Molecular Microbiology (1999) 31(2), 511–520
Cloning of two genes encoding potassium transporters in Neurospora crassa and expression of the corresponding cDNAs in Saccharomyces cerevisiae Rosario Haro, Loreto Sainz, Francisco Rubio and Alonso Rodrı´guez-Navarro* Departamento de Biotecnologı´a, Escuela Te´cnica Superior de Ingenieros Agro´nomos, Universidad Polite´cnica de Madrid, 28040 Madrid, Spain. Summary Two Neurospora crassa genes, trk-1 and hak-1, encode K þ 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 trk1D trk2D mutant of S. cerevisiae exhibited a high affinity for Rbþ and K þ. Northern blot analysis and comparison of the kinetic characteristics of the two transporters in the trk1D trk2D mutant with the kinetic characteristics of K þ uptake in N. crassa cells allowed TRK1 to be identified as the dominant K þ transporter and HAK1 as a transporter that is only expressed when the cells are K þ starved. The HAK1 transporter showed a high concentrative capacity and is identified as the K þ –Hþ symporter described in N. crassa, whereas TRK1 might be a K þ uniporter. Although the co-existence of K þ 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. Introduction Terrestrial environments are normally dilute and highly variable in nutrient composition. Therefore, non-animal cells thriving in these environments are protected by a cell wall to support the turgor pressure and are equipped with a great diversity of concentrative transporters. K þ is the nutrient maintained at the highest concentration in the cells and one of the nutrients that requires being transported against the highest transmembrane concentration Received 5 August, 1998; revised 28 September, 1998; accepted 30 September, 1998. *For correspondence. E-mail
[email protected]; Tel. (91) 336 5751; Fax (91) 336 5757. Q 1999 Blackwell Science Ltd
gradients when the external medium is dilute. Possibly because of this and the variability of the K þ concentrations in different environments, different types of K þ transporters exist in bacteria (Bakker, 1993a). A similar diversity may also exist in plants and fungi, but in these organisms our knowledge is less comprehensive and needs to be extended. These studies are not only of biological interest but also of biotechnological use, especially in relation to plant nutrition and salt tolerance. In free-living, cell-walled eukaryotic cells and in plant roots, three different types of K þ transporters, TRK, HKT and HAK, and an inward-rectifying K þ channel have been reported. The TRK type of K þ transporters was first identified in Saccharomyces cerevisiae (Gaber et al., 1988), in which K þ uptake is mediated by two transporters of this type (Ko et al., 1990; Ko and Gaber, 1991). Two TRK K þ transporters are also present in Schizosaccharomyces pombe (Soldatenkov et al., 1995; Lichtenberg-Frate´ et al., 1996; GenBank/EMBL accession number 1351299), but in this case the existence of other types of K þ transporters has not yet been ruled out. The wheat HKT1 K þ –Naþ transporter (Schachtman and Schroeder, 1994; Rubio et al., 1995) is distantly related to the TRK type of K þ transporters (Schachtman and Schroeder, 1994; LichtenbergFrate´ et al., 1996) but is functionally different (Gassmann et al., 1996). The last type of K þ transporter, HAK, is related to K þ transporters in bacteria (Santa-Marı´a et al., 1997) and has been found in the soil yeast Schwanniomyces occidentalis (Ban˜uelos et al., 1995) and in plants (Quintero and Blatt, 1997; Santa-Marı´a et al., 1997; Fu and Luan, 1998; Kim et al., 1998). Isoforms of this type of transporter are encoded by a large family of genes in barley and Arabidopsis that may operate in many parts of the plants (Santa-Marı´a et al., 1997). Finally, an inwardrectifier K þ channel (Sentenac et al., 1992) mediates K þ uptake in Arabidopsis roots (Hirsch et al., 1998). Neurospora crassa is a model organism in which extensive genetic, physiological and electrophysiological research has been carried out for a long time (Perkins, 1992). It is also the cell-walled eukaryotic organism on which pioneering work on membrane potential (Slayman and Slayman, 1962), on the function of the Hþ pump ATPase (Slayman et al., 1973; Scarborough, 1976), and on the mechanism and electrophysiology of K þ uptake (Rodrı´guez-Navarro
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et al., 1986; Blatt et al., 1987) were performed. This background knowledge makes N. crassa an ideal model organism for molecular studies on K þ uptake. However, in contrast with the extensive work on the genetics of K þ transporters carried out on S. cerevisiae (Gaber et al., 1988; Ko et al., 1990; Ko and Gaber, 1991), no similar studies have been reported on N. crassa. Certainly, S. cerevisiae has significant advantages over any other organism for molecular studies, but this is not the only aspect to consider in the selection of a model organism. In part, the interest of the studies on K þ uptake in fungi is because they may help to understand K þ uptake in plant roots, and for this purpose S. cerevisiae may not be the best organism, because the environments where S. cerevisiae is found (Yarrow, 1984) are quite different from soil. In the search for a more convenient organism, K þ uptake has been studied in S. occidentalis, a soil yeast, to which most of the molecular techniques developed for S. cerevisiae can be applied (Klein and Favreau, 1988; Klein and Roof, 1988; Klein et al., 1989; Claros et al., 1993). These studies allowed the discovery of the HAK1 transporter (Ban˜uelos et al., 1995), which is not present in S. cerevisiae and may be a good representative of plant K þ transporters (Quintero and Blatt, 1997; Santa-Marı´a et al., 1997; Fu and Luan, 1998; Kim et al., 1998). In spite of this success, more extensive work on K þ uptake with S. occidentalis is difficult, because there is a lack of genetic and physiological background information on this organism, which is incidental to biological research. To the general reasons expressed above supporting the usefulness of N. crassa as a fungal model of K þ uptake in plants, two specific reasons can be added. First, K þ uptake in N. crassa exhibits kinetics almost identical to the extensively discussed dual-uptake isotherm found in plant roots (Epstein et al., 1963; Ramos and Rodrı´guez-Navarro, 1985; Rodrı´guez-Navarro and Ramos, 1986), suggesting that clarifying the molecular basis of this kinetics in N. crassa may shed light on long-standing questions about this type of kinetics in plants. Second, recent evidence points out that some K þ transporters are involved in the control of the membrane potential in S. cerevisiae (Madrid et al., 1998), a function that can only be properly investigated in organisms amenable to electrophysiological techniques, as is N. crassa (Slayman, 1965; Blatt et al., 1987). Here we report that two types of K þ transporters, TRK and HAK, co-exist in N. crassa. The kinetic characteristics of the HAK transporter and the pattern of expression of the hak-1 mRNA suggest that this transporter is the K þ –Hþ symporter described in N. crassa (Rodrı´guez-Navarro et al., 1986; Blatt et al., 1987), which operates only when the cells are exposed to very low K þ concentrations.
Results
Cloning of the trk-1 gene and expression of the trk-1 cDNA in S. cerevisiae By transforming a trk1D trk2D mutant of S. cerevisiae, which is strongly defective for K þ uptake (Gaber, 1992; Ramos et al., 1994), with a genomic DNA library of N. crassa and plating the transformants at low K þ, we isolated a plasmid (pLS5) that weakly suppressed the defect of the mutant. Further tests showed that pLS5 increased the Rbþ uptake capacity of the mutant, although this increase was very low. Sequence analysis of the insert in this plasmid showed the presence of an open reading frame which could encode a protein with a homology of 41% and 46% to ScTrk1p and ScTrk2p respectively. The study of the insert in plasmid pLS5 suggested that its weak suppresser effect on the trk1D trk2D mutant could be explained by expression difficulties of the heterologous gene, because the orientation of the open reading frame was opposite to the orientation of the PGK1 promoter present in the vector in which the library was constructed. Furthermore, upstream from the open reading frame we suspected the existence of an intron interrupting the coding region of the gene. Sequences at the 58 splice site and branchpoint of this putative intron were GTAAGT and ACTAACT, significantly different from those normally used by S. cerevisiae, GTATGT and TACTAAC respectively (Woolford, 1989). Therefore, to improve the expression of the transporter in S. cerevisiae, we obtained the corresponding cDNA by RT-PCR (reverse transcriptase polymerase chain reaction) and inserted it in the correct orientation in plasmid pYPGE15 (Brunelli and Pall, 1993), a yeast expression vector with the PGK1 promoter. Transformation of the resulting plasmid (pYP3) into the trk1D trk2D mutant produced a complete suppression of the defective growth of the mutant at low K þ. Sequence analyses of the cDNA obtained by RT-PCR and the DNA fragment in the original clone confirmed the presence of a gene containing a 53 bp intron, starting at position þ116, and that the gene could encode a 975amino-acid polypeptide with high homology to ScTrk1p and ScTrk2p. Therefore, we named the N. crassa gene isolated in plasmid pLS5 trk-1. ScTrk1p and ScTrk2p are highly homologous proteins for which the most significant difference is the length of a putative cytoplasmic loop of the polypeptide chain located between transmembrane fragments 3 and 4 (Ko and Gaber, 1991). The special sequence characteristics of this region (Gaber et al., 1988; Ko and Gaber, 1991) suggest that some of the functional differences between ScTrk1p and ScTrk2p may reside in it. As shown in Fig. 1, the putative K þ transporter of N. crassa showed that the polypeptide fragment located between transmembrane fragments 3 and 4 was the short type as in ScTrk2p. Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 511–520
Potassium transport genes in N. crassa 513 S. cerevisiae (R. Haro and A. Rodrı´guez-Navarro, unpublished), the trk-1 mRNA could not be detected. trk-1 encodes a high-affinity K þ transporter different from the one expressed by low K þ N. crassa cells
Fig. 1. Comparison of the hydrophobicity plots of the TRK1 (Sc1) and TRK2 (Sc2) transporters of S. cerevisiae and the TRK1 (Nc1) transporter of N. crassa. The hydrophobicity plots were generated by the DNASTRIDER1.4 program, using the algorithm of Kyte and Doolittle with 19 amino acid windows. In the plots of the TRK2 transporter of S. cerevisiae and the TRK1 in N. crassa three gaps have been introduced manually for a good coincidence of the profiles.
