K⁺ fluxes in Schizosaccharomyces pombe

Abstract

All living cells accumulate high concentrations of K⁺ in order to keep themselves alive. To this end they have developed a great diversity of transporters. The internal level of K⁺ is the result of the net balance between the activities of the K⁺ influx and the K⁺ efflux transporters. Potassium fluxes have been extensively studied and characterized in Saccharomyces cerevisiae. However, this is not the case in the fission yeast and, in addition, the information available indicates that both yeasts present substantial and interesting differences. In this paper we have reviewed and summarized the information on K⁺ fluxes in Schizosaccharomyces pombe. We have included some unpublished results recently obtained in our laboratory and, in particular, we have highlighted the significant differences found between the well-known yeast S. cerevisiae and the fission yeast Sch. pombe.

Potassium transport, Schizosaccharomyces pombe, Yeast

1 Introduction

Potassium is the most abundant cation in living cells, playing a fundamental role in the maintenance of osmotic pressure and electrical neutrality. Potassium is also involved in the regulation of a variety of biological processes (intracellular pH, membrane potential). In order to fulfill these functions this cation is accumulated in the cells at concentrations several orders of magnitude higher than those found in the medium, although the levels of K⁺ in the different natural environments are highly divergent. This is also true in the case of yeasts, which can survive in the presence of very different concentrations of external K⁺ from low micromolar to molar levels. Yeast cells have developed powerful transport systems, tightly regulated in order to keep the internal concentrations of this cation quite constant, in a process known as K⁺ homeostasis. The coordinated functioning of the influx and efflux transporters is required to maintain the physiology of the cells and to avoid problems caused by lack or excess of K⁺.

Schizosaccharomyces pombe can be usually isolated from habitats such as arak mash, molasses, apples, grape juice or palm wine [1,2]. This yeast is type species of the genus Schizosaccharomyces, included in the family Schizosaccharomycetaceae, order Schizosaccharomycetales and phylum Ascomycota (‘Archiascomycetes’) [2]. From a scientific point of view, the fission yeast, Sch. pombe, is a powerful system in the study of numerous biological processes such as cell cycle, metabolism or regulatory cascades. The sequencing of the genome of Sch. pombe is a great advance in the study and comprehension of different physiological problems [3].

Saccharomyces cerevisiae probably is the best-known eukaryotic cell and is considered as a model organism. The information about K⁺ transport in S. cerevisiae is abundant. Influx and efflux of K⁺ in budding yeast have been studied from a biochemical and an electrophysiological point of view. The genes ending the transporters are well known [4] and, in addition, many factors affecting transport and regulatory proteins have also been studied. However, this is not the case in Sch. pombe since the information about K⁺ fluxes and their regulation is very fractional. Several papers describing aspects of K⁺ influx have been published but the information on K⁺ efflux and on the regulation of the transporters is scarce and we lack a complete understanding of the whole process. In this work we review the information available at this moment and try to present a clearer view of the state of the art.

2 K⁺ influx

Potassium influx in the fission yeast is acceptably well known. The process has been studied from both a biochemical and an electrophysiological viewpoint and, in addition, the genes involved in the uptake of K⁺ have been identified. Moreover, mutants in these genes have been obtained and further characterized.

In S. cerevisiae two TRK genes have been isolated and studied: ScTRK1 and ScTRK2. ScTRK1 is the most important gene involved in K⁺ transport. The trk1 strains require higher K⁺ concentrations to grow and have a clearly impaired K⁺ transport process [5,6]. ScTRK2 could be a defective copy of ScTRK1. Its role in wild-type cells is not clear since its transport activity has not been detected in the presence of ScTRK1[7,8]. In Sch. pombe, in 1964 Megnet has reported the isolation of mutants requiring a high concentration of K⁺ for growth [9]. The mutants were grouped into several classes according to the growth rates at different concentrations of external K⁺, but the nature of the lesions was not clarified. Thirty years later, Rothe and Höfer [10] defined the growth characteristics at different external K⁺ concentrations and the kinetics of K⁺ transport. They measured values of intracellular K⁺ in the range of 175–300 mM and identified a K⁺ transport process with a K_m for K⁺ of 0.4 mM and a V_max of 16 nmol K⁺ (mg dry wt)⁻¹min⁻¹.

