From Departamento de Biotecnología, Escuela
Técnica Superior de Ingenieros Agrónomos, Universidad
Politécnica de Madrid, 28040 Madrid, Spain and the
§ Departamento de Microbiología, Escuela
Técnica Superior de Ingenieros Agrónomos, Universidad de
Córdoba, 14071 Córdoba, Spain
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ABSTRACT |
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Null trk1 trk2 mutants of
Saccharomyces cerevisiae exhibit a low-affinity uptake of
K+ and Rb+. We show that this low-affinity
Rb+ uptake is mediated by several independent transporters,
and that trk1 cells and especially trk1
trk2
cells are highly hyperpolarized. Differences in the membrane
potentials were assessed for sensitivity to hygromycin B and by flow
cytometric analyses of cellular DiOC6(3) fluorescence. On
the basis of the latter analyses, it is proposed that Trk1p and Trk2p
are involved in the control of the membrane potential, preventing
excessive hyperpolarizations. K+ starvation and nitrogen
starvation hyperpolarize both TRK1 TRK2 and trk1
trk2
cells, thus suggesting that other proteins, in addition
to Trk1p and Trk2p, participate in the control of the membrane
potential. The HAK1 K+ transporter from
Schwanniomyces occidentalis suppresses the
K+-defective transport of trk1
trk2
cells
but not the high hyperpolarization, and the HKT1 K+
transporter from wheat suppresses both defects, in the presence of
Na+. We discuss the mechanism involved in the control of
the membrane potential by Trk1p and Trk2p and the causal relationship
between the high membrane potential (negative inside) of trk1
trk2
cells and its ectopic transport of alkali cations.
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INTRODUCTION |
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Potassium is an indispensable element, which is accumulated against large transmembrane concentration gradients in cells living in diluted environments. Probably because of the central role of this element in all forms of life, different mechanisms mediating K+ uptake have evolved in different types of cells. In plants and fungi, the cellular uptake of K+ is probably always an electrophoretic process, which takes place in response to the membrane potential created by the H+-pump ATPase (1, 2), as described for Neurospora crassa (3).
Among all eucaryotic non-animal cells, the maximum information about
potassium transport has been obtained in Saccharomyces cerevisiae. In this fungus, the biochemistry (4-7) and the
genetics of K+ transport (8-11) and H+-pump
ATPase (12) have been extensively studied. The TRK1 gene of
S. cerevisiae encodes a notable K+ transporter,
which is adapted to provide the required amount of K+ in
many different nutritional conditions. To perform this function, this
transporter changes its Km, which shows values in the millimolar range in cells growing at millimolar concentrations of
K+, and as low as 20-30 µM in
K+-starved cells (5, 8, 13). The
Vmax is also variable, depending on the cellular
pH and K+ content of the cells (5, 6). A second gene,
TRK2 (10, 11), encodes a second K+ transporter
structurally related to Trk1p (11). However, unlike Trk1p, Trk2p shows
a very low Rb+ influx Vmax in
trk1 cells with a low K+ content, and the
influx is undetectable in TRK1 cells or in
trk1
cells with a normal K+ content (7).
Using a trk1
strain overexpressing Trk2p, it has been
found that the affinity of the TRK2 transporter for K+ and
Rb+ is also regulated by the K+ content of the
cell, and that the Km in K+-starved
cells is only slightly higher than that of TRK1 (7). Because the
Vmax of TRK2 is very low in comparison to that
of TRK1 and the Km is higher than the
Km of TRK1, the function of Trk2p seems to be
superfluous as an independent K+ transporter.
Deletion of the TRK1 and TRK2 genes results in
cells that grow normally at high concentrations of K+, and
slowly at relatively low concentrations, exhibiting a Rb+
influx kinetics with a Km of 60 mM and a
Vmax of 9 and 16 nmol mg1
min
1, in the two strains studied so far (7). This
low-affinity Rb+ uptake of trk1
trk2
cells
also constitutes the major pathway for Rb+ uptake in
trk1
TRK2 cells, in which Trk2p mediates a minor pathway, as already mentioned. However, in contrast with the relevance of the
low-affinity K+ uptake in trk1
TRK2 and
trk1
trk2
cells, this uptake has never been detected
in wild strains (4, 5). Because the Km of the
low-affinity Rb+ influx is much higher than the
Km of TRK1 and the Vmax not
insignificant in comparison to the Vmax of TRK1,
it can be concluded that the low-affinity Rb+ uptake has
not been detected in TRK1 cells because it does not exist in
these cells.