The significance of this fact and the significance of fragments with high sequence homology among the three proteins are difficult to evaluate because of the lack of structure–function studies for the TRK transporters. Southern blot analyses of total DNA at high and low stringency demonstrated that the isolated trk-1 gene was a N. crassa gene, and that very likely only one copy of the gene was present in the genome (Fig. 2). The expression of trk-1 mRNA was then studied by Northern hybridization analyses in N. crassa cells grown in different conditions. As previously found with the TRK2 mRNA of
Fig. 2. Southern blot analysis of total DNA from N. crassa probed with a DNA fragment of the trk-1 gene between positions þ1624 and þ2763 (Dra I– Sph I fragment in plasmid pYP3). Bam HI (B), Eco RI (E), Hin dIII (H) and Xba I (X) digested DNA (4 mg), fractionated, and transferred to a nylon membrane was hybridized at 428C in the presence of 50% formamide. Identical results were obtained at 20% formamide. Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 511–520
Consistent with the excellent growth at low K þ of the trk1D trk2D mutant transformed with pYP3, we found that this strain very actively depleted the K þ present in the growth medium, leaving a final concentration of 0.3 mM (see Fig. 6), which is a lower concentration than that left by wild S. cerevisiae strains (Rodrı´guez-Navarro and Ramos, 1984; Ban˜uelos et al., 1995). To investigate whether the N. crassa TRK1 transporter was functionally similar to the K þ transporters of S. cerevisiae, we performed an extensive kinetic study of Rbþ influx in K þ-starved cells of the trk1D trk2D mutant transformed with the N. crassa trk-1 cDNA. Analysis of the kinetic data at low Rbþ concentrations (Fig. 3A) showed that the N. crassa TRK1 transporter exhibited a 100 mM Rbþ K m and that low K þ concentrations competitively inhibited Rbþ influx (9 mM K þ K i ). It is worth observing that the endogenous Rbþ uptake of the trk1D trk2D mutant (Madrid et al., 1998) did not preclude the study of the N. crassa transporter, because the latter showed a much higher affinity (Fig. 3B). One characteristic of the kinetics of K þ and Rbþ influxes in S. cerevisiae is the deviation from Michaelis–Menten kinetics at low cation concentrations (below 10–20 mM K þ and 40–80 mM Rbþ ), at which the rates are lower than expected. These deviations occur because K þ and Rbþ, in addition to being transported, activate their own transport by binding an activation site (Borst-Pauwels, 1981). This kinetic behaviour can explain the failure of S. cerevisiae to take up K þ at concentrations below 2 mM (Rodrı´guez-Navarro and Ramos, 1984), assuming the absence of thermodynamic restrictions. To test whether the presence of an activation site was characteristic of all TRK transporters, we investigated its presence in the transporter encoded by trk-1, finding that, in this case, Rbþ influx did not deviate from Michaelis–Menten kinetics (not shown). Thus, permanent activation (lack of an activation site) of the N. crassa TRK1 transporter could determine that the steady state of the internal–external concentrations mediated by this transporter is closer to thermodynamic equilibrium than in S. cerevisiae. The most striking characteristic of K þ uptake both in S. cerevisiae and N. crassa is the adaptability of the K m values of the transporters, which vary depending on the K þ status of the cells and on the K þ content of the external medium (Ramos and Rodrı´guez-Navarro, 1985; 1986). However, this type of regulation was not shown by the N. crassa TRK1 transporter when expressed in the trk1D trk2D mutant. In this case, Rbþ influx in cells grown at different K þ concentrations or K þ starved for different periods
514
R. Haro, L. Sainz, F. Rubio and A. Rodrı´guez-Navarro Fig. 3. Kinetic analyses of Rbþ influx in trk1D trk2D cells transformed with the N. crassa trk-1 cDNA (plasmid pYP3). A. Competitive inhibition of Rbþ influx by K þ, tested up to 0.5 mM Rbþ. B. Eadie–Hofstee plot of Rbþ influx up to 50 mM Rbþ. Data in (A) were fitted without constraints to Michaelis–Menten functions, which showed that K þ was a competitive inhibitor of Rbþ influx. The Michaelis–Menten parameter values are Vmax ¼ 10 nmol mg¹1 min¹1, K m ¼ 100 mM Rbþ, K i ¼ 9 mM K þ.
of time always exhibited a high-affinity state, as shown in Fig. 3A. Although the excellent performance of the transporter encoded by trk-1 in the trk1D trk2D mutant at low K þ could explain the K þ uptake capacity of low K þ N. crassa cells, the notable difference between the Rbþ K m and K þ K i exhibited by this transporter (Fig. 3A) indicated that it was not the transporter mediating K þ uptake in these cells (K m ¼ 100 mM for Rbþ and K i ¼ 9 mM for K þ in this report, compared with K m ¼ 6 mM for Rbþ and K i ¼ 5 mM for K þ in Ramos and Rodrı´guez-Navarro, 1985). In contrast to the similarity between the Rbþ K m and K þ K i in N. crassa low K þ cells, normal K þ cells show a much higher Rbþ K m /K þ K i ratio (3:1 or 4:1 in Ramos and Rodrı´guez-Navarro, 1985). This suggested that the transporter encoded by trk-1 could contribute to K þ uptake in normal K þ cells, but that it could not make any significant contribution to the uptake in low K þ cells. Therefore, with the aim of cloning the gene encoding the second transporter in N. crassa, we did an extensive screening in our library, testing the complementation of the trk1D trk2D mutant. Unfortunately, this procedure did not produce any clone different from pLS5.
