RESEARCH ARTICLE
Conservation and dispersion of sequence and function in fungal TRK
potassium transporters: focus on Candida albicans
Manuel Miranda1, Esther Bashi2, Slavena Vylkova3, Mira Edgerton3, Clifford Slayman2 & Alberto
Rivetta2
1
Department of Genetics, Yale School of Medicine, New Haven, CT, USA; 2Department of Cellular and Molecular Physiology, Yale School of Medicine,
New Haven, CT, USA; and 3Departments of Oral Biology and Restorative Dentistry, School of Dental, Medicine, State University of New York at Buffalo,
Buffalo, NY, USA
Correspondence: Clifford Slayman,
Department of Cellular and Molecular
Physiology, Yale School of Medicine, New
Haven, CT 06520, USA. Tel.: +1 203 785
4478; fax: +1 203 785 4951; e-mail:
[email protected]
Present addresses: Manuel Miranda,
Department of Biological Sciences and Border
Biomedical Research Center, University of
Texas at El Paso, El Paso, TX 79968, USA.
Slavena Vylkova, Department of Microbiology
and Molecular Genetics, University of Texas
Health Science Center, Houston, TX 77030,
USA.
Received 16 September 2008; revised 6
November 2008; accepted 14 November 2008.
First published online 19 January 2009.
DOI:10.1111/j.1567-1364.2008.00471.x
Editor: André Goffeau
Abstract
TRK proteins – essential potassium (K1) transporters in fungi and bacteria, as well
as in plants – are generally absent from animal cells, which makes them potential
targets for selective drug action. Indeed, in the human pathogen Candida albicans,
the single TRK isoform (CaTrk1p) has recently been demonstrated to be required
for activity of histidine-rich salivary antimicrobial peptides (histatins). Background for a detailed molecular investigation of TRK-protein design and function
is provided here in sequence analysis and quantitative functional comparison of
CaTrk1p with its better-known homologues from Saccharomyces cerevisiae. Among
C. albicans strains (ATCC 10261, SC5314, WO-1), the DNA sequence is essentially
devoid of single nucleotide polymorphisms in regions coding for evolutionarily
conserved segments of the protein, meaning the four intramembranal [membrane
–pore–membrane (MPM)] segments thought to be involved directly with the
conduction of K1 ions. Among 48 fungal (ascomycete) TRK homologues now
described by complete sequences, clades (but not the detailed order within clades)
appear conserved for all four MPM segments, independently assessed. The primary
function of TRK proteins, ‘active’ transport of K1 ions, is quantitatively conserved
between C. albicans and S. cerevisiae. However, the secondary function, chloride
efflux channeling, is present but poorly conserved between the two species, being
highly variant with respect to activation velocity, amplitude, flickering (channellike) behavior, pH dependence, and inhibitor sensitivity.
Keywords
Candida albicans ; potassium transport;
chloride channeling; TRK proteins; MPM motifs;
sequence dispersion.
Introduction
Whereas coupled exchange of potassium (K1) for sodium
(Na1), mediated by a P-type ATPase in cell plasma membranes, is the principal means for K1 accumulation by
animal cells, several quite different kinds of transporters
impel K1 accumulation in plants, fungi, and bacteria. The
fact that resting membrane voltages (Vm) in non-animal
systems are often very negative to the K1 equilibrium
voltage (EK; see Slayman, 1982) means that pure channel
structures can facilitate net K1 uptake and accumulation
in many circumstances. ATP-coupled K1-influx pumps also
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exist, for example the Kdp system in Escherichia coli
(Epstein, 1985; Siebers & Altendorf, 1993), but the major
devices for K1 accumulation are gradient-driven coupledion transporters and uniporters. Best known of these are the
so-called TRK and HAK proteins, which – in plants and
fungi – are homologues of bacterial Ktr and Kup transporters, respectively (Stumpe et al., 1996).
The TRK proteins had been assumed to underlie highaffinity K1 accumulation in fungi such as Neurospora
(Rodriguez-Navarro et al., 1986; Blatt et al., 1987; but see
Haro et al., 1999). They were also recognized, 4 10 years
ago, as having sequence homology with bona fide K1
FEMS Yeast Res 9 (2009) 278–292
279
The Candida potassium transporter
channels (Stumpe et al., 1996; Jan & Jan, 1997; Durell et al.,
1999), and were subsequently demonstrated to fold as
internal tetramers, thus forming a channel-like pathway for
K1 transit (Durell & Guy, 1999; Kato et al., 2001; Zeng et al.,
2004). The selectivity of this pathway, as explored in both
higher plants (Arabidopsis thaliana: Diatloff et al., 1998; Liu
et al., 2000) and bacteria (Vibrio alginolyticus: Tholema
et al., 1999, 2005) has been shown to depend critically on
specific amino-acid residues, whose counterparts in KcsA –
the crystallized K1 channel from Streptomyces lividans
(Doyle et al., 1998) – contribute to actual K1-binding sites.
Comparisons among the first few fungal TRK sequences
emerging from the genome data revealed an unexpected
degree of conservation for residues expected to reside at the
surface of the folded structure. This led Durell & Guy (1999)
to suggest oligomerization of folded monomers into tetrads,
within cell plasma membranes, resulting in an overall
configuration similar to that for aquaporins.
Subsequent patch-clamp experiments, on the yeast Saccharomyces cerevisiae and several mutant strains thereof
(Bihler et al., 1999; Kuroda et al., 2004), identified strange
ionic currents mediated via the two TRK proteins in that
organism (Trk1p and Trk2p; S. cerevisiae has no homologue
of the HAK gene). These currents are not visible as singlechannel events, but do display macroscopic channel-like
properties in whole-cell records: they are strongly dependent
on extracellular pH (pHo), with a ‘gating’ voltage of
267 mV at pHo = 7.5 and 157 mV at pHo = 4.5; they are
very small for Vm’s positive to 100 mV, but can be more
than 10-fold larger than expected transporter currents at
large negative voltages; and they have proven proportional
to the intracellular (pipette) chloride (Cl) concentration at
all values of pHo. These currents are evidently carried by Cl
efflux, and detailed kinetic analysis has suggested that they
flow through the central ‘pore’ in the assembled tetrads of
Trk1p/Trk2p (Kuroda et al., 2004; Rivetta et al., 2005).
