(Received for publication, October 9, 1996, and in revised form, March 23, 1997)
From the Departments of Biochemistry, Molecular
Biology, and Cell Biology, Northwestern University, Evanston,
Illinois 60208, and the ¶ Departamento de Microbiologia, Escuela
Técnica Superior de Ingenieros Agrónomos y de Montes,
14071 Cordoba, Spain
Deletion of the potassium transporter genes
TRK1 and TRK2 impairs potassium uptake in
Saccharomyces cerevisiae, resulting in a greatly increased
requirement for the ion and the inability to grow on low pH medium.
Selection for mutations that restored growth of trk1
trk2
cells on low pH (3.0) medium led to the isolation of a
dominant suppressor that also partially suppressed the increased
K+ requirement of these cells. Molecular analysis revealed
the suppressor to be an allele of BAP2 that encodes a
permease for branched chain amino acids. The suppressor mutation
(BAP2-1) converts a phenylalanine codon, highly conserved
among the amino acid permease genes, to a serine codon in a region
predicted to lie within the sixth membrane-spanning domain. Generation
of the analogous mutation in the histidine permease produced an allele,
HIP1-293, that similarly suppressed the low pH sensitivity
of trk1
trk2
cells. Suppression of trk1
trk2
phenotypes by BAP2-1 or HIP1-293
was correlated with increased Rb+ uptake. The
presence of the substrate amino acids enhanced but was not essential
for suppression of trk1
trk2
phenotypes and increased
Rb+ uptake. The conserved site altered by the suppressor
mutations appears to be important; his4 HIP1-293 cells show
an increased requirement for histidine compared with his4
HIP1 cells.
Potassium transport in Saccharomyces cerevisiae
is primarily dependent on the product of the TRK1 gene.
Mutations in TRK1 lead to a significant increase in the
requirement for potassium due to decreased uptake of the ion (1, 2).
Cells in which TRK1 is deleted lose the ability to grow on
medium containing micromolar concentrations of potassium but readily
grow on medium containing millimolar concentrations of the ion due to
the presence of a second, highly related transporter encoded by
TRK2 (3, 4). Under conditions where transcription of
TRK2 is genetically derepressed, trk1 TRK2
cells regain the ability to grow on micromolar potassium (5, 6).
Consistent with the ability of Trk2 under these conditions to suppress
the trk1
phenotype, kinetic analysis showed that cells
expressing either TRK1 or TRK2 exhibit high affinity K+ uptake compared with trk1
trk2
cells (7). Thus, the dual affinity potassium uptake activities in
S. cerevisiae identified by Rodriguez-Navarro and Ramos (8)
appear to require Trk1 and Trk2 for high affinity transport and other,
unidentified pathways for low affinity transport.
Proteins highly related to Trk1 have been identified in
Saccharomyces uvarum (9), Schizosaccharomyces
pombe (10), and Neurospora,1 but few details
have been discerned regarding their mechanism of transport. The Trk
proteins do not contain sequences that are conserved in
ion-transporting ATPases, nor do they contain the signature sequences
of K+ channels. Recent electrophysiological analysis has
addressed the participation of Trk proteins in K+ transport
more directly. Whole cell patch-clamp experiments with wild-type
S. cerevisiae cells detected Trk-dependent
inward K+ currents in response to hyperpolarizing membrane
potentials (11). These results strongly suggest that K+
transport via the Trk system is a passive process driven by the membrane potential. Trk-dependent K+ currents
were not detected until the membrane potential was increased beyond
100 mV (11), which is consistent with previous estimates of the
membrane potential in S. cerevisiae (12, 13).
Disruption of TRK1 and TRK2 significantly impairs
the ability of the cell to take up K+, resulting in a
1,000-fold increase in the K+ requirement for growth (3).
The hyperpolarization-dependent inward K+
currents observed with wild-type cells by whole cell patch clamp analysis were essentially abolished in trk1 trk2
cells
(11). However, the membrane potential that appears to be the driving force for K+ uptake is intact. This is supported by the
functional expression of Arabidopsis K+ channels
in trk1
trk2
cells. Expression of Kat1 restores the ability of trk1
trk2
cells to grow on very low
concentrations of potassium (14). Because the inward
Kat1-dependent K+ channels are also activated
by hyperpolarization beyond
100 mV (11, 15), the membrane potential
in trk1
trk2
cells is probably at least of this
magnitude and thus can provide sufficient driving force for the uptake
of K+ ions provided a portal of entry is available.