Cloning of the hak-1 gene Unlike the TRK transporters, the kinetics of Rbþ influx in HAK transporters show similar Rbþ K m and K þ K i values (Ban˜uelos et al., 1995; Santa-Marı´a et al., 1997). Therefore, the high-affinity K þ uptake exhibited by low K þ N. crassa cells could be mediated by a HAK transporter. Taking this as a starting hypothesis, we carried out RT-PCR amplifications on mRNA obtained from low K þ N. crassa cells, using oligonucleotides designed to amplify cDNA fragments encoding conserved regions of HAK transporters (Santa-Marı´a et al., 1997). By this procedure we isolated a 0.76 kb cDNA fragment whose translated sequence showed high homology to other HAK transporters. Using this fragment as a probe for colony hybridization in
the N. crassa library, we isolated plasmid pRH1.1, which contained the probe sequence. As expected from our previous screening with the N. crassa library, plasmid pRH1.1 did not suppress the K þ uptake deficiency of the trk1D trk2D mutant, although a putative hak-1 gene was complete in the insert. Therefore, as described above for the trk-1 gene, we cloned the corresponding cDNA by RT-PCR and inserted it in plasmid pYPGE15 (Brunelli and Pall, 1993). The resulting plasmid (pNH14.3) was then transformed into the trk1D trk2D mutant. Growth tests showed that the transformant strain grew vigorously at low K þ, indicating that this cDNA, which we named hak-1, encoded an efficient K þ transporter belonging to the HAK type. Analyses of the hak-1 gene and cDNA sequences revealed that the gene contained two introns of 91 and 49 bp, starting at positions þ435 and þ714 respectively, and a translated sequence of 861 amino acids, which showed high homology to other HAK-type K þ transporters (Table 1). Southern blot analysis of genomic N. crassa DNA, at high and low stringency, demonstrated that hak-1 was present in the genome of N. crassa, and suggested that other genes identical or homologous to hak-1 did not exist (Fig. 4). Northern blots of RNA obtained from N. crassa cells grown at high and low K þ detected the hak-1 transcripts only in K þ-starved cells (Fig. 5). Table 1. Binary comparison scores for members of the Kup-HAK family of proteins.a Proteinsb
AtKT1
HvHAK1
SoHAK1
Kup
NcHAK1 AtKT1 HvHAK1 SoHAK1
33 (44)
31 (45) 39 (50)
27 (38) 30 (42) 27 (39)
30 30 31 25
(41) (41) (45) (37)
a. Percentage of identity is given first and percentage of similarity is given in parenthesis. b. GenBank/EMBL accession numbers are: N. crassa HAK1, AJ009759; Arabidopsis thaliana KT1, AF012656; Hordeum vulgare HAK1, AF025292; S. occidentalis HAK1, U22945; Escherichia coli Kup, X68551. Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 511–520
Potassium transport genes in N. crassa 515
Fig. 6. K þ depletion of the external medium by trk1D trk2D cells transformed with the N. crassa hak-1 (plasmid pNH14.3) and trk-1 (plasmid pYP3) cDNAs. Both type of cells were grown in the arginine medium at 3 mM K þ and transferred to fresh medium containing 15 mM K þ. Fig. 4. Southern blot analysis of total DNA from N. crassa probed with a the 0.76 kb DNA fragment of the hak-1 gene obtained by RT-PCR (insert in plasmid pRH8.1, as described in Experimental procedures ). Bam HI (B), Eco RI (E), Hin dIII (H) and Xba I (X) digested DNA (4 mg), fractionated, and transferred to a nylon membrane was hybridized at 428C in the presence of 50% formamide. Identical results were obtained at 20% formamide.
hak-1 probably encodes the high-affinity K þ transporter of N. crassa cells As previously reported for the S. occidentalis HAK1 K þ transporter (Ban˜uelos et al., 1995), the transporter encoded by hak-1 showed an extremely high ability to deplete the external K þ. Thus, when K þ-starved cells of the trk1D trk2D mutant transformed with the hak-1 cDNA were suspended in an ammonium-free medium with low K þ, they exhausted the K þ, leaving less than 0.05 mM K þ. The same strain transformed with trk-1 left 0.3 mM (Fig. 6). As for the N. crassa TRK1 transporter, we made a kinetic
Fig. 5. Northern blot analysis of the hak-1 transcripts in N. crassa. Total RNA extracted from cells grown in the ammonium medium with 37 mM K þ (þK þ ) and 0.25 mM K þ (¹K þ ). A. Total RNA (10 mg) was fractionated, transferred to a nylon membrane, and probed with a 0.76 kb labelled antisense RNA (obtained from plasmid pRH8.1 as described in Experimental procedures ). The position of the ribosomal RNAs is indicated on the right. B. The filter was stripped and stained with 0.04% methylene blue in 0.5 M Naþ acetate (pH 5.2) as a loading control. Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 511–520
analysis of Rbþ influx in the trk1D trk2D mutant transformed with the hak-1 cDNA (Fig. 7), finding that the Rbþ K m (3 mM) and the K þ K i (3 mM) were almost coincident with the corresponding values observed in low K þ N. crassa cells (Ramos and Rodrı´guez-Navarro, 1985).
