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Journal of Bacteriology, October 1999, p. 6456-6462, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Biochemical and Genetic Analyses of the Role of
Yeast Casein Kinase 2 in Salt Tolerance
Eulàlia
de
Nadal,1
Fernando
Calero,2
José
Ramos,2 and
Joaquín
Ariño1,*
Departamento Bioquímica i Biologia
Molecular, Universitat Autònoma de Barcelona, Bellaterra
08193, Barcelona,1 and Departamento
de Microbiología, Escuela Técnica Superior de
Ingenieros Agrónomos y Montes, 14080 Córdoba,2 Spain
Received 27 May 1999/Accepted 6 August 1999
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ABSTRACT |
Saccharomyces cerevisiae cells lacking the regulatory
subunit of casein kinase 2 (CK-2), encoded by the gene
CKB1, display a phenotype of hypersensitivity to
Na+ and Li+ cations. The sensitivity of a
strain lacking ckb1 is higher than that of a calcineurin
mutant and similar to that of a strain lacking HAL3, the
regulatory subunit of the Ppz1 protein phosphatase. Genetic analysis
indicated that Ckb1 participates in regulatory pathways different from
that of Ppz1 or calcineurin. Deletion of CKB1 increased the
salt sensitivity of a strain lacking Ena1 ATPase, the major determinant
for sodium efflux, suggesting that the function of the kinase is not
mediated by Ena1. Consistently, ckb1 mutants did not show
an altered cation efflux. The function of Ckb1 was independent of the
TRK system, which is responsible for discrimination of
potassium and sodium entry, and in the absence of the kinase regulatory
subunit, the influx of sodium was essentially normal. Therefore, the
salt sensitivity of a ckb1 mutant cannot be attributed to
defects in the fluxes of sodium. In fact, in these cells, both the
intracellular content and the cytoplasm/vacuole ratio for sodium were
similar to those features of wild-type cells. The possible causes for
the salt sensitivity phenotype of casein kinase mutants are discussed
in the light of these findings.
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INTRODUCTION |
As for many cell types, sodium
cations are rather toxic for yeast cells, and consequently, the
maintenance of suitable intracellular concentrations of Na+
is a strong requirement for survival (see reference
42 for a review). Intracellular sodium levels are
the result of influx and efflux processes that are subjected to
regulation. Saccharomyces cerevisiae actively extrudes
sodium through the Na+-ATPase encoded by the gene
ENA1, the first member of the ENA (also called
PMR2) locus (13, 18, 40, 47). ENA1 is
barely expressed under normal growth conditions, but its expression is sharply increased by osmotic and saline (sodium or lithium) stresses, as well as by alkaline pH (13, 23, 25). As a consequence, cells lacking ENA1 are highly sensitive to sodium and
lithium. Several components of the regulatory network that controls
ENA1 expression have been identified in the last few years.
Interestingly, this regulation involves phospho-dephosphorylation
mechanisms. For instance, the Ser/Thr protein phosphatase PP2B
(calcineurin) is needed for full response to sodium stress (25,
28). On the other hand, the Ppz1 protein phosphatase represses
ENA1 expression through a mechanism that is independent from
that of calcineurin. This repression of ENA1 results in
phosphatase mutants that are hypertolerant to sodium (32).
Recent work has shown that HAL3, initially identified as a
halotolerant determinant that influences ENA1 expression
(11), is a negative regulatory subunit of Ppz1 and thus
defines a novel regulatory pathway (8). Hal1, a conserved salt-induced protein (14), has been defined as an effector
of ENA1 expression (39). Recently,
HAL8 and HAL9 have been determined to be genes
encoding putative transcriptional activators of the ENA1
response to salt stress (24).
In S. cerevisiae the uptake of K+ and
Na+ is mediated by the Trk1-Trk2 transport system, being
the Trk1 function predominant under normal growth conditions (12,
19, 20, 36). The TRK system discriminates between Na+
and K+, thus preventing the entry of an excess of
Na+ when the levels of the cation in the medium are too
high. Therefore, a proper functioning of this cation uptake system
ought to be important for salt tolerance, as demonstrated by the
observation that trk1 trk2 mutants are hypersensitive to
sodium ions (17, 19). In addition, intracellular
sequestration of sodium can also be an efficient method of improving
salt tolerance, and confinement of Na+ in the vacuole has
been proposed as a mechanism that reduces the cytosolic levels of this
cation (19). It has been documented that the putative
Na+-H+ exchanger encoded by the gene
NHX1 is involved in the vacuolar compartmentalization of
sodium ions (29, 30).