Such routes of monovalent cation transport have been previously
described for bacterial systems. Dosch et al. (16) showed that expression of the wild-type tetracycline/H+ antiporter
resulted in partial suppression of a K+ transport-defective
Escherichia coli mutant by increasing K+ uptake.
More recently, it was shown that expression of the wild-type Ca2+/H+ antiporter gene chaA from
E. coli and the wild-type tetracycline/H+
antiporter gene tetA(L) from Bacillus subtilis
restore Na+ transport activity in
Na+/H+ antiport-defective mutants of E. coli (17, 18). By selecting for suppressors in trk1
trk2
cells that restore the ability to grow on low potassium
medium, we have obtained dominant mutations in genes encoding glucose
transporters (19), the galactose
transporter,2 and one of the inositol
transporters3 among such revertants.
A second phenotype of trk1 trk2
cells is their
hypersensivity to low pH. Whereas wild-type cells can grow quite well
on medium adjusted below pH 3.0, growth of trk1
trk2
cells is severely inhibited below pH 4.5. This phenotype appears to be
the consequence of the impaired potassium uptake because high
concentrations of K+, but not sodium or sorbitol, restore
growth of trk1
trk2
at low pH (4). Further support of
this interpretation is provided by the observation that K+
uptake by the K+-selective channel Kat1 fully suppresses
the low pH sensitivity of trk1
trk2
cells (14). In
this report, we describe the isolation and analysis of mutations at a
highly conserved site in the amino acid permease genes BAP2
and HIP1 that suppress the low pH hypersensitivity and, to a
lesser degree, the potassium requirement of trk1
trk2
cells by conferring increased K+ (Rb+) uptake.
We present evidence that suggests wild-type amino acid permeases are
also somewhat permeable to K+ ions.
S. cerevisiae and E. coli strains used in this study are listed in Table I. YPD, YNB, sporulation medium, and routine genetic manipulations are described by Sherman et al. (20). LS medium was made as described previously (1, 2). YNB media containing amino acids as nitrogen sources were made by omitting ammonium sulfate from the recipe and adding the appropriate amount of filter-sterilized amino acid stock solution to medium after sterilization. Yeast and bacterial transformations were performed by electroporation (21).
|
Yeast genomic DNA, miniprep DNA,
restriction endonuclease analysis, and gel electrophoresis were
prepared or performed as described by Sambrook et al. (22).
DNA hybridization analysis was performed using Quikhyb (Stratagene)
according to the directions of the manufacturer. DNA probes were
prepared by incorporation of [-32P]dCTP by random
priming of denatured DNA fragments isolated by gel electrophoresis
(23). Random hexamers were obtained from Pharmacia Biotech Inc. and
[
-32P]dCTP was obtained from DuPont NEN.
A S. cerevisiae genomic library was
constructed by methods previously described (24) from a trk1
trk2
RPD4-1 (BAP2-1) mutant strain (MS40).
Basically, size-selected (10-15-kb),4
partially Sau3AI-digested genomic DNA fragments were cloned
into the yeast centromeric shuttle vector pRS316 (25), and the ligation reaction was transformed directly into a trk1
trk2
recipient strain (CY152; Table I). Transformants were selected on
permissive medium (pH 5.9) lacking uracil and then replica-plated to
identify potential RPD4-1 (BAP2-1) clones by
their ability to suppress the negative growth phenotype of
trk1
trk2
cells on low pH medium (pH 4.0). A single
plasmid, pGM200, was obtained that conferred suppression of the low pH
sensitivity of trk1
trk2
cells. pGM200 was rescued by
transformation in E. coli and was tested to confirm that its
reintroduction into trk1
trk2
cells by transformation led to suppression of the low pH sensitivity. A 2.6-kb
EcoRV-HindIII fragment from pGM200 was subcloned
into pRS316 (25) producing pKM19-1, which was capable of suppressing
the trk1
trk2
phenotype. The sequence of both strands
of this cloned fragment was determined using synthetic oligonucleotide
primers (Beckman Oligo 1000 DNA Synthesizer) and the Sequenase kit
(U. S. Biochemical Corp.).