The N. crassa TRK1 and HAK1 transporters show low affinity for Na þ Naþ is taken up by N. crassa cells (Ortega and Rodrı´guezNavarro, 1986), and Naþ produces a pure competitive inhibition on Rbþ influx (Ramos and Rodrı´guez-Navarro, 1985). This suggested that if TRK1 and HAK1 mediated the main pathways of K þ and Rbþ uptake in N. crassa, they could be also the main pathways for the Naþ uptake detected in N. crassa cells. To address this possibility we studied the inhibition of Rbþ influx by Naþ in the trk1D trk2D mutant transformed with the trk-1 and hak-1 cDNAs. Unfortunately, the kinetics of Naþ influx mediated by the heterologous transporters cannot be studied in these transformants because the intrinsic Naþ uptake of the mutant is high and may vary when a heterologous transporter is expressed, if the expressed transporter affects the membrane potential of the transformant (Madrid et al., 1998). However, the inhibition of Rbþ influx by Naþ, which shows a pure competitive inhibition (Fig. 8), suggested that both the TRK1 and HAK1 transporters of N. crassa mediate Naþ uptake. In both cases, the Ki values were in the millimolar range (8 mM for TRK1 and 5 mM for HAK1). Discussion We have cloned the trk-1 and hak-1 genes of N. crassa, which encode two different types of K þ transporters. These types of transporters, TRK and HAK, have been found separately in different species of fungi, the former in S. cerevisiae (Gaber et al., 1988; Ko and Gaber,
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Fig. 7. Kinetic analyses of Rbþ influx in trk1D trk2D cells transformed with the N. crassa hak-1 cDNA (plasmid pNH14.3). Competitive inhibition of Rbþ influx by K þ, tested up to 25 mM Rbþ. Data were fitted without constraints to Michaelis–Menten functions, which showed that K þ was a competitive inhibitor of Rbþ influx. The Michaelis–Menten parameter values are Vmax ¼ 15 nmol mg¹1 min¹1, K m ¼ 3 mM Rbþ, K i ¼ 3 mM K þ.
1991) and S. pombe (Soldatenkov et al., 1995; Lichtenberg-Frate´ et al., 1996; GenBank/EMBL accession number 1351299), and the latter in S. occidentalis (Ban˜uelos et al., 1995), and now we demonstrate that they co-exist in N. crassa. Regarding the physiological significance of the co-existence of these two transporters, it is relevant that a gene encoding a TRK transporter has also been cloned in S. occidentalis (R. Madrid and A. Rodrı´guezNavarro, unpublished), the soil yeast in which the first HAK transporter was described (Ban˜uelos et al., 1995). This coincidence between N. crassa and S. occidentalis, the only two soil fungi in which the genes encoding the K þ transporters have been cloned, suggests that the co-existence of HAK and TRK transporters may be common among other soil fungi. This notion raises the question of whether the K þ uptake characteristics of S. cerevisiae, and possibly S. pombe, having two genes encoding TRK transporters, but lacking a HAK transporter, may be exceptional. Although the question cannot be answered, exceptionality is a likely possibility, because S. cerevisiae is adapted to colonize fruit environments with high sugar contents, which also present high K þ contents.
In contrast, the HAK transporters so far found in fungi are specialized for extremely low K þ concentrations that S. cerevisiae may never find in its habitat. Assuming that a HAK transporter is unnecessary in S. cerevisiae, it is interesting that the lack of a HAK gene is compensated by the presence of an additional TRK gene (Ko and Gaber, 1991). The existence of two TRK genes encoding very similar K þ transporters may fulfil the same function as the PMA1 and PMA2 genes encoding two Hþ pump ATPases (Schlesser et al., 1988). In both cases the second gene has a lower expression and encodes a protein with slightly different properties (Supply et al., 1993a,b; Ramos et al., 1994). For fungi thriving occasionally in environments with a very low concentration of K þ, a HAK transporter may represent an ecological advantage. Consistent with previous results in S. occidentalis (Ban˜uelos et al., 1995), we have found that the most striking characteristic of the N. crassa HAK1 K þ transporter is its capacity to mediate uptake at extremely low K þ concentrations (Fig. 6). However, at higher concentrations of K þ, a HAK transporter may not be more efficient than a TRK transporter, and possibly for that reason the mRNA of the HAK transporter is expressed only in cells that have suffered K þ starvation (Fig. 5, and Ban˜uelos et al., 1995). The proposal that two or more transporters with formally similar roles alternate their functions in any organism requires the identification of the transporters encoded by the cloned genes with the transporters expressed in the original organism. Although this can be done by comparing the kinetic constants, this approach poses difficulties that must be taken into consideration. In the present case, comparison of the K þ or Rbþ K m values in N. crassa cells and in trk1D trk2D S. cerevisiae cells expressing the transporter must be done with caution. The K ms of the K þ transporters vary within a wide range, both in S. cerevisiae (Rodrı´guez-Navarro and Ramos 1984; Ramos and Rodrı´guez-Navarro, 1986) and in N. crassa (Ramos and Rodrı´guez-Navarro 1985), and one of the factors regulating the K m , at least in low K þ N. crassa cells, is the membrane Fig. 8. Competitive inhibition of Rbþ influx by Naþ in trk1D trk2D cells transformed with the N. crassa hak-1 (plasmid pNH14.3) and trk-1 (plasmid pYP3) cDNAs. The double reciprocal plot shows the competitive inhibition of Rbþ influx at millimolar Naþ concentrations. The Naþ K i values were 5 mM and 8 mM respectively.
