the journal of broloclcfi chemistry vol. no. for and … · molecular cloning and expression of the...

THE JOURNAL OF BroLoClcfi CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 6, Issue of February 11, pp. 4180-4186, 1994 Printed in U.S.A.

Molecular Cloning and Expression of the Saccharomyces cerevisiae STSl Gene Product A YEAST ABC TRANSPORTER CONFERRING MYCOTOXIN RESISTANCE*

(Received for publication, August 9, 1993, and in revised form, October 20, 1993)

Peter H. BissingerSS and Karl Kuchled /I From the Wepartment of Molecular Genetics, University and Biocenter of Vienna, A-1030 Vienna, Austria and

Research Ltd., Crown Research Institute of Pastoral Agricultural Research, Hamilton 3123, New Zealand

We have cloned a yeast gene that confers a multidrug resistance phenotype on Saccharomyces cerevisiae when present in multiple copies. The STSl (for Spo- ridesmin Toxicity fuppressor) gene encodes a 1511-residue protezn whose predicted structural organization is characterized by 12 a-helical membrane segments and two domains containing consensus sites for ATP binding, indicating that STSl is a new yeast ATP-binding cassette (ABC) transporter. A chromosomal deletion of STSl leads to viable Astsl cells of both mating types, suggesting that STSl is not essential for cell growth. However, Astal cells exhibit supersensitivity to sporidesmin and to other structurally unrelated drugs such as cycloheximide. Conversely, overexpression of STSl leads to increased resistance to the same drugs. Al- though Northern analysis showed that STSl mRNA is present in all yeast cell types, its drastically reduced level in a-factor-arrested cells indicates that expression of STSl is regulated by mating pheromones. Subcellular fractionation and immunoblotting using monoclonal antibodies, which recognize a fully functional epitope- tagged Stsl protein, showed that Stsl is a 175-kDa membrane protein localized mainly to intracellular membranes.

ABC transporters (for ATP-binding cassette) constitute a novel and rapidly growing superfamily of membrane transport proteins that are found operating from microorganisms to man (reviewed in Refs. 1 and 2). The characteristic features of all ABC proteins include the presence of two domains for ATP binding (ABC), and two membrane domains each containing usually six membrane spanning a-helices (TMS). These four domains are normally arranged in an (TMS6-ABC)2 configura- tion, but “half-size” transporters with an TMS6-ABC or ABC- TMS6 topology are also frequently found (1, 2).

Overexpression of certain ABC proteins in prokaryotes and eukaryotes is linked to drug and antibiotic resistance phenomena. For example, the well characterized mammalian P-glycoprotein or Mdrl (3) is associated with the development of a

* This work was supported by FRST Grant 92MTT05-323 (to P. H. B.)

Austrian National Bank Grant OENB-4486 ( to K. K.). The costs of and in part by Austrian Science Foundation Grant MOB-09537 and

publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession numbeds) X74113. The nucleotide sequence(s) reported in this paper has been submitted

0. Box 219, North Ryde, New South Wales 2113, Australia. Q Present address: Bums Philp Technology and Research Centre, P.

1) To whom correspondence and reprint requests should be addressed: Dept. of Molecular Genetics, University and Biocenter Vienna, Dr. Bohr-Gasse 9/2, A-1030, Vienna, Austria. Tel.: 43-1-79515-2007; Fax: 43-1-79515-2900; e-mail: [email protected].

multidrug resistance (MDR)l phenotype in tumor cells (4). Fur- ther, overexpression of LtpgpA from Leishmania is responsible for methotrexate and heavy metal resistance (5, 6), and P2as- modium pfMdr has been implicated in chloroquine resistance of the malarial parasite (7). Likewise, bacterial erythromycin resistance in Staphylococcus is caused by MsrA overexpression (81, and the ABC protein DrrAB of Streptomyces appears to be a daunomycin resistance determinant (9).

The yeast Ste6 a-factor transporter (10) was the first ABC transporter to be identified in this lower eukaryote (11, 12). Ste6 is closely related to mammalian P-glycoproteins, yet overexpression of Ste6 is generally not associated with a MDR phenotype. However, yeast has been reported to contain several MDR determinants, some of which are encoded by ABC transporters (13, 14). For instance, the recently cloned SNQ2 gene encodes an ABC protein whose expression is associated with a MDR phenotype (15). In some cases, like that of the putative translation elongation factor Yef3, overexpression leads to drug hypersensitivity toward the aminoglycoside antibiotics hygro- mycin and paromomycin rather than to drug resistance (16). Expression of several yeast MDR genes appears to be under transcriptional control of dedicated transcription factors because mutations in PDRl (171, PDR3 (18, 191, and the allelic SNQ3IYApl /PAR1 genes (20-221, all of which encode putative transcription factors, lead to multidrug resistance. Taken to- gether, numerous yeast genes may comprise a distinct gene family implicated in MDR and drug hypersensitivity phenomena (13, 16). Individual members of such a gene family may be necessary for specialized drug transport or cellular detoxification of poisonous metabolites.