The wild-type allele of the suppressor gene was recovered by an
integration and excision experiment with plasmid pMW131. pMW131 was
constructed by subcloning a 745-bp HindIII-ClaI
fragment that encompassed the 5 end of RPD4
(BAP2) from pKM22-1 into the integrative plasmid pRS306
(25). Only about 60 bp of the subcloned fragment overlapped with the
coding region of RPD4 (BAP2). pMW131 was
linearized with SphI and used to transform strain CY152 to
Ura+. Genomic DNA from one transformant was prepared,
digested with XhoI, recircularizd with T4 DNA ligase, and
used to transform E. coli to ampicillin resistance. The DNA
sequence of the 2.6-kb EcoRV-HindIII region of
the cloned fragment in the resulting plasmid (pMW243) was determined
using the same panel of oligonucleotide primers used for the
RPD4-1 (BAP2-1) allele. DNA sequence analyses and
comparisons were performed with the DNA Inspector IIe software program
(Textco), Geneworks (Intelligenetics), and the package of
programs of the Genetics Computer Group (26).
A
deletion of 1.25 kb of the BAP2 coding region was made by
the integration-replacement method (gamma deletion) (25). A plasmid
capable of disrupting BAP2 (pMW133) was created by
subcloning the 745-bp HindIII-ClaI fragment
described above and a 450-bp XbaI-HindIII
fragment corresponding to the region immediately 3 to the
BAP2 coding region into the yeast integrative vector pRS306
(25). Plasmid pMW133 was linearized by HindIII digestion and
used to transform both diploid (MW156) and haploid (CY152 and MS40)
recipient strains to Ura+. About half of the trk1
trk2
BAP2-1 Ura+ transformants lost the suppressor
phenotype, indicating that they contained a disruption of the
BAP2 locus. The other half had become His
evidently due to recombination between pMW133 and the resident plasmid
at the trk2
::HIS3 locus resulting in
replacement of the HIS3 marker with URA3. The
presence of the bap2
deletion in individual transformants
was verified by Southern blot analysis (data not shown). The
bap2
::URA3 haploid strains MW158 and MW159 are
pMW133 integrants of strains CY152 and MS40, respectively (Table
I).
To determine if
suppression of the trk1 trk2
phenotypes by
RPD4-1 (BAP2-1) required amino acids in the
growth medium ura3-52 trk1
trk2
bap2
strains
lacking further auxotrophic requirements were generated. Briefly,
strain MW158 was crossed with strain CY152, and meiotic segregants were
dissected yielding strains MW79A-2D and MW79A-3B, which require only
tryptophan (Table I). Selectable marker replacement (27)
was performed to convert bap2
:URA3 alleles to
bap2
:TRP1 alleles by transformation of MW79A-2D and
MW79A-3B with HindIII-digested plasmid pRS304 (25). All
Trp+ transformants were tested to identify those that had
become Ura
by marker replacement at the BAP2
locus. This strategy yielded strains MW178 and MW179 that are able to
grow on yeast minimal medium after transformation with
URA3-containing plasmids. MW178 and MW179 were transformed
with BAP2 and BAP2-1 alleles carried by
centromeric (pKM22-1 and pMW136) and multicopy (pMW137 and pMW138)
plasmids.
The 2.2-kb
HindIII-XbaI fragment of plasmid pPL241
containing the functional HIP1 gene (kindly provided by P. Ljundahl and G. Fink) was subcloned into plasmid pSelect1 (Promega),
and oligonucleotide-directed mutagenesis was performed according to the
suggested protocol. The oligonucleotide 5-CGGAATAAGAAGAGGCAGCGGT-3
,
which is complementary to the HIP1 coding strand, was used
to introduce a single T to C transition in the second nucleotide of
amino acid codon 293. The presence of the mutation was verified by
determining the DNA sequence in this region. Both the wild-type
HIP1 allele and the mutant HIP1-293 allele were
recovered as 2.2-kb HindIII-XbaI fragments from
plasmid pSelect1 and subcloned into the yeast centromeric and multicopy
plasmids pRS316 (25) and pRS426 (28).
Cells were grown in either YNB and uracil or YNB and uracil with all amino acids supplemented with 30 mM KCl. After 20 h of incubation, cells were harvested by centrifugation and suspended in uptake buffer (10 mM MES adjusted to pH 5.8-6.0 with Ca(OH)2, containing 0.1 mM MgCl2, 2% glucose, and either uracil alone or uracil with a mixture of all amino acids (totaling 2%)). After 2-3 min of incubation in uptake buffer, RbCl (100 mM) was added, and the velocity of uptake was determined during the first 7-10 min. Samples of cells were harvested by filtration, washed with 20 mM MgCl2, and solubilized in HCl. The rubidium content of each sample was analyzed by atomic absorption spectrophotometry.
