cloning of two genes encoding potassium transporters in neurospora crassa and expression of the...

Cloning of two genes encoding potassium transportersin Neurospora crassa and expression of thecorresponding cDNAs in Saccharomyces cerevisiae

Rosario Haro, Loreto Sainz, Francisco Rubio andAlonso Rodrı ´guez-Navarro *

Departamento de Biotecnologıa, Escuela TecnicaSuperior de Ingenieros Agronomos, UniversidadPolitecnica de Madrid, 28040 Madrid, Spain.

Summary

Two Neurospora crassa genes, trk-1 and hak-1, encodeKþ transporters that show sequence similarities to theTRK transporters described in Saccharomyces cere-visiae and Schizosaccharomyces pombe , and to theHAK transporters described in Schwanniomyces occi-dentalis and barley. The N. crassa TRK1 and HAK1transporters expressed by the corresponding cDNAsin a trk1 D trk2 D mutant of S. cerevisiae exhibited ahigh affinity for Rb þ and K þ. Northern blot analysisand comparison of the kinetic characteristics of thetwo transporters in the trk1 D trk2 D mutant with thekinetic characteristics of K þ uptake in N. crassa cellsallowed TRK1 to be identified as the dominant K þ trans-porter and HAK1 as a transporter that is only expressedwhen the cells are K þ starved. The HAK1 transportershowed a high concentrative capacity and is identi-fied as the K þ–Hþ symporter described in N. crassa ,whereas TRK1 might be a K þ uniporter. Althoughthe co-existence of K þ transporters of the TRK andHAK types in the same species had not been reportedformerly, we discuss whether this co-existence maybe the normal situation in soil fungi.

Introduction

Terrestrial environments are normally dilute and highlyvariable in nutrient composition. Therefore, non-animalcells thriving in these environments are protected by acell wall to support the turgor pressure and are equippedwith a great diversity of concentrative transporters. Kþ isthe nutrient maintained at the highest concentration inthe cells and one of the nutrients that requires being trans-ported against the highest transmembrane concentration

gradients when the external medium is dilute. Possiblybecause of this and the variability of the Kþ concentrationsin different environments, different types of Kþ transportersexist in bacteria (Bakker, 1993a). A similar diversity mayalso exist in plants and fungi, but in these organismsour knowledge is less comprehensive and needs to beextended. These studies are not only of biological interestbut also of biotechnological use, especially in relation toplant nutrition and salt tolerance.

In free-living, cell-walled eukaryotic cells and in plantroots, three different types of Kþ transporters, TRK, HKTand HAK, and an inward-rectifying Kþ channel have beenreported. The TRK type of Kþ transporters was first iden-tified in Saccharomyces cerevisiae (Gaber et al., 1988), inwhich Kþ uptake is mediated by two transporters of thistype (Ko et al., 1990; Ko and Gaber, 1991). Two TRKKþ transporters are also present in Schizosaccharomycespombe (Soldatenkov et al., 1995; Lichtenberg-Frate et al.,1996; GenBank/EMBL accession number 1351299), butin this case the existence of other types of Kþ transportershas not yet been ruled out. The wheat HKT1 Kþ–Naþ

transporter (Schachtman and Schroeder, 1994; Rubio etal., 1995) is distantly related to the TRK type of Kþ trans-porters (Schachtman and Schroeder, 1994; Lichtenberg-Frate et al., 1996) but is functionally different (Gassmannet al., 1996). The last type of Kþ transporter, HAK, isrelated to Kþ transporters in bacteria (Santa-Marıa etal., 1997) and has been found in the soil yeast Schwannio-myces occidentalis (Banuelos et al., 1995) and in plants(Quintero and Blatt, 1997; Santa-Marıa et al., 1997; Fuand Luan, 1998; Kim et al., 1998). Isoforms of this typeof transporter are encoded by a large family of genes inbarley and Arabidopsis that may operate in many partsof the plants (Santa-Marıa et al., 1997). Finally, an inward-rectifier Kþ channel (Sentenac et al., 1992) mediates Kþ

uptake in Arabidopsis roots (Hirsch et al., 1998).Neurospora crassa is a model organism in which exten-

sive genetic, physiological and electrophysiological researchhas been carried out for a long time (Perkins, 1992). It isalso the cell-walled eukaryotic organism on which pioneer-ing work on membrane potential (Slayman and Slayman,1962), on the function of the Hþ pump ATPase (Slaymanet al., 1973; Scarborough, 1976), and on the mechanismand electrophysiology of Kþ uptake (Rodrıguez-Navarro

Molecular Microbiology (1999) 31(2), 511–520

Q 1999 Blackwell Science Ltd

Received 5 August, 1998; revised 28 September, 1998; accepted 30September, 1998. *For correspondence. E-mail [email protected]; Tel. (91) 336 5751; Fax (91) 336 5757.