We have used Saccharomyces cerevisiae as a model organism to search for genes that can mediate resistance to sporidesmin, a highly toxic epidithiodioxopiperazine mycotoxin produced in the spores of the fungus Phytomyces chartarum. Its toxicity is believed to be mediated by superoxide and hydroxyl radicals generated during intracellular thiol oxidation of reduced sporidesmin. Sporidesmin intoxication leads to “facial excema,” a hepatogenous photosensitivity disease that afflicts grazing ru- minants (23).

In this report, we describe the isolation and characterization of the S. cerevisiae S T S l gene (for Sporidesmin Toxicity sup- pressor) that encodes a membrane protein with extensive homology to other ABC proteins. Overexpression of S T S l leads to a compound MDR phenotype with increased resistance to sporidesmin and other structurally unrelated drugs such as cycloheximide. Comparison of the nucleotide sequences of S T S l and PDR5 (24), a pleiotropic drug resistance gene previously char-

1 The abbreviations used are: MDR, multidrug resistance; kb, kilobase(s); bp, base pair(s); ER, endoplasmic reticulum; NQQ, 4-nitroquinoline N-oxide; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine.

4180

Multidrug Resistance nansporters from Yeast 4181

acterized as a cycloheximide resistance determinant (14), demonstrated that the STSl and PDR5 genes are allelic.

MATERIALS AND METHODS

Yeast Strains and Culture Conditions-The following yeast strains were used in this study. WKK7-A5 (MATa Aste6::HISS Asts1:TRPl) is a isogenic derivative of WKK7 (MATa AsteG::HIS3) described earlier (11). Strain A2-200 (25) was the recipient strain for the high copy number Yepl3-based genomic library of S. cereuisiae (26). YKKD-1 is a heterozygous diploid (Asts1:TRPl ISTSl) derived from parent strain YPH5Ol (27). YKKB-13 (MATar Asts1:TRPl) and YKKA-7 (MATa Asts1:TRPl) are isogenic haploids derived from YKKD-1. Synthetic medium (SD), supplemented with appropriate nutrients for maintenance of plasmids, or rich medium (YPD), were prepared exactly as described elsewhere (28). Yeast cultures were grown routinely at 30 "C.

Drug Resistance Assays-Sporidesmin was isolated from cultures of F? chartarum as previously described (23). Agar plates containing various drugs (sporidesmin, 4-nitroquinoline-N-oxide, chloramphenicol, or N-methyl-N'-nitro-N-nitrosoguanidin) at desired concentrations were prepared by adding a stock solution of the drug (usually 10 mg/ml in dimethyl sulfoxide) to 2 ml of sterilized YPD agar equilibrated at 50 "C before pouring into small Petri dishes. An equal number of logarithmically growing cells were spotted onto these plates. Growth was monitored after incubation for 4S72 h at 30 "C.

Genomic Screening and Mapping-Yeast strain A2-200 (25) was transformed with a Yepl3-based genomic library to LEU' prototrophy. Sporidesmin resistant transformants able to grow on YPD medium containing 0.5-0.8 mg/ml of the mycotoxin were selected for further studies. Plasmids were recovered from resistant yeast colonies and propagated in Escherichia coli DH5a by routine methods (29). Yeast chromosomes (strain S288C) were purchased from Promega and re- solved in a 1% agarose gel with the CHEF DR-I1 program under conditions recommended by the manufacturer (Bio-Rad). Chromosomal fine mapping of STSl utilized a set of filters carrying 95% of the yeast genome as A-phage genomic clones (kindly provided by L. Riles).

Recombinant DNA Manipulations and Epitope Tagging-DNA sequence analysis was performed on double-stranded plasmids by the Sanger dideoxy chain termination method (30) using a sequenase kit (United States Biochemical Corp.). Plasmid templates were generated by exonuclease I11 treatment (31) of plasmid pYLS181 that contains a 7-kb HindIII fragment from pYSTSl cloned into the HindIII restriction site of Yeplacl81 (32). Remaining gaps were sequenced using plasmid pYSTSl and synthetic oligonucleotide primers.

A one-step gene disruption of STSl was carried out according to a standard method (28). In this deletion mutant, about 90% of the STSl coding region (fmmAf2II at n+399 to BsmI at n+4456) were replaced by a 800-bp StuVSnabI fragment containing the TRPl gene. Transforma- tion of yeast cells was carried out by the LiOAc procedure (28). All strains carrying a Astsl allele were analyzed by Southern blotting to confirm the chromosomal STSl deletion (29).

Total RNA and poly(A)+-enriched mRNA was isolated from yeast cells by the hot phenol extraction method as described elsewhere (28). RNA from a-factor pheromone-arrested cells was isolated after treating a logarithmically growing yeast culture for 45 min with synthetic a-factor (Peninsula Laboratories) at a final concentration of 5 JIM as described previously (10).

