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

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This is the published version of a paper published in Journal of Biological Chemistry.

Citation for the original published paper (version of record):

Larsson, K., Böhl, F., Sjöström, I., Akhtar, N., Strand, D. et al. (1998)

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.

Journal of Biological Chemistry, 273(50): 33610-33618

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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*

(Received for publication, July 29, 1998, and in revised form, September 24, 1998) Katrin Larsson‡, Florian Bo¨ hl§, Ingrid Sjo¨ stro¨ m‡, Noreen Akhtar‡, Dieter Strand§,

Bernard M. Mechler§, Reiner Grabowski‡, and Lennart Adler‡¶

From the ‡Department of Cell and Molecular Biology, Microbiology, Go¨teborg University, Box 462, SE 40530 Go¨teborg, Sweden and the §Department of Developmental Genetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany

By complementation of a salt-sensitive mutant of

Sac-charomyces cerevisiae, we cloned the SOP1 gene,

encod-ing a 114.5-kDa protein of 1033 amino acids. Cells de-leted for SOP1 exhibited sensitivity to sodium stress, but showed no sensitivity to general osmotic stress.

Fol-lowing exposure of sop1D cells to NaCl stress, the

intra-cellular Na1 level and the Na1/K1 ratio rose to values

significantly higher than in wild type cells. Deletion of

SOP2, encoding a protein sharing 54% amino acid

iden-tity with Sop1p, produced only slight Na1 sensitivity.

Cells carrying a sop1Dsop2D double deletion became,

however, hypersensitive to Na1and exhibited increased

sensitivity also to Li1and K1, suggesting involvement of

both SOP1 and SOP2 in cation homeostasis. The pre-dicted 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 fraction-ation studies showed that a significant fraction of Sop1p was recovered in a sedimentable fraction of the cytoso-lic material. Expression of a Drosophila l(2)gl cDNA in

the sop1Dsop2D strain partially restored the Na1

toler-ance of the cells, indicating a functional relationship between the Sop proteins and the tumor suppressor pro-tein, and a novel function in cell homeostasis for this family of proteins extending from yeast to human.

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 Na1is generally abun-dant and K1 scarce, transport must occur against concentra-tion gradients. Genetic analysis of salt tolerance in

Saccharo-myces 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 K1uptake is constituted by the TRK1- and

TRK2-encoded membrane proteins (2– 4), which appear to contribute to the uptake of K1in 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 Na1, while under NaCl stress, the uptake system has the capacity of increasing its selectivity for K1over Na1(6).

In yeast cells, influx of Na1is counteracted by Na1efflux, 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 Na1or Li1stress or cells exposed to alkaline pH (7). An additional sodium trans-porter encoded by the NHA1 gene and acting as a putative Na1/H1antiporter was recently identified in S. cerevisiae (9). Disruption of the NHA1 gene displays only minor effects in wild type cells but elicits increased Na1sensitivity in S.

cer-evisiae 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 character-ized 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 Na1 tolerance and is required for (i) induced expression of the

PMR2A gene and (ii) modulating the K1 uptake system to display increased K1versus Na1discrimination. Further evi-dence that protein phosphorylation and dephosphorylation reg-ulate Na1tolerance 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, en-coding 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 spe-cifically sensitive to high concentrations of Na1(13).

By isolation and functional complementation a NaCl-sensi-tive 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 span-ning regions or other characteristics of previously isolated de-terminants for Na1tolerance, and displays significant homol-ogy 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 * This work was supported by grants from the Swedish National

Science Research Council, the Swedish Council for Forestry and Agri-cultural Research, the Swedish Research Council for Engineering Sci-ences, 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. This 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@gmm.gu.se.

This paper is available on line at http://www.jbc.org

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SOP1 and a second related gene, designated as SOP2, are

involved in regulating cation homeostasis in S. cerevisiae. Fur-thermore, we demonstrate that the expression of a Drosophila

l(2)gl cDNA sequence in the S. cerevisiae sop1_{Dsop2D double}

mutant can partially restore Na1tolerance. 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

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 120mg/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

con-centration of 120mg/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 DH5a (17) was used for cloning and amplification of DNA. Bacterial cultures were grown in 23 LB medium (18).

Plasmids—The YCp50-based yeast genomic library (19) was used for

complementation of the osg4 mutant. Transformants were screened on

YNB agar plates containing 1.4MNaCl, and plasmids from

comple-mented 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 2m-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

hu-man c-Myc epitope was removed from the pBsMYC plasmid. This

prod-uct was subcloned into pBSH/B (pBluescript KS1containing the

Hin-dIII/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 anal-ysis and sequencing.