Mutations that suppress the
low pH hypersensitivity of trk1 trk2
cells were
isolated by selecting for spontaneous revertants able to grow on YPD
medium supplemented with 100 mM KCl but adjusted to pH 4.0. Genetic analysis of these mutants indicated the presence of both
recessive (to be reported elsewhere) and dominant suppressors. The
dominant suppressors also partially suppressed the increased potassium
requirement conferred by the trk1
trk2
mutations and were thus designated RPD for their reduced potassium
requirement. Recombination tests between the dominant suppressors
revealed a single representative, RPD4-1, of the locus
described here. Tetrad analysis of meiotic segregants from a cross
between the trk1
trk2
RPD4-1 mutant and a compatible
trk1
trk2
strain revealed that the suppressor mutation
segregated as a single Mendelian factor (not shown).
To identify the RPD4
gene, a library of genomic DNA fragments made from the dominant
RPD4-1 strain MS40 was screened for clones that could
suppress the low pH sensitivity and potassium requirement of
trk1 trk2
cells. A single suppressor clone was
obtained, and a smaller subclone (pKM22-1) that conferred suppression
of the trk1
trk2
phenotypes was chosen for molecular
analysis. The wild-type RPD4 gene, recovered on a 2.6-kb
HindIII fragment by an integration and excision experiment
(see "Materials and Methods"), was subcloned onto both single copy
and multi-copy plasmids (pMW136 and pMW137) and transformed into a
trk1
trk2
recipient (CY152). Both plasmids failed to
suppress the trk1
trk2
phenotypes, confirming that the
cloned fragment contained the wild-type allele of the suppressor
locus.
DNA sequence analysis of the 2.6-kb HindIII-EcoRV fragment that contained the functional suppressor revealed a single long open reading frame of 609 amino acid codons, which predicts a protein of 67 kDa. Hydropathy analysis (29) of the protein sequence revealed 12 putative membrane-spanning domains (M1-M12). A search of the S. cerevisiae genome revealed that the open reading frame encoding the RPD4-1 allele was essentially identical to open reading frame YBR068c discovered through the genomic sequencing project (30) and independently identified as BAP2, an amino acid permease capable of transporting branched chain amino acids (31). A comparison with related sequences revealed that Rpd4, henceforth referred to as Bap2, shares greatest sequence identity with the putative amino acid permease Pap1 (78%) (32) and least sequence identity (32%) with the proline permease, Put4 (33).
Effects of bap2The ability of trk1
trk2
cells to grow on K+-supplemented medium
suggests that plasma membrane proteins other than Trk1 or Trk2 must be
capable of mediating K+ uptake. To determine if Bap2 plays
a major role in non-Trk-mediated potassium uptake, a null allele of
BAP2 containing a 1.25-kb deletion of the open reading frame
(bap2
) was generated in trk1
trk2
ura3-52
BAP2 and trk1
trk2
ura3-52 BAP2-1 recipient cells
(strains CY152 and MS40; see "Materials and Methods" for details).
As predicted, deletion of the BAP2 sequences in the
trk1
trk2
BAP2-1 host abolished suppression of the
trk1
trk2
phenotype. The trk1
trk2
bap2
cells exhibited no apparent growth defects compared with
the pretransformed cells confirming that BAP2 is
nonessential (31). In addition, the potassium requirement of
trk1
trk2
bap2
cells, assessed by testing for
growth on medium containing various concentrations of the ion, was not
increased compared with the isogenic trk1
trk2
BAP2
cells (data not shown). Thus, wild-type Bap2 does not provide a major
avenue of K+ uptake in trk1
trk2
cells and
is not responsible for their ability to grow on 100 mM
K+.