Therefore, sodium homeostasis in yeast appears to be a complex process,
still poorly understood at the molecular level. Casein kinase 2 (CK-2)
has been proposed as an additional component of this regulatory system.
CK-2 is a highly conserved Ser/Thr protein kinase that has also been
related to cell polarity and cell cycle progression (for a recent
review, see reference 16). In yeast, CK-2 is an
oligomer composed of two related catalytic subunits (
and '),
encoded by the genes CKA1 and CKA2 (6,
31), and two regulatory polypeptides (
and '), encoded by
the genes CKB1 and CKB2 (5, 37),
respectively. In order to survive, yeast cells require at least one of
the catalytic subunits (31). On the contrary, the regulatory
subunits do not appear to be necessary for growth under normal
conditions. Interestingly, deletion of either CKB1 or
CKB2 results in the same phenotype of hypersensitivity to
Na+ and Li+ (5). The effect of the
mutations is not additive and does not affect the tolerance to
potassium cations (5).
Our laboratories are interested in the analysis of the role of protein
phosphorylation in the regulation of salt tolerance in yeast cells.
Therefore, to gain insight into the mechanism responsible for the role
of CK-2 in yeast biology, we undertook a genetic and biochemical study
of the effects of the absence of Ckb1 on the different cell processes
that affect the sensitivity to Na+ and Li+. Our
results indicate that CK-2, in contrast with recently reported data,
does not regulate the influx or the efflux of sodium, thus suggesting
that this kinase might be involved in the regulation of a putative
target for sodium toxicity.
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MATERIALS AND METHODS |
Strains and growth conditions.
Escherichia coli NM522
and DH5 were used as hosts for DNA cloning. Bacterial cells were
grown at 37°C in Luria-Bertani medium containing 50 µg of
ampicillin per ml, when needed, for plasmid selection. Yeast cells were
grown at 28°C in yeast extract-peptone-dextrose (YPD) medium or, when
indicated, in synthetic minimal (SD) or complete minimal medium
(43). The relevant genotypes of the strains described in
this work can be found in Table 1.
Recombinant DNA techniques, gene disruptions, and plasmids.
E. coli and S. cerevisiae cells were transformed
by standard techniques as previously described (8).
Restriction reactions, DNA ligations, and other standard recombinant
DNA techniques were carried out as described previously
(41). Gene disruptions were performed as follows. Disruption
of PPZ1 and CNB1 was as described in reference
32. Disruption of CKB1 with the
HIS3 marker was made by integration of plasmid pAPB17
linearized by digestion with EcoRI (5). To
disrupt CKB1 with the marker TRP1, plasmid pAPB17
was digested with XhoI and SacI and the insert
(about 1.0 kbp) was cloned into plasmid pRS304. This plasmid was
linearized as described above and used to transform yeast cells.
Disruption of the genes HAL1 and HAL3 with the
LEU2 marker was performed in manners similar to those
described in references 14 and
11, respectively.
To achieve high levels of expression of Hal1 and Hal2, the
corresponding open reading frames were cloned into high-copy-number vectors carrying the PMA1 promoter, as previously described
(26, 39).
-Galactosidase measurements.
To evaluate the effect of
the ckb1 mutation on ENA1 expression, wild-type
DBY746 and EDN1 (ckb1
) cells were transformed with the
multicopy plasmid pKC201 (1, 7), which contains
ENA1 sequences from 
1385 to +35 (relative to the starting
initiating Met), fused to lacZ. Cells (5 ml) were grown to
an optical density at 660 nm of 0.5 to 1.0, solid NaCl was added to
achieve a final concentration of 0.75 M, and growth was resumed for 60 min. Cells were then centrifuged, and -galactosidase activity was
measured as described in reference 38.
Determination of cation influx and efflux.
For influx
experiments, cells grown in SD medium were potassium starved by
incubation in the minimal ammonium-phosphate medium (35).
After 5 h, cells were harvested and resuspended in buffer containing RbCl or LiCl (50 mM). Samples were taken at regular time
intervals, filtered immediately, and treated for determination of
intracellular ion content.