For epitope-tagging the STSl gene product, a gel-purified 7.5-kb SacVSphI fragment, obtained from plasmid pYSTS1, was religated (with the SphI site blunt-ended with T4 DNA polymerase) into the SacVSmaI sites of pRS315 (27) to yield the CEN-based plasmid pCKSTS1. A double-stranded 41-mer linker encoding the FLU-epitope (YPYDVPDYAAFL) (33) was inserted at the N terminus of the STSl coding region into a unique AvaI site of pCKSTSl to produce plasmid pCKSF1. In this construct, the first tyrosine of the FLU-epitope be- comes amino acid 4 in the FLU::STSl gene (MPEYPYDVPDYM- FLAE..). To obtain an epitope-tagged multicopy version of STSl, an 800-kb BamHI restriction fragment in pYLS181 was exchanged by a 1500-bp BamHI fragment (isolated from pCKSF1) harboring the STSl promotor including the epitope-tagged Stsl N terminus to yield plasmid pYFS18. Correct in-frame insertion of the FLU-epitope into the STSl coding sequence was confirmed by DNA sequence analysis. Drug resistance assays demonstrated that all FLU-Stsl constructs fully restored cycloheximide and sporidesmin susceptibility in a Astl deletion strain when compared with the wild-type STSl strain.

Preparation of Membranes and Cell Fractionation-Membrane fractions and total extracts were prepared by glass bead lysis from yeast cells grown to mid-exponential phase OD,,,, = 0.5-1) in SD or YPD

medium exactly as previously described (10). Isolation of subcellular membrane fractions was carried by an established procedure (34) using minor modifications as described elsewhere (10). Briefly, a 12,000 x g P1 membrane fraction enriched for plasma membrane vesicles, a 200,000 x g P2 light membrane fraction containing mainly ER and Golgi vesicles (34), and the corresponding high speed supernatant fractions S1 and S2 were prepared by differential centrifugation of a total cell-free extract that was isolated from gently lysed yeast spheroblasts (10). Protein concentrations were measured by the Lowry method (35) in the presence of SDS to solubilize membrane proteins and with bovine serum albumin as a standard. SDS-polyacrylamide gel electrophoresis of protein extracts and immunoblotting was carried out exactly as previously described (10).

RESULTS

Cloning a n d Sequence Analysis of the STSl Gene-We have devised a genetic screen allowing for the selection of yeast cells resistant to sporidesmin up to 500 pglml(1.1 mM). About 20,000 prototrophic LEU+ transformants, obtained by introducing a genomic multicopy library into strain "200 (25), were pooled and plated on YPD plates containing 0.5 mg/ml sporidesmin. The plasmids recovered from three independent transformants were analyzed by restriction mapping and partial sequence analysis (data not shown). All plasmids were identical and had a 9.5-kb DNA insert and, upon retransformation into sensitive yeast cells, were able to suppress the sporidesmin toxicity (data not shown). The plasmid recovered from one transformant, pYSTS1, was further analyzed by DNA sequence analysis. We detected an open reading frame of 4533 bp that can encode a protein of 1511 amino acids with a calculated molecular weight of 170.243 (Fig. 1, panel A). The 5'-end preceding the open reading frame contained typical promotor-like sequences such as two potential TATA boxes (36) at position -93 and -232 relative to the first base of the initiating ATG codon which was numbered +l. Moreover, the sequence around the putative ATG start codon with an A at position -3 and a C at position +4 is consistent with the consensus for translation initiation in yeast (37). At the 3'-end of the gene, the open reading frame is ter- minated by two consecutive stop codons. Consensus sequences for transcription termination in yeast, W A T A (381, and TAGN,TAGTN,"T (39) were detected at positions 4770 and 4774, respectively (Fig. 1, panel A).

Hydrophobicity analysis of the STSl coding region using the algorithms of Kyte and Dolittle (401, Eisenberg et al. (411, and Klein et al. (42) allowed the prediction of a total of 12 putative a-helical segments (TMS) of sufficient length to span a lipid bilayer (Fig. 1, panels B and C). In addition, two domains containing consensus sequences for binding and hydrolysis of ATP (43), previously recognized to be diagnostic for ABC transporters (21, were detected within the STSl coding region (Fig. 1, panels B and C). The predicted membrane topology suggests a (ABC-TMS& topology for Stsl and places both ABC domains on one side of a membrane (Fig. 1, panel B ) . Furthermore, we detected a total of 10 consensus sites (-Asn-X-Ser/Thr-) for the addition ofN-linked carbohydrate (441, two of which are located in predicted external hydrophilic loops of Stsl.