et al., 1986; Blatt et al., 1987) were performed. Thisbackground knowledge makes N. crassa an ideal modelorganism for molecular studies on Kþ uptake. However,in contrast with the extensive work on the genetics of Kþ

transporters carried out on S. cerevisiae (Gaber et al.,1988; Ko et al., 1990; Ko and Gaber, 1991), no similarstudies have been reported on N. crassa. Certainly, S.cerevisiae has significant advantages over any otherorganism for molecular studies, but this is not the onlyaspect to consider in the selection of a model organism.In part, the interest of the studies on Kþ uptake in fungiis because they may help to understand Kþ uptake inplant roots, and for this purpose S. cerevisiae may notbe the best organism, because the environments whereS. cerevisiae is found (Yarrow, 1984) are quite differentfrom soil. In the search for a more convenient organism,Kþ uptake has been studied in S. occidentalis, a soilyeast, to which most of the molecular techniques deve-loped for S. cerevisiae can be applied (Klein and Favreau,1988; Klein and Roof, 1988; Klein et al., 1989; Claros etal., 1993). These studies allowed the discovery of theHAK1 transporter (Banuelos et al., 1995), which is notpresent in S. cerevisiae and may be a good represen-tative of plant Kþ transporters (Quintero and Blatt, 1997;Santa-Marıa et al., 1997; Fu and Luan, 1998; Kim et al.,1998). In spite of this success, more extensive work onKþ uptake with S. occidentalis is difficult, because thereis a lack of genetic and physiological background infor-mation on this organism, which is incidental to biologicalresearch.

To the general reasons expressed above supporting theusefulness of N. crassa as a fungal model of Kþ uptake inplants, two specific reasons can be added. First, Kþ uptakein N. crassa exhibits kinetics almost identical to the exten-sively discussed dual-uptake isotherm found in plant roots(Epstein et al., 1963; Ramos and Rodrıguez-Navarro,1985; Rodrıguez-Navarro and Ramos, 1986), suggestingthat clarifying the molecular basis of this kinetics in N.crassa may shed light on long-standing questions aboutthis type of kinetics in plants. Second, recent evidencepoints out that some Kþ transporters are involved in thecontrol of the membrane potential in S. cerevisiae (Madridet al., 1998), a function that can only be properly inves-tigated in organisms amenable to electrophysiologicaltechniques, as is N. crassa (Slayman, 1965; Blatt et al.,1987).

Here we report that two types of Kþ transporters, TRKand HAK, co-exist in N. crassa. The kinetic characteris-tics of the HAK transporter and the pattern of expres-sion of the hak-1 mRNA suggest that this transporteris the Kþ–Hþ symporter described in N. crassa (Rodrı-guez-Navarro et al., 1986; Blatt et al., 1987), whichoperates only when the cells are exposed to very low Kþ

concentrations.

Results

Cloning of the trk-1 gene and expression of the trk-1cDNA in S. cerevisiae

By transforming a trk1D trk2D mutant of S. cerevisiae,which is strongly defective for Kþ uptake (Gaber, 1992;Ramos et al., 1994), with a genomic DNA library of N.crassa and plating the transformants at low Kþ, we iso-lated a plasmid (pLS5) that weakly suppressed the defectof the mutant. Further tests showed that pLS5 increasedthe Rbþ uptake capacity of the mutant, although thisincrease was very low. Sequence analysis of the insertin this plasmid showed the presence of an open readingframe which could encode a protein with a homology of41% and 46% to ScTrk1p and ScTrk2p respectively.

The study of the insert in plasmid pLS5 suggested thatits weak suppresser effect on the trk1D trk2D mutant couldbe explained by expression difficulties of the heterologousgene, because the orientation of the open reading framewas opposite to the orientation of the PGK1 promoter pre-sent in the vector in which the library was constructed.Furthermore, upstream from the open reading frame wesuspected the existence of an intron interrupting thecoding region of the gene. Sequences at the 58 splicesite and branchpoint of this putative intron were GTAAGTand ACTAACT, significantly different from those normallyused by S. cerevisiae, GTATGT and TACTAAC respec-tively (Woolford, 1989). Therefore, to improve the expres-sion of the transporter in S. cerevisiae, we obtained thecorresponding cDNA by RT-PCR (reverse transcriptasepolymerase chain reaction) and inserted it in the correctorientation in plasmid pYPGE15 (Brunelli and Pall, 1993),a yeast expression vector with the PGK1 promoter. Trans-formation of the resulting plasmid (pYP3) into the trk1D

trk2D mutant produced a complete suppression of thedefective growth of the mutant at low Kþ.

Sequence analyses of the cDNA obtained by RT-PCRand the DNA fragment in the original clone confirmed thepresence of a gene containing a 53 bp intron, starting atposition þ116, and that the gene could encode a 975-amino-acid polypeptide with high homology to ScTrk1pand ScTrk2p. Therefore, we named the N. crassa geneisolated in plasmid pLS5 trk-1.

ScTrk1p and ScTrk2p are highly homologous proteinsfor which the most significant difference is the length ofa putative cytoplasmic loop of the polypeptide chainlocated between transmembrane fragments 3 and 4 (Koand Gaber, 1991). The special sequence characteristicsof this region (Gaber et al., 1988; Ko and Gaber, 1991)suggest that some of the functional differences betweenScTrk1p and ScTrk2p may reside in it. As shown inFig. 1, the putative Kþ transporter of N. crassa showedthat the polypeptide fragment located between transmem-brane fragments 3 and 4 was the short type as in ScTrk2p.