For determination of efflux rate, cells were grown in SD medium up to
optical density at 660 nm of 0.3 to 0.4 and then LiCl
or NaCl (100 mM)
was added. Growth was resumed for 3 h in order
to load the cells
with the cation. After this time, cells were
harvested and resuspended
in buffer {10 mM MES [2-(
N-morpholino)ethanesulfonic
acid] brought to pH 5.8 with Ca(OH)
2 and containing 0.1 mM
MgCl
2 and 2% glucose}, supplemented with 10 mM KCl to
trigger the efflux
process. Samples were taken at regular time
intervals, filtered,
and treated for determination of intracellular ion
content.
The intracellular ion content of the cells was determined as previously
described (
34,
36). Briefly, samples of cells
were filtered,
washed with 20 mM MgCl
2, and treated with acid
and the
cations were analyzed by atomic absorption
spectrophotometry.
Other methods.
Salt sensitivity assays were performed with
freshly prepared YPD plates containing different concentrations of the
compound (drop tests) or with liquid cultures as described in reference 32. Measurement of proton fluxes were performed as
described previously (2), except that cells were grown in
YPD medium. Differential extraction of potassium and sodium ions from
the cytoplasm and vacuole was essentially achieved as previously
described (10), with minor modifications, including a
treatment of the cells with 0.1 mg of digitonin per ml for 5 min.
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RESULTS |
CK-2 regulates sodium tolerance by a mechanism independent from
that of calcineurin and Ppz1.
It has been reported that deletion
of CKB1 results in a phenotype of sensitivity to sodium and
lithium ions (5). To evaluate the potency of this phenotype,
we deleted the CKB1 gene in the DBY746 background and
compared the sensitivities to sodium and lithium of the
ckb1 mutant with those of cells lacking other genes known
to be involved in salt sensitivity, such as the regulatory subunit of
calcineurin (CNB1), HAL1, and HAL3.
Strains with mutations in the HAL1 gene displayed a very
weak salt sensitivity phenotype. Deletion of CKB1 resulted
in a phenotype that was stronger than that of calcineurin mutants and
almost as strong as that of cells lacking HAL3 (not shown).
Dose-response experiments performed with SD liquid cultures showed that
the tolerance to lithium ions of a ckb1 mutant was
reduced by about 30% compared to the tolerance of the wild-type strain
(50% inhibitory concentration 18 mM versus 26 mM).
Because
CKB1 encodes a regulatory subunit of a protein
kinase, we considered it interesting to test the possibility of genetic
interaction between this gene and the pathways defined by the
calcineurin and Ppz1 phosphatase genes. To this end, we disrupted
the
CKB1 gene in cells lacking
CNB1, the gene
encoding the regulatory
subunit of calcineurin, and tested the
sensitivity of these cells
to Li
+. As shown in Fig.
1, lack of Ckb1 resulted in an additional
increase
in sensitivity to lithium cations, indicating that the kinase
and the phosphatase do not share a common regulatory pathway.
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FIG. 1.
Additive effects of the calcineurin and ckb1
mutations. Strains DBY746 (wild type [wt]), JA40 (cnb1),
EDN1 (ckb1), and EDN22 (cnb1 ckb1) were plated on
YPD plates containing the indicated concentrations of LiCl. Plates were
incubated at 28°C, and growth was scored after 2 days.
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Disruption of the protein phosphatase Ppz1 resulted in increased salt
tolerance. As shown in Fig.
2, the
absence of Ckb1 decreased
the tolerance of a
ppzl strain,
as would be expected if Ckb1
and Ppz1 regulate independent pathways.
Hal3 has been defined
as a regulatory subunit of Ppz1, thus placing
Ppz1 and Hal3 in
the same regulatory pathway. To confirm our
observation, we generated
a
ckb1 hal3 double mutant and
analyzed its sensitivity to lithium
ions. As shown in Fig.
2, the
ckb1 hal3 double mutant was more
sensitive than a single
hal3 or
ckb1 deletion mutant.
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FIG. 2.