The product of the
SHR3 gene has been shown to be localized within the
endoplasmic reticulum and is believed to be required for the processing
of most, if not all, newly synthesized amino acid permeases in S. cerevisiae (34). Although amino acid permeases are retained in the
endoplasmic reticulum in shr3 mutants, other membrane
proteins, including the H+-ATPase are apparently
unaffected. Thus, the role of Shr3 appears to be specific for the
processing and/or targeting of amino acid permeases. To assess whether
or not processing of Bap2 is dependent on SHR3, an
shr3 null allele was generated in a trk1 trk2
BAP2-1 strain to test for epistasis, i.e. loss of the
BAP2-1 suppressor phenotype. The shr3
mutation
abolished the ability of trk1
trk2
BAP2-1 cells
(strain MW210) to grow on low pH and on low potassium medium but only
modestly increased the K+ requirement of trk1
trk2
cells expressing the wild-type BAP2 allele
(strain MW211; data not shown). In addition, the K+
requirement of TRK1 trk2
cells containing the
shr3
mutation (strain MW236) was indistinguishable from
that of TRK1 trk2
cells harboring the wild-type
SHR3 gene (strain MW235; data not shown), suggesting that
the activity of Shr3 is not required for maturation of Trk-related
proteins.
In S. cerevisiae
amino acid permeases apparently function as H+/amino
acid-coupled symporters. To test whether or not the BAP2-1 mutation might have altered the specificity of an ion-binding site
while leaving the amino acid transport coupling mechanism unaffected,
the ability of BAP2-1 to suppress trk1 trk2
phenotypes was assessed in the absence of amino acids in the growth
medium. A ura3-52 trk1
trk2
strain containing the
dominant BAP2-1 suppressor on a centromeric plasmid
exhibited growth on low potassium or low pH medium both in the presence
and the absence of amino acids (Fig. 1), indicating that
suppression of the trk1
trk2
phenotypes is not
dependent on the uptake of amino acids. Thus, if the suppressor mutation in BAP2-1 alters the putative proton translocation
capability of the permease to allow potassium uptake, this new
capability is not obligatorily coupled to amino acid symport.
The BAP2-1 Suppressor Mutation Alters a Highly Conserved Site
The DNA sequence of the 2.6-kb HindIII fragment
of pMW243 containing the wild-type BAP2 gene was determined
and compared with the BAP2-1 sequence. A single difference
between the two sequences was found: a T (BAP2) to C
(BAP2-1) transition of the second nucleotide of codon 299 resulting in a phenylalanine to serine substitution in the deduced
amino acid sequence. This site is located within the putative sixth
transmembrane domain of the transporter and occurs at a site highly
conserved among S. cerevisiae amino acid permeases. All but
one of the members of the amino acid permease gene family in S. cerevisiae contain phenylalanine at this site (Fig.
2). In the case of Tat2, the tyrosine permease, a
conservative substitution to tyrosine is present at this position. The
conservation of this site suggests that it plays an important role in
the structure/function of amino acid permeases.
The Analogous F
If the conserved phenylalanine residue that is changed
to serine in BAP2-1 plays an important role in transport, an
equivalent mutation in another amino acid permease might also suppress
the trk1 trk2
phenotypes. To test this hypothesis, the
analogous mutation was made in the histidine permease gene,
HIP1 (Fig. 2). Codon 293 was converted from a phenylalanine
to a serine codon by site-directed mutagenesis. The resulting allele
(HIP1-293) was subcloned onto both centromeric and
multi-copy plasmids (pRS316 and pRS426). The HIP1-293
plasmids were able to suppress both the sensitivity to low pH and the
potassium requirement of a trk1
trk2
recipient (Fig.
3A). Like BAP2-1, suppression of
the trk1
trk2
low potassium and low pH phenotypes
could be detected on medium lacking amino acids, indicating that
suppression could occur independently of histidine transport. Cells
harboring the HIP1-293 allele on the multi-copy plasmid
exhibited slightly stronger suppression of the trk1
trk2
potassium requirement compared with cells that expressed
the suppressor from the centromeric plasmid (Fig. 3A),
suggesting that transport of K+ by Hip1-293 was
rate-limiting for growth when expressed as a single copy gene.
