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J Biol Chem, Vol. 273, Issue 50, 33610-33618, December 11, 1998
From the By complementation of a salt-sensitive mutant of
Saccharomyces cerevisiae, we cloned the
SOP1 gene, encoding a 114.5-kDa protein of 1033 amino
acids. Cells deleted for SOP1 exhibited sensitivity to
sodium stress, but showed no sensitivity to general osmotic stress.
Following exposure of sop1 Ions are continuously transported across the cell membrane, the
net flux being adjusted to satisfy the requirement for a cytosol rich
in potassium and scarce in sodium. Control of the intracellular concentration of these major monovalent cations is crucial to generate
a biochemically-functional intracellular milieu. Since in natural
environments Na+ is generally abundant and K+
scarce, transport must occur against concentration gradients. Genetic
analysis of salt tolerance in Saccharomyces cerevisiae has
identified a number of cation transporters which interact with multiple
regulatory components in a largely unidentified fashion (1). In
particular, a major system involved in K+ uptake is
constituted by the TRK1- and TRK2-encoded
membrane proteins (2-4), which appear to contribute to the uptake of
K+ in symport with protons (1). The proton gradient
providing the driving force for secondary transport is generated by the PMA1-encoded plasma membrane ATPase, a major membrane
protein whose activity shows little sensitivity to high extracellular NaCl concentration (5). The TRK1/TRK2-dependent transport
system also permits influx of Na+, while under NaCl stress,
the uptake system has the capacity of increasing its selectivity for
K+ over Na+ (6).
In yeast cells, influx of Na+ is counteracted by
Na+ efflux, the primary pathway being mediated by the
P-type ATPase encoded by the PMR2A gene (also known as
ENA1) (6, 7). The PMR2A gene is part of a gene
cluster, containing tandem repeats of 2-5 nearly identical genes (8).
However, only PMR2A appears to be significantly expressed
(7, 8), and transcription of this gene is induced in cells subjected to
Na+ or Li+ stress or cells exposed to alkaline
pH (7). An additional sodium transporter encoded by the NHA1
gene and acting as a putative Na+/H+ antiporter
was recently identified in S. cerevisiae (9). Disruption of
the NHA1 gene displays only minor effects in wild type cells but elicits increased Na+ sensitivity in S. cerevisiae cells lacking the PMR2 genes.
To identify components that are crucial for salt tolerance, the
isolation of recessive, salt-sensitive mutations is an obvious approach. However, the only S. cerevisiae mutant
characterized so far by this procedure is the calcineurin-defective
strain isolated by Mendoza et al. (10). These authors
demonstrated that the protein phosphatase calcineurin, is involved in
Na+ tolerance and is required for (i) induced expression of
the PMR2A gene and (ii) modulating the K+ uptake
system to display increased K+ versus
Na+ discrimination. Further evidence that protein
phosphorylation and dephosphorylation regulate Na+
tolerance in S. cerevisiae is provided by the increased
cellular tolerance to sodium ions following inactivation of the
PPZ1 and PPZ2 encoded serine-threonine
phosphatases (11). In addition, increased dosage of the YCK1
or YCK2 gene, encoding yeast homologues of casein kinase I,
enhances sodium tolerance (12), while cells defective in either of the
a or b subunits of the yeast casein kinase II homologue become
specifically sensitive to high concentrations of Na+
(13).
By isolation and functional complementation a NaCl-sensitive mutant, we
cloned the SOP1 gene. Here we report the initial
characterization of the gene product and show that the predicted
sequence of Sop1p lacks apparent membrane spanning regions or other
characteristics of previously isolated determinants for Na+
tolerance, and displays significant homology with the
Drosophila p127 protein encoded by the lethal(2)giant
larvae (l(2)gl) tumor suppressor gene and its
homologues in mouse and man. Our results demonstrate that
SOP1 and a second related gene, designated as SOP2, are involved in regulating cation homeostasis in
S. cerevisiae. Furthermore, we demonstrate that the
expression of a Drosophila l(2)gl cDNA sequence in the
S. cerevisiae sop1 Strains, Media, and Yeast Genetic Methods--
S.
cerevisiae strains and genotypes are listed in Table
I. Cells were routinely grown at 30 °C
in either YEPD medium supplemented with 120 µg/ml adenine or in a
synthetic yeast nitrogen base (YNB) medium (Difco) supplemented with
2% glucose and necessary amino acids and nucleotides, to a final
concentration of 120 µg/ml for each. The procedure used to isolate
the osg4 mutant has been described previously (14). Cell
density was estimated by measuring optical density at 610 nm in 1-cm
cuvettes (OD610). Standard yeast genetic methods were used
throughout (15, 16).
Escherichia coli DH5 Plasmids--
The YCp50-based yeast genomic library (19) was
used for complementation of the osg4 mutant. Transformants
were screened on YNB agar plates containing 1.4 M NaCl, and
plasmids from complemented cells were amplified in E. coli.
Restriction fragments of a
7.5-kb1 insert of a YCp50
plasmid complementing the osg4 salt sensitivity were
subcloned in the 2µ-based shuttle vector pRS326 (20). A 4.7-kb
HindIII/BamHI fragment retaining the
complementing capacity was subcloned into pRS316 to generate the
pH/B316 plasmid. Overexpression of the SOP1 gene was
achieved by subcloning the 4.7-kb HindIII/BamHI fragment into the YEplac195 multicopy vector (21), yielding the YEpH/B vector.