The effect of CK-2 on salt tolerance is not mediated by
the Hal3/Ppz1 pathway. (A) YPD medium containing the indicated
concentrations of LiCl was inoculated (initial
A660, 0.007) with wild-type strain DBY746 ( )
or its derivatives EDN1 (ckb1) ( ), EDN4 (hal3)
( ), and EDN22 (ckb1 hal3) ( ). Cultures were grown for
18 h, and the densities of the cultures were then measured.
Relative growth was calculated as the ratio between growth in the
presence and growth in the absence of added salts and expressed as a
percentage. (B) Cultures of DBY746 (), JA30 (ppz1) ( ),
and EDN11 (ppz1 ckb1) () cells were grown as indicated
above. Data are means ± standard errors of the means of results
from four independent experiments.
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Both calcineurin and Ppz1 are known to affect sodium tolerance by
regulating the expression of
ENA1, a gene encoding the
Na
+-ATPase which represents the major mechanism for
Na
+ efflux in budding yeast.
HAL1 had been
defined as a gene that,
when it is expressed in multicopy numbers, was
able to increase
ENA1 expression. We considered that if Ckb1
was placed downstream
of Hal1, high levels of Hal1 would not confer
sodium tolerance
to the mutant. However, as shown in Fig.
3, high-copy-number expression
of
HAL1 clearly increased the tolerance of a
ckb1
strain, indicating
that the effect of Hal1 is independent of the
presence of Ckb1.
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FIG. 3.
High-copy-number expression of HAL1 increases
salt tolerance in a ckb1 background. Strains DBY746
(CKB1) and EDN1 (ckb1) were transformed with the
high-copy-number plasmid pRS699-HAL1 (denoted YEpHAL1) (+) or the empty
plasmid YEplac195 (). Positive cultures were plated on YPD plates
containing the indicated concentrations of LiCl, and growth was
monitored as described in the legend to Fig. 1.
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Analysis of the sodium efflux mechanisms in a ckb1
mutant.
Because of the relatively strong phenotype of the
ckb1 mutation, we decided to explore in a systematic way the
possible effect of Ckb1 on the expression of ENA1 and,
therefore, on sodium efflux. To this end, we disrupted the
CKB1 gene in an RH16.6 strain that lacks the
ENA1-ENA4 gene cluster. This strain has a very reduced sodium efflux, and therefore it is highly sensitive to Li+
and Na+. Interestingly, the deletion of CKB1
further increased the sensitivity to Li+ of the
ena1-ena4 mutant (Fig. 4A).
This result was somewhat unexpected because it indicated that, in
contrast to preliminary published data (16), the function of
CK-2 does not involve the Ena1 ATPase. To confirm this possibility, we
transformed wild-type and ckb1 strains with plasmid pKC201,
which bears the entire ENA1 promoter fused to
-galactosidase. The cells were stressed with 0.75 M NaCl for 1 h, and the -galactosidase activity was measured. As shown in Fig.
4B, cells lacking Ckb1 showed a response essentially identical to that
of wild-type cells whereas, under the same conditions, hal3
and ppz1 mutants displayed decreased (hal3) and
increased (ppz1) responses, respectively, as previously
described (11, 32). Therefore, our data did not support the
notion that CK-2 is an effector of ENA1 transcription and
suggested that cation efflux might not be affected in Cbk1-deficient
yeast cells. This possibility was directly tested by loading wild-type,
cnb1, and ckb1 cells with lithium and
measuring the efflux of this cation. As shown in Fig.
5, whereas the cation efflux of
calcineurin mutants was reduced (as previously described), the efflux
of the ckb1 strain was essentially identical to that of
wild-type cells. Therefore, from our data it can be concluded that the
increased sensitivity of the ckb1 mutant to sodium and
lithium cannot be attributed to a reduced efflux of these cations.
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FIG. 4.
The ckb1 mutation is additive to those of
ENA1 to ENA4 and does not alter the expression of
the ATPase. (A) Strains DBY746 (), RH16.6 (ena1 to
ena4) ( ), and EDN25 (ena1 to ena4
ckb1) () were tested for LiCl sensitivity in liquid cultures as
described for Fig. 2. Data are means ± standard errors of the
means of results from four independent experiments. (B) DBY746 (wild
type [wt]), EDN1 (ckb1), EDN4 (hal3), and JA30
(ppz1) were transformed with the multicopy plasmid pKC201,
which allows expression of the -galactosidase protein from the
ENA1 promoter. Cells were grown as described in Materials
and Methods, and -galactosidase activity was measured in
permeabilized cells treated with (+) or without () 0.75 M NaCl for 60 min. Data are means ± standard errors of the means of results
from 16 to 18 independent experiments performed with five independent
clones (DBY746 and EDN1) or eight independent experiments performed
with four independent clones (EDN4 and JA30).