The STSl Gene Encodes a n ABC transporter-The deduced structural organization of the STSl gene product identifies Sts l as a eukaryotic member of the ABC protein superfamily (Fig. 1). Comparison of the Stsl primary sequence with avail- able databases revealed that Stsl shares extensive homology with both endogenous and heterologous ABC proteins (Fig. 2). The C-terminal and N-terminal ABC-domains of Stsl are most closely related to the recently cloned 1501-residue Snq2 transporter (15) (35% identical over the entire length) and to yeast Adpl (45) (42% identical in a 150 residue overlap). Moreover, S ts l is also closely related to Drosophila White (46) and Brown (47) (30 and 35% in a 400- and 150-residue overlap, respec-

4182 Multidrug Resistance Dansporters from Yeast

A

qurnce o f t h r STSI grnr . Putat ive TATA FIG. 1. pnnrl A. complr.tc. nucleotide se-

boxrs in thr 5"promotor r rbon and t ran- scription termination sikmals at t h r 3 ' - rnd a r r hoxrd. I'rrdictrd t ransmem- b r a n e s e p r n t s arr undrrlinrd. Nuclro- t idr +1 is t h r A o f t h r ATG translational s ta r t codon. Areas shndrcf in grnv demar- catr the rrgions o f int r rnal Stsl homology that arr consrrvrd within thr suprrfamlly of ARC t ransportrrs . (hnsrnsus sites for thr addition of N-linkrd oligosaccharides (hn-.Y-SrrlThrl arr indicatrd hy sold t n - angles. A total of 5250 hp wrre srquenced on both strands. I'nnrl R . schematic dr- piction of thr prrdictrd mrmhranr topology o f S t s l . Solid hlnck lrnm r rpresrnt thr polvprptidr chain. Putativr trans- m r m h r a n r s r p r n t s arr shown as L w t i - m l hlnck hnrs. Thr two hydrophilic ARC- domains arr markrd hy thr hlnck o c d s and ATP. Ihttrtl o l d hnlls indicate potrn- tial N-linkrd cnrhohydratr. Thr site o f in- srr t ion o f n 12-amino-acid influenza virus hrmagglutinin t1Al epitopr is marked at t h r N t r rminus o f S t s l (FLU). fnnr l C . structural organization o f t h r STSI gene. A partial rrstriction map of the STSI genr and thr construct usrd for the onr- s tep genr r rplacrmrnt of t h r STSI gene is shown schrmatically. Drtails arc de- scr ihrd undrr 'MM;ttrrials and Methods." The horizontnl whitc hnr r rpr rsen ts the oprn reading frame o f S T S I . Shndcd hmrs mark thr conserved ARC domains. and hlnck hnrs indicatr putative trans- m r m h r a n r s r p r n t s . Arrows indicate the dirrction of trnnscriptian o f the STSI and THI'I grnrs .

B

Y

C

tively). Significant sequence conservation (ranging from 18 to P-glycoproteins (3) . the human cystic fibrosis protein (491. and 25'T sequence identity) was also observed when stretches of the the yeast St& a-factor transpnrter (11 ) (Fig. 2 ). S t s l ARC domains were aligned to other ARC proteins, such as STSI Is Not Essentinl /i)r Viability-To detmmine if yrast the E. coli HlyR hemolysin transporter (48). the mammalian cells require a functional STSI gene for viahilitv. wr cnn-

Multidrug Resistance Dansporters from Yeast 4183

Flc. 2. Stsl is an ABC protein. Stretches from the C-terminal and N-terminal ATP-binding domains of Stsl (Stsl-C and Stsl-N) representing the Walker A, Walker B, and ABC signature domains are aligned to the corresponding regions of other ABC proteins including Snq2, Adpl, White, Brown, Ste6, Mdrl, HIyB. Amino acid positions are numbered according to the primary sequence of each protein with the methionine being 1. Amino acids identical to either the C-terminal or N-terminal ABC domain are shaded if conserved in at least four proteins. Computer analysis of nucleotide sequences and multiple protein alignments were done using the Geneworks 2.1 and MacVector 4.0 software packages as avail- able from Intelligenetics and IBI, respectively.

188-Sts l -N 900-Sts1-c 884-Snqt-C 418-Adpl 61-Brown 125-While 108Z-S1e6-C 1065-hMdrl-C 497-HylB

302-Slr l -N 1 0 0 3 - S l s l - c 983-SnqZ-C 521-Adpl 160-Brown 234-White 1183-S1e6-C 1169-hMdrl-C 599-HyIB

structed strains carrying chromosomal deletions of STSl. About 90% of the STSl coding region was replaced by the TRPl gene (Fig. 1, panel C ) . Appropriate diploid p H 5 0 1 (27) recipient cells were transformed with a linear HincIVEcoRI fragment carrying the Asts1::TRPl allele to generate a one-step gene replacement by homologous recombination. Sporulation of heterozygous diploid STSl 1 Asts1::TRPl transformants yielded four viable spores indicating that STSl is not required for viability (data not shown). However, Asts1::TRPl cells were hypersensitive to sporidesmin, while overexpression of STSl from a multicopy plasmid rendered cells resistant to sporidesmin toxicity up to concentrations greater than 500 pg/ml (Fig. 3). The minimal inhibitory concentration of sporidesmin to wild-type yeast cells is 125 pg/ml (0.275 mM), whereas growth inhibition in Astsl cells can be observed at sporidesmin concentrations as low as 50 pg/ml (0.110 mM), as determined by agar plate drug resistance assays (Table I). The YKKD-1 heterozygous diploid STSllAsts1::TRPl strain exhibited a semi- dominant phenotype, for it was hypersensitive to sporidesmin when compared with the haploid STSl progeny (Fig. 3, Table I).