Disruption of SOP1 and SOP2—The 4.7-kb HindIII/BamHI

frag-ment harboring the SOP1 gene subcloned into pBluescriptKS1was 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 (59-TTCCGCTTCATAGGAGGAGA-39 and

59-GGGGATCC-GTCGACCTGCAGCGTACCATTTATAAAATTTTTGTAT -39) 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

(59-AAC-GAGCTCGAATTCATCGATGATATAGTCCATAAATAGTTTTTA-39

and 59-ACGGTTCATCATTCGGAAAA-39) was used to amplify a 377-bp

fragment immediately downstream of the SOP2 ORF stop codon. The 59 end of the primers adjacent to the insertion site carried 25 nucleotide

extensions homologous to the 59 and 39 region of the his5MX6

disrup-tion cassette of plasmid pFA6a-his5MX6 (26). In the second PCR reac-tion, pFA6a-his5MX6 was used as template and the 59- and 39-homol-ogous 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

transfor-mants able to grow on his2plates 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, 59-GGGGTACCCTCTGCGCCACCCACAC TTA-39; reverse, 59-TTCTGCAGATTCCGTATTTGCCAGTT-39) hybridizing upstream and downstream, respectively, of the disruption cassette was used to amplify chromosomal DNA. The length of the PCR products was veri-fied by agarose-gel electrophoresis, as was the length of the SphI and

XbaI restriction fragments of the PCR product.

Determination of Stress Tolerance—Tolerance to salt or osmotic

stress was examined by spotting 10-fold dilutions (10ml) of an overnight

culture diluted to OD610;1.0 onto YEPD plates (pH 6.9, unless

other-wise 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 OD610of approximately 0.2. The

cells were left to adjust to the new environment for 2 h, whereupon

H2O2was 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 Na1_{and K}1_{—Cells were grown} over-night 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 53 107_{cells. These cultures were}

centri-fuged at 35003 g for 5 min and then re-suspended in 40 ml of YEPD

medium supplemented with 0, 0.7, or 1.0MNaCl. After incubation for

6 h, triplicate samples of 5 ml of cell suspension were filtered through

0.22-mm filters and washed with three volumes of iso-osmotic CaCl2.

The filters were transferred to 5 ml of 20mMCa(OH)2and extracted by

heating for 10 min at 95 °C. The cell suspension was centrifuged and

the supernatant stored at220 °C until analyzed. Na1_{and K}1

concen-trations of the supernatants were measured with K1or Na1specific

electrodes (F2312K potassium selectrode and G502Na sodium selec-trode; Radiometer Denmark).

Northern Blot Analysis—Exponentially growing cells were

trans-ferred to fresh YEPD medium and the same medium supplemented

with 1.0MNaCl or 1.5Msorbitol, 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

1_{The abbreviations used are: kb, kilobase pair(s); bp, base pair(s);}

ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

TABLE I

Yeast strains

Strain Genotype Source

W303–1A MATa ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1 R. Rothstein

W303–1B MATa ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1 R. Rothstein

WKL-1A MATa ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1sop1D::URA3 This study

WKL-2A MATa ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1sop1D::LEU2 This study

WKL-2B MATa ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1sop1D::LEU2 This study

WKL-3A MATa ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1sop2D::HIS3 This study

WKL-23 MATa ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1sop1D::LEU2sop2D::HIS3 This study

K603 MATa ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1cnb1::LEU2 (57)

CRY1 MATa ade2oc can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1 (8)

YN10 MATa ade2oc can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1pmr2–2::HIS3 (8)

JGY148 MATa ade2oc can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1cmd1–5 (8)

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hexanucleotides and Klenow fragment from Boehringer Mannheim and

[32_{P]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 OD6100.5–1.0 in YEPD medium. Cells were washed by

cen-trifugation and resuspended in twice the pellet volume of resuspension

buffer (R-buffer), containing 50 mMTris (pH 7.5), 5 mMEDTA, complete

protease inhibitor mixture (Boehringer Mannheim; 1 tablet/50 ml of cell

extract), and either 0.1Mor 1MKCl. 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 5003 g for 5 min at 4 °C. The lysate

was then centrifuged at 100,0003 g for 1 h in a Beckman TL-100

ultracentrifuge. The pellets were resuspended in 100 mMKCl R-buffer

and the lysate desalted by gel filtration (Microcolumns, Bio-Rad). Ali-quots 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 manufac-turers’ 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

addi-tion of formaldehyde to a final concentraaddi-tion 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(ab9)2

frag-ment 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.