Because of the direct medical importance of the yeast
Candida albicans, particularly for immunocompromised
patients, and because of the importance of K1 regulation
for multiple cellular functions, we undertook to clone the
TRK gene(s) in Candida by expression in a double-knockout
strain of Saccharomyces, and to characterize the TRK protein(s) in Candida itself. Two related events occurred
in the same time-frame: (1) sequencing of the Candida
genome, which yielded a defective sequence for the single
TRK gene (http://www.candidagenome.org/), and (2)
discovery that this K1 transporter is a critical element in
the killing of Candida by the oral antimicrobial peptide,
histatin 5 (Baev et al., 2004). In the present study, we report
analysis of the TRK1 gene sequence, comparative analysis of
the protein sequence across fungal species, and a partial
physiological characterization of the protein CaTrk1p, in
C. albicans.
FEMS Yeast Res 9 (2009) 278–292
Materials and methods
Strains and maintenance
HY483, a double-TRK knockout strain of S. cerevisiae
(MATa leu2-3,112 ura3-1 trp1-1 his3-11,15 ade2-1 can1-100
GAL1 SUC21 trk1D<HIS3 trk2D<HIS3; S288C background; Ko & Gaber, 1991) was used for expression cloning
of the Candida TRK1 gene. The strain was maintained
routinely on plates in YPAD 1100 mM KCl at 30 1C, and
was grown for transformation in liquid YPAD 150 mM KCl
(Sherman, 1991; Kaiser et al., 1994). A standard C. albicans
library, prepared from strain American Type Culture Collection (ATCC) 10261 in the centromeric vector YCp50 (Rose
et al., 1987; Smith et al., 1992) was amplified in E. coli strain
DH5af1. Transformation of HY483 was carried out with the
BIO-101 kit for yeast (MP Biochemicals, Irvine, CA) plus
10 mg of the C. albicans library DNA. The plasmid DNA
from recovered colonies was reisolated using the yeast
Teeny-prep protocol (http://www.bs.jhmi.edu/MBG/boeke
lab/Resources/YGM/Protocols/TeenyPrepGenDNA.html).
After functional confirmation (growth on low K1), the
TRK1 insert was subcloned into YCplac33 (Gietz & Sugino,
1988), for later use.
Other yeast strains, used for functional comparisons,
were S. cerevisiae PLY232 (MATa his3-D200 leu2-3,112 trp1D901 ura3-52 suc2-D9; Bertl et al., 2003), BS202 (MATa
ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 lys2DNheI; Smith & Roeder, 2000); and C. albicans SC5314
(provided by Dr P.T. Magee, University of Minnesota), CAI4
(Dura3<imm434/Dura3<imm434; Fonzi & Irwin, 1993),
SGY243 (ade2/ade2 Dura3<ADE2/Dura3<ADE2; Kelley
et al., 1987), CaTK1 (Dura3<imm434/Dura3<imm434
Dtrk1/TRK1; Baev et al., 2004), and DBT3 (Dura3<
imm434/Dura3<imm434 Dtok1/Dtok1; Baev et al., 2003).
Ion flux measurements
Functional characterization of the endogenous C. albicans
TRK protein was carried out in two ways: first, by measurement of chemical fluxes in suspensions of intact C. albicans
yeast cells, using rubidium (especially 86Rb1) as a plausible
label for K1 influx (Love et al., 1954; Armstrong & Rothstein, 1967; Läuchli & Epstein, 1970; Aiking & Tempest,
1977; Rodriguez-Navarro, 2000) and second, by measurement of TRK-dependent ion currents via patch-clamping of
yeast-cell spheroplasts.
For chemical flux measurements, C. albicans (strain
CAI4) was grown in shaking cultures at 37 1C to
OD600 nm 1 (c. 3 107 cells mL1), in commercial YNB
medium (QBIOgene, Irvine, CA) plus 200 mM uridine and
140 mM KCl. The resulting log-phase cells were harvested by
centrifugation (500 g for 5 min), washed twice with glassdistilled water, and then subjected to a period of general
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280
starvation, patterned on that formerly used to ‘stabilize’
S. cerevisiae for ion-flux experiments (Armstrong & Rothstein, 1964; Eddy & Hopkins, 1989). The washed cells were
resuspended (at OD 1) in 140 mM glucose plus 1 M
sorbitol and incubated at room temperature (c. 23 1C) for
5 h on a rotary shaker (250 r.p.m.). The resulting starved
cells were washed twice, resuspended in transport buffer
(50 mM Tris-succinate, pH 5.9, plus 140 mM glucose) at a
density of 5 08 cells mL1, and equilibrated for 15 min.
Rubidium uptake was initiated by injecting 1 mL of this
suspension with 25 mL of transport buffer containing 43 mM
RbCl (final concentration = 1.07 mM) and 0.1 mCi of 86Rb1.
Labeled cells were then harvested at intervals, in 200-mL
aliquots, by rapid filtration on Durapore membranes
(0.45 mm pore diameter; Millipore Corp., Bedford, MA),
and rinsed three times with 2 mM MgCl2 to flush out the
extracellular 86Rb1. Pellets and filters were immersed in
Ecoscint fluid (National Diagnostics, Atlanta, GA) and
counted on a Beckman-Coulter scintillation counter (model
LS6500; Fullerton, CA). Data were collected as counts min1
per 108 cells, and converted to mM (mmol L1 cell volume)
via the measured specific activity plus a standard cell volume
of 47 fL per cell (Baev et al., 2002).
Transport (influx) proved essentially linear for the first c.
5 min of sampling, and sampling was routinely carried out
for 3 min.
Patch-clamp measurements
The whole-cell ‘patch’-clamp technique was used, slightly
modified from the standard methods for Saccharomyces
(Bertl et al., 1998; Baev et al., 2004). Cells were grown in
log-phase cultures as described above, but in YPD medium,
washed twice, and resuspended (at OD 1) in 3 mL of
50 mM KH2PO4 brought to pH 7.2 with KOH, plus 25 mM
b-mercaptoethanol. These suspensions were incubated on a
slow orbital shaker (64 r.p.m.), for 30 min at 30 1C, then
recentrifuged, and resuspended in 6 mL of the same buffer,
plus 3.6 U of zymolyase 20T (ICN Biomedicals Inc., Irvine,
CA), and incubated for 45 min at 30 1C. The resulting
spheroplasts were spun down (500 g for 5 min), gently
resuspended in stabilizing buffer1 and incubated stationary,
at room temperature (c. 23 1C) until use. A single batch of
spheroplasts could be used for patch recording over a 6–8-h
period. For actual recording, 1–10 mL of stabilizing suspension was injected into c. 700 mL of sealing buffer2, gently
mixed, and then allowed to settle for 10 min in the recording
chamber, so that a small number of spheroplasts adhered
lightly to the chamber bottom.