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 511–520

512 R. Haro, L. Sainz, F. Rubio and A. Rodrıguez-Navarro

The significance of this fact and the significance of frag-ments with high sequence homology among the three pro-teins are difficult to evaluate because of the lack ofstructure–function studies for the TRK transporters.

Southern blot analyses of total DNA at high and lowstringency demonstrated that the isolated trk-1 gene wasa N. crassa gene, and that very likely only one copy ofthe gene was present in the genome (Fig. 2). The expres-sion of trk-1 mRNA was then studied by Northern hybrid-ization analyses in N. crassa cells grown in differentconditions. As previously found with the TRK2 mRNA of

S. cerevisiae (R. Haro and A. Rodrıguez-Navarro, unpub-lished), the trk-1 mRNA could not be detected.

trk-1 encodes a high-affinity K þ transporter differentfrom the one expressed by low K þ N. crassa cells

Consistent with the excellent growth at low Kþ of the trk1D

trk2D mutant transformed with pYP3, we found that thisstrain very actively depleted the Kþ present in the growthmedium, leaving a final concentration of 0.3 mM (seeFig. 6), which is a lower concentration than that left bywild S. cerevisiae strains (Rodrıguez-Navarro and Ramos,1984; Banuelos et al., 1995). To investigate whether theN. crassa TRK1 transporter was functionally similar to theKþ transporters of S. cerevisiae, we performed an exten-sive kinetic study of Rbþ influx in Kþ-starved cells of thetrk1D trk2D mutant transformed with the N. crassa trk-1cDNA. Analysis of the kinetic data at low Rbþ concen-trations (Fig. 3A) showed that the N. crassa TRK1 trans-porter exhibited a 100 mM Rbþ Km and that low Kþ

concentrations competitively inhibited Rbþ influx (9 mMKþ Ki). 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 Kþ and Rbþ influxesin S. cerevisiae is the deviation from Michaelis–Mentenkinetics at low cation concentrations (below 10–20 mMKþ and 40–80 mM Rbþ), at which the rates are lowerthan expected. These deviations occur because Kþ andRbþ, in addition to being transported, activate their owntransport by binding an activation site (Borst-Pauwels,1981). This kinetic behaviour can explain the failure of S.cerevisiae to take up Kþ at concentrations below 2 mM(Rodrıguez-Navarro and Ramos, 1984), assuming theabsence of thermodynamic restrictions. To test whetherthe presence of an activation site was characteristic ofall TRK transporters, we investigated its presence in thetransporter 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 activa-tion site) of the N. crassa TRK1 transporter could deter-mine that the steady state of the internal–externalconcentrations mediated by this transporter is closer tothermodynamic equilibrium than in S. cerevisiae.

The most striking characteristic of Kþ uptake both in S.cerevisiae and N. crassa is the adaptability of the Km

values of the transporters, which vary depending on theKþ status of the cells and on the Kþ content of the externalmedium (Ramos and Rodrıguez-Navarro, 1985; 1986).However, this type of regulation was not shown by theN. crassa TRK1 transporter when expressed in the trk1D

trk2D mutant. In this case, Rbþ influx in cells grown at differ-ent Kþ concentrations or Kþ starved for different periods


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 generatedby the DNASTRIDER1.4 program, using the algorithm of Kyte andDoolittle with 19 amino acid windows. In the plots of the TRK2transporter of S. cerevisiae and the TRK1 in N. crassa three gapshave been introduced manually for a good coincidence of theprofiles.

Fig. 2. Southern blot analysis of total DNA from N. crassa probedwith a DNA fragment of the trk-1 gene between positions þ1624and þ2763 (DraI–SphI fragment in plasmid pYP3). BamHI (B),EcoRI (E), HindIII (H) and XbaI (X) digested DNA (4 mg),fractionated, and transferred to a nylon membrane was hybridizedat 428C in the presence of 50% formamide. Identical results wereobtained at 20% formamide.

Potassium transport genes in N. crassa 513

of time always exhibited a high-affinity state, as shown inFig. 3A.

Although the excellent performance of the transporterencoded by trk-1 in the trk1D trk2D mutant at low Kþ

could explain the Kþ uptake capacity of low Kþ N. crassacells, the notable difference between the Rbþ Km and Kþ

Ki exhibited by this transporter (Fig. 3A) indicated that itwas not the transporter mediating Kþ uptake in thesecells (Km ¼ 100 mM for Rbþ and Ki ¼ 9 mM for Kþ in thisreport, compared with Km ¼ 6 mM for Rbþ and Ki ¼ 5 mMfor Kþ in Ramos and Rodrıguez-Navarro, 1985). In con-trast to the similarity between the Rbþ Km and Kþ Ki inN. crassa low Kþ cells, normal Kþ cells show a muchhigher Rbþ Km/Kþ Ki ratio (3:1 or 4:1 in Ramos and Rodrı-guez-Navarro, 1985). This suggested that the transporterencoded by trk-1 could contribute to Kþ uptake in normalKþ cells, but that it could not make any significant contribu-tion to the uptake in low Kþ cells. Therefore, with the aim ofcloning the gene encoding the second transporter in N.crassa, we did an extensive screening in our library, test-ing the complementation of the trk1D trk2D mutant. Unfor-tunately, this procedure did not produce any clone differentfrom pLS5.

