From the
Protein phosphatases PPZ1 and PPZ2 represent a novel form of
Ser/Thr phosphatases structurally related to type 1 phosphatases and
characterized by an unusual amino-terminal region. We have found that
the deletion of PPZ1 gene results in increased tolerance to
Na
The yeast Saccharomyces cerevisiae is able to maintain
a suitable intracellular concentration of Na
Reversible protein
phosphorylation is a major regulatory mechanism for the modulation of
protein function, and protein phosphorylation mechanisms are likely to
be involved in the control of Na
Protein phosphatases reverse the action of kinases. Traditionally,
Ser/Thr phosphatases have been classified into four groups, namely
types 1, 2A, 2B, and 2C (see Cohen(1989) for a review), and this
classification is useful for a large variety of organisms, including
yeast. A role for the Ca
In addition to the mentioned Ser/Thr
phosphatases, the genome of S. cerevisiae encodes a number of
proteins structurally related to (but clearly different from) type 1
and 2A phosphatases. Almost without exception, the regulation and the
biological functions of these novel phosphatases are largely unknown. A
few years ago our laboratory identified and cloned a gene named
PPZ1, encoding a 692-residue putative Ser/Thr phosphatase
characterized by a carboxyl-terminal half related to the protein
phosphatase-1 family and a large amino-terminal extension unrelated to
protein phosphatases (Posas et al., 1992). We found that the
simultaneous disruption of PPZ1 and a second, related gene
designated PPZ2 results in increased sensitivity to caffeine
that lead to cell lysis, pointing to a role for these phosphatases in
the maintenance of cellular integrity (Posas et al., 1993).
PPZ2 also encodes a large protein (710 residues) sharing most
of the structural characteristics of PPZ1 (Lee et al., 1993a;
Hughes et al., 1993). It has been recently suggested that the
PPZ1/PPZ2 phosphatases might be related to the SLT2/MPK1 pathway, since
overexpression of gene PPZ2 suppresses the lytic defect of
mpk1
In this paper we present evidence that the PPZ phosphatases
are important determinants in salt tolerance by regulating the efflux
of cations and that these phosphatases and calcineurin play opposite
functional roles.
Escherichia coli NM522 strain
was used as bacterial host for plasmids and was grown in LB medium
supplemented with the appropriate antibiotic when needed.
The possibility that an altered cation uptake
might be responsible for the observed differences was tested by
determining the initial rate of Li
In the last few years, a number of genes involved in the
modulation of cellular sensitivity to Na
It is remarkable
that, in all cases described so far, the lack of function of the
identified genes results in increased sensitivity to salt. Here, we
present the opposite situation and, as far as we know, this is the
first case of genes that, when disrupted, yields a phenotype of
strongly increased salt tolerance. Both PPZ1 and PPZ2 genes encode very similar proteins, characterized by a
carboxyl-terminal half related to type 1 phosphatases and by a large
amino-terminal half rich in Ser/Thr residues. Despite this similarity,
our results suggest that both proteins do not contribute equally to the
phenotype studied, since deletion of PPZ1, but not of
PPZ2, already results in a noticeable increase in salt
tolerance. However, since the double disruption gives a more dramatic
phenotype, it has to be concluded that the lack of PPZ2 also provokes
alterations in salt homeostasis. This situation is very much alike to
the one previously observed in our laboratory regarding cell integrity
of the ppz mutants under caffeine stress (Posas et
al., 1993) and might be due to a number of factors, including
lower levels or activity of the PPZ2 gene product.
We postulate that
the increased cation efflux observed in ppz1 ppz2 cells is due
to the fact that the PPZ phosphatases negatively control the function
of the ENA1 gene. This notion is based on several lines of
evidence. First, we have observed that growth is improved in
ppz
It has been reported recently that calcineurin plays a
role in salt homeostasis and that, at least in part, this occurs
through the control of cation efflux. Loss of calcineurin function can
be provoked by deletion of the single CNB1 gene (encoding the
regulatory subunit of the phosphatase) or by deletion of the two genes
encoding the catalytic polypeptides (CNA1/CMP1 and
CNA2/CMP2). Both types of mutation result in decreased cation
efflux (Nakamura et al., 1993; Mendoza et al., 1994),
most probably as a result of a decrease in the expression of ENA1 (Mendoza et al., 1994). Our observations that the lack of
PPZ phosphatases can counteract the lack of calcineurin by increasing
ENA1 mRNA levels indicates that the effect of the PPZ
phosphatases on sodium and lithium efflux is not mediated by
calcineurin and provides a very interesting example of two different
phosphatases playing opposites roles in a given biological system. This
situation is reminiscent of the growth restoration observed in
cAMP-dependent protein kinase-deficient strains upon disruption of the
gene encoding the YAK1 kinase (Garret and Broach, 1989; Ward and
Garret, 1994) and is compatible with the notion of both phosphatases
acting in parallel pathways, with overlapping but antagonistic effects.