Although expression of the wild-type HIP1 allele from a
centromeric plasmid did not suppress either of the trk1
trk2
phenotypes, expression from a multi-copy plasmid modestly
suppressed the low pH phenotype (Fig. 3A), suggesting that
wild-type amino acid permeases may transport small amounts of
potassium. In further support of this hypothesis, deletion of
SHR3 in trk1
trk2
cells resulted in
slightly weaker growth on medium containing 100 mM
potassium compared with congenic trk1
trk2
SHR3
cells.5 This was not due to a K+
transport-independent effect on growth because deletion of
SHR3 in TRK1 cells had no effect on growth even
under potassium-limiting conditions.5
To determine if BAP2-1 and
HIP1-293 increase potassium uptake, Rb+ uptake
assays were performed using the trk1 trk2
recipient strain that was transformed with either the vector (pRS316) or plasmids
expressing the wild-type or suppressor alleles of the amino acid
permeases. Because Bap2 transports branched-chain amino acids and the
presence of these substrates in the growth medium results in greatly
increased BAP2 transcription (35), Rb+ uptake
assays were performed using cells grown either in the presence or the
absence of amino acids in the growth medium and in the uptake assay
bath. The velocity of Rb+ uptake in trk1
trk2
cells expressing the BAP2-1 allele was indistinguishable from trk1
trk2
cells harboring the
wild-type allele when the cells were cultured in minimal medium
supplemented only with uracil (to satisfy their auxotrophic
requirement). In contrast, the rate of Rb+ uptake increased
approximately 2-3-fold in trk1
trk2
BAP2-1 cells when
cultured under inducing conditions (see Table II). Increased Rb+ uptake was not observed with trk1
trk2
cells expressing the wild-type BAP2 gene under
these conditions. The presence of amino acids in the assay buffer
slightly increased Rb+ uptake by trk1
trk2
BAP2-1 cells.
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The expression of HIP1-293 in trk1 trk2
cells conferred increased Rb+ uptake compared with cells
expressing the wild-type HIP1 allele (Table II). The ability
to detect this increase was again dependent on culturing the cells in
the presence of amino acids and again enhanced slightly by the addition
of amino acids (or histidine) to the bath during the assay. The
greatest effect was obtained when histidine was added in the absence of
other amino acids (Table II), suggesting that amino acid transport
increased K+ uptake by the mutant permease. The requirement
of amino acids in the growth medium to detect Bap2-1- or
Hip1-1-mediated Rb+ uptake is in contrast with the ability
of BAP2-1 or HIP1-1 to suppress the trk1
trk2
phenotypes on minimal medium. Evidently the threshold for
increased growth of trk1
trk2
cells on
potassium-limiting or low pH medium is lower than that required to
detect an increase in Rb+ uptake by the methods used in our
assays.
The requirement for an aromatic amino acid at the conserved site altered by the BAP2-1 and HIP1-293 mutations was addressed by comparing the ability of plasmid-born HIP1 and HIP1-293 alleles to suppress the histidine auxotrophy of a his4 mutant containing the hip1-1 mutation (strain R770; Table I). Compared with his4 HIP1 cells, his4 hip1-1 cells require elevated levels of histidine (>2.0 mM versus 0.03 mM) to support growth in synthetic medium supplemented with all amino acids (36). Suppression of the histidine requirement by HIP1-293 expressed from a single copy plasmid was very weak, indicating that histidine transport is significantly impaired but not abolished by mutation at Phe293 (Fig. 3B). Consistent with this interpretation, overexpression of HIP1-293 from a multi-copy plasmid in the hip1-1 recipient resulted in growth indistinguishable from cells expressing the wild-type HIP1 allele.
The loss of high affinity K+ transport, mediated by
the products of the TRK1 and TRK2 genes, confers
strong negative phenotypes including a thousand-fold increase in
potassium required to support growth and the inability to grow on
medium below pH 4.5. The negative phenotypes associated with
trk1 or trk1
trk2
cells have allowed several genetic approaches to be taken toward understanding
K+ transport. These include the molecular and functional
analysis of heterologous proteins that restore K+ uptake
and the analysis of suppressor mutations. The first approach has led to
the identification of K+ transporters from other yeasts (9,
37) and wheat (38) and voltage-gated K+ channels from
Arabidopsis (14, 39), whereas the second approach is
revealing that a variety of S. cerevisiae membrane proteins, including sugar transporters
(19),6,6 the inositol
transporter,3 and amino acid transporters (this report) can
be genetically altered to confer increased potassium transport.