Cloning of the hak-1 gene

Unlike the TRK transporters, the kinetics of Rbþ influx inHAK transporters show similar Rbþ Km and Kþ Ki values(Banuelos et al., 1995; Santa-Marıa et al., 1997). There-fore, the high-affinity Kþ uptake exhibited by low Kþ N.crassa cells could be mediated by a HAK transporter. Tak-ing this as a starting hypothesis, we carried out RT-PCRamplifications on mRNA obtained from low Kþ N. crassacells, using oligonucleotides designed to amplify cDNAfragments encoding conserved regions of HAK trans-porters (Santa-Marıa et al., 1997). By this procedure weisolated a 0.76 kb cDNA fragment whose translated sequ-ence 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, whichcontained the probe sequence.

As expected from our previous screening with the N.crassa library, plasmid pRH1.1 did not suppress the Kþ

uptake deficiency of the trk1D trk2D mutant, although aputative hak-1 gene was complete in the insert. Therefore,as described above for the trk-1 gene, we cloned the cor-responding cDNA by RT-PCR and inserted it in plasmidpYPGE15 (Brunelli and Pall, 1993). The resulting plasmid(pNH14.3) was then transformed into the trk1D trk2D

mutant. Growth tests showed that the transformant straingrew vigorously at low Kþ, indicating that this cDNA, whichwe named hak-1, encoded an efficient Kþ transporterbelonging to the HAK type. Analyses of the hak-1 geneand cDNA sequences revealed that the gene containedtwo introns of 91 and 49 bp, starting at positions þ435and þ714 respectively, and a translated sequence of861 amino acids, which showed high homology to otherHAK-type Kþ transporters (Table 1).

Southern blot analysis of genomic N. crassa DNA, athigh and low stringency, demonstrated that hak-1 waspresent in the genome of N. crassa, and suggested thatother genes identical or homologous to hak-1 did not exist(Fig. 4). Northern blots of RNA obtained from N. crassacells grown at high and low Kþ detected the hak-1 tran-scripts only in Kþ-starved cells (Fig. 5).


Fig. 3. Kinetic analyses of Rbþ influx in trk1Dtrk2D cells transformed with the N. crassatrk-1 cDNA (plasmid pYP3).A. Competitive inhibition of Rbþ influx by Kþ,tested up to 0.5 mM Rbþ.B. Eadie–Hofstee plot of Rbþ influx up to50 mM Rbþ. Data in (A) were fitted withoutconstraints to Michaelis–Menten functions,which showed that Kþ was a competitiveinhibitor of Rbþ influx. The Michaelis–Mentenparameter values areVmax ¼ 10 nmol mg¹1 min¹1, Km ¼ 100 mMRbþ, Ki ¼ 9 mM Kþ.

Table 1. Binary comparison scores for members of the Kup-HAKfamily of proteins.a

Proteinsb AtKT1 HvHAK1 SoHAK1 Kup

NcHAK1 33 (44) 31 (45) 27 (38) 30 (41)AtKT1 39 (50) 30 (42) 30 (41)HvHAK1 27 (39) 31 (45)SoHAK1 25 (37)

a. Percentage of identity is given first and percentage of similarity isgiven in parenthesis.b. GenBank/EMBL accession numbers are: N. crassa HAK1,AJ009759; Arabidopsis thaliana KT1, AF012656; Hordeum vulgareHAK1, AF025292; S. occidentalis HAK1, U22945; Escherichia coliKup, X68551.


hak-1 probably encodes the high-affinity K þ transporterof N. crassa cells

As previously reported for the S. occidentalis HAK1 Kþ

transporter (Banuelos et al., 1995), the transporter encodedby hak-1 showed an extremely high ability to deplete theexternal Kþ. Thus, when Kþ-starved cells of the trk1D

trk2D mutant transformed with the hak-1 cDNA were sus-pended in an ammonium-free medium with low Kþ, theyexhausted the Kþ, leaving less than 0.05 mM Kþ. Thesame strain transformed with trk-1 left 0.3 mM (Fig. 6).As for the N. crassa TRK1 transporter, we made a kinetic

analysis of Rbþ influx in the trk1D trk2D mutant trans-formed with the hak-1 cDNA (Fig. 7), finding that theRbþ Km (3 mM) and the Kþ Ki (3 mM) were almost coinci-dent with the corresponding values observed in low Kþ

N. crassa cells (Ramos and Rodrıguez-Navarro, 1985).

The N. crassa TRK1 and HAK1 transporters show lowaffinity for Naþ

Naþ is taken up by N. crassa cells (Ortega and Rodrıguez-Navarro, 1986), and Naþ produces a pure competitiveinhibition on Rbþ influx (Ramos and Rodrıguez-Navarro,1985). This suggested that if TRK1 and HAK1 mediatedthe main pathways of Kþ and Rbþ uptake in N. crassa,they could be also the main pathways for the Naþ uptakedetected in N. crassa cells. To address this possibility westudied 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 theheterologous transporters cannot be studied in thesetransformants because the intrinsic Naþ uptake of themutant is high and may vary when a heterologous trans-porter is expressed, if the expressed transporter affectsthe membrane potential of the transformant (Madrid etal., 1998). However, the inhibition of Rbþ influx by Naþ,which shows a pure competitive inhibition (Fig. 8), sug-gested that both the TRK1 and HAK1 transporters of N.crassa mediate Naþ uptake. In both cases, the Ki valueswere in the millimolar range (8 mM for TRK1 and 5 mMfor HAK1).