Therefore, two important questions remain unanswered about
K+ transport in S. cerevisiae, the function of
Trk2p and the identity, and consequently the function, of the
transporter mediating the low-affinity uptake of K+ and
Rb+ observed in trk1 cells. For the latter
question two hypotheses have been put forward, that this transport
takes place mediated by several non-K+ transporters, which
harbor intrinsic K+ transport capabilities (14-16), and
that it is mediated by a part of the normal K+ uptake
system, which is multimeric and loses its normal properties in the
absence of Trk1p (7). The former hypothesis is supported by well
documented studies with strains carrying mutations in sugar and amino
acid transporters, and with strains overexpressing amino acid permeases
(14-16). The latter has been advanced to explain why the low-affinity
Rb+ uptake does not exist in wild strains, and to explain
why glucose activates the low-affinity Rb+ influx of
trk1
mutants in the same fashion as it does in wild strains (7, 17).
In this paper we report the characteristics of Rb+ uptake, tolerance to hygromycin B, and cellular DiOC6(3) fluorescence, as determined by flow cytometry, in strains carrying different combinations of wild and null alleles of the TRK1 and TRK2 genes. The results suggest that Trk1p and Trk2p are involved in the regulation of the electrical membrane potential. Deletion of these proteins brings the cells to a very high membrane potential, negative inside, which could drive an ectopic uptake of alkali cations.
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EXPERIMENTAL PROCEDURES |
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Strains, Plasmids, Growth Conditions, and Methods--
The
S. cerevisiae strains used in this study are all isogenic to
W303.1A (Mata ura3 his3 leu2 ade2 trp1) (Table
I).
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Rb+ Uptake--
Cells were separated from the
culture medium by centrifugation, washed, and suspended in testing
buffer composed of 10 mM MES1 buffer brought to pH 6.0 with Ca(OH)2, containing 0.1 mM
MgCl2 and 2% glucose. In some experiments with
trk1 trk2
-pRSHAK1 strain carrying the
pGAL1-HAK1 gene, galactose-substituted glucose, and in the
experiments designed to test the effect of divalent cations on
Rb+ influx, the pH of the testing buffer was adjusted with
NaOH, and MgCl2 was withdrawn. To start the experiments,
Rb+ was added to the cell suspension and, at short
intervals, samples of cells were removed by filtration from the testing
buffer. To determine the Rb+ content, the cells were
treated with HCl and the extracts analyzed by atomic emission
spectrophotometry, as described previously (5). In all cases, we
determined the initial rate of Rb+ uptake from the time
course of the net accumulation.
Flow Cytometry--
S. cerevisiae and N. crassa cells were grown as described in each case. S. cerevisiae was always grown at low cell density (<0.3 mg dry
weight ml1), and as a consequence the concentration of
glucose was very similar in all experiments (close to 2%). Cells were
harvested from the culture medium, suspended in testing buffer (2 × 106 cells ml
1), and exposed to
DiOC6(3) cyanine dye (3,3'-dihexyloxacarbocynine iodide,
Molecular Probes, Eugene, OR) at 1 nM for 30 min at
28 °C, in the dark. To test yeast cell viability, propidium iodide
was added at the moment of the analysis. Flow cytometric analyses were
performed in a FACScan (Becton Dickinson) and EPICS XL (Coulter Electronics) flow cytometers, equipped with argon lasers. For the
determination of the DiOC6(3) fluorescence, excitation at 488 nm and a 525-nm dichroic LP filter were used. For propidium iodide,
a 590-nm dichroic filter and a 610-nm LP absorbance filter were added.
Because the cellular fluorescence was size dependent, we normalized all
the results measuring the fluorescence of single small cells (25). In
all experiments, a control sample of wild-type cells (W303.1A strain)
grown in the arginine medium at 0.5 mM K+ was
analyzed in parallel with the other samples, and the fluorescence values given by the flow cytometer were always referred to the fluorescence of the control cells and expressed as a percentage. All
measurements were repeated at least in three different days.