To construct a plasmid that expresses a c-Myc-tagged SOP1,
an XmaI-SalI fragment containing three copies of
the 11-amino acid human c-Myc epitope was removed from the pBsMYC
plasmid. This product was subcloned into pBSH/B (pBluescript
KS+ containing the HindIII/BamHI
fragment) at the Bst11071 site, 190 bp upstream the stop
codon of the SOP1 ORF, generating an in-frame fusion. The
final construct was verified by sequencing. A 2.0-kb
HindIII/SphI fragment containing the c-Myc-tagged
carboxyl-terminal half of the SOP1 gene was then excised and
subcloned into the multicopy plasmid YEpH/B, or the single-copy plasmid
pH/B316, generating the YEpH/Bmyc and pH/B316myc plasmids, respectively.
To express the Drosophila l(2)gl gene in S. cerevisiae, a construct for the constitutive expression of the
l(2)gl gene was generated as follows. A 5269-bp
EcoRI fragment of the cDNA Ec173 inserted in pGEM4 (22)
was subcloned into the polylinker of the pYX212 and pYX112 plasmids
(R&D Systems Inc.), and constructs were controlled by restriction
analysis and sequencing.
Disruption of SOP1 and SOP2--
The 4.7-kb
HindIII/BamHI fragment harboring the
SOP1 gene subcloned into pBluescriptKS+ was cut
with BglII and NheI, and the resulting 2.3-kb
internal fragment of SOP1 was replaced with the
URA3 or LEU2 selectable marker. The resulting
constructs were amplified, and the HindIII/BamHI fragment was used to transform S. cerevisiae W303-1A and
W303-1B by the LiAc method (23) to generate
sop1::URA3 and sop1::LEU2 null mutants.
Deletion of the SOP2 gene was accomplished by the long
flanking homology PCR-targeting technique (24, 25). In the first step,
a set of primers (5'-TTCCGCTTCATAGGAGGAGA-3' and
5'-GGGGATCCGTCGACCTGCAGCGTACCATTTATAAAATTTTTGTAT -3') was used to
amplify 300 bp of genomic DNA from S. cerevisiae W303,
immediately upstream of the second codon of the SOP2 ORF. A
second set (5'-AACGAGCTCGAATTCATCGATGATATAGTCCATAAATAGTTTTTA-3' and 5'-ACGGTTCATCATTCGGAAAA-3') was used to amplify a 377-bp
fragment immediately downstream of the SOP2 ORF stop codon.
The 5' end of the primers adjacent to the insertion site carried 25 nucleotide extensions homologous to the 5' and 3' region of the
his5MX6 disruption cassette of plasmid
pFA6a-his5MX6 (26). In the second PCR reaction, pFA6a-his5MX6 was used as template and the 5'- and
3'-homologous regions of the first PCR reaction were fused to the
disruption cassette by serving as primers, together with the upstream
forward and downstream reverse primers of the flanking regions, thus
producing the ORF targeting cassette. This cassette was transformed
into a diploid S. cerevisiae W303 strain, and independent
transformants were selected for sporulation and verification of
SOP2 replacement. Diploid transformants able to grow on
his Determination of Stress Tolerance--
Tolerance to salt or
osmotic stress was examined by spotting 10-fold dilutions (10 µl) of
an overnight culture diluted to OD610 ~1.0 onto YEPD
plates (pH 6.9, unless otherwise stated) supplemented with NaCl, LiCl,
KCl, sorbitol, or glycerol, as indicated in the text. Tolerance to heat
stress and N-starvation was performed as described by Sass et
al. (27). To monitor tolerance to oxidative stress, cells grown
overnight in YEPD medium were diluted into fresh YEPD medium to yield
an OD610 of approximately 0.2. The cells were left to
adjust to the new environment for 2 h, whereupon H2O2 was added to a final concentration of 5 mM. Cultures were then incubated at 30 °C and growth
monitored. To determine tolerance to acidic or alkaline conditions,
10-fold dilutions of exponentially growing cells were spotted onto YEPD
agar plates buffered at pH 4.8 or 8.6, and growth was assessed after
incubation at 30 °C for 24 h.
Determination of Intracellular Na+ and
K+--
Cells were grown overnight at 30 °C in 500 ml
of YEPD medium to mid-exponential phase. The culture was divided into
three portions that were diluted with fresh medium to a cell density of
5 × 107 cells. These cultures were centrifuged at
3500 × g for 5 min and then re-suspended in 40 ml of
YEPD medium supplemented with 0, 0.7, or 1.0 M NaCl. After
incubation for 6 h, triplicate samples of 5 ml of cell suspension
were filtered through 0.22-µm filters and washed with three volumes
of iso-osmotic CaCl2. The filters were transferred to 5 ml
of 20 µM Ca(OH)2 and extracted by heating for
10 min at 95 °C. The cell suspension was centrifuged and the supernatant stored at Northern Blot Analysis--
Exponentially growing cells were
transferred to fresh YEPD medium and the same medium supplemented with
1.0 M NaCl or 1.5 M sorbitol, and incubated for
4 h before isolation and blotting of RNA, as described previously
(28). The SOP1 probe used was the 1.1-kb NheI
fragment labeled by random priming (29), using hexanucleotides and
Klenow fragment from Boehringer Mannheim and [32P]ATP
from Amersham Pharmacia Biotech. An actin probe was used for loading
control (28).