Here we describe a dominant, spontaneous mutation in the amino acid
permease gene BAP2, isolated as a suppressor of the low pH-sensitive phenotype of trk1 trk2
cells. Consistent
with previous observations that either high concentrations of potassium
in the growth medium (3) or the expression of a heterologous
K+ transporter (14) can completely suppress this phenotype,
we show that Bap2-1 confers a 3-4-fold increase in the rate of
K+ uptake by trk1
trk2
cells. Thus,
suppression of the low pH sensitivity by Bap2-1 appears to reflect the
acquisition of an alternative K+ transport pathway in these
cells. A mutation analogous to BAP2-1 was generated in the
histidine permease (HIP1), and it similarly suppressed
trk1
trk2
phenotypes by conferring a severalfold increase in the rate of K+ uptake, suggesting that the
ability of a single amino acid substitution to convert amino acid
permeases into proteins capable of transporting K+ ions may
be a general property of this family of transporters.
The BAP2-1 and HIP1-293 mutations substitute
serine for a highly conserved phenylalanine predicted to reside within
the sixth membrane-spanning domain. We have shown that this site is
important for amino acid transport in the case of the histidine
permease. The suppressor mutations replace a large, hydrophobic
side-chained amino acid for a small polar one. The increased
permeability to K+ conferred by this mutation could be a
consequence of the disruption of closely packed transmembrane helices
or a change in the polarity at this position. The ability of calcium to
inhibit suppression of trk1 trk2
phenotypes by
BAP2-1 and HIP1-2937
suggest that the site of this newly gained entry for cations is not
specific for K+.
The uptake of amino acids in S. cerevisiae can be coupled with the uptake of protons via a symport mechanism. This was originally observed as increased H+ uptake induced by specific amino acids in metabolically starved cells (40). Estimates of H+/amino acid stoichiometry vary from 1:1 to 3:1, depending on the species transported (41-43). Thus, many amino acid permeases in S. cerevisiae have inherent ion-transporting capacity, but the role of conserved residues or motifs in H+ binding or translocation has not been demonstrated. The presence of a basic residue at the beginning of the sixth predicted transmembrane domain is a conserved feature of various transporters known to catalyze uptake by substrate/H+ symport. It is not present in transporters known to mediate facilitated diffusion (44). Both Bap2 and Hip1 contain a lysine two amino acids beyond the boundary of their predicted sixth transmembrane domain. These observations are consistent with Bap2 and Hip1 functioning by a H+/amino acid symport mechanism.
We speculate that the amino acid altered by the BAP2-1 and
HIP1-293 mutations may normally constitute part of a
H+-binding or translocation site important to a symport
mechanism. The Phe Ser mutation may substitute K+ for
H+ as a co-substrate for amino acid transport, or it might
increase K+ permeability while simultaneously uncoupling
ion and amino acid transport. Two observations suggest that
K+ transport by the mutants is uncoupled to amino acid
uptake. First, increased K+ transport appears to occur
independently of amino acid uptake because both BAP2-1 and
HIP1-293 cells suppress the trk1
trk2
growth phenotypes in the absence of their substrate amino acids. Second, although the presence of amino acids during growth was required
to detect the increased Rb+ uptake, the presence of
substrate amino acids in the assay buffer only slightly increased
Rb+ transport by either Bap2-1 or Hip1-293. However, the
observation that the HIP1-293 mutant exhibits impaired
histidine uptake indicates that the mutation could directly affect the
transport mechanism.
Kat1, a voltage-dependent K+ channel from
Arabidopsis (15), completely suppresses the low pH
hypersensitivity and potassium requirement of trk1
trk2
cells (14) by restoring K+ uptake (11).
Because Kat1 is activated only upon membrane hyperpolarization in
excess of
100mV (11, 15) the membrane potential of trk1
trk2
cells is large enough to drive cation accumulation and is likely to provide the major driving force for K+ uptake via
Bap2-1 and Hip1-293.
Overexpression of the wild-type histidine permease was observed
to weakly suppress the low pH sensitivity of
trk1 trk2
cells. In addition,
disruption of SHR3 in trk1
trk2
cells
slightly increased their K+
requirement.8 Thus, it is likely that one
or more amino acid permeases in S. cerevisiae harbor
intrinsic K+ transport capability. An attractive
possibility is that H+/amino acid symporters may not have
an absolute requirement for H+ as a co-substrate and may be
capable of coupling K+ ions to drive amino acid uptake. If
so, the analysis of mutations that confer increased K+
uptake by amino acid permeases may identify residues or domains important for the ion-coupled symport mechanism.
We thank P. Ljundahl and G. Fink for plasmids and strains.