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FIG. 5.
Measurement of the efflux of lithium cations in
ckb1 cells. Wild-type DBY746 (), as well as EDN1
(ckb1) () and JA40 (cnb1) (), cells were
loaded with LiCl for 3 h and washed, and the efflux of
Li+ was monitored as described in Materials and Methods.
Data are means ± standard errors of the means of results from
three independent experiments.
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Mutation of CKB1 does not alter sodium or potassium
influx.
Changes in the influx of sodium and potassium, mediated by
the TRK system, can be responsible for salt sensitivity
phenotypes. To test the possibility that CK-2 affects the
TRK system, we introduced the ckb1 deletion in a
strain lacking TRK1 and TRK2. When this strain
was tested for sodium sensitivity, we observed that it was more
sensitive than the trk double mutant (Fig.
6). This result supported the notion that
CK-2 is required for normal salt resistance in trk1 trk2
cells and suggests that the TRK system is not regulated by
CK-2. In fact, we have measured Li+ and Rb+
influx (the latter being used as an analog of K+ for
transport experiments) in wild-type and ckb1 cells (Fig. 7). The time course of the uptake of
these cations showed that the initial velocities of influx were
virtually identical in both strains but that, as expected, it was
dramatically reduced in a trk1 mutant, which is defective in
high-affinity potassium transport. Consequently, the salt sensitivity
phenotype of the ckb1 mutant cannot be attributed to changes
in the influx of these cations. Because changes in proton efflux can
affect salt tolerance, we determined this parameter in wild-type and
ckb1 cells, obtaining values of 15 ± 2 and 13 ± 1.5 nmol of H+/mg (dry weight) of cells. Therefore, our
results indicate that the ckb1 mutation does not modify
proton pumping.
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FIG. 6.
The deletion of CKB1 increases the salt
sensitivity of a trk strain. The CKB1 gene was
disrupted in the wild-type (wt) strain W303.1A and in its isogenic
strain W3 (trk1 trk2) to yield EDN42 and EDN44,
respectively. The sensitivities to NaCl of these strains were tested on
plates as described in the legend to Fig. 1.
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FIG. 7.
Influx of Li+ and Rb+ in a
ckb1 strain. Potassium-starved wild-type W303.1A () and
EDN1 (ckb1) () were incubated with RbCl (upper panel) or LiCl (lower
panel), and the influxes of these cations were determined as described
in Materials and Methods. Data from strain W59 (trk1) (),
which is known to have a decreased potassium transport, is included for
comparison. Data are means ± standard errors of the means of
results from four independent experiments.
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The data presented so far indicate that the mutation of
CKB1
does not alter the normal influx and efflux of Na
+ and
K
+. Consistently with this evidence, we have observed that,
after
the cells were challenged with a range of NaCl concentrations
(from 0.25 to 1 M), the intracellular contents of Na
+ and
K
+, as well as the Na
+/K
+ ratio,
were virtually identical in wild-type cells and
ckb1 mutants
(data not shown). Finally, we have examined the possibility that
CK-2
is somehow involved in the process of sequestration of sodium
into the
vacuole. To this end, we measured the cytoplasmic and
vacuolar contents
for Na
+ and K
+, before and after 6 h of
incubation of the cells with 1 M NaCl.
As shown in Fig.
8, the intracellular distributions of
both cations
were very much alike in wild-type and
ckb1
cells.
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FIG. 8.
Cytoplasmic and vacuolar distributions of sodium and
potassium ions in wild-type and ckb1 yeast cells. Wild-type
(filled bars) and EDN1 (ckb1) (open bars) cells were grown
on YPD medium and incubated for 6 h with or without 1 M NaCl. The
contents of Na+ and K+ in the cytoplasm (Cit.)
and the vacuole (Vac.) were determined as described in Materials and
Methods. Data are means ± standard errors of the means of results
from three experiments.