While the involvement of calcineurin in adaptation to high salt stress
conditions clearly suggests a role for a calcium-mediated signaling
pathway, very little is known at present about the activity of
PPZ1/PPZ2 and their regulation.
We recently described that
disruption of both PPZ genes resulted in cell lysis upon
caffeine stress (Posas et al., 1993). Cell lysis is also a
characteristic phenotype of mutants in the PKC/MAP kinase pathway
(Levin and Errede, 1993). The role of the PPZ phosphatases in the
maintenance of cell integrity was reinforced after the isolation of
PPZ2 as a multicopy suppressor of the lytic effect of the
mpk1
Two main conclusions can be drawn from the results
presented here. First, the PPZ phosphatases negatively regulate the
efflux of cations in yeast cells, most probably through the repression
of the ENA1 gene. Second, this event is independent of the
existence of calcineurin, thus suggesting that PPZ and calcineurin are
two Ser/Thr phosphatases acting on parallel pathways. A tentative model
for PPZ function is depicted in Fig. 7. In this model we favor
the idea that the PPZ phosphatases do not directly regulate the
expression of the ENA1 gene, since these phosphatases do not
show the structural features of known transcriptional regulatory
proteins and, in addition, we have found that PPZ1 is not present in
nuclear fractions.(
Tolerance is expressed as
the concentration of Li
We thank Drs. M. S. Cyert and D. E. Levin for strains,
Dr. T. Miyakawa and Dr. J. M. Pardo for plasmids, and Dr. R. Serrano
for helpful discussion. We acknowledge the skillful technical support
of A. Vilalta and R. Martnez. We express our gratitude to Dr. A.
Rodrguez-Navarro for the generous gift of strains, plasmids, and
continuous scientific advice.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and Li
cations. Simultaneous
deletion of PPZ2 gene results in an additional increase in
salt tolerance. After exposure to high concentration of
Li
, the intracellular content of the cation was
markedly decreased in ppz1
ppz2
mutants when
compared to wild type cells. No significant differences were observed
between both strains when the Li
influx was measured,
but ppz1
ppz2
mutants eliminated Li
more efficiently than wild type cells. This can be explained by
the fact that expression of the ENA1 gene, which encodes the
major component of the efflux system for these cations, is strongly
increased in ppz1
ppz2
cells. As expected, the
disruption of the PPZ genes did not complement the
characteristic hypersensitivity for Na
and
Li
of a ena1
strain. The lack of protein
phosphatase 2B (calcineurin) has been found to decrease salt resistance
by reducing the expression of the ENA1 gene. We have observed
that the disruption of the PPZ genes substantially enhances
the resistance of the hypersensitive calcineurin-deficient mutants.
Since PPZ phosphatases have been found to be functionally related to
the protein kinase C/mitogen-activated kinase pathway, we have tested
bck1 or mpk1/slt2 deletion mutants and found that
they do not display altered salt sensitivity. However, disruption of
PPZ1 fails to increase salt resistance in a mpk1/slt2 background. In conclusion, we postulate the existence in yeast of
a novel PPZ-mediated pathway involved in salt homeostasis that is
opposite to and independent of the recently described
calcineurin-mediated pathway.
, even in
the present of relatively high concentrations of this cation in the
medium, through the coordinate regulation of uptake and efflux systems.
Influx of Na
(and Li
) occurs through
the K
uptake system. Exposure to sodium stress causes
the conversion of the K
uptake system to a high
affinity mode, in which the entrance of Na
is reduced
(Haro et al., 1993). Two components of this system, TRK1 and TRK2, have been identified and cloned (Gaber et
al., 1988, Ko and Gaber, 1991), and it has been postulated that
TRK1 is required for the expression of the high K
affinity mode. The efflux of Na
is mainly
mediated by a putative P-type ATPase encoded by the gene ENA1 (for a recent review, see Rodrguez-Navarro et al.(1994)).
