion of the CgCYN1 ORF (CAGL0M00154g) in C. glabrata met15Δ strains caused abrogation of growth on cystine with growth being restored when CgCYN1 was reintroduced. The CgCYN1 protein belongs to the amino acid permease family of transporters, with no similarity to known plasma membrane cystine transporters of bacteria and humans, or lysosomal cystine transporters of humans/yeast. Kinetic studies revealed a K(m) of 18 ± 5 μM for cystine. Cystine uptake was inhibited by cystine, but not by other amino acids, including cysteine. The structurally similar cystathionine, lanthionine, and selenocystine alone inhibited transport, confirming that the transporter was specific for cystine. CgCYN1 localized to the plasma membrane and transport was energy-dependent. Functional orthologues could be demonstrated from other pathogenic yeast like Candida albicans and Histoplasma capsulatum, but were absent in Schizosaccharomyces pombe and S. cerevisiae.     

Cloning of two genes encoding potassium transporters in Neurospora crassa and expression of the corresponding cDNAs in Saccharomyces cerevisiae

Two Neurospora crassa genes, trk-1 and hak-1, encode K+ transporters that show sequence similarities to the TRK transporters described in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and to the HAK transporters described in Schwanniomyces occidentalis and barley. The N. crassa TRK1 and HAK1 transporters expressed by the corresponding cDNAs in a trk1 delta trk2 delta mutant of S. cerevisiae exhibited a high affinity for Rb+ and K+. Northern blot analysis and comparison of the kinetic characteristics of the two transporters in the trk1 delta trk2 delta mutant with the kinetic characteristics of K+ uptake in N. crassa cells allowed TRK1 to be identified as the dominant K+ transporter and HAK1 as a transporter that is only expressed when the cells are K+ starved. The HAK1 transporter showed a high concentrative capacity and is identified as the K(+)-H+ symporter described in N. crassa, whereas TRK1 might be a K+ uniporter. Although the co-existence of K+ transporters of the TRK and HAK types in the same species had not been reported formerly, we discuss whether this co-existence may be the normal situation in soil fungi.     

Immunity to K1 killer toxin: internal TOK1 blockade

K1 killer strains of Saccharomyces cerevisiae harbor RNA viruses that mediate secretion of K1, a protein toxin that kills virus-free cells. Recently, external K1 toxin was shown to directly activate TOK1 channels in the plasma membranes of sensitive yeast cells, leading to excess potassium flux and cell death. Here, a mechanism by which killer cells resist their own toxin is shown: internal toxin inhibits TOK1 channels and suppresses activation by external toxin.     

A potassium transporter of the yeast Schwanniomyces occidentalis homologous to the Kup system of Escherichia coli has a high concentrative capacity.

The yeast Schwanniomyces occidentalis has a high-affinity K+ uptake system with a high concentrative capacity, which is able to deplete the external K+ to < 0.03 microM. We have cloned the gene HAK1 of S.occidentalis which complements defective K+ uptake by trk1 and trk1 trk2 mutants of Saccharomyces cerevisiae. When HAK1 was expressed in a trk1 trk2 S.cerevisiae mutant, transport affinities for K+ and other alkali cations resembled those of S.occidentalis. The predicted amino acid sequence of the HAK1 protein shows significant homology with the hydrophobic region of the Kup transporter of Escherichia coli. In S.occidentalis HAK1 expresses in K(+)-limiting conditions. Our data indicate that in K(+)-starved cells the system encoded by HAK1 is the major K+ transporter of S.occidentalis.     

Individual functions of the HAK and TRK potassium transporters of Schwanniomyces occidentalis

We have cloned the gene encoding the TRK transporter of the soil yeast Schwanniomyces occidentalis and obtained the HAK1 trk1 delta and the hak1 delta TRK1 mutant strains. Analyses of the transport capacities of these mutants have shown that (i) the HAK1 and the TRK1 potassium transporters are the only transporters operating at low and medium K+ concentrations (< 1 mM); (ii) the HAK1 transporter is functional at low pH but fails at high pH; and (iii) the TRK1 transporter functions at neutral and high pH and fails at low pH. At neutral pH, both transporters are functional, but HAK1 is not expressed, except at very low K+ concentrations (< 50 microM) where HAK1 is very effective. TRK1 is also involved in the control of the membrane potential.     

Saccharomyces cerevisiae BY4741 and W303-1A laboratory strains differ in salt tolerance

Saccharomyces cerevisiae yeast cells serve as a model to elucidate the bases of salt tolerance and potassium homeostasis regulation in eukaryotic cells. In this study, we show that two widely used laboratory strains, BY4741 and W303-1A, differ not only in cell size and volume but also in their relative plasma-membrane potential (estimated with a potentiometric fluorescent dye diS-C3(3) and as Hygromycin B sensitivity) and tolerance to alkali-metal cations. W303-1A cells and their mutant derivatives lacking either uptake (trk1 trk2) or efflux (nha1) systems for alkali-metal cations are more tolerant to toxic sodium and lithium cations but also more sensitive to higher external concentrations of potassium than BY4741 cells and their mutants. Moreover, our results suggest that though the two strains do not differ in the total potassium content, the regulation of intracellular potassium homeostasis is probably not the same in BY4741 and W303-1A cells.     

Use of synchronously excited fluorescence to assess the accumulation of membrane potential probes in yeast cells

Evaluation of emission spectra of fluorescent probes used for the monitoring of membrane potential in microbial cells can be greatly facilitated by using synchronously excited spectroscopy (SES). This method permits the suppression of undesirable spectrum components (contributions due to scattered light or cell autofluorescence) and leads to considerable increase in monitored emission intensity and to narrowing of spectral peaks. It allows an efficient fractional decomposition of the probe fluorescence spectra into their free and bound dye fluorescence components. The usefulness of the method was tested by monitoring the accumulation of the fluorescent membrane potential probe diS-C3(3) in yeast cells, which serves as a qualitative measure of the membrane potential.