Patch pipettes were manufactured as described for Saccharomyces (Bertl et al., 1998) and filled with an artificial
intracellular buffer3. A reference electrode, consisting of a
chlorided silver wire (Ag–AgCl) immersed in 1 M KCl, was
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M. Miranda et al.
connected to the efflux-end of the recording chamber via a
1 M KCl–agar bridge.
Light suction on a patch pipette, placed near a clean
spheroplast on the chamber bottom, would usually draw the
cell onto the pipette tip. With very light further suction, a
seal of 10–35 GO would normally develop within 4–6 min.
The whole-cell configuration was obtained by breaking the
membrane patch in the pipette tip, via a brief high-voltage
pulse (c. 750 mV for 100 ms). Before recording, a 10-min
period was allowed for equilibration between pipette contents and the spheroplast cytoplasm.
The three solutions noted above were as follows: (1)
stabilizing buffer = 220 mM KCl, 10 mM CaCl2, 5 mM
MgCl2, 5 mM 2[N-morpholino]ethanesulphonic acid
(MES) titrated to pH 7.2 with Tris base, 11 mM glucose,
and 230 mM sorbitol; (2) sealing buffer = 150 mM KCl,
20 mM CaCl2, 5 mM MgCl2, 1 mM MES titrated to pH 7.5
with Tris base, and 140 mM sorbitol; and (3) intracellular
buffer = 175 mM KCl, 1 mM EGTA, 0.15 mM CaCl2 (free
Ca21 = 100 nM), 4 mM MgCl2, and 4 mM ATP titrated to
pH 7.0 with KOH.
A standard staircase of voltage clamp pulses, covering the
Vm range of 1100 mV to 180 mV, was adopted to
generate current–voltage data. Stimulus delivery (the voltage pulses), current recording, and preliminary data analysis were carried out via an EPC9 patch-clamp amplifier
(HEKA Elektronik, Lambrecht, Germany) controlled by a
PowerMac G4 Computer (Apple Computer Inc., Cupertino,
CA), as already described (Bertl et al., 1998). Data were
collected at 2 kHz, filtered at 250 Hz, corrected for nonspecific leakage currents (Kuroda et al., 2004), and analyzed in
detail via Microsoft EXCEL and/or IGOR PRO software (WaveMetrics Inc., Lake Oswego, OR).
Results
Cloning of the C. albicans K1 transporter gene
TRK1
Transformation of S. cerevisiae HY483 with the C. albicans
DNA library and selection of transformants were performed
in a medium lacking uracil but containing 50 mM K1
(SC150K; see Sherman, 1991; Kaiser et al., 1994), then
replicated to a low-K1 medium (SC without added K1;
actual [K1]o = 5.7 mM). Five independent clones were obtained, and the plasmid DNA was isolated (Teeny-prep
recipe; see Materials and methods) and transferred to E. coli.
Only two plasmids proved capable of supporting growth on
the low-K1 medium, after retransformation of HY483.
Restriction digests of those two plasmids yielded similar
patterns with all enzymes tested, and were consistent with
the original library construct. One clone (designated YCp50TRK) having an c. 8-kb insert was selected for subcloning;
FEMS Yeast Res 9 (2009) 278–292
281
The Candida potassium transporter
the 8.0-kb fragment was excised with HindIII–BamH1, and
then ligated into HindIII–BamHI-digested YCplac33 (Gietz
& Sugino, 1988). The resulting construct was amplified in E.
coli, reisolated, and transformed into yeast strain HY483 in
order to certify its complementation of K1 transport in
Saccharomyces.
Figure 1 demonstrates complementation, by support of
robust growth of HY483 on K1 concentrations as low as
0.3 mM, which compares well with wild-type strains of both
Saccharomyces and Candida. The entire YCplac33-TRK
Fig. 1. Drop test to demonstrate that TRK1 from Candida albicans
complements the K1-transport deficit in Saccharomyces cerevisiae
deleted of both TRK1 and TRK2. The central experiment is represented
in columns 3, 4, and 5. Strain HY483 is the TRK1,2DD strain provided by
Ko & Gaber (1991). Untransformed (column 5), or transformed by the
empty vector (YCplac33; column 4), HY483 does not grow robustly at
[K1]o o 30 mM. Transformation by YCplac33-CaTRK1 (column 3)
confers robust growth at [K1]o as low as 0.3 mM, nearly equivalent to
wild-type Saccharomyces (strain PLY232, column 6), to wild-type Candida (strain CAI4; column 1), or to Candida deleted of a single allele of
TRK1 (strain CaTK1; column 2). All strains were grown overnight (to
OD600 nm2) at 30 1C in YPD150 mM KCl, harvested by centrifugation,
washed twice in sterile glass-distilled water, resuspended at OD 1, and
serially diluted to give concentrations of roughly 107, 106, 105, and
104 cells mL1. Single drops (7 mL) were then spotted onto agar containing K1-free low-salt medium (recipe L86: Ramos et al., 1985; Gaber
et al., 1988) supplemented with KCl, as indicated at the bottom of each
panel. Plates were incubated for 2 days at 30 1C, and then recorded on a
digital scanner.
FEMS Yeast Res 9 (2009) 278–292
plasmid was then sequenced. In addition, the TRK1 segments from two other strains of C. albicans were sequenced
as controls for comparison with the Candida genome
databases.
DNA sequence variations
For comparative purposes, our C. albicans TRK1 sequence
from strain ATCC 10261 (NCBI database # AF267125) has
been taken as the default sequence. This sequence includes
the TRK1 ORF plus 5 0 (937 bp) and 3 0 (666 bp) untranslated
regions (UTRs), as listed in Supporting Information, Fig.
S1A, along with the translation provided by the curators
(Fig. S1B). Single nucleotide polymorphism (SNP) changes
between ATCC 10261 and the genome sequences of strain
SC5314 or WO-1 are summarized in Table 1 (listing the
shaded residues in Fig. S1). Annotations for Assembly 21,
strain SC5314, indicate a 5 0 UTR for TRK1 (orf19.600c)
much longer than 937 bases, but a 3 0 UTR of only 74 bases,
followed by the coding sequence for a small ribonuclear
protein (orf19.603w). The combined UTRs sequenced, 1011
bases, contain 20 SNPs, or c. 2%, which are roughly equally
distributed among the three strains when the majority
residue at each site is taken as reference. We assume such
variations to be random.
A measure of ‘typical’ nucleotide variability, for referencing Table 1, was obtained by comparing a region of the
genome-database sequences for SC5314 and WO-1, spanning from 6000 bases upstream through 6000 bases downstream of TRK1. This region in chromosome R, described
especially for SC5314, includes five more putative ORFs
with a total coding sequence of 8087 bases, five noncoding
intervals with 3250 bases, and 88% of the centromere (3945
bases). Single-base changes are found at 1% of residues
in the noncoding intervals, which is not significantly
different from 2% in the TRK1 UTRs, combined for the
three strains. (The centromere region is more variable,
however, with 2.8% of sites differing between SC5314
and WO-1.)