Discussion

We have cloned the trk-1 and hak-1 genes of N. crassa,which encode two different types of Kþ transporters.These types of transporters, TRK and HAK, have beenfound separately in different species of fungi, the formerin S. cerevisiae (Gaber et al., 1988; Ko and Gaber,


Fig. 4. Southern blot analysis of total DNA from N. crassa probedwith a the 0.76 kb DNA fragment of the hak-1 gene obtained byRT-PCR (insert in plasmid pRH8.1, as described in Experimentalprocedures). BamHI (B), EcoRI (E), HindIII (H) and XbaI (X)digested DNA (4 mg), fractionated, and transferred to a nylonmembrane was hybridized at 428C in the presence of 50%formamide. Identical results were obtained at 20% formamide.

Fig. 5. Northern blot analysis of the hak-1 transcripts in N. crassa.Total RNA extracted from cells grown in the ammonium mediumwith 37 mM Kþ (þKþ ) and 0.25 mM Kþ (¹Kþ ).A. Total RNA (10 mg) was fractionated, transferred to a nylonmembrane, and probed with a 0.76 kb labelled antisense RNA(obtained from plasmid pRH8.1 as described in Experimentalprocedures). The position of the ribosomal RNAs is indicated onthe right.B. The filter was stripped and stained with 0.04% methylene bluein 0.5 M Naþ acetate (pH 5.2) as a loading control.

Fig. 6. Kþ depletion of the external medium by trk1D trk2D cellstransformed with the N. crassa hak-1 (plasmid pNH14.3) and trk-1(plasmid pYP3) cDNAs. Both type of cells were grown in thearginine medium at 3 mM Kþ and transferred to fresh mediumcontaining 15 mM Kþ.


1991) and S. pombe (Soldatenkov et al., 1995; Lichten-berg-Frate et al., 1996; GenBank/EMBL accession num-ber 1351299), and the latter in S. occidentalis (Banueloset al., 1995), and now we demonstrate that they co-existin N. crassa. Regarding the physiological significance ofthe co-existence of these two transporters, it is relevantthat a gene encoding a TRK transporter has also beencloned in S. occidentalis (R. Madrid and A. Rodrıguez-Navarro, unpublished), the soil yeast in which the firstHAK transporter was described (Banuelos et al., 1995).This coincidence between N. crassa and S. occidentalis,the only two soil fungi in which the genes encoding theKþ transporters have been cloned, suggests that theco-existence of HAK and TRK transporters may be com-mon among other soil fungi. This notion raises the ques-tion of whether the Kþ uptake characteristics of S.cerevisiae, and possibly S. pombe, having two genesencoding TRK transporters, but lacking a HAK trans-porter, may be exceptional. Although the question cannotbe answered, exceptionality is a likely possibility, becauseS. cerevisiae is adapted to colonize fruit environments withhigh sugar contents, which also present high Kþ contents.

In contrast, the HAK transporters so far found in fungi arespecialized for extremely low Kþ 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 iscompensated by the presence of an additional TRK gene(Ko and Gaber, 1991). The existence of two TRK genesencoding very similar Kþ transporters may fulfil the samefunction as the PMA1 and PMA2 genes encoding two Hþ

pump ATPases (Schlesser et al., 1988). In both cases thesecond gene has a lower expression and encodes a proteinwith slightly different properties (Supply et al., 1993a,b;Ramos et al., 1994).

For fungi thriving occasionally in environments with avery low concentration of Kþ, a HAK transporter mayrepresent an ecological advantage. Consistent with pre-vious results in S. occidentalis (Banuelos et al., 1995),we have found that the most striking characteristic of theN. crassa HAK1 Kþ transporter is its capacity to mediateuptake at extremely low Kþ concentrations (Fig. 6). How-ever, at higher concentrations of Kþ, a HAK transportermay not be more efficient than a TRK transporter, and pos-sibly for that reason the mRNA of the HAK transporter isexpressed only in cells that have suffered Kþ starvation(Fig. 5, and Banuelos et al., 1995).

The proposal that two or more transporters with formallysimilar roles alternate their functions in any organismrequires the identification of the transporters encoded bythe cloned genes with the transporters expressed in theoriginal organism. Although this can be done by compar-ing the kinetic constants, this approach poses difficultiesthat must be taken into consideration. In the presentcase, comparison of the Kþ or Rbþ Km values in N. crassacells and in trk1D trk2D S. cerevisiae cells expressing thetransporter must be done with caution. The Kms of the Kþ

transporters vary within a wide range, both in S. cere-visiae (Rodrıguez-Navarro and Ramos 1984; Ramos andRodrıguez-Navarro, 1986) and in N. crassa (Ramos andRodrıguez-Navarro 1985), and one of the factors regulatingthe Km, at least in low Kþ N. crassa cells, is the membrane


Fig. 7. Kinetic analyses of Rbþ influx in trk1D trk2D cellstransformed with the N. crassa hak-1 cDNA (plasmid pNH14.3).Competitive inhibition of Rbþ influx by Kþ, tested up to 25 mM Rbþ.Data were fitted without constraints to Michaelis–Menten functions,which showed that Kþ was a competitive inhibitor of Rbþ influx.The Michaelis–Menten parameter values areVmax ¼ 15 nmol mg¹1 min¹1, Km ¼ 3 mM Rbþ, K i ¼ 3 mM Kþ.