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RESULTS |
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Characteristics of the Low-affinity K+ Transport of
trk1 trk2
Cells--
The kinetic study of the influx of the
alkali cations in trk1
trk2
cells with different
K+ contents and the competitive inhibitions exerted between
them indicated that these cells took up alkali cations with little differences in affinities (we determined Km values
of 60 mM K+, 60 mM Rb+,
110 mM Na+, and 100 mM
Li+ in the W
3 strain), and without regulation by the
K+ content. Although this uptake shows homogeneous
kinetics, experiments with different inhibitors showed that it involves
different pathways. Ammonium inhibited 45% of the low-affinity
Rb+ uptake of trk1
trk2
cells, but showed
no effect over the remaining 55%; low concentrations of
Mn2+, and to a lesser extent other divalent cations
(Mg2+, Ca2+, and Ba2+), inhibited
30%, but showed no effect on the remaining 70% (Fig. 1); and ammonium plus Mn2+
(20 mM each) inhibited 60% of the uptake. These results
suggest the existence of at least three pathways for Rb+
uptake in trk1
trk2
cells, one inhibited by
Mn2+, another inhibited by ammonium and insensitive to
Mn2+, and a pathway insensitive to both ions. It is worth
mentioning that the described inhibition of Rb+ uptake by
ammonium or Mn2+ in trk1
trk2
cells was
different from that found in wild-type cells. In the latter, ammonium
was a competitive inhibitor of Rb+ uptake, and
Mn2+ and other divalent cations did not show any
significant effect (not shown).
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Overexpression of Hxt3p and Hnm1p Partially Suppresses the
Phenotype of trk1 trk2
Cells--
In a different part of the
study of the low-affinity K+ uptake of S. cerevisiae, we had isolated mutants suppressing the trk1
trk2
phenotype, and found that many of them mapped on the
HXT3 gene, as is consistent with previous reports (14, 15,
26). The sequence of the HXT3 mutated genes revealed that
some of these mutations were located in the 5' non-coding region. This
suggested that these mutations produced overexpression of the encoded
protein, and hence that the wild HXT3 transporter was able to transport K+. To test this possibility, we inserted the
HXT3 gene in a multicopy plasmid and transformed it into the
trk1
trk2
strain, finding that the growth at low
K+ was improved significantly (not shown).
The Lack of Trk1p Triggers the Low-affinity Uptake of
Rb+--
Two different reasons could trigger the general
response that furnished several different transporters with the
capacity of transporting K+, the loss of Trk1p by itself or
the K+ deficiency generated by its loss. To distinguish
between these two possibilities, we used the trk1 trk2
strain transformed with the HAK1 gene of
Schwanniomyces occidentalis (28). This gene encodes a
K+ transporter not related to Trk1p and Trk2p, that
restores K+ uptake, and consequently a normal growth at low
K+, in trk1
trk2
cells (28). Therefore, if
trk1
trk2
cells expressing Hak1p still kept the
low-affinity K+ and Rb+ uptake, the mediation
of the K+ deficiency in this uptake could be ruled out.
trk1 trk2
Strains Are Hypersensitive to Hygromycin
B--
The results obtained with the HAK1 transporter indicated that
Hak1p performed the K+ transport functions of Trk1p, but
that only in very special conditions did it prevent the low-affinity
Rb+ uptake. Among all the possible causes for this
behavior, changes in the membrane potential were the most likely. The
working hypothesis was that trk1
trk2
cells could be
hyperpolarized, and that the low-affinity Rb+ uptake
required such hyperpolarization. This hypothesis gave a satisfactory
explanation to the high sensitivity of the low-affinity Rb+
uptake to uncouplers (Fig. 2), and was testable by determining the
resistance of the different strains to hygromycin B, an amino glycoside
antibiotic for which, as in the case of Dio-9 (29, 30), the resistance
of the cells depends on their membrane potential (31, 32). The tests
were performed in YPD, which contains sufficient K+ (15 mM) for normal growth of the trk1
trk2
strain, using the different mutants in the TRK1 and
TRK2 genes, and the trk1
trk2
strain
transformed with the pPGK1-HAK1 gene or the HKT1
cDNA under the control of the PMA1 promoter (Table
II). HKT1 encodes a
K+-Na+ symporter in wheat (33-35), which is
related to the TRK transporters (36). Transformation with
HKT1 suppressed the K+ uptake deficiency of our
trk1
trk2
strain, consistent with previous results
(33-35). The tests of hygromycin B resistance (Table II) showed that
the trk1
strains, either TRK2 or
trk2
, were inhibited by one-fifth of the concentration
tolerated by the TRK1 strains, and that HKT1 but
not HAK1 suppressed the hypersensitivity of the
trk1
trk2
strain. In pma1 mutants the
sensitivity to hygromycin B depends on the K+ content of
the medium (31), and we found the same effect in trk
mutants. However, even after the addition of 50 mM
K+ to the YPD testing medium, the differences were still
appreciable (Table II).