Within the coding sequence itself, SNPs are less frequent,
occurring at 26 sites out of 3180 bases, or c. 0.8%, which
compares with 0.5% of residues in all six ORFs, between
SC5314 and WO-1. Furthermore, the SNP variations within
the TRK1 ORF are nonrandomly distributed in at least two
respects. First, from strain to strain: four changes from
majority in WO-1, four in ATCC 10261, and 18 in SC5314.
Second, location within the gene: 25 of the 26 identified
SNPs occur in codons for putative cytoplasmic residues
(viz., 693 amino acids out of 1059 total), regions that are
very poorly conserved across fungal species. The threeamino acid deletion in SC5314 (486-Asp.Asp.Asp.-488)
also maps to the major cytoplasmic loop of the protein.
Only a single SNP, 2364T in WO-1, maps within the
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282
M. Miranda et al.
transmembrane or extracellular segments of the protein,
which are well conserved across fungal species. Three
apparent SNPs in SC5314 have proven to be sequencing
errors in the genome database (see boxed residues in Table 1;
Fig. S1). Only four SNPs in SC5314 TRK1 and two in WO-1
TRK1 are nonsilent mutations. All these map to unconserved cytoplasmic segments of the protein, where they are
predicted to have little or no effect on function. Finally, the
silent mutation at base 465 (A/G, Table 1) has been
identified as SNP marker 1772/2368 in the Candida SNP
map constructed by Forche et al. (2004).
Amino acid conservation across species
As noted in the Introduction, TRK proteins in plants, fungi,
and bacteria are sequence-similar to the selectivity-filter
core of K1 channels, and have been postulated to fold in a
similar manner. This folding is shown in the bead diagram
of Fig. 2, by the clusters just below the membrane–pore–membrane (MPM) numbers (#1, #2, #3, and #4). The index
of sequence mutability (m) across fungal species (calculated
by Dr H.R. Guy, National Cancer Institute) is represented in
Fig. 2 by colors according to the figure key, with red
Table 1. Summary of polymorphisms in Candida albicans TRK1
Strain
Base
883
869
843
786
776
655
573
537
436
416
411
402
353
348
298
162
152
144
083
014
ORF19.600c
0465
0471
0474
0642
0801
0912
0966
0989
1097
1152
1182
1193
1195
1350
1383
1401
1455
1463
1634
2169
Codon
1553
1573
1583
2143
2673
3043
3223
3302
3662
3843
3943
3982
3991
4503
4613
4673
4853
4882
5452
7233
WO-1
10261
SC5314
A
T
T
C
T
A
T
A
C
T
T
A
C
C
C
T
A
T
T
T
A
T
T
C
T
A
T
–
A
C
A
G
A
T
C
A
T
T
G
C
C
G
C
T
A
G
A
A
A
T
A
G
C
C
T
T
T
A
G
C
AGA
GGG
TCG
GAG
CCA
ATT
GAA
GAT
GGA
AAG
ATC
AAG
CGC
GAT
AGC
GGA
GAC
GAT
GTA
ATC
AGA
GGG
TCG
GAG
CCA
ATC
GAA
GAT
GGA
AAG
ATC
AAG
CGC
GAC
AGT
GGG
GAC
GAT
GCA
ATA
AGG
GGA
TCT
GAA
CCG
ATT
GAG
GGT
GAA
AAA
ATT
ACG
TGC
GAT
AGC
GGA
GA – w
G –Tw
GCA
ATC
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Translation
WO-1
10261
SC5314
Arg
Gly
Ser
Glu
Pro
Ile
Glu
Asp
Gly
Lys
Ile
Lys
Arg
Asp
Ser
Gly
Asp
Asp
Val
Ile
Arg
Gly
Ser
Glu
Pro
Ile
Glu
Asp
Gly
Lys
Ile
Lys
Arg
Asp
Ser
Gly
Asp
Asp
Ala
Ile
Arg
Gly
Ser
Glu
Pro
Ile
Glu
Gly
Glu
Lys
Ile
Thr
Cys
Asp
Ser
Gly
Aspw
DDDw
Ala
Ile
FEMS Yeast Res 9 (2009) 278–292
283
The Candida potassium transporter
Table 1. Continued.
Strain
Translation
Base
Codon
WO-1
10261
SC5314
2274
2364
2503
2514
2550
2561
2568
3006
3102
ORF19.603w
1129
1366
1579
1666
7583
7883
8351
8383
8503
8542
8563
10023
10343
GTC
TTT
TCT
CCC
GAG
TTG
GTG
ACG
AAA
GTC
TTC
TCT
CCC
GAA
TTG
GTA
ACA
AAC
GTT
TTC
CTz
CCCCz
GAA
T#G
GTA
ACA
AAC
A
G
G
A
G
A
T
C
G
G
T
A
WO-1
10261
SC5314
Val
Phe
Ser
Pro
Glu
Leu
Val
Thr
Lys
Val
Phe
Ser
Pro
Glu
Leu
Val
Thr
Asn
Val
Phe
Ser
Pro
Glu
D
Val
Thr
Asn
The sequences listed for strain ATCC 10261 are taken as reference. Discrepancies in the genome databases, as reported for strain WO-1 (columns 3 and
6) and for strain SC5314 (columns 5 and 8), are shaded. See Fig. 2 legend for the genome sources. Subscripts for each codon (second column in the
ORF) indicate whether the shaded SNP is in the 1st, 2nd, or 3rd base of the codon.
The nucleotide sequence for strain SC314 was obtained from http://www.candidagenome.org/cgi-bin/locus.pl?locus=TRK1: contig19-10057.
Sequence for the 2nd allele (orf19.8233), contig19.20057, differs only slightly in the ORF itself, but is very close to ATCC 10261 in the upstream UTR.
Residue 465 has been described as an SNP marker site in Candida albicans (Forche et al., 2004). Nucleotide sequence for strain WO-1 obtained from
http://www.broad.mit.edu/annotation/genome/candida_albicans/Assembly.html; supercontig1.2.
w
A deletion of nine bases in strain SC5314, coding for three aspartate residues = DDD.
z
–CT and CCCC = compensated frame shifts, which would convert Ser.Val.Leu.Pro to Ser.Phe.Cys.Pro.