This gene is the first unit of a tandem array of four closely related
genes, namely ENA1-ENA4 (Rudolph et al., 1989;
Haro et al., 1991; Martnez et al., 1991).
ENA2, ENA3, and ENA4 present a very weak
constitutive expression, while ENA1, although poorly expressed
on basal conditions, is induced upon exposure to high concentrations of
Li
or Na
, as well as to alkaline pH.
Therefore, deletion of ENA1 results in cells hypersensitive to
Na
or Li
(Haro et al., 1991;
Garciadeblás et al., 1993).
homeostasis. For
instance, increased dosage of YCK1 or YCK2, the genes
encoding casein kinase I homologues, has been reported to increase
tolerance to salt (Robinson et al., 1992). Similarly,
disruption of the YCR101 gene, encoding a predicted protein
kinase, confers sensitivity to salt (Skala et al., 1991).
-dependent Ser/Thr protein
phosphatase 2B (calcineurin) in the regulation of salt homeostasis has
been suggested very recently (Nakamura et al., 1993; Mendoza
et al., 1994), since disruption of the genes encoding either
the catalytic (CMP1/CNA1 and CMP2/CNA2) or the
regulatory (CNB1) subunits of this phosphatase results in
increased sensitivity to salt. It has been proposed that, at least in
part, this increase in sensitivity is due to the reduced expression of
the ENA1 gene in a calcineurin-deficient background (Mendoza
et al., 1994).
strains and deletion of PPZ1/PPZ2 genes is
additive with the mpk1
defect (Lee et al.,
1993b).
Strains, Media, and Growth Conditions
S.
cerevisiae strains used in this work are listed in .
Strains were grown in S.D. or in complex YPD medium (1% yeast extract,
2% peptone, 2% glucose) at 28 °C for routine work and storage
(Sherman et al., 1986). YPD was supplemented with 20
mM TAPS()
when used at pH 8.5. For
Northern blot experiments YPD was supplemented with 50 mM
HEPES and adjusted to pH 7.0.
Gene Disruption Methods
The one-step gene
disruption method (Rothstein, 1983) was used in all cases. ppz1 and ppz2 deletion mutants were obtained as described
previously (Posas et al., 1992, 1993). cnb1 disruption mutants were obtained in our laboratory exactly as
described previously (Mendoza et al., 1994). Yeast cells were
transformed by using a modification of the lithium acetate method (Hill
et al., 1991). All mutations were confirmed by Southern blot
analysis using digoxigenin-labeled DNA probes.
Salt Sensitivity Assays
The sensitivity of the
cells to NaCl, LiCl, or other salts was evaluated on freshly prepared
YPD medium containing various concentrations of the salts. For drop
tests cells were grown for 2 days in liquid culture without salt and
then 3 µl of a 1/100 dilution were plated on YPD-agar medium with
salt. Growth was recorded after 3 days at 28 °C. Relative growth
was measured in liquid cultures as growth, recorded by measuring the
A, of overnight cultures in different salt
concentrations in comparison with growth monitored in the same medium
without added salts.
Measurement of Intracellular Li
For measurement of ion fluxes, cells
were grown in YPD until an AConcentrations
of 1 was reached.
Then LiCl was added from a 5 M stock solution to achieve a
final concentration of 200 mM (only of 50 mM in the
case of strain RH16.6, because of its very high sensitivity to this
cation). At the indicated intervals, samples were harvested by
filtration and washed with three volumes of ice-cold water containing
1.5 M sorbitol and 20 mM MgCl
, rinsed
with cold deionized water, and extracted by incubation at 95 °C for
30 min as described (Gaxiola et al., 1992). Li
concentrations of the clarified extract were determined by flame
spectrophotometry using a Corning 810 apparatus.
Northern Blot Analysis
For Northern blot analysis,
cells from a stationary culture were grown as described until the
culture reached an optical density of 1. Total RNA was prepared as
described (Treco, 1989), electrophoresed on 0.7% agarose-formaldehyde
gels (20-30 µg/lane) and transferred to nylon membranes
(Hybond N, Amersham Corp.) under vacuum. Membranes were hybridized at
42 °C in the presence of 50% (v/v) formamide and 10 cpm/ml of the appropriate
P-labeled DNA fragment.
The probes used were the 3.7-kilobase pair XhoI-PstI
fragment of the ENA1 gene, kindly provided by Prof. A.