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Differences in the Membrane Potential Can Be Estimated by DiOC6(3) Fluorescence and Flow Cytometry-- The electrical membrane potential of yeast cells is not known, but comparative assessments of membrane potentials in different conditions may be obtained with fluorescent cyanine dyes (37, 38). The cyanine dye DiOC6(3) has been used to stain internal membranes in yeast (39) and to detect dysfunctional mitochondria (18, 25, 40). To use this dye as a probe for the membrane potential, avoiding the interference of the fluorescence of the internal membranes, we reduced the concentration of the dye in the staining medium (testing buffer) to 1 nM and tested only fermenting cells in which the function of the mitochondria is highly reduced (41). Fermenting cells stained with 1 nM DiOC6(3) and analyzed by flow cytometry showed size-dependent fluorescence (Fig. 3A). Taking the whole population or dividing it into two subpopulations, roughly corresponding to single cells and cells with small buds, and large cells and cells with large buds, K+-starved cells showed a much higher fluorescence than cells with a normal K+ content (Fig. 3, A and B).
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trk1 Cells Are Highly Hyperpolarized--
Flow-cytometric
analyses of the cellular DiOC6(3) fluorescence in
TRK1 TRK2, TRK1 trk2
, trk1
TRK2,
trk1
trk2
, trk1
trk2
HAK1, and
trk1
trk2
HKT1 cells, either normal in K+
or K+-starved, before and after the addition of KCl or
NaCl, revealed that the cellular DiOC6(3) fluorescence, and
hence the membrane potential, depended on the genotype (Table
IV). Comparing first the TRK1
TRK2 and trk1
trk2
strains, it was clear that the
latter was hyperpolarized with reference to the former, both in
conditions of a normal K+ supply and in
K+-starved conditions. In K+-starved
trk1
trk2
cells, the addition of 10 mM KCl
or NaCl produced further hyperpolarization, whereas in TRK1
TRK2 cells NaCl showed no effect and KCl depolarized. Deletion of
TRK2 did not produce any significant effect with reference
to the wild strain, but TRK1-deleted cells (trk1
TRK2 strain) showed a hyperpolarized state between the TRK1
TRK2 and trk1
trk2
cells. Interestingly, in the
trk1
TRK2 strain neither NaCl nor KCl showed any
appreciable effect.
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Chloride Is Not Involved in the Hyperpolarizing Effects of NaCl and
KCl--
One intriguing result among those summarized in Table IV is
the hyperpolarizing effect of KCl and NaCl in the trk1
trk2
strain. This effect could result from the high diffusion
potential created by the increase in the external concentration of
chloride if a chloride channel exists in the plasma membrane. To
address this possibility we repeated the experiments summarized in
Table IV substituting Na+-MES and K+-MES for
NaCl and KCl, respectively. The change of the anion did not change the
results, thus indicating that Na+ and K+, and
not chloride, were involved in the hyperpolarizing effect.
Trk1p and Trk2p Are Involved in the Response of the Membrane
Potential to Changes in the External pH--
Two reasons could explain
why trk1 trk2
cells were hyperpolarized with reference
to TRK1 TRK2 cells, that the H+-pump ATPase was
more active in the mutant than in the wild strain, because Trk1p and
Trk2p exercise some degree of control on the pump, or that Trk1p and
Trk2p depolarized the membrane by mediating a flux of ions at high
membrane potentials. Because the difference between the membrane
potentials of these strains persisted in a buffer with only MES,
Ca2+, and H+, the most likely ion fluxes were
H+ inward or anions outward. To test if H+
influx was involved, we studied the response of the cellular DiOC6(3) fluorescence of the different genotypes to changes
in the external pH and to the addition of uncouplers. These tests were
performed with K+-starved cells in the complete absence of
K+, thus eliminating the differences in depolarizing
currents that this cation may introduce in genotypes with different
K+ uptake capacities. The data summarized in Table
V (data at pH 6.0 correspond to basal
conditions, K+-starved cells, in Table IV) show clearly
that the membrane potential decreased with the decrease of the external
pH only when Trk1p or Trk2p are present. In contrast, the response to
the addition of CCCP, which should increase unspecific H+
conductance, was not appreciably different in the different genotypes, producing in all cases a progressive decrease in the cellular DiOC6(3) fluorescence with the increase in the CCCP
concentration. Taken together, these results suggest that Trk1p and
Trk2p somehow increase the permeability to H+.