Fig. 8. Competitive inhibition of Rbþ influx byNaþ in trk1D trk2D cells transformed with theN. crassa hak-1 (plasmid pNH14.3) and trk-1(plasmid pYP3) cDNAs. The double reciprocalplot shows the competitive inhibition of Rbþ

influx at millimolar Naþ concentrations. TheNaþ Ki values were 5 mM and 8 mMrespectively.


potential (Blatt et al., 1987). Because trk1D trk2D mutantsare in a highly hyperpolarized state (Madrid et al., 1998),one should expect that only the lowest Km of the transpor-ter would be expressed in trk1D trk2D cells. In the case ofHAK1, which is expressed only in low Kþ cells, the almostcoincident Rbþ Km values found in N. crassa low Kþ cellsand in the N. crassa HAK1 transporter expressed in trk1D

trk2D cells is consistent with the observation that themembrane potential of N. crassa low Kþ cells almost satu-rates the response of the transporter (Blatt et al., 1987).Therefore, the Rbþ Km of the N. crassa HAK1 transporterexpressed in trk1D trk2D cells cannot be much lower thanthe Rbþ Km exhibited by the low Kþ N. crassa cells (3 mMin trk1D trk2D cells expressing HAK1, Fig. 7, versus 6 mMin low Kþ N. crassa cells, Ramos and Rodrıguez-Navarro,1985). By contrast, the Km of TRK1 in trk1D trk2D cellscould be much lower than in normal Kþ cells of N. crassa,where it is expressed, because the membrane potential ofnormal Kþ cells of N. crassa is considerably less negativethan that of trk1D trk2D cells (Madrid et al., 1998).

A more useful characteristic than the Rbþ Km values fordistinguishing between HAK and TRK transporters is thedifference between the Rbþ Km/Kþ Ki ratios exhibited bythe two systems: in HAK transporters this ratio is closeto 1 (Banuelos et al., 1995; Santa-Marıa et al., 1997)and much higher in TRK transporters (see data in Rodrı-guez-Navarro and Ramos, 1984, and consider that thesystem involved is ScTRK1, as shown in Gaber et al.,1988). Our proposal that in N. crassa the HAK1 trans-porter operates in low Kþ cells and the TRK1 transporterin normal Kþ cells is supported by the Rbþ Km/Kþ Ki

ratios shown by these two transporters. In low Kþ cellsthe ratio is close to 1 (Ramos and Rodrıguez-Navarro,1985), suggesting the presence of the HAK1 transporter,whereas cells grown at high Kþ show a higher ratio(Ramos and Rodrıguez-Navarro, 1985), which is a charac-teristic of the TRK transporters.

A Kþ–Hþ symport mediates Kþ uptake in low Kþ cellsof N. crassa (Rodrıguez-Navarro et al., 1986). Therefore,one conclusion of the above discussion is that hak-1 prob-ably encodes this Kþ–Hþ symporter. According to kineticsimilarities and concentrative capacities, the same mechan-ism may operate in the HAK transporters of S. occidentalis(Banuelos et al., 1995) and barley (Santa-Marıa et al.,1997). Whether this mechanism operates in all trans-porters belonging to the Kup–HAK type is unknown atthis moment. However, it is worth observing that the Kupsystem of E. coli also mediates ‘active’ transport (Bakker,1993b). The Kup–HAK transporters have evolved consid-erably among bacteria, fungi and plants, and the aminoacid sequences of the proteins show significant variability(Santa-Marıa et al., 1997). However, the structure of theprotein regarding the transmembrane segments and frag-ments connecting the transmembrane segments is well

conserved (Banuelos et al., 1995). Therefore, an attractivehypothesis is that the mechanism has also been con-served, and that most of the Kup–HAK-type transportersare Kþ–Hþ symporters.

We have discussed the ecological advantage of anHAK1 transporter over a TRK transporter for fungi thrivingin poor environments. The question now is why the TRKsystem is conserved together with the HAK1 transporterin N. crassa and why TRK1 is the dominant transporterin the fungal cells except when they are suffering Kþ star-vation. An explanation for the prevalence of the TRK1transporter would be energy saving, assuming that theTRK1 transporter is a Kþ uniporter, which is a likely pos-sibility. Although this is contradictory with previous propo-sals regarding TRK transporters as Kþ–Hþ symporters,the support for this hypothesis is insufficient. In S. cere-visiae, in which Kþ uptake is mediated only by TRK trans-porters (Ko and Gaber, 1991; Ramos et al., 1994; Madridet al., 1998), it has been considered that a Kþ uniportercannot mediate Kþ uptake because the membrane poten-tial is not negative enough to account for the internal–external ratio of the cation (Boxman et al., 1984). Inthat study, however, the reported membrane potential,¹100 mV or more positive, was estimated from the distri-bution of the tetraphenylphosphonium cation, a methodthat may produce false results (Eraso et al., 1984).Furthermore, a membrane potential more negative than¹300 mV has been demonstrated in a trk1.1 TRK2 strainexpressing the Arabidopsis AKT1 inward rectifier Kþ