Rodrguez-Navarro (Haro et al., 1991) and a 1.75-kilobase pair
fragment of the ACT1 gene (Gallwitz and Sures, 1980). Filters
were washed in 0.1
SSC (1
SSC is 150 mM NaCl,
15 mM sodium citrate, pH 7.0), 0.1% SDS at 55 °C.
Disruption of PPZ Genes Results in Increased Tolerance
to Salt
During our studies on the role of the PPZ phosphatases
in the maintenance of cellular integrity, we observed that the
ppz1 ppz2
double mutant JA20-1C displayed a
surprisingly vigorous growth compared to the wild type JA20-1D when
exposed to moderate saline stress. This initial observation was further
confirmed when the double disruption was carried out in other genetic
backgrounds. The effect of increasing concentrations of salt on cell
growth was studied by growing wild type as well as ppz1
and ppz1
ppz2
cells in plates containing
different concentrations of LiCl. Lithium was used in these and other
experiments instead of NaCl because these cations share the same uptake
and efflux systems. Because Li
is more toxic than
Na
, the former cation could be used at lower
concentrations, thus avoiding the osmotic effects derived of the use of
very high concentrations of NaCl. As can be observed in
Fig. 1
(panelA), wild type DBY746 cells grows
poorly at 100 mM LiCl and cannot grow at all at 300
mM, whereas strain JA30 (which lacks a functional copy of gene
PPZ1) can still grow at 300 mM LiCl. Simultaneous
disruption of PPZ1 and PPZ2 genes, as in the case of
strain JA31, allows cell growth even in the presence of 600 mM
LiCl. This double disruptant was able to grow in YPD plates containing
up to 800 mM LiCl (not shown). The effect of lack of PPZ
phosphatases has also been observed in other genetic backgrounds, as in
the case of the haploid cells derived from the diploid strain DL790
(ppz1
/PPZ1 ppz2
/PPZ2). Interestingly, in all cases
the disruption of PPZ2 alone resulted in an almost negligible
increase in salt tolerance when compared with wild type cells (not
shown).
Figure 1:
Growth of wild type and
ppz mutant strains in the presence of salt. PanelA, wild type DBY746 cells as well as ppz1
PPZ2 (JA30) and ppz1
ppz2
(JA31) strains were
inoculated in YPD plates, containing the indicated concentrations of
LiCl, as described under ``Materials and Methods.'' PanelB, wild type DBY746 (
), JA30 (
), and JA31
(
) cells were grown in YPD medium to saturation and then
aliquots transferred to YPD supplemented with the indicated
concentrations of LiCl or NaCl. Growth was monitored
spectrophotometrically at 660 nm and is expressed as percentage of the
growth measured in the absence of added salts. Data are presented as
the mean ± S.E. of three to five independent
experiments.
The increased tolerance to lithium conferred to the cells by
the disruption of the PPZ1 and PPZ2 genes can be also
observed in liquid cultures (Fig. 1, panelB).
Growth was also improved in the mutants in the presence of increasing
concentrations of NaCl (rightpanel). When the pH of
the medium was raised to 8.5, the resistance to Li and
Na
of the ppz
mutants was similar to
than that observed when the medium was not buffered at alkaline pH (not
shown). The described effects of the ppz1
ppz2
mutations were specific for Li
or
Na
, since growth was not improved in the presence of
equivalent concentrations of KCl.
The Lack of PPZ Phosphatases Leads to Decreased Internal
Li
To learn whether the increased salt tolerance of
ppz Content as a Result of Increased Cation
Efflux
mutants was due to the fact that these cells contain
a reduced amount of cations, we grew wild type and ppz1
ppz2
cells for 90 min in YPD medium containing 200
mM LiCl. When the intracellular content of Li
was measured, it became evident that mutant cells contained a
lower amount of the cation (30.6 ± 1.9 nmol/mg dry cells) than
wild type cells (48.1 ± 2.5 nmol/mg dry cells). The
intracellular concentration of Na
was similarly
reduced in the mutant strain when cells were preloaded for 90 min with
0.96 M NaCl.
uptake in both wild
type and ppz1
ppz2
cells. As presented in
Fig. 2
, both strains take up Li
at approximately
the same rate (about 2
nmol
mg
min
),
suggesting that alterations in the uptake mechanisms are not
responsible for the observed differences in the intracellular content
of Li
. Changes in accumulation might also be caused by
an altered efflux rate. To test this possibility, cells were incubated
for 90 min in YPD medium in the presence of 200 mM LiCl and
then centrifuged and resuspended in Li
-free medium.