We then tested a representative tetrad derived from YKKD-1 for a resistance phenotype to various other compounds including cycloheximide, chloramphenicol, 4-NQQ, MNNG, and ethidium bromide. The results from the cycloheximide test are shown in Fig. 4. Markedly increased cycloheximide resistance was observed in both MATa and MATa Astsl cells only in the presence of multiple copies of STSl (2pSTS1, Fig. 4). Again, the heterozygous STSl lAsts1::TRPl diploid exhibited a semi- dominant cycloheximide resistance phenotype, because i t was more sensitive to cycloheximide than the haploid STSl strain (Fig. 4). In contrast, Astsl cells were supersensitive to the drug (Fig. 4, Table I). The only other drug that gave a slight but significant resistance phenotype was chloramphenicol (data not shown).

Because Stsl is closely related to the multidrug resistance determinant Snq2, and to a lesser extent to the Ste6 a-factor transporter (Fig. 2), we wanted to test for overlapping function of Stsl , Ste6, and Snq2. Overexpression of S ts l did not result in resistance toward the drugs 4-NQQ and MNNG (data not shown) as observed for overexpression of Snq2 (15). Likewise, strains overexpressing Snq2 (kindly provided by Dr. Martin Brendel) did not exhibit elevated resistance to either cycloheximide or sporidesmin (data not shown). Moreover, both quan- titative mating tests and drug resistance assays of appropriate WKK7-A5 Astsl Aste6 double mutants that overexpress either Stsl or Ste6 showed that there is no functional overlap between Ste6 and Stsl (data not shown).

ABC-Motif and Walker B

O N D I V R ~ V S O O S ~ X R V E X A - S V E I C O S K ~ Q C , ~ D N A T R O L D ~ A ~ ~ ~ V A O ~ O L N V ~ ~ ~ K ~ L T I ~ ~ S L T A K P ~ K L L ~ ~ ~ L D ~ P ' T ~ ~ ~ L D ~ ~

~ N ~ ? D ~ O I ~ O O ~ ~ ~ ~ Y ~ I ~ ' ~ ~ L V T ~ P - L V L ~ L D ~ P + ~ ~ L D A S

. . . ' ,, i

V O 1 V O C O L N V I Q R X X L B I O V l L V A K P D L L L ? L D B P T 8 O L D B Q

1 P O R V K O L 8 0 0 X R X R L A P A 8 S A L T D P - P L L I C D S P T S O L D E ? A U T R I Q Q L S O O ~ ~ K R L ~ L A I S L I T D P - I ? L ? C D S P T T O L D ~ ~ ~

T R I D T T L L B O O Q A Q R L C I A R A L L R X S - K I L I L D S C T S A L D B V V O D ~ O T Q L S O O ~ X ~ P I A ~ A R A L V R ~ P ' - ~ I L L L D ~ A T ~ A L D T ~ V O 1 Q O A O L 8 0 0 Q R Q R I A I A R A L V N N P - X I L I ? D l A ~ S A L D ~ 1

0.30 lllg/llll 0.35 m</ml

0.4Il I11~iml 0.50 l l l ~ / l l l l

FIG. 3. Sporidesmin susceptibili'ty of isogenic Ants1 and STSl

YPD plates containing indicated amounts of sporidesmin (from 0.30 to cells. An equal number of logarithmically growing cells were spotted on

0.50 mg/ml) or on a control YF'D plate (Control) lacking sporidesmin. Diploid AstsllSTSl, YKKD-1; STSI , YPH499; Astsl, YKKB-13; ZpSTSl, YKKB-13 containing pYSTS1; vector, YKKB-13 carrying plasmid Yepl3. Cell growth was monitored after incubation for 48-72 h a t 30 "C.

ZYanscription of STSl Is under Hormonal Control- Northern blotting experiments using poly(A)+-rich RNA isolated from MATa, MATa, and a/a diploid cells indicated that transcription of STSl is not restricted to a certain cell type (Fig. 5). The observed STSl transcript size of 5.2 kb is consistent with the size of the STSl coding region as predicted from the nucleotide sequence (Fig. 1). When Northern blots were probed with an N-terminal HindIIYPstI restriction fragment from pYSTS1, which contained only sequences from the STSl open reading frame, we consistently observed cross-hybridization to an unknown transcript X with an apparent size of about 3.6 kb even at high stringency conditions (Fig. 5). This cross- hybridization indicates the presence of a t least one mRNA that is highly homologous to STSl mRNA. Transcript X , although its nature is unknown at present, serves as a useful internal standard for comparing the actual RNA amounts in individual lanes.