RESULTS

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 gen-erally osmosensitive. Transformation of this mutant with a YCp50-based yeast genomic library (19) resulted in the isola-tion of clones that restored growth of the mutant at high salinity (Fig. 1A), and restriction endonuclease analysis iden-tified a 4.7-kb HindIII/BamHI DNA fragment, which fully com-plemented 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. cerevi-siae Protein and to the p127 Protein Encoded by the l(2)gl Tumor Suppressor Gene of Drosophila—Homology searches

re-vealed 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 b subunit of trimeric GTP-binding proteins (35, 36), and might represent domains for protein-protein interactions.

The sop1D Mutant Displays No General Stress Sensitivity but Is Specifically Sensitive to Na1Stress—To determine the

phe-notype of a cell lacking the SOP1 gene, null alleles were con-structed by one-step gene disruption (37). A BglII/NheI frag-ment 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).

The sop1_{D 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 Li1and Na1 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 Li1tolerance (Fig. 3), suggesting that SOP1 is highly specific for Na1ion homeostasis of S. cerevisiae.

FIG. 1. A, complementation of the salt sensitivity of the osg4 mutation

by SOP1 in the YCp50 centromeric vector. Growth on YPED medium

containing 1.4MNaCl 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.

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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.

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To examine whether the sop1D 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, indicat-ing that a loss of the SOP1 gene causes no general stress response defect (data not shown). However, the Na1sensitivity of the sop1_{D mutant proved strongly pH-dependent, as} indi-cated by a considerably stronger tolerance to Na1 at pH 4.5 than at 6.9 (Fig. 3A).

SOP1 Expression Is Not Controlled by Environmental Salin-ity, and Na1 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 over-expression of SOP1 gene from its own promoter on a YEplac195 multicopy vector caused no increase in Na1tolerance by com-parison to control cells, carrying a vector without insert (data not shown). Likewise, overexpression of SOP1 exerted no effect on the Na1tolerance of a pmr2A null mutant lacking the Na1 extruding ATPase activity, or could not bypass the salt sensi-tivity of either cnb1 cells (10) lacking calcineurin or cmd1-5 cells (38) carrying a mutant form of calmodulin unable to bind Ca21.

Na1Accumulates in sop1_{D Cells at High Extracellular NaCl} Concentrations—To examine the effect of SOP1 inactivation on

Na1and K1homeostasis, 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 Mor 1.0M NaCl. The intracellular Na1 concentration in the mutant cells increased strongly with

sa-linity to become about 3 times higher (0.4M) than in wild type cells maintained in medium containing 1MNaCl (Fig. 4). The K1 concentration in sop1D cells decreased proportionally so that the internal K1level reached 40% (0.06M) of the wild type level at the highest salinity. Thus, under these conditions, not only the Na1concentration but also the Na1/K1ratio 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.

Possible Genetic Interaction between SOP1 and PMR2A—

Since the PMR2A gene is reported to be the most important determinant of Na1 tolerance in S. cerevisiae (1, 6, 39), we examined whether we could detect a genetic interaction be-tween SOP1 and PMR2A. For this purpose, a sop1D::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_{D::LEU2 pmr2A::HIS3 clones proved identical to that of a} pmr2A single mutant strain (Fig. 5). The absence of

enhance-ment of the NaCl sensitivity of a pmr2A strain by a simulta-neous inactivation of SOP1 indicates that SOP1 may contrib-ute to the same transport mechanisms as PMR2.

Cytoplasmic Distribution of Sop1p—To determine the

sub-cellular localization of Sop1p, three tandem repeats encoding the c-Myc epitope were fused in frame near the COOH termi-nus of Sop1p and expressed from a single-copy (pH/B316myc) or a multicopy plasmid (YEpH/Bmyc). The SOP1::myc deriva-tive fully complemented the salt sensitivity of the sop1D 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

dia-FIG. 3. Growth of the wild type,

W303–1A strain, the sop1D, sop2D, and sop1Dsop2D mutants at various solute concentrations. Cells were grown overnight in YEPD medium,

ad-justed to OD6105 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, 0.7MNaCl (pH 4.5), 1Msorbitol,

0.7MKCl, or 150 mMLiCl, as indicated.

Plates were incubated for 3 days at 30 °C prior to photography.