The internal Li
content was then determined in both
wild type and ppz1
ppz2
cells at different times
after removal of the cation. As presented in Fig. 3, the rate of
elimination of Li
was clearly higher in mutant cells,
suggesting that the decreased intracellular levels observed in these
cells are essentially the result of an increased Li
efflux.
Figure 2:
Time dependence of Li
accumulation in DBY746 and JA31 strains. Wild type DBY746 (
) and
JA31 (
) cells were grown in YPD until the optical density
reached about 1-2. The medium was then made 200 mM LiCl,
and samples were removed at the indicated times for determination of
intracellular Li
concentration. Data are presented as
the mean ± S.E. of four independent
experiments.
Figure 3:
Determination of lithium efflux in wild
type and ppz cells. DBY746 (
) and JA31 (
)
cells were grown as described in Fig. 2. Then, the medium was made 200
mM LiCl and growth resumed for 90 min. The cells were
centrifuged and resuspended in fresh YPD medium, samples were taken at
the indicated times, and the intracellular lithium content determined
as described. Data are presented as the mean ± S.E. of four
independent experiments.
Disruption of the PPZ Genes Results in Increased
Expression of the ENA1 Gene
The putative P-type ATPase encoded
by the ENA1 gene is believed to constitute the major system
for Na (and Li
) efflux in yeast
cells. We compared the sensitivity to Na
and
Li
of strains RH16.6 (ena1-ena4
PPZ1
PPZ2) and JA35 (ena1-ena4
ppz
ppz2
) in
both YPD liquid cultures and YPD-agar plates. We found that in a
ena1
background, the ppz1
ppz2
mutation was unable to increase the tolerance to the cations
(). Furthermore, when RH16.6 and JA35 cells were loaded
for 45 min in YPD medium containing 50 mM LiCl and the amount
of internal Li
was determined, no significant
differences were found between both strains (32.4 ± 0.5 nmol/mg
dry cells versus 30.2 ± 0.7, respectively). Both
results were compatible with the notion of an involvement of the
ENA1 gene product in the mechanism of action of the PPZ
phosphatases. Since it is known that exposure to salt stress results in
induction of gene ENA1, which otherwise is strongly repressed,
we tested by Northern blot experiments the mRNA levels of the ENA1 gene in wild type and ppz1
ppz2
mutants. As
shown in Fig. 4, exposure of wild type cells to Li
cations results in a transient increase of ENA1 mRNA
levels, which is clearly higher in ppz1
ppz2
cells.
Remarkably, ppz-deficient cells contain more ENA1 message even in the absence of salt stress, suggesting an effect
of the ppz1
ppz2
mutation in basal conditions.
Figure 4:
Effect of lack of PPZ phosphatases in
ENA1 mRNA levels. Wild type DBY746 cells (openbars) as well as JA31 mutants (ppz1 ppz2
) (stripedbars) were grown in YPD medium
supplemented with 50 mM HEPES (pH 7.0) as described and then
made 200 mM LiCl. Growth was resumed, and cells collected at
the indicated times for total RNA extraction. Northern blots were
performed, membranes hybridized with a
P-labeled ENA1 probe, and films exposed for 48 h. The membranes were then
stripped and reprobed with the ACT1 gene as control for the
amount of RNA transferred. The levels of ENA1 mRNA were
determined by densitometric scanning of the films. Data are referred to
the signal obtained from wild type cells collected immediately prior to
the addition of LiCl and is expressed as the mean ± S.E. from
four independent experiments. Inset shows an autoradiogram
obtained from a typical experiment.