# = Uncompensated frame shift, which would result in premature termination and deletion of the last 202 amino acid residues of the protein. The
corresponding protein sequence in the database is ‘corrected’ by deletion (D) of residue 854, thus preserving alignment of the C-terminal 202 amino
acid residues.
Boxed and shaded residues designate bases (2503, 2514, and 2561) for which independent resequencing of TRK1 DNA in strains SC5314 and CAI4
failed to confirm the previous three variations listed in the genome database for SC5314. That is, the reported frame-shift mutations appear to have
been genome-sequencing errors.
Five CTG codons translated as serine (Ohama et al., 1993) are located at residues 202, 328, 647, 675, and 1043 in strains ATCC 10261 and WO-1, and
at 202, 328, 644, 672, and 1040 in strain SC5314.
The predominant base changes throughout are A () G, and T () C, which together account for 64% of all the SNPs.
designating best conserved (least mutable), gray designating
very poorly conserved, and colorless designating the absence
of conservation.
The majority of cytoplasmically localized residues, including the N terminus, the C terminus, and the long
hydrophilic loop (L23) show little conservation, whereas
the transmembrane helices tend to be well conserved,
especially the so-called pore loops (P1, P2, P3, and P4).
Indeed, the ‘signature’ glycine residues within the putative
filter sequences, QAGLN, DLGLT, TVGFS, and TVGMS,
appear to be absolutely conserved, not only between species,
but also among the separate MPM motifs within each
species. More broadly, among the four MPM motifs, the
segment TM7 through TM8 is the best conserved.
A detailed view of these results, extended to 48 TRK
sequences that are now complete in the fungal (ascomycete)
genome databases, is provided in the Fig. S2 (1–4). This
information is analyzed and summarized in Fig. 3, via
FEMS Yeast Res 9 (2009) 278–292
phylogenetic trees for the four separate MPM motifs. The
colors designate seven distinct clades, which are roughly
conserved in the four MPM motifs. Trk1p for C. albicans
(marked by a white dot) relates most closely with the same
six TRK proteins (the red block) in all four MPMs, for
Ashbya gossypii, Debaryomyces hansenii, Debaryomyces occidentalis, Pichia guilliermondii, Pichia stipitis, and Yarrowia
lipolytica, although nearest neighbor arrangements within
that group differ considerably among the four MPM motifs.
The closest adjacent clade (the blue block), containing S.
cerevisiae (two isoforms), Saccharomyces uvarum, Candida
glabrata, Vandervaltozyma polyspora (two isoforms), and
Kluyveromyces lactis, is also consistent in all four MPM
motifs. Despite the obvious variance, these distributions of
sequence are approximately compatible with the current
understanding of phylogenetic relationships among the
ascomycete fungi (Barr, 2001; Berbee & Taylor, 2001; Kurtzman & Sugiyama, 2001). They also emphasize that residue
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M. Miranda et al.
Fig. 2. Representation of high sequence conservation within the MPM segments of TRK proteins. Whole-protein alignments and index-of-conservation
calculations were carried out by H.R. Guy according to the procedures described by Durell et al., (1999) and Shrivastava et al. (2004), for the first 19
fungal TRK sequences obtained from genomic data: Candida albicans, Aspergillus nidulans (two isoforms), Debaryomyces occidentalis, Ashbya gossypii,
Gibberella zeae (three isoforms), Kluyveromyces lactis, Magnaporthe grisea (three isoforms), Neurospora crassa, Podospora anserina, Schizosaccharomyces pombe (two isoforms), Saccharomyces cerevisiae (two isoforms), and Saccharomyces uvarum. The triplet diagonal arrays designate a helices,
and the extended doublets designate b strands, predicted by means of the PREDICT PROTEIN software, available at http://www.expasy.org. The bead clusters
directly below each MPM number (#1, #2, #3, and #4) represent the pore loops, with each a-helical segment on the left and each filter sequence on the
right, just above P1, P2, P2, and P4. In the intact, folded protein, the four filter sequences would cluster radially around a pore, thus forming several
binding sites for K1 ions being transported.
dispersion across species has occurred at very different rates
in different portions of the TRK molecule; in particular,
MPM4 has been much more stable than the other three
MPM motifs, requiring c. 50% fewer nucleotide substitutions to source the entire set of 48 fungal sequences. The
possible significance of this finding is treated further in the
Discussion.
The primary function of CaTrk1p: K1 uptake
Transport functions at yeast plasma membranes are well
demonstrated to be stabilized by preconditioning of the cells
under generalized starvation, for example incubation for
several hours in distilled water or lightly buffered glucose
solution (Armstrong & Rothstein, 1964; Eddy & Hopkins,
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1989). Influx measurements on such preconditioned cells of
Candida were routinely initiated by injecting cell suspensions with 86Rb1 in c. 1 mM extracellular chemical Rb1 (but
nominally zero K1). Averaged results from six experiments
are displayed in Fig. 4a, showing a bound component of
2.6 1.5 mM (ordinate intercept 1 SE) and a stable influx
(slope) of 6.4 1.2 mM min1. The dependence of this
influx upon the TRK gene/protein was demonstrated previously by means of severe haploid insufficiency: deletion of
only one of the two alleles of CaTRK1 reduced Rb1 influx by
fivefold (Baev et al., 2004).
Concentration dependence of the uptake process was
assessed from similar measurements made with 10 mM,
100 mM, and 1 mM extracellular rubidium, as shown in Fig.
4b, from which the linear slopes describe a simple saturation
FEMS Yeast Res 9 (2009) 278–292
285
The Candida potassium transporter
Fig. 3. Comparison of the separately computed phylogenetic trees of the four MPM motifs in TRK proteins from ascomycete fungi. Sequence data
assembled in Fig. S2, aligned via the Clustal V algorithm. Trees constructed via the MegAlign algorithm in the LASERGENE software (DNASTAR Inc.,
Madison, WI). Note that distances (hundreds of nucleotide substitutions) are reckoned from the common trunk, rather than from the present, and that
the scale of major branches, earlier than 8000 substitutions, is compressed fourfold for MPM1, MPM2, and MPM3. The full list of species names,
abbreviated in each panel above, is given in the legend of Fig. S2.
Fig. 4. Parameters of K1 uptake by Trk1p in Candida albicans. (a)
Average results for six independent experiments at 1 mM extracellular
RbCl. (b) Separate experiment for kinetic parameters, using three
different extracellular concentrations of RbCl. Experimental details are
given in Materials and methods.