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The fact that mutation of Ckb1 affects Na
+ and
Li
+ tolerance in the absence of increased levels of these
cations drew our attention
to the
HAL2 (also called
MET22) gene, which codes for an Na
+- and
Li
+-sensitive phosphohydrolase identified as a putative
target for
the toxicities of these cations. Overexpression of
HAL2 in a wild-type
background resulted in a relatively weak
increase in salt tolerance.
As shown in Fig.
9, high levels of Hal2 also increase the
tolerance
of a
ckb1 strain, indicating that the regulatory
subunit of CK-2
does not mediate Hal2 function.
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FIG. 9.
Overexpression of HAL2 increases the
Li+ tolerance of a ckb1 strain. Wild-type DBY746
(circles) and EDN1 (ckb1) (triangles) cells were transformed
with plasmid pRS699-HAL2 (open symbols) or the empty plasmid YEplac185
(filled symbols), and their sensitivities to LiCl were measured in
liquid cultures as described for Fig. 2. Data are means ± standard errors of the means of results from four independent
experiments.
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DISCUSSION |
The finding that the mutation of the regulatory subunits of CK-2
(CKB1 and CKB2) results in a phenotype of
sensitivity to sodium and lithium ions (5) raised the key
question of what type of cellular process, relevant for cation
tolerance, involves this kinase. Because of the equivalence in potency
and the lack of an additive phenotype, the disruption of only one gene,
CKB1, was chosen as a working model. Our data indicated that
the potency of the mutation is relatively strong, thus suggesting that
this cellular process is highly relevant for salt tolerance. In
S. cerevisiae, a key factor for salt tolerance is the proper
function of the major determinant for sodium efflux, the Ena1
Na+-ATPase (13, 19, 47). In addition, the
expression of the ENA1 gene is regulated by mechanisms
involving phospho-dephosphorylation reactions. Therefore, it was
reasonable to assume that the function of CK-2 is related to that of
the Hal3 and Ppz1 (8) or the calcineurin (25)
regulatory system. However, our data clearly show that CK-2
participates in a mechanism that is independent from the mentioned
phosphatases. In addition, we demonstrate that the function of Hal1,
which, when expressed at high levels, results in increased
ENA1 expression (39), does not require Ckb1.
While the above-mentioned results are still compatible with the notion
that CK-2 regulates a novel Ena1-regulatory pathway, we provide here
biochemical and genetic data demonstrating that the function of Ckb1 in
salt tolerance does not involve Ena1 and that the expression of the
ENA1 ATPase gene is not altered by the absence of Ckb1.
These conclusions are in sharp contrast with recently published data
suggesting that the kinase regulates the transcription of the
ENA1 gene (45). Tenny and Glover (45) derived their conclusions essentially from experiments with a -galactosidase reporter system (similar to what is shown in our Fig.
4B). Although, at this moment, we cannot account for these contradictory results, it is worth noting that the experimental conditions differed in a number of circumstances, including strain background and concentration (0.4 instead 0.75 M) and time of exposure
to NaCl (30 min instead 60 min). However, we performed -galactosidase experiments after stressing the cells for different times with 0.4 M NaCl and still could not find differences between wild-type and ckb1 strains. As an additional proof for the
involvement of Ena1 in the mechanism of action of the kinase, Tenny and
Glover invoked the fact that the overexpression of ENA1
suppresses the salt sensitivity of the ckb1 mutants.
However, it seems evident that overexpression of Ena1 would result in
active extrusion of sodium and lithium cations and, probably,
sequestration in intracellular compartments (4). This
overexpression would also alleviate the salt sensitivity phenotype of
an Ena1-independent mutation, simply by reducing the cytosolic amount
of the cations. Furthermore, our analysis of cation efflux clearly
shows that the output of sodium or lithium ions is not modified by the
absence of Ckb1. In contrast, and consistently with reported data
(25), a cnb1 mutant (which has a weaker salt
sensitivity phenotype), shows a clear-cut decrease in efflux rate. This
decrease can be considered further evidence against an involvement of
ENA1 in CK-2 function. We feel that our conclusions are
further strengthened by the fact that they are sustained by a
combination of both genetic and biochemical evidence. A consequence of
the independence of Ckb1 and Ena1 is that the function of CK-2 would
also be independent from that of the SOP1 and
SOP2 gene products, which have been shown to require ENA1 for function (21). In addition, from our
efflux data, one might expect that the absence of Ckb1 would not affect
the function of the Nha1 antiporter, a protein that under specific
circumstances also plays a role in sodium efflux (3, 33,
44).