The Lack of PPZ Phosphatases Counteracts the Defect
Associated to the Absence of Calcineurin
Since it has been shown
recently that the lack of the type 2B phosphatase (calcineurin) leads
to a salt-sensitive phenotype, we constructed strains JA40 (PPZ1
PPZ2 cnb1) and JA41 (ppz1
ppz2
cnb1
)
and tested their sensitivity to LiCl in comparison with wild type and
JA31 (ppz1
ppz2
CNB1) cells (Fig. 5, upperpanel). As reported previously, the lack of CNB1 (encoding the regulatory subunit of calcineurin) in an otherwise
wild type background leads to hypersensitivity to salt. Simultaneous
disruption of PPZ1 and PPZ2 in a cnb1 background results in higher tolerance than wild type cells, but
this mutant is less resistant than a ppz1
ppz2
CNB1
strain, indicating that the effects of these mutations are independent
and additive. Furthermore, exposure of strain YPH499 carrying the
double disruption cna1
cna2
(the genes encoding the
catalytic subunits of calcineurin) to 0.8 M NaCl prevents
growth in rich medium plates. However, when these cells carry the
ppz1
disruption, growth was restored (Fig. 5,
middlepanel). Growth under higher concentrations of
NaCl indicated that cna1
cna2
ppz1
cells were
slightly less tolerant than wild type cells (data not shown). When the
levels of ENA1 mRNA were determined, it was observed that, as
expected, they were lower in cnb1
than in wild type
cells. However, the levels of ENA1 message were dramatically
increased when the cnb1
cells also contains the
ppz1
ppz2
mutation (Fig. 5, lowerpanel). These observations indicate that the lack of PPZ
phosphatases is able to increase salt tolerance in calcineurin-depleted
cells and suggest that the mechanism of action of the PPZ phosphatases
is not mediated by calcineurin.
Figure 5:
Complementation of the salt sensitivity
phenotype of calcineurin-deficient cells by the ppz
mutations. Upperpanel, wild type DBY746 (PPZ1
PPZ2 CNB1), JA40 (PPZ1 PPZ2 cnb1
), JA31
(ppz1
ppz2
CNB1), and JA41 (ppz1
ppz2
cnb1
) were inoculated on YPD plates or YPD plates containing
the indicated concentrations of LiCl. Growth was scored after
incubation at 28 °C for 3 days. Strain JA40 was constructed in our
laboratory but should be identical to strain
DBY746cnb1
described previously (Mendoza et
al., 1994). Middle panel, wild type YPH499 (CNA1 CNA2
PPZ1) and its isogenic derivatives cna1
cna2
PPZ1 (Cyert et al., 1991) and cna1
cna2
ppz1
(strain JA45) were streaked onto YPD plates or YPD
plates supplemented with 0.8 M NaCl. Growth was scored after
incubation at 28 °C for 4 days. Lower panel, wild type
DBY746 (lanesA) as well as JA40 (lanesB), JA31 (lanesC), and JA41 (lanesD) mutants were grown and incubated with 200 mM LiCl as described in Fig. 4. Samples were taken at the indicated times,
total RNA prepared, and Northern blot experiments performed using
P-labeled ENA1 and ACT1 probes.
Saline Stress and the Relationship between PPZ
Phosphatases and the PKC/MAP Kinase Pathway
PPZ phosphatases
appear to be related to some of the effects derived of blockage of the
PKC/MAP kinase pathway (i.e. cellular lysis under temperature
or caffeine stress). However, when strain DL456, which carries a
disruption of the MAP kinase gene (SLT2/MPK1), is grown on YPD
plates containing different concentrations of LiCl, it does not present
a significantly altered salt tolerance when compared with the wild type
strain (Fig. 6). Disruption of the bck1 gene, encoding a
kinase acting upstream the MAP kinase gene product, equally results in
absence of phenotypic change (not shown). Interestingly, the disruption
of PPZ1 in a mpk1 background (strain DL823) does
not result in improved growth in the presence of different
concentrations of LiCl. Since DL823 cells must be grown in plates
supplemented with 10% sorbitol, it is important to note that the
failure of these cells to grow was due to Li
toxicity
and not to the rather high osmolarity of the medium, since this strain
did survive when identical concentrations of the less toxic salt NaCl
were used instead of LiCl (Fig. 6).
Figure 6:
Comparison of the effect of the
ppz mutation on salt resistance in wild type and
mpk1
backgrounds. The wild type strain 1788 (PPZ1
PPZ2 MPK1), as well as strains DL823 (PPZ1 PPZ2
mpk1
) and DL456 (ppz1
ppz2
mpk1
),
were inoculated in YPD plates or YPD plates containing different
concentrations of LiCl or NaCl. In all cases 10% sorbitol was included
in the plates. A haploid ppz1
MPK1 strain derived from
strain 1788 is included for comparison. Plates were incubated at 28
°C for 3 days.
and
Li
toxicity have been identified in yeast. Some of
these genes encode components of the uptake and efflux systems located
at the plasma membrane, as it is the case of genes TRK1/TRK2 and ENA1, respectively. Other genes would include
regulatory components of the above mentioned systems, as it has been
suggested in the case of calcineurin (Mendoza et al., 1994).