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midinophenylindole 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 stron-ger than in control cells, lacking the Myc epitope.

We also used subcellular fractionation to determine the in-tracellular localization of Sop1p expressed from YEpH/Bmyc. The distribution of Sop1p between the supernatant and the 100,0003 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 pel-letable structures was further analyzed by increasing the ion strength. Treatment with 1MKCl 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 back-ground staining due to the much weaker signal (data not shown).

Deletion of the SOP2 Gene and Characterization of sop2_D and sop1Dsop2D 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 sop2D::HIS3 clones were verified by PCR and examined for sensitivity to high concentrations of Na1, Li1, K1, sorbitol, or glycerol. This examination revealed a slight Na1-specific sensitivity of the sop2D mutant that was much less pronounced than for the sop1D strain (Fig. 3). How-ever, unlike sop1_{D cells, mutants lacking SOP2 displayed} ob-vious sensitivity to Li1.

To produce a sop1Dsop2D double null mutant strain, a

sop1D::LEU2 mutant was mated with a sop2D::HIS3 strain and

the resultant diploid sporulated and subjected to tetrad dissec-tion. His1 Leu1 segregants were isolated and the expected deletion of the SOP1 and SOP2 loci verified by PCR. All of the confirmed sop1Dsop2D mutated cells grew more slowly than wild type cells in basal YEPD medium and showed hypersen-sitivity toward Na1(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 sop1Dsop2D 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.

Complementation of the sop1_{D and the sop1Dsop2D} Pheno-type by the Drosophila l(2)gl Gene—The strong salt sensitivity

of the sop1D and the sop1Dsop2D 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 intro-duced 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 ex-pressed by the l(2)gl1-transformed sop1Dsop2D S. cerevisiae cells. Only slight complementation by p127 of the yeast sop1_D mutation was obtained (data not shown), whereas p127 clearly decreased the Na1sensitivity of the sop1Dsop2D cells (Fig. 7, B and C). Three independently isolated sop1Dsop2D clones were transformed with the l(2)gl cDNA, all being complemented to a similar extent, while mutants transformed with an empty vec-tor 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.

DISCUSSION

Role of Sop1p in Cellular Tolerance toward Na1—By

com-plementing the NaCl sensitivity of a previously isolated mu-tant, we cloned the SOP1 gene, encoding a 114.5-kDa cytosolic protein that is required for growth at high Na1concentrations. Although deletion of SOP1 strongly decreases Na1tolerance,

SOP1 overexpression results in no protection toward increased

concentration of Na1. In this respect, SOP1 differs from the series of HAL genes (1, 40, 41), which improve Na1tolerance in a dose-dependent fashion. SOP1 differs also from the genes involved in the general osmoregulatory response. The sop1D cells remain osmoresistant, and the production and accumula-tion of the compatible solute, glycerol, is similar to that of wild type cells at high salinity (data not shown).

The strict Na1sensitivity of sop1D cells and the enhanced Na1levels 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 Na1, due to defec-tive transporter function(s). Considering the drastic effect on cellular Na1 sensitivity caused by the SOP1 deletion, it is possible that that Sop1p may contribute to the transporter system controlled by the major determinant for Na1tolerance FIG. 4. Intracellular concentration of Na1(open bars) and K1

(shaded bars) in the sop1D mutant and the W303–1A wild type

strain after conditioning for 6 h in YEPD or YEPD plus 0.7Mor 1.0MNaCl. Triplicate samples taken from each culture varied within

610%.

FIG. 5. Genetic interaction between SOP1 and PMR2A. A wild

type CRY1 strain, a pmr2–2 mutant, a sop1D mutant, and a

sop1Dpmr2–2 double mutant were cultured overnight in liquid YEPD

medium. The OD610was adjusted to 1 and serial 10-fold dilutions

spotted onto YEPD plates supplemented with 0.1MNaCl or 0.3MNaCl,

as indicated. Plates were incubated for 2 days at 30 °C prior to photography.

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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 Na1tolerance profile of the

pmr2A mutant (Fig. 5), suggesting direct or indirect

interac-tions of Sop1p with the Na1pumping ATPase. Furthermore, the strong pH dependence of the Na1 sensitivity of sop1_D mutants indicates that the cells might have become dependent upon the alternative Na1exporting system, the NHA1-encoded Na1/H1 antiporter (9). Since the activity of this system be-comes gradually inhibited by increasing pH, cells with an de-fective PMR2A function will display a drastic decrease of the Na1tolerance at high pH.