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Potassium transport genes in N. crassa 517 potential (Blatt et al., 1987). Because trk1D trk2D mutants are in a highly hyperpolarized state (Madrid et al., 1998), one should expect that only the lowest K m of the transporter would be expressed in trk1D trk2D cells. In the case of HAK1, which is expressed only in low K þ cells, the almost coincident Rbþ K m values found in N. crassa low K þ cells and in the N. crassa HAK1 transporter expressed in trk1D trk2D cells is consistent with the observation that the membrane potential of N. crassa low K þ cells almost saturates the response of the transporter (Blatt et al., 1987). Therefore, the Rbþ K m of the N. crassa HAK1 transporter expressed in trk1D trk2D cells cannot be much lower than the Rbþ K m exhibited by the low K þ N. crassa cells (3 mM in trk1D trk2D cells expressing HAK1, Fig. 7, versus 6 mM in low K þ N. crassa cells, Ramos and Rodrı´guez-Navarro, 1985). By contrast, the K m of TRK1 in trk1D trk2D cells could be much lower than in normal K þ cells of N. crassa, where it is expressed, because the membrane potential of normal K þ cells of N. crassa is considerably less negative than that of trk1D trk2D cells (Madrid et al., 1998). A more useful characteristic than the Rbþ K m values for distinguishing between HAK and TRK transporters is the difference between the Rbþ K m /K þ K i ratios exhibited by the two systems: in HAK transporters this ratio is close to 1 (Ban˜uelos et al., 1995; Santa-Marı´a et al., 1997) and much higher in TRK transporters (see data in Rodrı´guez-Navarro and Ramos, 1984, and consider that the system involved is ScTRK1, as shown in Gaber et al., 1988). Our proposal that in N. crassa the HAK1 transporter operates in low K þ cells and the TRK1 transporter in normal K þ cells is supported by the Rbþ K m /K þ K i ratios shown by these two transporters. In low K þ cells the ratio is close to 1 (Ramos and Rodrı´guez-Navarro, 1985), suggesting the presence of the HAK1 transporter, whereas cells grown at high K þ show a higher ratio (Ramos and Rodrı´guez-Navarro, 1985), which is a characteristic of the TRK transporters. A K þ –Hþ symport mediates K þ uptake in low K þ cells of N. crassa (Rodrı´guez-Navarro et al., 1986). Therefore, one conclusion of the above discussion is that hak-1 probably encodes this K þ –Hþ symporter. According to kinetic similarities and concentrative capacities, the same mechanism may operate in the HAK transporters of S. occidentalis (Ban˜uelos et al., 1995) and barley (Santa-Marı´a et al., 1997). Whether this mechanism operates in all transporters belonging to the Kup–HAK type is unknown at this moment. However, it is worth observing that the Kup system of E. coli also mediates ‘active’ transport (Bakker, 1993b). The Kup–HAK transporters have evolved considerably among bacteria, fungi and plants, and the amino acid sequences of the proteins show significant variability (Santa-Marı´a et al., 1997). However, the structure of the protein regarding the transmembrane segments and fragments connecting the transmembrane segments is well Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 511–520
conserved (Ban˜uelos et al., 1995). Therefore, an attractive hypothesis is that the mechanism has also been conserved, and that most of the Kup–HAK-type transporters are K þ –Hþ symporters. We have discussed the ecological advantage of an HAK1 transporter over a TRK transporter for fungi thriving in poor environments. The question now is why the TRK system is conserved together with the HAK1 transporter in N. crassa and why TRK1 is the dominant transporter in the fungal cells except when they are suffering K þ starvation. An explanation for the prevalence of the TRK1 transporter would be energy saving, assuming that the TRK1 transporter is a K þ uniporter, which is a likely possibility. Although this is contradictory with previous proposals regarding TRK transporters as K þ –Hþ symporters, the support for this hypothesis is insufficient. In S. cerevisiae, in which K þ uptake is mediated only by TRK transporters (Ko and Gaber, 1991; Ramos et al., 1994; Madrid et al., 1998), it has been considered that a K þ uniporter cannot mediate K þ uptake because the membrane potential is not negative enough to account for the internal– external ratio of the cation (Boxman et al., 1984). In that study, however, the reported membrane potential, ¹100 mV