By using Rb⁺ as a K⁺ analog we have shown that the characteristics of K⁺ influx greatly depend on the K⁺ status of the cell. In cells grown under non-limiting K⁺ concentrations the content of this cation was about 400 nmol (mg dry weight)⁻¹ of cells and the kinetics of Rb⁺ influx were monophasic with low affinity for the cation (Table 1). K⁺-starved cells of Sch. pombe can be obtained by suspending cells with a normal K⁺ content in K⁺-free medium for several hours. Under these conditions, the K⁺ content of the cells decreases to half the initial concentration and the kinetic characteristics of Rb⁺ transport drastically change. A biphasic pattern can be observed: a transport process saturated in the micromolar range, and a second phase with much lower affinity that works at high concentrations of Rb⁺ (Table 1) [11]. The regulation of transport by the internal K⁺ level has been previously reported in different living organisms and the existence of a high-affinity process of transport in K⁺-starved cells of S. cerevisiae has been extensively studied [12,13]. In plants [14] and fungi [15,16] biphasic kinetics for K⁺ transport have been described but this is not the case for yeasts. S. cerevisiae[12,13], Schwanniomyces occidentalis[17] and Debaryomyces hansenii[18] show monophasic K⁺ transport, and Sch. pombe is the only yeast reported so far with a biphasic pattern for Rb⁺ (K⁺) influx [11].

1

Kinetic parameters for Rb⁺ transport in a Sch. pombe wild-type strain

	K+-starved cells		Normal-K+ cells
	Micromolar range	Millimolar range
Vmax (nmol min−1 mg−1)	11	17	15
Km (mM)	0.25	6.8	35

Normal-K⁺ cells were obtained by growing the cells in a medium with high levels of the cation (mM). K⁺-starved cells were obtained by suspending normal-K⁺ cells in a medium without added K⁺ during 5 h.

The first gene ending a K⁺ transporter in Sch. pombe was reported in 1995 [19]. The gene was named Sptrk, since it shows high homology with other TRK genes previously isolated from yeasts ([20] and references therein). The Sptrk gene (here indicated as Sptrk1⁺) is located on chromosome I and encodes a protein of 833 amino acids, around 40% similar to S. cerevisiae TRK genes (Table 2). The heterologous expression of Sptrk1⁺ in a trk1 trk2 mutant strain of S. cerevisiae complemented both the growth defect and the K⁺ transport characteristics at low concentrations of the cation [21]. The same authors have also confirmed the presence of inward currents mediated by the Sptrk1⁺ gene product. On the basis of increased currents observed after acidification of the extracellular medium, a K⁺::H⁺ cotransport was proposed as the mechanism mediating K⁺ influx, and the protein encoded by the Sptrk1⁺ gene was named Tkh (for K⁺–H⁺ transporter) [21]. However, the observation (increased current upon acidification) would also be consistent with the existence of a K⁺ uniporter modulated by external pH. In fact, this is the case for inward-rectifying K⁺ channels in plants which show acid-induced current increase but work as uniporters [22,23]. The Sptrk1⁺ gene was disrupted and the mutant characterized [24]. Unlike the S. cerevisiae trk1 mutant, the Sch. pombe trk1 strain grows well at low K⁺ concentrations and does not show any K⁺ (Rb⁺) transport deficiency. In fact, the disruption of the Sptrk1⁺ gene only results in a slightly increased sensitivity to sodium ions [24]. These results have been considered as an indication of the existence of an additional efficient K⁺ transport system.