A very relevant finding regarding the role of CK-2 in salt tolerance is
that the absence of Ckb1 does not increase the intracellular sodium
content or alter the intracellular Na+/K+
ratio. These facts are in agreement with our findings that the lack of
Ckb1 has very little effect on Li+ and Rb+
uptake and that the ckb1 mutation shows, as far as sodium
tolerance is concerned, an additive effect on the trk1 trk2
mutation. Our data also rule out the possibility that the absence of
Ckb1 alters the ability of the cell to reduce the cytoplasmic levels of
sodium cations through vacuolar sequestration.
Therefore, our findings define a scenario in which ckb1
cells are substantially more sensitive to sodium and lithium than wild-type cells in the absence of an increased intracellular cation content. A reasonable hypothesis would be that the absence of Ckb1
results in increased sensitivity to sodium and lithium cations of an
important component of the cellular machinery. This component would be
a direct or an indirect target for CK-2 phosphorylation, being the
dephosphorylated salt-sensitive protein and, therefore, a cellular
target for salt toxicity.
So far, only Hal2/Met22 has been characterized through genetic and
biochemical methods as a target for lithium toxicity (15, 26,
27). Hal2 degrades adenosine 3', 5'-bisphosphate (pAp) and
3'-phosphoadenosine, 5'-phosphosulphate (pApS). These compounds are
intermediates of the sulfate assimilation pathway, which is needed
mainly for the synthesis of sulfur-containing amino acids (see
references 46 for a review). Overexpression of
HAL2 increases Li+ tolerance because this enzyme
is inhibited by Li+, and pApS is highly toxic for yeast.
While at this moment we cannot rule out the possibility that the salt
sensitivity phenotype of ckb1 mutants is related to
alterations in the sulfate uptake pathway, several lines of reasoning
suggest that this does not occur through the regulation of Hal2. For
instance, Hal2 does not appear to contain consensus sequences for CK-2
phosphorylation. We show here that overexpression of HAL2
still increases Li+ tolerance even in the absence of Ckb1
and that the ckb1 mutant has a rather strong phenotype but
that the hal2 deletion has almost no effect on salt
tolerance (15). Finally, hal2 mutants display an auxotrophy for methionine (15), presumably because Met
supplementation greatly reduces the need for sulfate intake and, hence,
pAp and pApS formation, whereas we have found that ckb1
mutants grow well in the absence of Met (data not shown). It has been
recently suggested that RNA processing might be a target function for
lithium toxicity and that this would be the result of the existence of
several Li+-sensitive components displaying synergistic
toxicity (9). Certain components would be inhibited by an
excess of pAp or pApS (as a result of Hal2 inhibition), whereas others,
such as the RNase MRP ribonucleoprotein, would be directly inhibited by
lithium ions. A conceivable hypothesis would be that one of the latter components is phosphorylated by CK-2 and that the absence of Ckb1 would
yield a dephosphorylated protein, hypersensitive to Li+ and
Na+ ions.
| |
ACKNOWLEDGMENTS |
We thank C. V. Glover for the CKB1 disruption
cassettes, A. Rodríguez-Navarro for the RH16.6 strain, R. Haro
for the W59 and W3 strains, and R. Serrano for the HAL1
and HAL2 plasmids. The skillful technical help of Anna
Vilalta and Mireia Zaguirre is acknowledged.
This work was supported by grants PB95-0663 and PB95-0976 from the
Dirección General de Investigación Científica y
Técnica, Spain, to J.A. and J.R., respectively; by an Ajut de
Suport als Grups de Recerca de Catalunya (SGR97-127) from the
Generalitat de Catalunya to J.A.; and by grant BIO4-CT97-2210 from the
European Union to J.R. E.d.N. is the recipient of a predoctoral
fellowship from the Ministerio de Educación y Cultura, Spain.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept.
Bioquímica i Biologia Molecular, Facultat de Veterinària,
Ed. V, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain. Phone: 34-93-5812182. Fax: 34-93-5812006. E-mail:
J.Arino{at}cc.uab.es.
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Abstract
Introduction