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Nitrogen Starvation Also Produces Hyperpolarization--
Before
addressing the question of whether Trk1p and Trk2p are H+
transporters or only regulate the H+ permeability of
another membrane protein, we addressed the more general question of
whether hyperpolarization of the plasma membrane was a specific
response to K+ starvation or a more general response to any
kind of starvation. With this purpose we studied the cellular
DiOC6(3) fluorescence of nitrogen and phosphate-starved
cells, finding that fluorescence increased as a consequence of nitrogen
starvation but not after phosphate starvation. We have described that
trk1 trk2
cells hyperpolarized in response to
K+ starvation, increasing their cellular
DiOC6(3) fluorescence over the already high normal value.
Unlike this response, nitrogen starvation did not show any effect on
the DiOC6(3) fluorescence of trk1
trk2
cells.
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DISCUSSION |
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The results presented in this report indicate that the growth of
trk1 trk2
mutants of S. cerevisiae is
supported by an ectopic K+ uptake mediated by several
transporters, which, as a whole, resembles a low-affinity single
transporter for K+, Rb+, Na+, and
Li+. The view is supported by the manner in which this
low-affinity uptake is inhibited by ammonium and by divalent cations
(Fig. 1), and by the number of different and unrelated transporters (amino acids, sugar, inositol, and choline transporters) (this report
and Refs. 14-16) that by overexpression partially suppress the
defective growth of trk1
trk2
strains at low
K+. Our results also indicate that in the absence of Trk1p,
or Trk1p and Trk2p, the membrane potential is exceptionally high,
negative inside. Taken together, these results pose two questions,
whether there is any causal relationship between the ectopic
low-affinity K+ and Rb+ uptake and the high
membrane potential, and what the cause of this high membrane potential
is? Regarding the former question, it is clear that both the
low-affinity Rb+ uptake and the high membrane potential are
the consequences of the deletion of the TRK1 gene.
Furthermore, the ectopic K+ uptake must result from a
change in a general property of the membrane, because it is unlikely
that the deletion of the TRK1 gene can modify the specific
properties of many different transporters, making all of them permeable
to alkali cations. Considering these observations and the high
sensitivity of the low-affinity Rb+ uptake to CCCP, the
most likely cause for the ectopic uptake of K+ is the
unusually high membrane potential exhibited by trk1
strains.
Regarding the high membrane potential of trk1 and
trk1
trk2
cells, our results reveal that Trk1p and
Trk2p are not only K+ transporters but also essential
regulators of the membrane potential. To fulfill this function they
must either counter the activity of the pump or control the conductance
of the plasma membrane. Before going further in the discussion of the
function of Trk1p and Trk2p, it is worth mentioning that a pitfall in
the use of the cellular fluorescence of the DiOC6(3) probe
to assess differences in membrane potentials is extremely unlikely,
considering the conditions for our assessments: (i) we used the
cellular DiOC6(3) fluorescence only for comparative
purposes in entirely isogenic cells, except for the genes considered in
each case; (ii) the contribution of the mitochondria to these changes
in fluorescence has been ruled out; (iii) we did a control using
N. crassa cells, for which the membrane potential has been
measured using intracellular electrodes (3). Furthermore, the
hyperpolarized state of the trk1
and trk1
trk2
cells can be also deduced by contrasting the phenotype of
these mutants with the phenotype of pma1 mutants. The latter
are more resistant to hygromycin B than the wild type, whereas
trk1
and trk1
trk2
mutants are
strikingly more sensitive (Table II). Assuming that the phenotype of
pma1 mutants is the result of a lower membrane potential
(32), it can be concluded that the phenotype of trk1
and
trk1
trk2
mutants is the result of a higher membrane
potential.