Subcellular Fractionation of Sop1p--
Strains harboring
plasmid YEpH/Bmyc or pH/B316myc and control plasmids without insert
were grown to OD610 0.5-1.0 in YEPD medium. Cells were
washed by centrifugation and resuspended in twice the pellet volume of
resuspension buffer (R-buffer), containing 50 mM Tris (pH
7.5), 5 mM EDTA, complete protease inhibitor mixture (Boehringer Mannheim; 1 tablet/50 ml of cell extract), and either 0.1 M or 1 M KCl. Four volumes of glass beads were
added, and each suspension was vortexed four times consecutively for
30 s at 4 °C, with incubation on ice between each vortex.
Unlyzed cells were removed by centrifugation at 500 × g for 5 min at 4 °C. The lysate was then centrifuged at
100,000 × g for 1 h in a Beckman TL-100
ultracentrifuge. The pellets were resuspended in 100 mM KCl
R-buffer and the lysate desalted by gel filtration (Microcolumns, Bio-Rad). Aliquots containing equal amounts of cell material were boiled for 1 min in Laemmli SDS buffer (16).
Western Blot Analysis--
Proteins were separated by SDS-PAGE
in 10% acrylamide gels at 125 V for 1.5 h using a Mini-PROTEAN II
electrophoresis system (Bio-Rad) and transferred overnight to Hybond
membranes (Amersham Pharmacia Biotech), according to the
manufacturers' protocols. The membranes were first incubated for
1 h at room temperature in 10 mM Tris (pH 7.4), 0.9%
NaCl, 0.05% Tween 20 (Buffer A), supplemented with 1% milk powder and
then for 2 h with mouse anti-c-Myc antibody (Boehringer Mannheim)
diluted 1:5000 in Buffer A with 1% milk powder. The anti-p127 C39
antibodies were as described by Strand et al. (30).
Membranes were washed once for 5 min in Buffer A, twice for 5 min in
0.2% SDS, 0.5% Triton X-100, 0.9% NaCl (Buffer B), and twice for 5 min again in Buffer A. The membranes were probed for 1 h with
1:20,000 dilution of anti-mouse peroxidase-linked antibodies (Amersham
Pharmacia Biotech) in Buffer A containing 5% milk powder. The blots
were washed as before, and antibody detection was performed using ECL
labeling system (Amersham Pharmacia Biotech).
Immunofluorescence--
Cells were fixed in growth media by the
addition of formaldehyde to a final concentration of 3.7%, and cells
were prepared for immunofluorescence according to Rose et
al. (31). Mouse anti-c-Myc antibodies and sheep anti-mouse
Ig-fluorescein, F(ab')2 fragment secondary antibodies were
from Boehringer Mannheim. Glass slides were coated with
poly-L-lysine, Mr >300,000 (Sigma),
to promote cell adhesion. Anti-c-Myc antibody concentration was 0.1 mg/ml, and the concentration of anti-mouse Ig-fluorescein was 0.02 mg/ml. Cells were stained with diamidinophenylindole (1 mg/ml) for 1 min and covered by mounting medium (50 mg of
p-phenylenediamine in 5 ml of phosphate-buffered saline (pH
9) and 45 ml of glycerol). Coverslips were then applied and sealed with
nail polish.
Complementation of a Salt-sensitive Yeast Mutant Identifies the
SOP1 Gene--
We previously reported the isolation of a set of
osmosensitive (osg) mutants of S. cerevisiae
(14). Among these mutants we also identified a strain (osg4)
that proved specifically sensitive to sodium chloride rather than being
generally osmosensitive. Transformation of this mutant with a
YCp50-based yeast genomic library (19) resulted in the isolation of
clones that restored growth of the mutant at high salinity (Fig.
1A), and restriction endonuclease analysis identified a 4.7-kb
HindIII/BamHI DNA fragment, which fully
complemented the mutation. Subcloning, partial sequencing, and
searching of DNA data bases tentatively identified a single large open
reading frame, assigned YPR032W by the Yeast Genome Sequencing Project
(EMBL accession no. Z49274). The 3102-bp open reading frame is
localized to the left arm of chromosome XVI and encodes a putative
protein of 1033 amino acids with a predicted molecular mass of 114.5 kDa. The codon bias (0.054) predicts a low expression of the gene, as
indicated by the weak signal observed on a Northern blot (data not
shown). Since this gene restored the salt sensitivity of the mutant, it
was named SOP1 for sodium
protection.
The Predicted Sop1p Shows Homology to Another S. cerevisiae Protein
and to the p127 Protein Encoded by the l(2)gl Tumor Suppressor Gene of
Drosophila--
Homology searches revealed that Sop1p possesses 54%
amino acid identity with another S. cerevisiae protein,
encoded by the open reading frame YBL106C (EMBL accession no. Z35867).
We designated this gene SOP2, since it shows structural and
functional (see below) similarities to SOP1. Significant
similarity scores were also recovered (Fig.