FEMS Yeast Res 9 (2009) 278–292
function having a maximal transport velocity (Vmax) of
19.0 mM min1, and a Michaelis constant (K0.5) of
0.64 mM. These results place the normal function of the
Trk1 protein in Candida in almost the same physiological
range as the combined actions of Trk1p and Trk2p in
Saccharomyces, for cells of that species similarly preconditioned (Armstrong & Rothstein, 1967). The two are directly
compared in Fig. 5 (lower two curves), with kinetic parameters in Saccharomyces of Vmax = 16.2 mM min1 and
K0.5 = 0.56 mM.
A long-recognized additional property of the yeast TRK
system(s), however, is that its detailed kinetic behavior
depends significantly on the regimen of preconditioning, in
a manner which defies simple separation into functionally
high-affinity and low-affinity systems (Borst-Pauwels, 1981,
1993; Rodriguez-Navarro & Ramos, 1984; Ramos & Rodriguez-Navarro, 1986; Ramos et al., 1994). Thus, for Saccharomyces cells grown overnight in medium limited only by low
K1, uptake of K1 (or Rb1) occurred with roughly 10-fold
higher affinity (K0.50.08 mM) and twofold higher velocity
(Vmax = 28 mM min1) than for cells stabilized by preincubation in distilled water. The explicit comparison is made in
Fig. 5, between the upper two curves for Saccharomyces, and
the bottom curve, all representing data from the established
literature. It is not known with certainty whether the
detailed conditions for K1 starvation similarly affect the
kinetics of transport in Candida, but that would be
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286
Fig. 5. K1-limited growth induces more vigorous TRK-dependent transport than does generalized starvation: comparison of Candida and
Saccharomyces. Data sources: curve 1 (Candida albicans), Fig. 4b; curve
2 (Saccharomyces cerevisiae), Armstrong & Rothstein (1967) (measured
flux of 42K1, not 86Rb1); curve 3, Ramos et al. (1985); curve 4,
Rodriguez-Navarro & Ramos (1984). Low-K1 growth medium contained
10 mM arginine brought to pH 6.5 with phosphoric acid, 2 mM MgSO4,
0.2 mM CaCl2, 110 mM glucose, standard vitamins1trace elements, and
20 mM K1 (Rodriguez-Navarro & Ramos, 1984). When [K1]O had fallen
to 2 mM, cells were harvested and prepared for the Rb1 influx measurements represented in curves 3 and 4. Kinetic parameters, for curves 1–4,
respectively: K0.5 = 0.64, 0.56, 0.086, and 0.078 mM; Vmax = 19.0, 16.2,
28.4, and 27.5 mM min1.
expected, as a mechanism to optimize resources under
conditions of varying nutrient stress. With regard to other
members of the C. albicans clade (red block in Fig. 3), data
on TRK-mediated K1 fluxes in K. lactis (Miranda et al.,
2002) and D. hansenii (Prista et al., 2007) qualitatively
resemble those for CaTrk1p and ScTRK1,2p, but do not
address the quantitative impact of varying methods of
starvation.
Characteristic secondary function of CaTrk1p: Cl
channeling
For cells the size of C. albicans, chemical fluxes of K1 or Rb1
such as reported in Figs 4 and 5 would imply ionic currents
in the range of 1–2 pA per cell, only marginally large enough
to be measured – as steady currents – by whole-cell patchclamp techniques. However, early patch-clamp studies of
Saccharomyces identified the ScTRK proteins with significantly larger currents, which were peculiarly insensitive to
extracellular K1 (Bihler et al., 1999; Kuroda et al., 2004).
Those currents were shown to arise from a stable anion
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M. Miranda et al.
Fig. 6. Voltage pulses trigger outward currents via Tok1p and inward
currents via Trk1p in the Candida plasma membrane. Patch-clamp traces
from whole-cell records, using 2.5-s voltage pulses from a holding value
of 40 mV, as shown superimposed in (b). (a and e) Wild-type strain
SGY243; (c) TOK1-knockout strain DBT3; (d) TRK1-single-allele knockout
strain CaTK1. Standard extracellular buffer (sealing buffer, pH = 7.5) was
used throughout, as described in Materials and methods. Standard
intracellular (pipette) buffer, containing 183 mM Cl, was used in the
experiments of (a), (c), and (d); Cl was replaced by gluconate, for the
experiment of (e).
permeability in both Trk1p and Trk2p, plus the action of Cl
ions introduced to cytoplasm by the pipette-filling solution
(Kuroda et al., 2004; Rivetta et al., 2005). Patch-clamp studies
on Candida have now demonstrated a similar Cl permeability in that organism, dependent upon the CaTRK1 protein.
Figure 6a shows a typical set of whole-cell patch-clamp
records from a single cell of C. albicans, wild-type strain
SGY243. Each of the superimposed traces represents current
required to clamp the membrane voltage suddenly from the
reference value of 40 mV to test values of 1100 mV (top
trace), 180, 160, . . ., 160, 180 mV (bottom trace).
Figure 6b depicts the actual voltage-clamp pulses (also
superimposed), each lasting for 2.5 s, after a 0.5-s ‘hold’ at
the reference value. The upward (outward) currents reflect
K1 efflux through Candida’s plasma-membrane K1 channel, Tok1p, and – as shown in Fig. 6c – those currents
disappeared when both alleles of the TOK1 gene were
deleted (Baev et al., 2003). The currents activated with time
constants of c. 120 ms (half times of c. 85 ms read from the
left end of each trace) at the onset of each voltage pulse,
reflecting molecular conformation changes that are
FEMS Yeast Res 9 (2009) 278–292
287
The Candida potassium transporter
customarily referred to as ‘gating movements’ in bona fide
channel proteins. The currents deactivated very much faster
when the clamp voltage was returned to its reference value
(see right end of each trace).
The downward (inward) current traces in Fig. 6a reflect
ion flow associated with Trk1p, the K1 transporter protein,
and these were nearly abolished by deletion of a single TRK1
allele, as demonstrated in Fig. 6d. This finding is fully
compatible with the severe reduction of cation influx,
produced by single-allele deletion (86Rb1; Baev et al.,
2004), in this diploid organism. (CaTRK1 appears to be an
essential gene, and C. albicans does not grow, even on K1rich medium, when both alleles have been deleted.) As had
been found in Saccharomyces, however, these inward currents proved insensitive to extracellular [K1] (data not
shown) and were roughly proportional to intracellular
chloride ([Cl] in the pipette solutions). Figure 6e demonstrates the nearly complete disappearance of inward currents
when [Cl]i was reduced to submillimolar levels.