Nitrogen starvation hyperpolarized TRK1 TRK2 cells but not
trk1 trk2
cells, whereas K+ starvation
hyperpolarized both types of cells. This suggests that at least two
parallel and additive routes control the membrane potential, one
dependent on Trk1p and Trk2p, and the other independent of these
proteins. Apparently, K+ starvation activates both routes,
whereas nitrogen starvation activates only that dependent on Trk1p and
Trk2p. The mechanisms involved in the control of the membrane potential
cannot be established at this moment. However, there are only two ways
to achieve this control, either modifying the activity of the pump or
triggering a "safety depolarizing current" when the membrane
potential reaches a certain value. In the latter case, because the
function can be performed in the absence of K+, or any
other alkali cation (Table IV), it is likely that the ions moving are
either H+ inward or anions outward. The Trk1p-Trk2p
dependence of low-pH depolarization (Table V) indicates that an inward
movement of H+ may be involved in the route of control
depending on Trk1p and Trk2p.
The capacity of Trk1p and Trk2p to control the membrane potential is not a general property of K+ transporters, because Hak1p cannot perform these functions (Tables III and IV). Although the results supporting this conclusion have been obtained using a heterologous expression, the high level of conservation in proteins and functions among fungi, and even among fungi and higher plants, suggests that it is correct. It is very interesting that in S. occidentalis,2 in N. crassa,3 and in Debaryomyces hansenii4 HAK1 type K+ transporters coexist with TRK2 K+ transporters. Whether the function of these TRK2 transporters in these species is more related with the control of the membrane potential than to K+ uptake is now under study. Interestingly, in barley plants K+ uptake is mediated by HAK1 transporters (43-46), and wheat plants have a HKT1 transporter whose function is not clear (47). Hkt1p shows homology with Trk1p and Trk2p (36) and, when expressed in S. cerevisiae, it produced strong depolarization in the presence of Na+ (Table IV). Therefore, the involvement of Hkt1p in the control of the membrane potential in higher plants is an attractive possibility.
The conclusion of this and previous reports (14-16) identifying
independent mechanisms for Rb+ influx in TRK1
TRK2 and trk1 trk2
cells indicates that the mechanisms involved in the activation of the
Vmax of Rb+ influx by glucose in
both types of cells (17) may be also different. Interestingly, we found
that the addition of glucose hyperpolarized both wild type and
trk mutant cells (not shown), which is consistent with the
known activating effect of glucose on the H+-pump ATPase
(48). The possibility that this hyperpolarization brings about the
increase of the Vmax of Rb+ influx
in all cases is an attractive idea. Unfortunately, this cannot be
tested with the techniques used in this report.
Finally, trk1 trk2
mutants have been extensively used
for cloning heterologous K+-transport genes. Our results
indicate that genes expressing low-rate, low-affinity K+
uptake in these mutants, may encode non-K+ transporters
that support ectopic K+ uptake.
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ACKNOWLEDGEMENTS |
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We thank R. Haro for providing W3, W
2,
and W59 strains; F. Rubio for providing plasmid pPMAHKT1; F. Bouillaud
for strain UCP
9; A. Alvarez and A. Vázquez (flow cytometry
service of the Departamento de Microbiología, Facultad de
Farmacia, UCM) and P. Lastres (flow cytometry service of Centro de
Investigaciones Científicas, CSIC) for helping with flow
cytometry experiments; and E. Rial for permitting the use of his
laboratory in preparing many experiments of flow cytometry.
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FOOTNOTES |
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* This work was supported by European Community Grants BI02-CT-0400 and BI04-CT96-0775 (to A. R.-N.) and BIO4-CT97-2210 (to J. R.), and Ministerio de Educación y Ciencia of Spain Grants PB92-0907 (to A. R.-N.) and PB95-0976 (to J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, 28040 Madrid, Spain. Tel.: 34-91-336-5751; Fax: 34-91-336-5757; E-mail: arodrignavar{at}bit.etsia.upm.es.
1 The abbreviations used are: MES, 2-(N-morpholino)ethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; CCCP, carbonyl cyanide p-chlorophenylhydrazone.
2 R. Madrid and A. Rodríguez-Navarro, unpublished results.
3 R. Haro, L. Sainz-Pastor, F. Rubio, and A. Rodriguez-Navarro, unpublished results.
4 C. Prista, M. C. Loureiro-Días, and J. Ramos, unpublished results.
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REFERENCES |
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