2

Some characteristics of the main K⁺ transporters identified in S. cerevisiae and Sch. pombe

	>Characteristic
Yeast	Gene	Length (bp)a	Introns (number)	Chromosome	Probable transmembrane domains	Molecular mass
S. cerevisiae	TRK1	3708	–	X	12	141
	TRK2	2670	–	XI	12	101
	TOK1	2076	–	X	8	77
	KHA1	2622	–	X	12	97
Sch. pombe	trk1+	2526	–	1	8	96
	trk2+	2643	12	1	8	100
	kha1+	2697	4	1	10	100
	cta3+	3114	–	2	9	115

a

The unspliced length of the trk2⁺ and kha1⁺ genes is 3377 and 2945 bp respectively.

A search of the databanks revealed the existence of a gene (Sptrk2⁺), located on chromosome I and homologous to Sptrk1⁺. This second gene contains multiple introns and encodes a protein of 880 amino acids, 34% identical to the Sptrk1⁺ gene product (Table 2). When Sptrk2⁺ was disrupted and the resulting strain characterized, the results were similar to those obtained with the trk1 mutant strain. The Sptrk2 mutant did not show increased K⁺ requirements and the kinetics of K⁺ (Rb⁺) transport were similar to those in the wild-type or in the single trk1 mutant [11]. These results indicate that, unlike S. cerevisiae, the presence of any of the Trk potassium transporters is enough to provide optimum growth and transport characteristics. However, non-published results obtained in our laboratory suggest that under several stress conditions, i.e. high external sodium or low pH, the presence of Trk1 confers tolerance to the stress factor more efficiently than Trk2.

Only the disruption of the two trk genes in Sch. pombe induced a clear K⁺ phenotype. Growth of the double mutant trk1 trk2 was severely affected at low K⁺ since this strain showed impaired K⁺ transport. The biphasic pattern for Rb⁺ transport changed and the high-affinity Rb⁺ transport basically disappeared. Other additional characteristics of the trk1 trk2 mutants are their sensitivity to Na⁺ ions, to hygromycin B and to acidic pH values. The double mutants can grow at a similar rate as the wild-type when the external K⁺ concentration is high enough (around 20 mM under standard conditions) and they are able to transport Rb⁺ with low affinity [11]. These results indicate that other transport systems are accepting the ion under such conditions. In S. cerevisiae, the characteristics of the transport process present in the double mutant do not significantly depend on the K⁺ status of the cells. It has been proposed that the transport system operating in the trk1trk2 strain could either be a non-selective cation channel [25], or be different non-specific transporters that could accept K⁺ under these limiting conditions [26,27]. In the case of Sch. pombe, we have observed that the remaining process of K⁺ transport present in the double mutant was also regulated by K⁺ starvation. As a consequence, the affinity for the cation increased with starvation (Fig. 1). A possible explanation for these results is the existence of a third K⁺ transporter, somehow regulated by the internal K⁺ content. This remaining K⁺ transporter would be significantly less efficient than Trk1 or Trk2. However, it is also possible that K⁺ uptake in the double mutant could be an ectopic process similar to the one described for S. cerevisiae[27], but tightly regulated by other processes affected by K⁺ starvation (internal pH or membrane potential) (Fig. 2). It is worth noting that the search of databanks did not confirm the existence of a third trk gene homologous to Sptrk1⁺ or Sptrk2⁺.

1

Double-reciprocal plot for Rb⁺ transport in normal-K⁺ cells and K⁺–starved cells of a Sch. pombe trk1 trk2 double mutant. Normal-K⁺ cells (squares) and K⁺-starved cells (triangles) were obtained as described in the legend of Table 1.

2

Putative K⁺ transporters in Sch. pombe. Putative K⁺ channel mediating K⁺ efflux (a). Potassium influx in the absence of Trk1 and Trk2 could be mediated by non-specific K⁺ transporters (b,c,d) or by a third K⁺ transporter not identified (e). See text for details.