Expression of Ste6 is under the positive control of the a-factor pheromone (lo), and STE6 transcription is restricted to MATa 4 1 s (11, 50). Since both Stsl and Ste6 are members of the ABC protein superfamily, we tested if transcription of STSl is also influenced by pheromone. While the level of transcript X is unchanged in a-factor pheromone-arrested cells, the amount of STSl mRNA is dramatically reduced in MATa cells that were exposed to a-factor pheromone (+a-f, Fig. 5), suggesting that transcription of the STSl gene is regulated by a-factor.


TABLE I Minimal inhibitory concentrations (ME) of cycloheximide and sporidesmin

The MIC was defined as the drug concentration in YPD agar at which inhibition of cell growth was observed when compared to the control plate lacking the drug. Drug concentrations were increased by 10 ng/ml and 25 pg/ml increments for cycloheximide and sporidesmin, respectively.

Strain (relevant genotype) STSI ISTSI STSl IAstsI Asts1 Astsl

YPH5Ol YKKD-1 YKKA-7 n a ( B - 1 3 YPH499 YKKR-13 YKKEi-13 YKKEi-13 STS I Astsl Asts1 1 S t S l

+ (pRS315) + (pCKSFll + (pCKSTS1) + (pYSTS1)

l1.2i !Ig/ml 0.5 pgiml C y c l o h e x i m i d e

FIG. 4. Cycloheximide susceptibility of isogenic Astsl and STSl cells. An equal number of logarithmically growing isogenic cells of both mating types were spotted on YPD agar plates containing indicated amounts of cycloheximide (0.25 and 0.5 pg/ml), or on a control YPD plate. Diploid Astsl ISTSI, YKKD-1; STSl, YPH499 and YPH501; Astsl, YKKA-7 and YKKB-13; 2pSTS2, YKKA-7 and YKKB-13 containing pYSTS1; vector, YKKA-7 and YKKI3-13 harboring Yepl3. Growth was monitored after incubation for 48-72 h at 30 "C.

MATa

L L

5.3 kb

FIG. 5. Northern analysis of yeast RNA. Poly(A)'-rich RNA was isolated from all yeast cell types (MATa, MATa, and da) and fractionated in a 1% agarose gel containing 2.2 M formaldehyde. For preparation of RNA from pheromone-arrested cells, a logarithmically growing culture ofMATa cells was split in half. Half was treated with 5 p~ a-factor pheromone (+a-f) for 45 min; the other half was chilled on ice to stop growth of cells (-a-f). Following pheromone treatment, poly(A)+-rich RNA was isolated from both cultures and analyzed by Northern blotting. Hybridization used a radiolabeled 3.5-kb HindIIIIPstI restriction fragment isolated from pYSTS1.

Immunodetection of the S ts l Protein-The STSl open reading frame predicts a 1511-residue protein. To determine if yeast cells actually produce a corresponding polypeptide, we have generated a FLU epitope-tagged Stsl derivative. A 12-amino- acid antigenic determinant from the influenza virus hemagglu- tinin protein HA1 (33) was attached to the extreme N terminus of S t s l (Fig. 2, panel B ). When expressed from a CEN plasmid in an Astsl background, cells that express FLU-Stsl or authentic Stsl are indistinguishable from wild-type STSl cells with

respect to drug susceptibility (Table I), indicating that the FLU epitope did not debilitate function or proper subcellular target- ing of FLU-Stsl (Table I).

Total extracts were prepared from strain YKKA-7 expressing both FLU-Stsl and authentic Stsl from the CEN-based plasmids pCKSFl and pCKSTS1, respectively, and analyzed by immunoblotting using monoclonal antibody 12CA5 (Fig. 6, panel A ). A protein band with an apparent mobility of 175 kDa (CEN, STSI'), which is not present in extracts derived from cells expressing the untagged protein (Astsl), is specifically recognized by antibody 12CA5 (Fig. 6, panel A). If FLU-Stsl was expressed from a multicopy plasmid (Zp, STSI'), the same band is markedly overexpressed, confirming that the 175-kDa protein is indeed the product of the STSl gene (Fig. 6, panel A 1. The apparent mobility of FLU-Stsl is in good agreement with the size as predicted from the open reading frame. However, FLU-Stsl typically migrated as a broad, fuzzy band suggesting that it could be a glycoprotein. Indeed, the STSl coding region contains two potential glycosylation sites located in predicted external hydrophilic loops and, thus, would be in a favorable position for the attachment of N-linked sugar (Fig. 1, panel B ).