The coordinate regulation of both PMR2A-encoded Na1 ef-flux system and TRK1-encoded Na1/K1influx system is con-trolled by a signaling pathway involving Ca21/calmodulin and the protein phosphatase, calcineurin (8, 10). This signaling system appears to play a crucial role in modulating intracellu-lar Na1and K1concentrations 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 Na1sensitivity of cmd1–5 or

cnb1 mutants, indicating that Sop1p does not operate

down-stream 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 Sup-pressor 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 architec-ture (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 par-tial homology to the WD-40 repeats are evolutionary conserved among the members of the l(2)gl protein family. The conserva-tion 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 sediment-able 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 be-tween Sop1p and p127 was provided by the immunolocalization studies, which indicated a predominantly cytoplasmic

localiza-FIG. 6. Immunofluorescence

local-ization of epitope-tagged Sop1p. The

sop1D 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 immuno-fluorescence microscopy using anti-c-Myc

and fluorescein

isothiocyanate-conju-gated anti-mouse antibodies. B, subcellu-lar localization of Sop1p. Cleared cell ly-sates of the sop1D mutant, transformed

with YEpH/Bmyc, containing 100 mMor 1

MKCl were centrifuged at 100,0003 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 sop1D mutant transformed with an empty vector gave no band at the

SOP1::myc position (data not shown).

Pel-let and supernatant refer to the two

frac-tions obtained after the 100,000 3 g

centrifugation.

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tion 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 Na1/Li1 sensitiv-ity, the sop1_{Dsop2D mutant exhibited a dramatic salt} sensitiv-ity, 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 re-lationships in cation homeostasis between Sop1p and Sop2p. Plausibly, SOP1 and SOP2 are involved in different but over-lapping functions in ion homeostasis; SOP1 is able to compen-sate well for loss of SOP2, while SOP2 is unable to adequately correct for a loss of SOP1.

A Link between Cytoskeletal Organization and Cation Home-ostasis—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 cy-toskeleton for proper surface growth of the yeast bud. A plau-sible 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 Na1and Li1(50). Inter-estingly, there are precedents in higher eukaryotes for a close relationship between ion transporters and the underlying cy-toskeleton. In particular, the gastric parietal cell H1/K1 ATPase (51) and the anion exchanger of the red cell (52) are associated with the membrane cytoskeleton via ankyrin, a com-ponent of the plasma membrane cytoskeleton. There is also evidence that interaction between the cadherin-catenin com-plex and the membrane cytoskeleton is required for localization of Na1/K1ATPases to sites of cell adhesion in epithelial cells (53, 54). In addition, Na1/K1exchangers 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 Na1/K1exchange 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 phylogeneti-cally distant cells (48). Our results showing a functional complementation of the sop1_{Dsop2D 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 Na1sensitivity 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.

Acknowledgments—We thank Dr. P. Sunnerhagen for providing the

pFA6a-hisGMX6 plasmid, Dr. H. Rudolph for the cmd1–3 and pmr2–2

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 de-tected by ECL. B, complementation of the NaCl sensitivity of the

sop1Dsop2D double mutant. The W303–1A wild type strain and the

sop1Dsop2D 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 0Mand 0.3MNaCl. C, complementation shown in

liquid medium. The transformed W303–1A wild type strain and

sop1Dsop2D mutant were grown overnight in YNB medium. Sidearm

flasks containing 25 ml of YEPD medium plus 0.1MNaCl were

inocu-lated with overnight cultures to give a final OD610of 0.1. The flasks

were incubated with agitation at 30 °C. Growth was monitored by

sidearm measurement of OD610of W303–1A1 pYX212 (●), sop1Dsop2D

plus pYX212 containing the l(2)gl cDNA construct (M), and sop1Dsop2D plus pYX212 (‚).

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strains, Dr. G. Fink for the Ycp50 library and the cnb1 strain, and Dr.

C. Schu¨ ller 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.

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Mechler, Reiner Grabowski and Lennart Adler

Katrin Larsson, Florian Böhl, Ingrid Sjöström, Noreen Akhtar, Dieter Strand, Bernard M.

Tumor Suppressor Gene

larvae

Drosophila lethal(2)giant

Homeostasis, Can Be Functionally Substituted by the

Genes, Which Act in Cation

SOP2

and

Saccharomyces cerevisiae SOP1

The

doi: 10.1074/jbc.273.50.33610

1998, 273:33610-33618.

J. Biol. Chem.

http://www.jbc.org/content/273/50/33610

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