channel. This strain, in which Kþ uptake is driven exclu-sively by the membrane potential, depletes the externalKþ down to 0.65 mM (Sentenac et al., 1992). Even assum-ing that this result may be biased because the trk1 muta-tion produces hyperpolarization (Madrid et al., 1998), theeffect cannot be very important because other resultssuggest that the membrane potentials in S. cerevisiaeand N. crassa are similar (Madrid et al., 1998). Thismeans a membrane potential of approximately ¹300 mV(Rodrıguez-Navarro et al., 1986) for Kþ-starved S. cere-visiae cells, potential that can account for an externalconcentration of 2 mM Kþ, the lowest concentration ofKþ that a wild-type S. cerevisiae strain leaves in theexternal medium (Rodrıguez-Navarro and Ramos, 1984).Even in the case of the trk1D trk2D mutant expressingthe N. crassa TRK1 transporter, which depleted theexternal Kþ to 0.3 mM (Fig. 6), a membrane potential of¹350 mV would account for an internal concentration of150 mM, and this membrane potential may be normal intrk1D trk2D mutants (Madrid et al., 1998). In S. pombe,one of the two TRK Kþ transporters has been expressedin a trk1D trk2D mutant of S. cerevisiae and studied bypatch clamp in the whole-cell configuration. The currentsmediated by SpTrkp are enhanced when the pH decreasesfrom 7.5 to 5.5, and this has been taken as support for a



Kþ–Hþ symport mechanism (Lichtenberg-Frate et al.,1996). This enhancement, however, can also be explainedby many different effects of the pH on the transporter, andis not sufficient to demonstrate a Kþ–Hþ symport mechan-ism. Finally, fungal TRK transporters show homology (35%)to the TrkG and TrkH Kþ transporters of E. coli and to otherbacterial Kþ transporters (Nakamura et al., 1998). Again,the mechanism involved in these transporters has beencontroversial. However, in bacteria, as already describedfor S. cerevisiae and S. pombe, the arguments in favourof a Kþ–Hþ symport or any other ‘active’ mechanism forthis type of transporters are far from sufficient (Bakker,1993b).

The cloning of the genes encoding the Kþ transportersof N. crassa opens new technical possibilities for the rig-orous study of many different plant and fungal Kþ trans-porters. The disruption of the trk-1 and hak-1 genes isnow in progress, and if the double disruptant is obtainedand other Kþ transporters do not exist, this mutant couldbe used to express genes encoding Kþ transporters inthe same way as S. cerevisiae trk1D trk2D mutants arenow used, but making the electrophysiological study ofthe transporters possible.

Experimental procedures

Strains, media and growth conditions

A S. cerevisiae trk1D trk2D mutant, WD3 (MAT a ade2 ura3trp1 trk1D::LEU2 trk2D::HIS3) was constructed from strainW303-1A by the single-step gene disruption method (Roth-stein, 1983). For TRK1 disruption, the 2.3 kb XbaI fragmentof this gene was replaced by the LEU2 gene, and for theTRK2 disruption, the 1.4 kb Bst EII fragment was replacedby the HIS3 gene. Double disruptants were confirmed throughSouthern blot analyses. Yeast strains were grown in a mineralmedium with ammonium or with arginine as the nitrogen source(Rodrıguez-Navarro and Ramos, 1984). The basic mediumcontained neither Kþ nor Naþ, which were added as indicated.Growth temperature was 288C.

The N. crassa strain used in this work (74-ORF-1VA) wasobtained from the Fungal Genetic Stock Center (FGSC 2489).General procedures for growing and handling the cells havebeen reported previously (Ramos and Rodrıguez-Navarro,1985; Rodrıguez-Navarro et al., 1986). Briefly, 106 conidiaper ml were inoculated in ammonium phosphate medium sup-plemented with either 0.25 mM or 37 mM KCl to prepare low ornormal Kþ cells respectively. The growth temperature was288C.

Escherichia coli DH5a (Gibco, BRL) was used in routinepropagation of plasmids.

Recombinant DNA techniques

Manipulation of nucleic acids was performed by standard pro-tocols (Sambrook et al., 1989) or, when appropriate, accord-ing to the manufacturer’s instructions.

A N. crassa genomic DNA library was constructed in yeastplasmid YEp91, a derivative of plasmid YEp24 in which theHindIII fragment of plasmid pMA91 (Mellor et al., 1983), con-taining the PGK1 promoter and terminator regions, was sub-stituted for the tetracycline resistance gene at the SmaI, PvuIIsites. N. crassa genomic DNA fragments ranging from 3 to6 kb were obtained by partial digestion with Sau 3A and insertedinto the Bgl II site of YEp91, generating 100 000 independentclones.