Sts l Is an Intrinsic Membrane Protein-To determine the subcellular localization of Stsl , we performed subcellular fractionation experiments. Total cell-free extracts obtained from cells that express chromosomal levels of FLU-Stsl from plasmid pCKSFl were subjected to differential centrifugation to generate the membrane fractions P1 and P2, and the corresponding supernatant fractions S1 and S2 (Fig. 6, panel B) . Equivalent amounts of each fraction were analyzed by immunoblotting using monoclonal 12CA5 anti-FLU antibodies, polyclonal anti-Sec61 antibodies (51), and polyclonal anti-Pmal antibodies (Fig. 6,panel B ) . The majority of Stsl was found in the 200,000 x g light membrane fraction (P2) that has been shown to contain predominantly ER and Golgi membranes (34), while the plasma membrane marker Pmal was found almost exclu- sively in the P1 membrane fraction (10,34) (Fig. 6,panel B ). No immunoreactivity was detected in the soluble high speed supernatant fraction (S2), suggesting that all of the cellular FLU- Stsl is intrinsically associated with the membrane fraction (Fig. 6, panel B ) . The Sec61 ER marker (51) fractionated mainly with the P2 light membrane fraction, although a significant amount was also found in P1 (Fig. 6, panel B).' The amount of FLU-Stsl in P1 appears to parallel the amount of Sec61, suggesting that Stsl in the P1 fraction is mainly due to cross-contamination with light membranes (Fig. 6, panel B ) . The results show that Stsl and the plasma membrane Pmal do not cofractionate, suggesting that Stsl may localize to the light density membrane fraction representing predominantly intracellular membranes.

DISCUSSION

We report here the molecular cloning of a yeast gene that encodes a new member of the ABC transporter superfamily. The gene was isolated by virtue of its ability to confer increased

C. Sterling, personal communication.

Multidrug Resistance Damporters from Yeast 4185

Stsl

Pmal

Secbl

4"*.

FIG. 6. panel A, immunodetection of Stsl gene product. Total cell extracts were prepared from yeast cells expressing FLU-Stsl from plasmid pCKSFl (CEN, STSI') or from plasmid pYKS18 (Zp,STSI+). A control extract was prepared from cells expressing authentic Stsl from plasmid pCKSTSl (As ts l ) . The extracts were fractionated by SDS-poly-

with monoclonal antibody 12CA5 that was purchased from the Berkeley acrylamide gel electrophoresis, transferred to nitrocellulose, and probed

Antibody Company. Proteins on immunoblots were detected using the enhanced chemiluminescence detection system (ECL). Panel B, Stsl and Pmal do not cofractionate. A total cell-free extract (TOT) was prepared from cells expressing FLU-Stsl from plasmid pCKSFl and subjected to differential centrifugation in a Sorvall RC-5B (rotor HB-4) to generate a 12,000 x g membrane pellet (PI ). The resulting supernatant (SI) was recentrifuged for 40 min at 200,000 x g in a Beckman Table Top TLX ultracentrifuge (rotor TLA100.3) to yield a light membrane fraction (P2) and a high speed supernatant fraction (S2). Equiv- alent aliquots of each fraction (about 100 pg of protein) were fractionated through SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting using monoclonal antibody 12CA5, polyclonal anti- Pmal antiserum, and polyclonal anti-Sec6l antibodies.

sporidesmin resistance to yeast cells when present in multiple copies. Strikingly, Stsl is highly homologous to White (46) and Brown (471, two previously identified half-size ABC proteins from Drosophila that are putatively involved in pteridine uptake during eye pigmentation of the fly. Yeast Stsl exhibits a similar domain organization as White and Brown, except that Stsl is a typical full-size ABC transporter with a predicted (ABC2-TMSs)2 topology (Fig. 1, panel B) . A putative counter- part of Stsl in mammalian cells has not yet been discovered.

Furthermore, Stsl is closely related to the multidrug resistance protein Snq2, another yeast ABC transporter that was recently shown to be responsible for a high copy-mediated MDR phenotype (15) and to the Ste6 a-factor transporter (Fig. 2) . However, there seems to be no functional overlap between Stsl and Snq2 because overexpression of Stsl did not result in resistance toward the Snq2 substrates 4-NQQ, and MNNG (data not shown). Likewise, yeast strains overexpressing Snq2 did not exhibit elevated resistance to either cycloheximide or spo-

ridesmin, and the fact that SNQ2 was not isolated in our sporidesmin resistance screen indicates a quite different substrate specificity for Stsl and Snq2. However, this does not exclude the possibility that Stsl and Snq2 have a common function in vivo, since the physiological substrates of both proteins are unknown. Chromosomal deletions of Stsl and Snq2 (15) are not lethal, indicating redundancy of Stsl and/or Snq2 function in yeast. Indeed, both Northern (Fig. 5) and Southern analysis indicated the presence of genes that are closely related to STSl but different from the SNQ2 gene (data not shown). I t will be of interest to see whether a Asnq2 Astsl double mutant exhibits a detrimental growth phenotype.