3 K⁺ efflux

The K⁺ efflux process in Sch. pombe has not been studied sufficiently and unfortunately the information available is very fractional. K⁺ efflux systems must play an important role in regulating the K⁺ content, especially at high K⁺ concentrations in the external medium. The existence of a K⁺ efflux process in the fission yeast has already been reported in 1977 by Foury et al. [28]. These authors showed that in the presence of glucose, Dio-9 induced an efflux of K⁺. They proposed that the exit of K⁺ down the concentration gradient would be balanced by the uptake of protons or other cations in order to neutralize the electrical potential generated across the cell membrane by the exit of K⁺. Moreover, they proposed that the K⁺ efflux was not a secondary effect due to the inhibition of the plasma membrane ATPase by Dio-9, since the efflux could be observed under conditions where the plasma membrane ATPase was either non-functional (absence of glucose) or not inhibited (low Dio-9 concentration). More recently, we have shown that it is possible to observe K⁺ efflux from wild-type cells suspended in a medium without added K⁺. This is also true in the case of the mutant lacking trk1 and trk2 genes, which suggests that both transporters (Trk1 and Trk2) are not involved in the main route mediating the efflux of the cation [11].

The presence of a putative K⁺ channel in the fission yeast has been reported by Vacata et al. [29] and by Golubnitchaya-Labudová et al. [30]. The channel exhibits several conductances with a maximum of 153 pS. It was calculated that about 35 channels of this kind are present in the plasma membrane of a single Sch. pombe cell. The expression of this voltage-gated K⁺ channel is regulated by pH, K⁺ and cAMP, at the transcription level. The presence or absence of external Na⁺ only slightly altered the transcription of the channel [30]. Interestingly, this channel is not homologous to the ScTOK1, supposed to be the most important K⁺ efflux system in the budding yeast (Table 2). Apparently, Sch. pombe is not endowed with a system homologous to the main K⁺ channel in S. cerevisiae.

A Sch. pombe gene named cta3⁺ mediates a K⁺ efflux process. The gene product has been proposed to work as an ATP-dependent calcium pump [31]. However, more recent work has shown that the gene is induced by salt stress [32] and the heterologous expression in a S. cerevisiae mutant (ena1-4, nha1) suppressed the sensitivity to high K⁺ and induced a rapid K⁺ efflux [33].

An additional K⁺ efflux system is probably present in the plasma membrane of Sch. pombe, since the search of databanks indicates the existence of a gene homologous to ScKHA1. The corresponding protein works as a putative K⁺/H⁺ antiporter in the plasma membrane of S. cerevisiae[34]. ScKHA1 encodes a protein of 873 amino acids located on chromosome X while putative Spkha1⁺ is located on chromosome I and is slightly longer (898 amino acids). Both genes share 45.5% identity in 420 amino acids but no additional biochemical or physiological information on the role of the protein has been published (Fig. 2 and Table 2).

Finally, it is interesting to note that the main Na⁺ efflux system in the fission yeast works as a Na⁺/H⁺ antiporter, and does not recognize K⁺ as a substrate. Yeast plasma membrane Na⁺/H⁺ antiporters share a high degree of similarity at the protein level. In S. cerevisiae, the antiporter encoded by NHA1 has a broad substrate specificity and accepts Na⁺ or K⁺. However, the results obtained after the expression of the Sch. Pombe antiporter (encoded by the sod2⁺ gene) in a S. cerevisiae mutant lacking its endogenous Na⁺ efflux systems indicated that Sod2 is very specific for Na⁺[35].

4 Regulation of K⁺ transport

Yeast cells have to regulate K⁺ for homeostasis, to cope with the very different K⁺ concentrations they can find in natural habitats. For this purpose they have developed an especially tight regulation of K⁺ fluxes. In S. cerevisiae different factors involved in the regulation of K⁺ transport are well known. However, this is not the case in the fission yeast where, as in the case of K⁺ efflux, little information about factors or pathways regulating K⁺ fluxes is available.