For trk-1 isolation, the yeast WD3 mutant was electro-porated (Becker and Guarente, 1991) with the genomic DNAN. crassa library. We obtained 30 000 Uraþ clones, sevenof which grew in the arginine medium supplemented with0.5 mM Kþ. All these clones contained plasmids with identicalinserts of 7.2 kb, and one of the plasmids, pLS5, was chosenfor further study. A trk-1 cDNA was obtained by RT-PCR fromlow Kþ cells RNA. For the reverse transcription, the First-Strand cDNA Synthesis kit (Pharmacia Biotech) was usedwith an anchored Not I(dT)18 primer. The reverse transcrip-tion products were amplified by PCR with the Expand High-Fidelity PCR system (Boehringer Mannheim) using a senseprimer starting in the first ATG triplet of the open readingframe corresponding to the trk-1 gene and an antisense pri-mer specific to the anchor. The N. crassa trk-1 cDNA wasthen inserted in the correct orientation in the BamHI site ofpYPGE15 (Brunelli and Pall, 1993), yielding plasmid pYP3.

For hak-1 isolation, a 0.76 kb cDNA fragment was obtainedfrom low Kþ cells RNA by RT-PCR (plasmid pRH8.1), usingprimers deduced from conserved regions of the kup andHAK1 genes, as described previously (Santa-Marıa et al.,1997). The PCR fragment was then used as a probe forscreening the genomic DNA N. crassa library. Five positiveclones were found to contain plasmids with inserts comprisingDNA fragments identical to the probe. One of these plasmids,pRH1.1, was chosen for further study. A full-length hak-1cDNA was obtained by RT-PCR from low-Kþ cell RNA,using the procedure described for the trk-1 gene, using ananchored oligo-dT primer and two nested primers, the secondstarting in the first ATG triplet of the open reading frame cor-responding to the hak-1 gene. This cDNA was cloned in theEcoRI site of pYPGE15, yielding plasmid pNH14.3.

PCR reactions were performed in a Perkin-Elmer thermo-cycler and the PCR products were first cloned into thePCR2.1-Topo vector using the TOPO TA Cloning kit(Invitrogen).

Sequencing was carried out by the dideoxy terminationmethod described by Sanger (Sanger et al., 1977) modifiedas for the use with Sequenase (USB). The sequences ofthe trk-1 and hak-1 cDNAs were compared with the genomicsequences of the genes. There were no differences except atposition 1259 of the hak-1 open reading frame, where therewas a C in the cDNA and a T in the genomic sequence.This base change did not give rise to an amino acid changein the deduced polypeptide sequence.

DNA sequence data for comparative analyses were obtainedfrom BLAST (NCBI, Bethesda, MD) (Altschul et al., 1990). Proteinalignments were performed using the PILEUP and GAP algorithmsfrom the University of Wisconsin Computer Group. (Devereuxet al., 1984, and updates).

Note on gene nomemclature: we have used the conventionalnomenclature for genes specific for each organism mentioned.



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. cere-visiae TRK1 gene.

Hybridization assays

Colonies from the genomic DNA N. crassa library were immo-bilized on nylon membranes and screened using the 0.76 kbhak-1 fragment (see above) as a deoxygenin-labelled probe,according to the manufacturer’s instructions (BoehringerMannhein). Membranes were hybridized in the presence of50% formamide at 428C.

Southern hybridization analyses were performed with DNAprobes labelled with [a-32P]-dATP by the random primingmethod at 428C in the presence of 20% or 50% of formamide.

For Northern blot analysis, total RNA was extracted fromlow and normal Kþ cells of N. crassa, fractionated throughformaldehyde 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 Transcrip-tion Kit (Ambion), at 608C in the presence of 50% of forma-mide. Membranes were then washed at high-stringencyconditions.

Membranes were exposed at ¹708C to Curix RP-2 (Agfa)films.

Transport assays

Yeast cells were grown in the arginine medium supplementedwith either 30 mM KCl (for WD3) or 3 mM KCl (for WD3 trans-formed with the trk-1 or hak-1 cDNAs) and then starved of Kþ

for 5 h in Kþ-free arginine medium. Cells were suspended in a2% glucose and 10 mM MES buffer brought to pH 6.0 withCa(OH)2. At intervals after the addition of the cations, sampleswere taken, filtered through 0.8 mm pore nitrocellulose mem-brane filters (Millipore) and washed with 20 mM MgCl2. Filterswere incubated overnight in 0.1 M HCl. Rbþ was determinedby atomic emission spectrophotometry of acid extracted cells(Rodrıguez-Navarro and Ramos, 1984). The initial rates ofRbþ uptake were determined from the time courses of the cel-lular Rbþ content and reported, on a cell dry weight basis, asthe means from at least four independent experiments.

Nucleotide accession numbers

The GenBank/EMBL accession numbers for the trk-1 andhak-1 genes are AJ009758 and AJ009759 respectively.

Acknowledgements

This work was supported by the European CommissionDG XII Biotechnology Programme, contract number BIO4CT960775, and by grant PB92–0907 from the Direccion Gen-eral de Investigacion Cientıfica y Tecnica, Spain. F.R. is a post-doctoral fellow funded by Ministerio de Educacion y Cultura.

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