2) between the yeast proteins and the p127 protein encoded by the Drosophila l(2)gl tumor
suppressor gene (22, 32), and its homologues from mouse (MGL) (33) and
man (HUGL) (34). A well conserved feature in this family of proteins is
the presence of WD-40-like motifs in the NH2-terminal half
of the proteins (Fig. 2). These motifs were first described for the The sop1
The sop1
To examine whether the sop1 SOP1 Expression Is Not Controlled by Environmental Salinity, and
Na+ Tolerance Is Not Enhanced by Increased Gene
Dosage--
As SOP1 inactivation confers sensitivity to
NaCl, it was of interest to examine whether the expression of the gene
is controlled by environmental salinity. Northern blot analysis
revealed a low abundance of SOP1 transcript in cells grown
in basal medium and no increased amount of transcript under salt stress
conditions (data not shown). We also observed that overexpression of
SOP1 gene from its own promoter on a YEplac195 multicopy
vector caused no increase in Na+ tolerance by comparison to
control cells, carrying a vector without insert (data not shown).
Likewise, overexpression of SOP1 exerted no effect on the
Na+ tolerance of a pmr2A null mutant lacking the
Na+ extruding ATPase activity, or could not bypass the salt
sensitivity of either cnb1 cells (10) lacking calcineurin or
cmd1-5 cells (38) carrying a mutant form of calmodulin
unable to bind Ca2+.
Na+ Accumulates in sop1 Possible Genetic Interaction between SOP1 and PMR2A--
Since
the PMR2A gene is reported to be the most important
determinant of Na+ tolerance in S. cerevisiae
(1, 6, 39), we examined whether we could detect a genetic interaction
between SOP1 and PMR2A. For this purpose, a
sop1 Cytoplasmic Distribution of Sop1p--
To determine the
subcellular localization of Sop1p, three tandem repeats encoding the
c-Myc epitope were fused in frame near the COOH terminus of Sop1p and
expressed from a single-copy (pH/B316myc) or a multicopy plasmid
(YEpH/Bmyc). The SOP1::myc derivative fully complemented the salt sensitivity of the sop1
We also used subcellular fractionation to determine the intracellular
localization of Sop1p expressed from YEpH/Bmyc. The distribution of
Sop1p between the supernatant and the 100,000 × g
pellet fraction was semiquantitatively assessed by immunoblotting (Fig.
6B), demonstrating that the protein was present in both the
supernatant and the high speed pellet fraction. The nature of the
association of Sop1p with the pelletable structures was further
analyzed by increasing the ion strength. Treatment with 1 M
KCl readily solubilized most of the Sop1p contained in the particulate
fraction, suggesting that Sop1p is non-covalently associated with other
components present in the pelleted material. Experiments expressing
SOP1::myc from a single-copy plasmid (pH/B316myc) gave qualitatively similar results, although with a marked background staining due to the much weaker signal (data not shown).
Deletion of the SOP2 Gene and Characterization of sop2
To produce a sop1 Complementation of the sop1 Role of Sop1p in Cellular Tolerance toward Na+--
By
complementing the NaCl sensitivity of a previously isolated mutant, we
cloned the SOP1 gene, encoding a 114.5-kDa cytosolic protein
that is required for growth at high Na+ concentrations.
Although deletion of SOP1 strongly decreases Na+
tolerance, SOP1 overexpression results in no protection
toward increased concentration of Na+. In this respect,
SOP1 differs from the series of HAL genes (1, 40,
41), which improve Na+ tolerance in a
dose-dependent fashion. SOP1 differs also from the genes involved in the general osmoregulatory response. The sop1
The strict Na+ sensitivity of sop1
The coordinate regulation of both PMR2A-encoded
Na+ efflux system and TRK1-encoded
Na+/K+ influx system is controlled by a
signaling pathway involving Ca2+/calmodulin and the protein
phosphatase, calcineurin (8, 10). This signaling system appears to play
a crucial role in modulating intracellular Na+ and
K+ concentrations following exposure of cells to NaCl
stress, and it is conceivable that Sop1p may interact with components
of this signaling system. However, overexpression of SOP1
was unable to rescue the Na+ sensitivity of
cmd1-5 or cnb1 mutants, indicating that Sop1p does not operate downstream from these components in the salt stress
signaling system controlled by these genes.
Relationship between the Yeast SOP Encoded Proteins and the
Drosophila p127 Protein Encoded by the l(2)gl Tumor Suppressor
Gene--
The deduced amino acid sequences of Sop1p and its close
yeast homologue Sop2p show a significant about 50% similarity,
extending over the entire coding sequence, with the p127 protein
encoded by the l(2)gl tumor suppressor gene of
Drosophila and its homologues from mouse (MGL) and man
(HUGL). Biochemical investigations and cell fractionation studies have
previously shown that both p127 and HUGL are intracellular proteins
diffusely distributed in the cytoplasm and associated with the
cytoskeletal matrix underlying the plasma membrane (30, 34, 42).