or more positive, was estimated from the distribution of the tetraphenylphosphonium cation, a method that may produce false results (Eraso et al., 1984). Furthermore, a membrane potential more negative than ¹300 mV has been demonstrated in a trk1.1 TRK2 strain expressing the Arabidopsis AKT1 inward rectifier K þ channel. This strain, in which K þ uptake is driven exclusively by the membrane potential, depletes the external K þ down to 0.65 mM (Sentenac et al., 1992). Even assuming that this result may be biased because the trk1 mutation produces hyperpolarization (Madrid et al., 1998), the effect cannot be very important because other results suggest that the membrane potentials in S. cerevisiae and N. crassa are similar (Madrid et al., 1998). This means a membrane potential of approximately ¹300 mV (Rodrı´guez-Navarro et al., 1986) for K þ-starved S. cerevisiae cells, potential that can account for an external concentration of 2 mM K þ, the lowest concentration of K þ that a wild-type S. cerevisiae strain leaves in the external medium (Rodrı´guez-Navarro and Ramos, 1984). Even in the case of the trk1D trk2D mutant expressing the N. crassa TRK1 transporter, which depleted the external K þ to 0.3 mM (Fig. 6), a membrane potential of ¹350 mV would account for an internal concentration of 150 mM, and this membrane potential may be normal in trk1D trk2D mutants (Madrid et al., 1998). In S. pombe, one of the two TRK K þ transporters has been expressed in a trk1D trk2D mutant of S. cerevisiae and studied by patch clamp in the whole-cell configuration. The currents mediated by SpTrkp are enhanced when the pH decreases from 7.5 to 5.5, and this has been taken as support for a
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K þ –Hþ symport mechanism (Lichtenberg-Frate´ et al., 1996). This enhancement, however, can also be explained by many different effects of the pH on the transporter, and is not sufficient to demonstrate a K þ –Hþ symport mechanism. Finally, fungal TRK transporters show homology (35%) to the TrkG and TrkH K þ transporters of E. coli and to other bacterial K þ transporters (Nakamura et al., 1998). Again, the mechanism involved in these transporters has been controversial. However, in bacteria, as already described for S. cerevisiae and S. pombe, the arguments in favour of a K þ –Hþ symport or any other ‘active’ mechanism for this type of transporters are far from sufficient (Bakker, 1993b). The cloning of the genes encoding the K þ transporters of N. crassa opens new technical possibilities for the rigorous study of many different plant and fungal K þ transporters. The disruption of the trk-1 and hak-1 genes is now in progress, and if the double disruptant is obtained and other K þ transporters do not exist, this mutant could be used to express genes encoding K þ transporters in the same way as S. cerevisiae trk1D trk2D mutants are now used, but making the electrophysiological study of the transporters possible.
Experimental procedures
Strains, media and growth conditions A S. cerevisiae trk1D trk2D mutant, WD3 (MAT a ade2 ura3 trp1 trk1D::LEU2 trk2D::HIS3 ) was constructed from strain W303-1A by the single-step gene disruption method (Rothstein, 1983). For TRK1 disruption, the 2.3 kb Xba I fragment of this gene was replaced by the LEU2 gene, and for the TRK2 disruption, the 1.4 kb Bst EII fragment was replaced by the HIS3 gene. Double disruptants were confirmed through Southern blot analyses. Yeast strains were grown in a mineral medium with ammonium or with arginine as the nitrogen source (Rodrı´guez-Navarro and Ramos, 1984). The basic medium contained neither K þ nor Naþ, which were added as indicated. Growth temperature was 288C. The N. crassa strain used in this work (74-ORF-1VA) was obtained from the Fungal Genetic Stock Center (FGSC 2489). General procedures for growing and handling the cells have been reported previously (Ramos and Rodrı´guez-Navarro, 1985; Rodrı´guez-Navarro et al., 1986). Briefly, 106 conidia per ml were inoculated in ammonium phosphate medium supplemented with either 0.25 mM or 37 mM KCl to prepare low or normal K þ cells respectively. The growth temperature was 288C. Escherichia coli DH5a (Gibco, BRL) was used in routine propagation of plasmids.
Recombinant DNA techniques Manipulation of nucleic acids was performed by standard protocols (Sambrook et al., 1989) or, when appropriate, according to the manufacturer’s instructions.