Additional yeast pleiotropic MDR determinants that were recently characterized include the PDRS gene that was isolated in a multicopy-mediated cycloheximide resistance screen (14). The PDR5 gene has been mapped to yeast chromosome XV (14). STSl also resides on chromosome XV as determined by chromosome blotting (data not shown). Moreover, chromosomal fine mapping using a set of filters containing the yeast genome in A-phages indicated that STSl maps about 120 kilobase pairs proximal to HIS3 and 100 kilobase pairs distal of the RASl gene (data not shown). Comparison of the MDR phenotype of 2pSTSl and Astsl cells (Fig. 4) with the MDR phenotype of pdr l -3 cells that overexpress Pdr5 (52), and with the proper- ties of pdr5::Tn5 cells (52) indicated that there was an almost identical resistance pattern, suggesting that STSl and PDRS could be allelic. Indeed, upon comparison of the nucleotide sequences of STSl and PDRS (kindly provided by A. Goffeau), allelism was confirmed (24).

Transcription of the PDR5ISTSl gene was previously demonstrated to be regulated by Pdrl (52). In addition, we have here shown that the amount of STSl transcript is dramatically reduced in a-factor growth-arrested MATa cells (Fig. 51, implying that Stsl function may be dispensable in non-growing cells. Thus, transcriptional events that control gene expression such as the hormonal induction of Ste6 expression (10) within the mating signaling pathway (531, may also control STS1, a gene that is outside the mating pathway. In turn, it was also specu- lated that Ste6 may be under the negative transcriptional control of PDRl (521, suggesting that distinct transcriptional control elements may somehow connect the mating pathway and MDR phenomena. However, at the moment we cannot entirely exclude the possibility that the STSl mRNA is simply unstable in pheromone-arrested cells. Further experiments will be required to resolve this issue.

The observed MDR phenotype of cells overexpressing Stsl raises the possibility that Stsl may be a component of an endogenous defense or detoxification machinery of growing yeast cells. Such a function of Stsl would suggest the plasma membrane to be the normal cellular location of Stsl. Indeed, it has been shown that Pdr5 is present in very high levels in the plasma membrane of pdrl mutants (52). By contrast, we here present evidence that chromosomal levels of Stsl are mainly found in a light density membrane fraction containing mostly intracellular membranes, although a significant portion of Stsl did cofractionate with Pmal, the plasma membrane ATPase (Fig. 5, panel B). A localization of Stsl in intracellular membranes would also be consistent with its proposed function in a cellular detoxification system. For instance, Stsl could be mov- ing back and forth within the secretory pathway from the ER to the plasma membrane. Notably, human Mdrl, when expressed in yeast, leads to a drug resistance phenotype and localizes to both ER membranes and to the cell surface (54). Thus, detoxification may involve Stsl-mediated uptake of various toxic metabolites into vesicular secretory compartments before being transported to the cell surface. A possible explanation for the apparent plasma membrane localization of Pdr5 (or Stsl) in a


pdrl-3 mutant (24) may simply be mislocalization of Pdr5 to the plasma membrane, sincepdrl-3 mutants overexpress Pdr5 almost two orders of magnitude (52). Such a massive overpro- duction of a membrane protein may elicit toxic effects which can only be prevented by shunting the excess protein to the plasma membrane. Conversely, a mislocalization of FLU-Stsl to membranes other than the plasma membrane caused by the epitope tag in FLU-Stsl (Fig. 1, panel B) seems highly unlikely because the same epitope has been used to determine the correct subcellular localization of another yeast membrane protein ( 5 3 , and because FLU-Stsl is functionally indistinguishable from authentic Stsl, implying that FLU-Stsl is properly localized.

An alternative yet highly speculative function for S ts l may be a role in glycosylation or cell wall biosynthesis. Intriguingly, S ts l is also homologous to the bacterial ABC transporters ChvA (56) and NdvA (571, both of which are required for the export of P-1,2-glucan in Agrobacterium tumefaciens and Rhizobium me- Ziloti, respectively. Thus, Stsl could serve a similar or related function in yeast. For instance, Stsl may transport sugar precursors such as UDP-GlcNac, GDP-Man, or UDP-Glc required for protein glycosylation in the ER lumen (44) or Stsl may mediate uptake of cell wall precursors such as (1-3)-P-glucan (58) into membrane compartments of the secretory pathway.

The physiological function of Stsl and its determinants of substrate specificity are unknown at present. The monoclonal antibody recognizing FLU-Stsl, and polyclonal anti-Stsl antibodies, which we are currently generating, represent invalu- able tools for further studies on Stsl using an in vitro vesicle transport system (59) to uncover a possible in vivo function of the yeast STSl gene product.

and Paul Attfield for critical and helpful comments on the manuscript. Acknowledgments-We thank Wolfgang Schneider, Robert G. Elkin,

The skillful technical assistance of Andrea Lamprecht is highly appre- ciated. We are greatly indebted to Andre Goffeau and Elisabetta Balzi for sharing data prior to publication. Thanks to Linda Riles for providing the yeast clone grid library and to Ramon Serrano and Colin Ster- ling for providing polyclonal anti-Pmal and anti-Sec61 antisera, respee- tively. Thanks to Jorg Servos and Martin Brendel for providing SNQ2 overexpressing strains and to Neil Towers for the gift of sporidesmin.

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