Finally, a number of genes have been described to produce a
salt-sensitive phenotype when mutated or to confer increased salt
tolerance when overexpressed, as it is the case of HAL1 and
HAL2 (Gaxiola et al., 1992; Gläser et
al., 1993; Murgua et al., 1995), LIS1/ERG6 (Welihinda et al., 1994), and the protein kinases encoded
by genes YCK1/YCK2 (Robinson et al., 1992)
and YCR101 (Skala et al., 1991).
mutants even when the extracellular pH is high enough
to block the function of the proposed Na
/H
antiporter system (Rodrguez-Navarro et al., 1981, 1994).
Therefore, involvement of this hypothetical antiporter in the mechanism
of action of PPZ phosphatases can be discarded. Second, the fact that
the lack of PPZ cannot improve growth in the presence of salt of a
hypersensitive ena1
-ena4
strain suggests that the
mechanism of action of PPZ might be mediated by the ENA1 gene
product. Finally, the observation that ENA1 mRNA levels are
increased in ppz
mutants suggests that the increase in
cation efflux is due, at least in part, to the existence of higher
amounts of the ENA1 protein. A remarkable fact is that an increase in
the expression of ENA1 can be detected in a PPZ-deficient
background even when the cells have not been exposed to saline stress.
This could be interpreted as if the PPZ phosphatases would negatively
affect the expression of ENA1 under basal conditions, thus
avoiding an unnecessarily excessive expression of ENA1, which
is, by itself, detrimental for cell growth.(
)
Exposure of ppz
cells to salt would trigger a
signaling pathway that would cause an additional increase in ENA1 mRNA levels. Of course, our present data cannot rule out the
possibility of a negative control of the activity of the ENA1 protein
by the PPZ phosphatases, conceivably through phospho-dephosphorylation
reactions.
mutation (Lee et al., 1993b), thus
suggesting that these phosphatases may function within this
PKC1-mediated pathway. However, our finding that neither the
lack of the BCK1 gene (Lee and Levin, 1992), encoding a
putative MAP kinase kinase kinase that is believed to act directly
downstream from PKC, nor the disruption of the SLT2/MPK1 gene,
encoding the MAP kinase homologue of the pathway (Torres et
al., 1991; Lee et al., 1993a), results in significant
changes in salt sensitivity suggests that this pathway is not involved
in the regulation of salt homeostasis. However, it is remarkable that
the disruption of PPZ1 fails to increase the tolerance for
Li
or Na
of a mpk1
strain. This observation suggests that a functional MAP kinase
would be a requisite for the phenotypic expression of the lack of PPZ
function and provides additional evidence for a functional link between
both proteins.
)
Instead, a regulatory
protein (denoted as X in Fig. 7) might act as a
transcriptional repressor when dephosphorylated by PPZ1/PPZ2.
Therefore, the lack of PPZ phosphatases would result in inactivation of
the repressor and in increase of the expression of the ENA1 gene even in the absence of salt stress. Under saline stress, an
independent signaling pathway would be activated and induce ENA1 expression, thus overriding the effect of the repressor. Finally,
the combination of saline stress and lack of PPZ would result in
stimulation of a positive effect and blocking of a negative one and
would account for the strong expression of ENA1. It is worth
noting that our results indicate that ENA1 is still induced in
significant extent in the absence of the CNB1 gene, suggesting
that calcineurin must not be solely responsible for salt-induced
increased in ENA1 transcription. Alternatively, the PPZ
phosphatases might inactivate a transcriptional activator. In this
situation, the absence of PPZ would also result in an increased
transcription of ENA1 in basal conditions that would be
further enhanced by activation of the salt-induced pathway upon
exposure to saline stress. In any case, the dramatic increase in salt
resistance featured by ppz
strains reveals that these
novel phosphatases, whose biological functions were essentially
unknown, are important determinants in salt homeostasis in yeast cells.
Figure 7:
A tentative model for PPZ function in salt
homeostasis. See details in the main text.
Table:
Saccharomyces cerevisiae strains used in
this work
Table:
Effect of ppz and ena1
mutations on Li
tolerance
that reduces 50% growth of
liquid cultures. Data are expressed as mean ± S.E. from 3 to 5
independent experiments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.