Neoplastic transformation of Drosophila larvae resulting
from the inactivation of the l(2)gl gene may stem from a
partial disruption of the cytoskeletal network, leading to reduced
potential for signal processing and alteration in the maintenance of
cell polarity and cell architecture (43). Biochemical and immunological
evidence show that p127 forms high molecular mass complexes made of
homo-oligomers (44) with which are associated other proteins. Among the
proteins interacting with p127, a few have been identified, including
non-muscle myosin II (45) and a putative serine kinase, whose
activation leads to a specific phosphorylatation of p127, resulting in
the dissociation of myosin II from p127 without affecting p127
oligomerization (46). Several potential motifs for protein-protein
interaction, such as repeated heptad units of hydrophobic amino acids
(42) and motifs showing partial homology to the WD-40 repeats are
evolutionary conserved among the members of the l(2)gl
protein family. The conservation of two putative WD-40 motifs in p127,
as well as in the other homologues, suggests that the Sop1p, like p127
and HUGL, may be a component of large protein complexes. Our
subcellular fractionation studies revealing that a significant fraction
of Sop1p is associated with a particulate fraction of the yeast cells support this contention. In particular, the sedimentable Sop1p proteins
could be readily solubilized by treatment with 1 M KCl, suggesting that Sop1p association within this material is dependent upon electrostatic interactions.
Additional information on possible functional overlaps between Sop1p
and p127 was provided by the immunolocalization studies, which
indicated a predominantly cytoplasmic localization of Sop1p, with a
preferential distribution to the periphery of the cell. The overall
intracellular distribution of Sop1p is highly reminiscent to that
previously described for p127 (30) and HUGL (34). Indication for a true
functional relationship between the yeast and Drosophila
proteins derives, however, from the results obtained by analyzing
double mutant cells in which SOP1 was deleted together with
its isogene SOP2. While deletion of SOP2 alone
produced only slight Na+/Li+ sensitivity, the
sop1 A Link between Cytoskeletal Organization and Cation
Homeostasis--
A possible relationship between the Sop proteins and
the yeast cytoskeleton is suggested by a recent entry in the SGD data base of the SRO7 gene, which corresponds to SOP1.
The SRO7 gene is one of the nine different isolated
multicopy suppressors of a rho3 cell polarity defect (47).
The GTP-binding Rho proteins are implicated in regulating various
actin-based events that are involved in cytoskeletal polarity (48). At
non-permissive temperature, conditional rho3 mutants lose
cell polarity during bud formation and display randomized actin and
delocalized chitin (49). These findings were interpreted as an
involvement of Rho3p in the organization of the actin cytoskeleton for
proper surface growth of the yeast bud. A plausible mechanism for
explaining the suppression of the rho3 defect by increased
dosage of SRO7/SOP1 would be that Sop1p acts downstream of
Rho3 by stabilizing the polarized actin skeleton. Among additional
genes that were also identified as rho3 multicopy suppressors (47), the CDC42 and BEM1 genes
deserve special mention because BEM1 encodes a bud site
assembly protein that binds to Cdc24p, a guanine nucleotide exchange
factor of the CDC42 encoded GTPase (48). A further
indication of a possible link between the polar organization of the
cytoskeletal elements and ion homeostasis stems from the recent
observation that certain temperature-sensitive alleles of
CDC24 exhibit specific sensitivity to Na+ and
Li+ (50). Interestingly, there are precedents in higher
eukaryotes for a close relationship between ion transporters and the
underlying cytoskeleton. In particular, the gastric parietal cell
H+/K+ ATPase (51) and the anion exchanger of
the red cell (52) are associated with the membrane cytoskeleton via
ankyrin, a component of the plasma membrane cytoskeleton. There is also
evidence that interaction between the cadherin-catenin complex and the
membrane cytoskeleton is required for localization of
Na+/K+ ATPases to sites of cell adhesion in
epithelial cells (53, 54). In addition, Na+/K+
exchangers of the NHE1 subtype are reported to be downstream targets of
Cdc42p in fibroblasts (55), and, interestingly, neoplastic transformation of these cells is correlated with an increased Na+/K+ exchange activity (56). Although the
consequences of the establishment of cell polarity may be different in
yeast and metazoan cells, the general mechanisms controlling cell
polarity and cell architecture may be highly conserved among
eukaryotes, even for phylogenetically distant cells (48). Our results
showing a functional complementation of the
sop1 We thank Dr. P. Sunnerhagen for providing the
pFA6a-hisGMX6 plasmid, Dr. H. Rudolph for the
cmd1-3 and pmr2-2 strains, Dr. G. Fink for the
Ycp50 library and the cnb1 strain, and Dr. C. Schüller
for the pBsMYC plasmid. We acknowledge the help of C. Carlsson in
subcloning and disruption of the SOP1 gene and Drs. S. Hohmann and R. Bill for valuable comments on the manuscript.
*
This work was supported by grants from the Swedish National
Science Research Council, the Swedish Council for Forestry and Agricultural Research, the Swedish Research Council for Engineering Sciences, and by EU Programs BIOL-CT 950161, ERB4061 PL95-0014, and
BMH1-CT94-1572.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. Tel.:
46-31-7732500; Fax: 46-31-7732599; E-mail:
Lennart.Adler{at}gmm.gu.se.
The abbreviations used are:
kb, kilobase pair(s); bp, base pair(s); ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
The Saccharomyces cerevisiae SOP1 and
SOP2 Genes, Which Act in Cation Homeostasis, Can Be
Functionally Substituted by the Drosophila lethal(2)giant
larvae Tumor Suppressor Gene*
,,,, and¶
Department of Cell and Molecular Biology,
Microbiology, Göteborg University, Box 462, SE 40530 Göteborg, Sweden and the § Department of Developmental
Genetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
cells to NaCl stress, the intracellular Na+ level and the
Na+/K+ ratio rose to values significantly
higher than in wild type cells. Deletion of SOP2, encoding
a protein sharing 54% amino acid identity with Sop1p, produced only
slight Na+ sensitivity. Cells carrying a
sop1sop2 double deletion became, however,
hypersensitive to Na+ and exhibited increased sensitivity
also to Li+ and K+, suggesting involvement of
both SOP1 and SOP2 in cation homeostasis. The
predicted amino acid sequences of Sop1p and Sop2p show significant homologies with the cytoskeletal-associated protein encoded by the
Drosophila lethal(2)giant larvae tumor suppressor gene.