It has been reported that the K⁺ content of the cells and the external pH are involved in the control of K⁺ fluxes in the fission yeast. We have shown that internal K⁺ directly or indirectly controls K⁺ influx. According to this idea, K⁺ starvation induces the appearance of the high-affinity Rb⁺ (K⁺) transport process [11]. In addition, as mentioned above, Lichtenberg-Fraté et al. have shown that acidification of the external medium increased the currents mediated by the trk1⁺ gene product [21].

On the other hand, two proteins involved in the regulation of K⁺ uptake have been described in Sch. pombe: Pzh1 and Tpr1. The Sppzh1⁺ gene encodes a 515-amino-acid protein highly homologous to the PPZ genes of S. cerevisiae. The gene product is a Ser/Thr protein phosphatase involved in salt tolerance and cation fluxes. Disruption of the gene renders the cells highly tolerant to Na⁺ or Li⁺, and hypersensitive to K⁺[36]. On the one hand, the increased tolerance to Na⁺ and Li⁺ is probably related to a decrease in the influx of these cations. On the other hand, the sensitivity to high K⁺ in the pzh1 mutants could be the consequence of a reduced K⁺ efflux [24]. The same report also showed that Trk1 does not mediate the effect of pzh1⁺ on Na⁺ or K⁺ tolerance [24]. The possible regulation of Trk2 by Pzh1 and its role in the tolerance to Na⁺ (and Li⁺) and in hypersensitivity to K⁺ has not been reported. From this point of view it would be of great interest to study salt tolerance in a double mutant trk2 pzh1 and in the triple mutant trk1 trk2 pzh1.

The second protein so far reported to be involved in the regulation of K⁺ uptake in Sch. pombe is the tpr1⁺ gene product. Tpr, for tetratrico peptide repeat, encodes a 1039-amino-acid protein with several reiterated Tpr units. The gene is homologous to the ScCTR9 and both genes could be part of a homologous and evolutionary conserved gene family. The tpr1⁺ gene was isolated as a suppressor of the S. cerevisiae trk1 trk2 double mutant phenotype but it does not seem to work as a transporter. The Tpr1 protein is indirectly involved in K⁺ transport. The analysis of its sequence and the putative binding proteins suggests that it mediates interspecies protein–protein interactions and thus may enhance K⁺ fluxes by altering non-specific transporters into K⁺ translocators [37].

More information is available on the regulation of the K⁺ efflux transporter Cta3. Curiously, some steps in the regulation of the corresponding gene have been studied before the real function of the product was clarified [32,33]. The Wisl1-Sty1 MAP kinase signaling pathway plays a role in salt stress response and regulates the expression of cta3⁺[32]; more recently Greenall et al. [38] have proposed a model for the regulation of cta3⁺ which is repressed by Tup11-12 and activated by Atf1-Pcr1 and Prr1.

5 Outlook

Sch. pombe is a model organism in the study of several physiological processes. In this paper we have summarized what is known on K⁺ fluxes in Sch. pombe and important differences with the well-studied S. cerevisiae have been emphasized. Electrophysiological, biochemical and genetic studies have been published in the last few years but more work is required to fully understand the characteristics of K⁺ transport in the fission yeast. In particular, questions such as the nature of the remaining K⁺ influx process in trk1 trk2 mutants or the gene and systems mediating K⁺ efflux should be clarified. In addition, little is known about the signaling pathways involved in the regulation of K⁺ transport. Hopefully, the recent sequencing of the Sch. pombe genome will be a great help for the different studies on this yeast. Finally, it is important to keep in mind that the significant differences found between Sch. pombe and S. cerevisiae strongly indicate that genetic studies have to be performed in parallel to physiological work.

Acknowledgements

Work in the laboratory of the authors was supported by grants PB98-1036 and BMC2002-0411 from the Ministerio de Ciencia y Tecnología and FEDER (Spain) to J.R.