More detailed experiments, however, have revealed several modes in which the Cl currents, mediated by CaTrk1p,
differ very significantly from those mediated by the two TRK
proteins in Saccharomyces. Most conspicuous is a large
difference in rates of activation during hyperpolarizing
voltage pulses. As shown in Fig. 7a, in Saccharomyces the
inward currents jumped (downward) essentially as fast as
the voltage clamp pulses were imposed. More specifically,
the maximal currents for each pulse were attained within a
single sampling interval, 63 ms, for all of the records in Fig. 7.
Figure 7b, closely resembling the records of Fig. 6, displays
much slower activation of the CaTrk1 currents, with time
constants of c. 150 ms. The traces in Fig. 7b also display
much larger amplitude noise at low frequencies than is
apparent for Saccharomyces (in Fig. 7a). Taken together, the
slow activation and relatively large low-frequency noise
suggest that bursts of Cl ions are admitted through CaTrk1p
by typical channel gating movements. For ScTrk1p and
ScTrk2p, by contrast, the nearly instantaneous activation
and low noise level (Fig. 7a) are more readily compatible
with single-ion jumps through the protein, viz., simple
Eyring-barrier events (Rivetta et al., 2005).
Three other properties distinguishing the Cl currents
through CaTrk1p from those through the Saccharomyces
proteins are their larger amplitude, their pH insensitivity,
and their ready blockade by anion-channel inhibitors.
Despite the fact that C. albicans cells routinely selected for
patch-clamp experiments were significantly smaller than
those of S. cerevisiae (diameters of 5–7 vs. 6–8 mm), the
measured TRK-mediated currents were conspicuously larger
in Candida, as is readily seen in Fig. 7 (cf. b and a). The
effects of elevating the pHo from 5.5 to 7.5 are also
demonstrated in Fig. 7: that is, a fourfold reduction of
current amplitude in Saccharomyces but no change of
FEMS Yeast Res 9 (2009) 278–292
Fig. 7. TRK-dependent Cl currents are larger, slower, noisier, less pH
sensitive, and more sensitive to 4,4 0 -diisothiocyano-2,2 0 -stilbene disulfonic acid (DIDS) in Candida than in Saccharomyces. Procedures as in Fig. 6,
except that voltage–pulse durations were only 1.5 s for some experiments with Saccharomyces. Extracellular buffer at pH 5.5 was similar to
sealing buffer, except that acidic MES was titrated only as far as pH 5.5,
with Tris base. For the experiments of (e) and (f), carried out at pHO = 5.5,
0.1 mM DIDS was injected into the pipette solution; similar results were
obtained with 1 mM extracellular DIDS. Wild-type strain BS202 of
Saccharomyces cerevisiae (a, c, and e), and strain SGY243 of Candida
albicans (b, d, and f).
amplitude in Candida (cf. Fig. 7c with Fig. 5a, and 7d with
7b). (However, the rate of activation was slowed in Candida
by about threefold.) Finally, the classic anion-channel inhibitor 4,4 0 -diisothiocyano-2,2 0 -stilbene disulfonic acid had
little effect on Cl currents through ScTrk1p1ScTrk2p (cf.
Fig. 7e with Fig. 7a), but nearly completely blocked the
currents through CaTrk1p (cf. Fig. 7f with Fig. 7d; see also
Baev et al., 2004). These results are further evidence that the
molecular events determining Cl permeability of the TRK
protein in Candida differ significantly from those in
Saccharomyces.
Discussion
Implications from sequence
Comparison of the TRK gene among strains of C. albicans,
as summarized in Table 1, shows the strain ATCC 10261 to
be more closely related to WO-1 than to SC5314 (which was
selected first for Candida genome sequencing), as judged by
the frequency of SNP variations in the coding region plus c.
800-base flanks. While the overall incidence of SNPs is
consistent with random single events, their actual distribution is clearly nonrandom; as is generally to be expected,
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288
SNPs occur in coding regions at only about half of the rate
observed in noncoding flanks. But, among the three strains,
DNA sequences that correspond to the ‘channel-forming’
MPM motifs – viz. 35% of the TRK protein – contain only
4% of SNPs (1/26) identified in the whole coding region. On
this basis alone, selection has clearly occurred for structural
stability in those domains of the protein that are directly
involved in K1 transport.
The same conclusion has emerged more conventionally
by comparison of amino-acid sequences among homologues of CaTrk1p, across fungal species (see Fig. S2). This
information reveals MPM4, the most C-ward component of
the protein that is folded into the transport structure, to be
especially strongly conserved (Figs 2 and 3), accumulating
fewer than half the mutations across species as in the other
three MPM segments. One possible interpretation of this
finding is that MPM1,2,3 have evolved separately from
MPM4; but that seems unlikely, because the primary function of TRK proteins in fungi – K1 accumulation – is
regarded as essential. However, if MPM4, but not the other
three MPM motifs, were involved in a separate function
(such as Cl channeling), simultaneous imposition of two
selective pressures could retard its evolution. A relevant
additional point may be that the selectivity of the actual
ionic pathway through TRK proteins, for K1 ions relative to
Na1 or other monovalent cations, is only modest (Armstrong & Rothstein, 1967) compared with the selectivity of
canonical K1 channels, for example.
A particularly surprising feature of interspecies sequence
comparisons for MPM4, as originally noted by Durell et al.
(1999), is conservation of residues along TM7 and TM8,
which ‘should’ be buried rather nonspecifically in the
plasma membrane’s phospholipid bilayer. This observation
led to a structural picture (see Modeling the unexpected,
below) which cogently anticipated the observed secondary
function of fungal TRK proteins.
Functions of Trk1p in Candida
Serious functional comparisons of Candida Trk1p can be
made with proteins from only one other yeast species thus
far, S. cerevisiae. As demonstrated in Table 2 for all four
MPM motifs, sequence identity is nearly 60% between
CaTrk1p and both Saccharomyces proteins, and similarity is
near 75% when conservative substitutions are included. The
numbers for MPM4 itself are close to 65% and 85%. While
many factors determine the actual functional capability of a
protein in situ – including other (binding) proteins and
small molecules that may differ from organism to organism
– extended identity/similarity between proteins in two
separate species is normally expected to reflect a quantitatively similar function. This expectation was certainly satisfied by the data on K1 transport per se (Fig. 5), when C.
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M. Miranda et al.
albicans and S. cerevisiae were similarly preconditioned by
generalized starvation.