Immunolocalization of Sop1p revealed a cytoplasmic distribution and
cell fractionation studies showed that a significant fraction of Sop1p
was recovered in a sedimentable fraction of the cytosolic material.
Expression of a Drosophila l(2)gl cDNA in the
sop1sop2 strain partially restored the
Na+ tolerance of the cells, indicating a functional
relationship between the Sop proteins and the tumor suppressor protein,
and a novel function in cell homeostasis for this family of proteins extending from yeast to human.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
sop2 double mutant can
partially restore Na+ tolerance. Our data provide evidence
for functional conservation between the Drosophila p127
protein and its yeast homologues, and reveal involvement of
l(2)gl proteins in a new aspect of cell homeostasis.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Yeast strains
(17) was used for cloning and
amplification of DNA. Bacterial cultures were grown in 2× LB medium (18).
plates were sporulated, and the progeny from a
complete tetrad was examined by PCR for correct integration of the
disruption cassette into one of the SOP2 alleles. A set of
primers (forward, 5'-GGGGTACCCTCTGCGCCACCCACAC TTA-3'; reverse,
5'-TTCTGCAGATTCCGTATTTGCCAGTT-3') hybridizing upstream and downstream,
respectively, of the disruption cassette was used to amplify
chromosomal DNA. The length of the PCR products was verified by
agarose-gel electrophoresis, as was the length of the SphI
and XbaI restriction fragments of the PCR product.
20 °C until analyzed. Na+ and
K+ concentrations of the supernatants were measured with
K+ or Na+ specific electrodes (F2312K potassium
selectrode and G502Na sodium selectrode; Radiometer Denmark).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
A, complementation of the salt
sensitivity of the osg4 mutation by SOP1 in the
YCp50 centromeric vector. Growth on YPED medium containing
1.4 M NaCl is illustrated for the wild type W 303-1A
strain, the mutant (osg4), and the mutant cells transformed
with an empty vector or a SOP1-containing vector.
B, I, partial restriction map of the 4.7-kb DNA
of the genomic SOP1 locus showing the SOP1-coding
region (arrow). II, replacement construct using
the LEU2 selectable marker. III, the
SOP1::myc construct, which contains an insert
encoding three tandem repeats of the c-Myc epitope (black
box) in the Bst11071 site, 190 bp upstream the
SOP1 stop codon.
subunit of trimeric GTP-binding proteins (35, 36), and might represent
domains for protein-protein interactions.
View larger version (88K):
[in a new window]
Fig. 2.
Alignment of the SOP1 and
SOP2 predicted amino acid sequences with the sequence of
the p127 protein encoded by the Drosophila l(2)gl gene
(DLGL) as well as the mouse (MGL1) and human (HUGL) homologues.
The comparison was generated by the BestFit and Gap programs of the GCG
Wisconsin package. Two WD-40-like motifs are indicated by
solid bars.
Mutant Displays No General Stress Sensitivity but Is
Specifically Sensitive to Na+ Stress--
To determine the
phenotype of a cell lacking the SOP1 gene, null alleles were
constructed by one-step gene disruption (37). A
BglII/NheI fragment of the SOP1 open
reading frame was replaced by the LEU2 (Fig. 1B)
or URA3 marker gene, and the
HindIII/SpeI fragment of the resulting constructs
was used to disrupt the SOP1 locus of the W303-1A and
W303-1B strains. Southern blot analysis of the genomic DNA prepared
from the putative disruptants confirmed the correct replacement of the
SOP1 gene with the marker gene constructs (data not shown).
mutant was screened for osmotic sensitivity on
agar plates supplemented with NaCl, KCl, LiCl, or sorbitol. Of these solutes, only NaCl strongly restricted the growth of the null mutant
(Fig. 3), whereas KCl and the non-ionic
agent sorbitol caused no significant decrease in the growth of the
mutant compared with that of the wild type. Since Li+ and
Na+ ions are relatively similar, it is generally believed
that these ions share the same uptake and efflux systems in the cell
(6). Interestingly, we found that SOP1 inactivation
conferred only minor changes in Li+ tolerance (Fig. 3),
suggesting that SOP1 is highly specific for Na+
ion homeostasis of S. cerevisiae.
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Fig. 3.