A N. crassa genomic DNA library was constructed in yeast plasmid YEp91, a derivative of plasmid YEp24 in which the Hin dIII fragment of plasmid pMA91 (Mellor et al., 1983), containing the PGK1 promoter and terminator regions, was substituted for the tetracycline resistance gene at the Sma I, Pvu II sites. N. crassa genomic DNA fragments ranging from 3 to 6 kb were obtained by partial digestion with Sau 3A and inserted into the Bgl II site of YEp91, generating 100 000 independent clones. For trk-1 isolation, the yeast WD3 mutant was electroporated (Becker and Guarente, 1991) with the genomic DNA N. crassa library. We obtained 30 000 Uraþ clones, seven of which grew in the arginine medium supplemented with 0.5 mM K þ. All these clones contained plasmids with identical inserts of 7.2 kb, and one of the plasmids, pLS5, was chosen for further study. A trk-1 cDNA was obtained by RT-PCR from low K þ cells RNA. For the reverse transcription, the FirstStrand cDNA Synthesis kit (Pharmacia Biotech) was used with an anchored Not I(dT)18 primer. The reverse transcription products were amplified by PCR with the Expand HighFidelity PCR system (Boehringer Mannheim) using a sense primer starting in the first ATG triplet of the open reading frame corresponding to the trk-1 gene and an antisense primer specific to the anchor. The N. crassa trk-1 cDNA was then inserted in the correct orientation in the Bam HI site of pYPGE15 (Brunelli and Pall, 1993), yielding plasmid pYP3. For hak-1 isolation, a 0.76 kb cDNA fragment was obtained from low K þ cells RNA by RT-PCR (plasmid pRH8.1), using primers deduced from conserved regions of the kup and HAK1 genes, as described previously (Santa-Marı´a et al., 1997). The PCR fragment was then used as a probe for screening the genomic DNA N. crassa library. Five positive clones were found to contain plasmids with inserts comprising DNA fragments identical to the probe. One of these plasmids, pRH1.1, was chosen for further study. A full-length hak-1 cDNA was obtained by RT-PCR from low-K þ cell RNA, using the procedure described for the trk-1 gene, using an anchored oligo-dT primer and two nested primers, the second starting in the first ATG triplet of the open reading frame corresponding to the hak-1 gene. This cDNA was cloned in the Eco RI site of pYPGE15, yielding plasmid pNH14.3. PCR reactions were performed in a Perkin-Elmer thermocycler and the PCR products were first cloned into the PCR2.1-Topo vector using the TOPO TA Cloning kit (Invitrogen). Sequencing was carried out by the dideoxy termination method described by Sanger (Sanger et al., 1977) modified as for the use with Sequenase (USB). The sequences of the trk-1 and hak-1 cDNAs were compared with the genomic sequences of the genes. There were no differences except at position 1259 of the hak-1 open reading frame, where there was a C in the cDNA and a T in the genomic sequence. This base change did not give rise to an amino acid change in the deduced polypeptide sequence. DNA sequence data for comparative analyses were obtained from BLAST (NCBI, Bethesda, MD) (Altschul et al., 1990). Protein alignments were performed using the PILEUP and GAP algorithms from the University of Wisconsin Computer Group. (Devereux et al., 1984, and updates). Note on gene nomemclature: we have used the conventional nomenclature for genes specific for each organism mentioned. Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 511–520
Potassium transport genes in N. crassa 519 For example, trk-1 refers to the N. crassa gene, TRK1 to the S. cerevisiae gene, and trk1D to a deletion mutant of the S. cerevisiae TRK1 gene.
Hybridization assays Colonies from the genomic DNA N. crassa library were immobilized on nylon membranes and screened using the 0.76 kb hak-1 fragment (see above) as a deoxygenin-labelled probe, according to the manufacturer’s instructions (Boehringer Mannhein). Membranes were hybridized in the presence of 50% formamide at 428C. Southern hybridization analyses were performed with DNA probes labelled with [a-32P]-dATP by the random priming method at 428C in the presence of 20% or 50% of formamide. For Northern blot analysis, total RNA was extracted from low and normal K þ cells of N. crassa, fractionated through formaldehyde gels and transferred to nylon membranes. The filters were hybridized with an RNA probe labelled with [a-32P]-UTP as indicated in the MAXIscript in Vitro Transcription Kit (Ambion), at 608C in the presence of 50% of formamide. Membranes were then washed at high-stringency conditions. Membranes were exposed at ¹708C to Curix RP-2 (Agfa) films.
Transport assays Yeast cells were grown in the arginine medium supplemented with either 30 mM KCl (for WD3) or 3 mM KCl (for WD3 transformed with the trk-1 or hak-1 cDNAs) and then starved of K þ for 5 h in K þ-free arginine medium. Cells were suspended in a 2% glucose and 10 mM MES buffer brought to pH 6.0 with Ca(OH)2 . At intervals after the addition of the cations, samples were taken, filtered through 0.8 mm pore nitrocellulose membrane filters (Millipore) and washed with 20 mM MgCl2 . Filters were incubated overnight in 0.1 M HCl. Rbþ was determined by atomic emission spectrophotometry of acid extracted cells (Rodrı´guez-Navarro and Ramos, 1984). The initial rates of Rbþ uptake were determined from the time courses of the cellular Rbþ content and reported, on a cell dry weight basis, as the means from at least four independent experiments.
Nucleotide accession numbers The GenBank/EMBL accession numbers for the trk-1 and hak-1 genes are AJ009758 and AJ009759 respectively.
Acknowledgements This work was supported by the European Commission DG XII Biotechnology Programme, contract number BIO4 CT960775, and by grant PB92–0907 from the Direccio´n General de Investigacio´n Cientı´fica y Te´cnica, Spain. F.R. is a postdoctoral fellow funded by Ministerio de Educacio´n y Cultura.
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