The effect of pure K1 starvation (growth in rich medium
containing only mM K1) still needs to be explored in
Candida, for comparison with data from Saccharomyces
(upper two curves of Fig. 5). Another important property
remaining to be explored, in both organisms, is the effect of
small changes of sequence on cation selectivity in transport,
particularly with respect to the selectivity-filter motifs
(QAGLN, DLGLT, TVGFS, TVGMS). Studies on bacterial
and plant TRK proteins have shown that cation permeability
varies greatly with sequence changes in these motifs, as is to
be expected from the large literature on bona fide K1channel molecules. In KtrB of V. alginolyticus, for example,
conversion of any of the four ‘signature’ glycine residues to
alanine, serine, or aspartate greatly reduced the absolute
transport rates of the protein, and conversion specifically to
serine resulted in preferential transport of Na1 rather than
K1 (Tholema et al., 1999, 2005).
The secondary function of TRK proteins, outward conduction of Cl ions (Fig. 6), also confirms general expectation based on similarity of sequence. However, the observed
quantitative differences from this function in Saccharomyces
are particularly interesting. As shown in Fig. 7, the Cl
currents associated with CaTrk1p are slowly activating (in
response to voltage shifts), large, noisy, insensitive to
changes of extracellular pH, and very sensitive to anion
channel blockers. Such differences might arise from any of
several general causes: detailed sequence differences between
the Saccharomyces and Candida proteins, differences of the
membrane environment in the two species, or different
cytoplasmic binding proteins and regulatory pathways.
Although this secondary function of fungal TRK proteins
may have been an important factor in the strong interspecies
sequence conservation of the MPM4 segment, the essential
physiological role of such Cl channeling is still speculative.
Glycophilic fungi seem to need only trace amounts of Cl,
and intracellular concentrations should be kept low –
compared with the extracellular solutions – by large steadystate membrane voltages (viz., in the range of 200 mV). In
this context, a TRK-mediated Cl pathway should serve as a
Cl escape route, perhaps even too efficiently, because
Saccharomyces, at least, appears to concentrate Cl (weakly)
by means of a formate transporter (Jennings & Cui, 2008).
But in yeasts that can adapt to very salty environments, this
pathway could become essential to Cl detoxification. The
clade containing C. albicans (red block, Fig. 3) is rich in
halophilic species, including D. hansenii, D. occidentalis, P.
guilliermondii, P. stipidis, and Y. lipolytica. Whether the
pathway plays that same role in sustaining C. albicans on
mammalian epithelial surfaces, or in physiological saline
solutions such as saliva (with o 150 mM salt), is not yet
known.
FEMS Yeast Res 9 (2009) 278–292
289
The Candida potassium transporter
Table 2. Summary of identities and similarities of primary structure, between CaTrk1p and the two Saccharomyces proteins, ScTrk1p and ScTrk2p
a
Identical residues are marked by (|) between each pair of sequences; conservative changes, by (:). Percentage ‘similarity,’ below, includes the sum of
identical and conservatively changed residues. The K1-channel signature glycine in each sequence is designated by the enlarged font. Percentages of
total residues, in the order (Identity/Similarity):
MPM:
ScTrk1p
ScTrk2p
#1
#2
#3
#4
Average
58/76
61/72
55/69
45/62
58/68
59/71
63/82
67/85
59/74
58/73
Modeling the unexpected
Potential insight into the origin of Cl channeling via the
TRK proteins, and specifically to the corresponding functional differences between C. albicans and S. cerevisiae, is
afforded by the structural model of fungal TRK proteins
originally proposed by Durell & Guy (1999) as an interesting
way to accommodate the unexpected degree of sequence
conservation in transmembrane segments TM7 and TM8:
intramembranal oligomerization of TRK molecules (see
Introduction). This model features specific close packing of
the TM7 helix from each of four molecules of Trk1p at the
center of the assembly, with the four TM8 helices forming a
supporting ring (see fig. 5 in Durell & Guy, 1999, and fig. 14
in Rivetta et al., 2005). The postulated tetrad assembly
would thus carry a central ‘channel’ in addition to the four
radially arranged K1 pathways formed by the selectivityfilter motifs in each individual molecule.
According to atomic coordinates provided by H.R. Guy,
this central channel would possess a wide, positively charged
vestibule at the intracellular surface of the yeast plasma
membrane, and two uncharged choke points along the
channel wall, buried within the membrane bilayer. This
structural model provides a way to account quantitatively
for Cl efflux currents in S. cerevisiae (see Rivetta et al.,
FEMS Yeast Res 9 (2009) 278–292
2005). The essential residues in TM7 of ScTrk1p correspond
to residues Arg879 (in the vestibule), Trp887, and Phe894 of
CaTrk1p. If this model is generally correct, three other
residues in TM7 may also contribute to the special properties of Cl currents in Candida, Asn880, Cys890, and Ala899,
which correspond to very different residues in Saccharomyces: lysine or arginine, phenylalanine, and cysteine, respectively.
This ‘central pore’ hypothesis remains to be tested by
mutational analysis, as well as by the effects – in Candida –
of other chaotropic ions (nitrate, thiocyanate, and bromide), whose permeability via Trk1,2p of Saccharomyces is
at least equal to that of Cl (A. Rivetta & T. Kuroda,
unpublished data).
Acknowledgements
This work was supported by research grants GM60696 from
the National Institute of General Medical Sciences (to C.S.)
and DE10641 from the National Institute of Dental and
Craniofacial Research (to M.E.). M.M. was supported in
part by grant 5G12 RR 008124, from the National Center for
Research Resources, to the Border Biomedical Research
Center/University of Texas at El Paso. The authors are
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290
indebted to Dr Carolyn W. Slayman, Dr Alan B. Mason, and
Mr Kenneth Allen (Yale Department of Genetics) and to Drs
Paul T. Magee and Beatrice B. Magee (Department of Genetics
and Cell Biology, University of Minnesota, St. Paul, MN) for
much helpful advice and provision of strains. We are also
indebted to Drs Richard F. Gaber (Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern
University, Evanston, IL) and Per Lungdahl (Karolinska
Institute, Stockholm, SE) for strains of S. cerevisiae, to Dr
John D. Reid (formerly of Glaxo IMB, Zurich, Switzerland) for
the C. albicans DNA library and to Drs Stewart R. Durell and
H. Robert Guy (National Cancer Institute, N.I.H., Bethesda,
MD) for the computed cross-species indices of conservation
of TRK proteins (Fig. 2), as well as for the coordinates of their
atomic-scale model of the yeast protein.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Gene and protein sequences for the Candida
albicans potassium transporter, CaTrk1p, as cloned by
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
292
expression in Saccharomyces cerevisiae, from a plasmid
library of C. albicans strain ATCC 10261.
Fig. S2. Alignments of the four MPM motifs across
fungal species.
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
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M. Miranda et al.
Please note: Wiley-Blackwell is not responsible for the
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should be directed to the corresponding author for the article.
FEMS Yeast Res 9 (2009) 278–292