Growth of the wild type, W303-1A strain, the
sop1 , sop2, and
sop1sop2 mutants at various solute
concentrations. Cells were grown overnight in YEPD medium,
adjusted to OD610 = 1, and serial 10-fold dilutions spotted
onto YEPD plates (pH 6.9), without solute addition (YEPD) or
supplemented with 0.3 M NaCl, 0.7 M NaCl, 0.7 M NaCl (pH 4.5), 1 M sorbitol, 0.7 M KCl, or 150 mM LiCl, as indicated. Plates
were incubated for 3 days at 30 °C prior to photography.
strain was sensitive to other
forms of stress, we compared the response of mutant and wild type cells
to heat stress, nitrogen starvation, oxidative stress, and high and low
pH. None of these conditions produced any growth difference between
wild type and mutant cells, indicating that a loss of the
SOP1 gene causes no general stress response defect (data not
shown). However, the Na+ sensitivity of the
sop1 mutant proved strongly pH-dependent, as
indicated by a considerably stronger tolerance to Na+ at pH
4.5 than at 6.9 (Fig. 3A).
Cells at High Extracellular
NaCl Concentrations--
To examine the effect of SOP1
inactivation on Na+ and K+ homeostasis, the
intracellular levels of these ions were measured in mutant and wild
type cells after conditioning the cells for 6 h in basal medium or
in the same medium containing either 0.7 M or 1.0 M NaCl. The intracellular Na+ concentration in
the mutant cells increased strongly with salinity to become about 3 times higher (0.4 M) than in wild type cells maintained in
medium containing 1 M NaCl (Fig.
4). The K+ concentration in
sop1 cells decreased proportionally so that the internal
K+ level reached 40% (0.06 M) of the wild type
level at the highest salinity. Thus, under these conditions, not only
the Na+ concentration but also the
Na+/K+ ratio became unfavorably high, reaching
a ratio of 5 in the mutant cells, while staying at about 1 in wild type
cells. These observations suggest a defective ion transporting
potential of the mutant at high salinity.
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Fig. 4.
Intracellular concentration of
Na+ (open bars) and K+
(shaded bars) in the sop1 mutant
and the W303-1A wild type strain after conditioning for 6 h in
YEPD or YEPD plus 0.7 M or 1.0 M NaCl.
Triplicate samples taken from each culture varied within ±10%.
::LEU2 strain was mated to a
pmr2A::HIS3 strain and the resultant diploid cells
were sporulated and dissected. Half of the 36 tetrads dissected
produced four viable spore clones, including double null mutant
segregants. The NaCl sensitivity of the
sop1::LEU2 pmr2A::HIS3
clones proved identical to that of a pmr2A single mutant
strain (Fig. 5). The absence of
enhancement of the NaCl sensitivity of a pmr2A strain by a
simultaneous inactivation of SOP1 indicates that
SOP1 may contribute to the same transport mechanisms as
PMR2.
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Fig. 5.
Genetic interaction between SOP1
and PMR2A. A wild type CRY1 strain, a
pmr2-2 mutant, a sop1 mutant, and a
sop1pmr2-2 double mutant were cultured
overnight in liquid YEPD medium. The OD610 was adjusted to
1 and serial 10-fold dilutions spotted onto YEPD plates supplemented
with 0.1 M NaCl or 0.3 M NaCl, as indicated.
Plates were incubated for 2 days at 30 °C prior to
photography.
null
mutant, indicating that the c-Myc-tagged Sop1p is functional.
Immunofluorescence staining of cells expressing Myc-tagged Sop1p from
pH/B316myc showed that Sop1p is present in the cytoplasm of the yeast
cells (Fig. 6A), and as
revealed by diamidinophenylindole staining absent from the nucleus
(data not shown). When examining the stronger signal obtained from the
YEpH/Bmyc plasmid, the protein appeared to be preferentially
distributed toward the periphery of the cell (data not shown). In all
cases the detected cytoplasmic staining was much stronger than in
control cells, lacking the Myc epitope.
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Fig. 6.
Immunofluorescence localization of
epitope-tagged Sop1p. The sop1 strain transformed
with a SOP1::myc centromeric vector (A,
left) and the wild type W303-1A harboring a vector without insert
(A, right) were grown to early exponential phase in YEPD
medium, harvested, fixed with formaldehyde, and processed for
immunofluorescence microscopy using anti-c-Myc and fluorescein
isothiocyanate-conjugated anti-mouse antibodies. B,
subcellular localization of Sop1p. Cleared cell lysates of the
sop1 mutant, transformed with YEpH/Bmyc, containing 100 mM or 1 M KCl were centrifuged at 100,000 × g for 1 h at 4 °C. Samples from the resulting
supernatant and pellet were subjected to SDS-PAGE and transferred to
Hybond membranes, and the blot incubated with anti-c-Myc antibody and
visualized by ECL. A sop1 mutant transformed with an
empty vector gave no band at the SOP1::myc
position (data not shown). Pellet and supernatant refer to the two
fractions obtained after the 100,000 × g
centrifugation.
and
sop1sop2 Mutants--
To obtain further clues as to the role of
SOP1 in cation homeostasis we deleted its homologue
SOP2 (YBL106C), using homology-directed replacement by PCR
(25). Isolated sop2::HIS3 clones
were verified by PCR and examined for sensitivity to high
concentrations of Na+, Li+, K+,
sorbitol, or glycerol. This examination revealed a slight
Na+-specific sensitivity of the sop2 mutant
that was much less pronounced than for the sop1 strain
(Fig. 3). However, unlike sop1 cells, mutants lacking
SOP2 displayed obvious sensitivity to Li+.
sop2 double null mutant
strain, a sop1::LEU2 mutant was
mated with a sop2::HIS3 strain and
the resultant diploid sporulated and subjected to tetrad dissection.
His+ Leu+ segregants were
isolated and the expected deletion of the SOP1 and
SOP2 loci verified by PCR. All of the confirmed
sop1sop2 mutated cells grew more slowly
than wild type cells in basal YEPD medium and showed hypersensitivity
toward Na+ (Fig. 3). The double mutant also exhibited
sensitivity toward solutes other than NaCl, as demonstrated by the
attenuated growth at increased concentrations of LiCl and KCl. However,
the tolerance toward the non-ionic osmoticum sorbitol of
sop1sop2 was identical to that of wild type
cells. These observations clearly indicate that both SOP1
and SOP2 play a role in cation homeostasis in S. cerevisiae.
and the sop1sop2 Phenotype by
the Drosophila l(2)gl Gene--
The strong salt sensitivity of the
sop1 and the sop1sop2 strains
provided an assay to explore the functional relationship between the
SOP1 and SOP2 gene products and the p127 protein
encoded by the l(2)gl tumor suppressor gene of
Drosophila. To this end we introduced the cDNA Ec173
encoding the p127 protein (22) in the multicopy pYX212 plasmid under
control of the constitutive yeast TPI promoter, and
determined whether the l(2)gl cDNA would complement the
sop1 or sop1sop2 mutations. As shown in Fig.
7A, Western blot analysis
confirmed that p127 is expressed by the l(2)gl+-transformed
sop1sop2 S. cerevisiae cells.
Only slight complementation by p127 of the yeast sop1
mutation was obtained (data not shown), whereas p127 clearly decreased
the Na+ sensitivity of the
sop1sop2 cells (Fig. 7, B and
C). Three independently isolated
sop1sop2 clones were transformed with the
l(2)gl cDNA, all being complemented to a similar extent,
while mutants transformed with an empty vector showed no improved salt tolerance. The complementation of the yeast double mutant by the Drosophila l(2)gl gene indicates functional conservation of
this family of proteins across species borders.
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Fig. 7.
A, immunoblot showing expression of the
Drosophila p127 protein in the S. cerevisiae
transformants. Cell lysates were subjected to SDS-PAGE and transferred
to Hybond membranes, and the blot was incubated with anti-p127 antisera
and immunoreactive species detected by ECL. B,
complementation of the NaCl sensitivity of the
sop1 sop2 double mutant. The W303-1A wild
type strain and the sop1sop2 mutant were
transformed with the Drosophila l(2)gl cDNA inserted in
the multicopy pYX212 plasmid, or the same plasmid without insert.
Serial dilutions was produced as described in Fig. 3 and spotted onto
YEPD plates of 0 M and 0.3 M NaCl.
C, complementation shown in liquid medium. The transformed
W303-1A wild type strain and sop1sop2
mutant were grown overnight in YNB medium. Sidearm flasks containing 25 ml of YEPD medium plus 0.1 M NaCl were inoculated with
overnight cultures to give a final OD610 of 0.1. The flasks
were incubated with agitation at 30 °C. Growth was monitored by
sidearm measurement of OD610 of W303-1A + pYX212 (
),
sop1sop2 plus pYX212 containing the
l(2)gl cDNA construct (
), and
sop1sop2 plus pYX212 (
).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
cells remain osmoresistant, and the production and
accumulation of the compatible solute, glycerol, is similar to that of
wild type cells at high salinity (data not shown).
cells and
the enhanced Na+ levels detected in a null mutant subjected
to exogenous NaCl stress suggest that inactivation of SOP1
leads to an increased influx and/or a decreased efflux of
Na+, due to defective transporter function(s). Considering
the drastic effect on cellular Na+ sensitivity caused by
the SOP1 deletion, it is possible that that Sop1p may
contribute to the transporter system controlled by the major
determinant for Na+ tolerance in S. cerevisiae,
the PMR2-encoded system. This conjecture agrees with the
observation that deletion of SOP1 in an pmr2A background does not aggravate the Na+ tolerance profile of
the pmr2A mutant (Fig. 5), suggesting direct or indirect
interactions of Sop1p with the Na+ pumping ATPase.
Furthermore, the strong pH dependence of the Na+
sensitivity of sop1 mutants indicates that the cells
might have become dependent upon the alternative Na+
exporting system, the NHA1-encoded
Na+/H+ antiporter (9). Since the activity of
this system becomes gradually inhibited by increasing pH, cells with an
defective PMR2A function will display a drastic decrease of
the Na+ tolerance at high pH.
sop2 mutant exhibited a dramatic salt sensitivity, which was partly restored by expressing the
Drosophila l(2)gl gene in the double mutant cells. In
addition, the much stronger phenotype of the double mutant, as compared
with the single mutants, indicates synergistic effects and functional
relationships in cation homeostasis between Sop1p and Sop2p. Plausibly,
SOP1 and SOP2 are involved in different but
overlapping functions in ion homeostasis; SOP1 is able to
compensate well for loss of SOP2, while SOP2 is
unable to adequately correct for a loss of SOP1.
sop2 defect by the Drosophila
l(2)gl tumor suppressor gene provide evidence for
phylogenetic conservation of a previously unrecognized function of a
family of proteins linked to maintenance of the cytoskeletal
architecture. The Na+ sensitivity of strains lacking
SOP1, or both SOP1 and SOP2, provides an avenue for further exploring the physiological role of these molecules and uncovering new biological functions.
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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