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A Ste6p/P-glycoprotein homologue from the asexual yeast Candida albicans transports the a-factor mating pheromone in Saccharomyces cerevisiae

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A Ste6p/P-glycoprotein homologue from the asexual yeast Candida albicans transports the a-factor mating pheromone in Saccharomyces cerevisiae
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  A Ste6p/P-glycoprotein homologue from the asexualyeast  Candida albicans   transports the a-factor matingpheromone in  Saccharomyces cerevisiae  Martine Raymond, 1 *  Daniel Dignard, 2 Anne-MarieAlarco, 1 Norman Mainville, 2 Beatrice B. Magee 3 andDavid Y. Thomas 2,4,5 1 Institut de recherches cliniques de Montre ´ al, 110 Pine Avenue West, Montre ´ al, Que ´ bec, Canada H2W 1R7. 2  National Research Council of Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montre ´ al,Que ´ bec, Canada H4P 2R2. 3  Department of Genetics and Cell Biology, University of Minnesota, 1445 Gortner Avenue, St Paul, MN 55108 1095, USA. 4  Department of Biology, McGill University, Montre ´ al,Que ´ bec, Canada H3A 1B1. 5  Department of Anatomy and Cell Biology, McGill University, Montre ´ al, Que ´ bec, Canada H3A 2B2. SummaryIn  Saccharomyces cerevisiae MAT  a cells, export of thea-factor mating pheromone is mediated by Ste6p, amember of the ATP-binding cassette (ABC) super-familyof transportersand aclosehomologue of mam-malian multidrug transporter P-glycoproteins (Pgps).We have used functional complementation of a  ste6   mutationtoisolateageneencodinganABCtransportercapable of a-factor export from the pathogenic yeast, Candida albicans  . This gene codes for a 1323-aminoacid protein with an intramolecular duplicated struc-ture, each repeated half containing six potential hydro-phobic transmembrane segments and a hydrophilicdomain with consensus sequences for an ATP-bind-ing fold. The predicted protein displays significantsequence similarity to  S. cerevisiae   Ste6p and mam-malian Pgps. The gene has been named  HST6  , forhomologue of  STE6  . A high degree of structural con-servation between the  STE6   and the  HST6   loci withrespect to DNA sequence, physical linkage and tran-scriptional arrangement indicates that  HST6   is the  C.albicans   orthologue of the  S. cerevisiae STE6   gene.We show that the  HST6   gene is transcribed in a hap-loid-specific manner in  S. cerevisiae  , consistent withthepresenceinitspromoterofaconsensussequencefor Mata1p-Mat  2p binding known to mediate therepression of haploid-specific genes in  S. cerevisiae  diploid cells. In  C. albicans  ,  HST6   is expressed con-stitutively at high levels in the different cell typesanalysed (yeast, hyphae, white and opaque), demon-strating that  HST6   transcription is not repressed inthis diploid yeast, unlike in diploid  S. cerevisiae  , andsuggesting a basic biological function for the Hst6ptransporter in  C. albicans  . The strong similarity bet-ween Hst6p and the multidrug transporter Pgps alsoraises the possibility that Hst6p could be involved inresistance to antifungal drugs in  C. albicans  .Introduction P-glycoproteins (Pgps) are membrane-associated proteinswhose overexpression in mammalian cells causes resis-tancetoseveraldifferent chemotherapeutic drugs, apheno-type known as multidrug resistance (MDR) (Germann,1996). Sequence analysis and topology mapping experi-ments have shown that Pgps are formed by two sequence-related symmetrical structural units, each containing sixtransmembrane (TM) domains and an intracellular loopwith consensus ATP-binding motifs (Germann, 1996). Pgpshave been shown to bind ATP and drug analogues andto have ATPase activity (Germann, 1996). Experimentalevidence indicates that Pgps function as ATP-dependentefflux transporters of cytotoxic drugs, resulting in reducedintracellular drug accumulation in resistant cells (Germann,1996).Pgpsbelongtoalargesuperfamilyofevolutionarilycon-served transport proteins, named the ABC (ATP-bindingcassette) superfamily of proteins, involved in the activetransport of a variety of structurally heterogeneous sub-strates across cellular membranes, such as proteins, smallpeptides, amino acids, cyclic sugars, ions and antibiotics(Higgins, 1992). In the yeast  Saccharomyces cerevisiae  ,a close homologue of the Pgps is the  STE6   gene product,Ste6p, which mediates the extracellular transport of a-fac-tor, a peptide pheromone required for mating (Kuchler  et al  ., 1989; McGrath and Varshavsky, 1989).  S. cerevisiae MAT  acellscarryinga ste6   chromosomaldeletionproduceno extracellular a-factor, accumulate intracellular pro- andmature a-factor and are therefore defective in mating Molecular Microbiology (1998)  27 (3), 587–598   1998 Blackwell Science Ltd Received 9 January, 1996; revised 13 October, 1997; accepted 3November, 1997.  * For correspondence. E-mail: raymonm@ircm.umontreal.ca; Tel. (514) 987 5770; Fax (514) 987 5732.   (sterile) (Kuchler  et al  ., 1989; McGrath and Varshavsky,1989; Berkower and Michaelis, 1991).Ste6p and Pgps display high sequence homology andhave similar length, predicted secondary structure and pro-posed membrane topology (Kuchler  et al  ., 1989; McGrathand Varshavsky, 1989). We have shown that the functionalexpressionina ste6   strainofPgpencodedby themurine mdr3   gene can substitute for Ste6p by partially restoringthe ability of the cells to export a-factor, therefore allowingcells to mate (Raymond  et al  ., 1992). Two other ABCtransporters homologous to Ste6p, the  pfmdr1  gene pro-duct of  Plasmodium falciparum   (Pgh1) associated withantimalarialdrugresistanceandthemammalianmultidrugresistance-associated protein Mrp have recently beenshown to complement a  ste6    mutation (Volkman  et al  .,1995; Ruetz  et al  ., 1996). Thus, the structural homologyobservedbetweenyeastSte6pandtheseABCtransporterstranslates into a functional homology conserved across alarge evolutionary distance. These results implied thatmating constitutes a phenotype that can be used in  S.cerevisiae   for the functional identification from evolution-arily distant species of Ste6p/Pgp homologues sharingthe ability to transport a-factor. Candida albicans  , the aetiological agent of candidiasis,is the major fungal pathogen in humans (Odds, 1987). Itis a naturally occurring diploid yeast with no known sexualstage and has, therefore, been classified as a Deutero-mycetes. It is also a dimorphic organism, capable ofgrowing either as yeast or as hyphae depending on theenvironmental conditions (Soll, 1997).  C. albicans   usuallyexists as a commensal of healthy individuals however, inindividuals with impaired immunity or in cancer patients,infections with  C. albicans   can be a serious medical prob-lem (Odds, 1987). Azole derivatives, such as fluconazole(FCZ), are often used for clinical treatment, but resistanceto this class of drugs is becoming increasingly common inpatients undergoing long-term or prophylactic treatment,mostlyAIDSpatients(Rex etal  .,1995).Studiesinvestigat-ingthe mechanismsof FCZ resistancein C. albicans   haveshown previously that resistant strains fail to accumulateFCZ because of an increased drug efflux, suggesting theparticipation of transporter-mediated drug resistance mech-anismsinthesestrains(Sanglard etal  ., 1995). C. albicans  genes encoding two transporters of the ABC superfamily, CDR1  and  CDR2  , and one transporter of the major facili-tator superfamily,  BEN  r  , have been shown to be over-expressed in  C. albicans   FCZ-resistant isolates from AIDSpatients with oropharyngeal candidiasis, suggesting thatthese genesare involvedin clinicalFCZ resistance(Sang-lard et al  ., 1995; 1997). Understandingthe molecular mech-anisms of transmembrane transport in general and ofantifungal drug resistance in particular is important forthe improved management of  C. albicans   infections. Inthe present report, we describe the isolation of a new  C.albicans   ABC transporter highly homologous to  S. cere- visiae   Ste6p and mammalian Pgps, using functional com-plementation of a  ste6    mutation in  S. cerevisiae  . Results Isolation of   HST6  by complementation of   ste6   in   S.cerevisiaeWehavelookedfor  C. albicans   genomic sequences encod-ing potential transporter molecules capable of complement-ing an  S. cerevisiae ste6   null mutation by restoring theability of the cells to export a-factor, as detected by patch-mating assay (Sprague, 1991). To this end, a high-copy URA3  -based genomic  C. albicans   library was introducedinto JPY201, a  MAT  a strain carrying a  ste6   deletion(McGrath and Varshavsky, 1989). These cells produceno extracellular a-factor and are, therefore, defective inmating (McGrath and Varshavsky, 1989). JPY201 trans-formants were selected on SD-ura plates and screenedfor their capacity to conjugate with the tester strainDC17.Ofabout10 4 coloniestested,onemetthatcriterion.The plasmid was recovered from diploids, retransformedinto JPY201 cells, along with plasmids YEp352-STE6 andYEp352 as positive and negative controls, respectively,and transformants were tested for mating (Fig. 1A). Thisexperiment confirmed that the mating defect of JPY201cells (YEp352) was corrected by the expression of theisolated  C. albicans   genomic clone (YEp352-HST6) in aplasmid-dependent manner and with an efficiency similarto that of JPY201 cells expressing  S. cerevisiae   Ste6p(YEp352-STE6)(Fig. 1A).Theabilityofthetransformantsto produce biologically active extracellular a-factor was alsodetermined by halo assay, a test for a-factor export inde-pendent of the mating assay (Fig. 1B). JPY201 cells trans-formed with the isolated plasmid (YEp352-HST6) produceda zone of growth inhibition of significant size, confirming theability of YEp352-HST6 to complement the  ste6    defect byrestoring the capacity to export substantial amounts ofa-factor pheromone (Fig. 1B). The size of the halo pro-duced by JPY201 cells transformed with YEp352-HST6was smaller than that produced by JPY201 cells trans-formed with YEp352-STE6, indicating a difference ina-factor export between these two transformants thatwas not detectable by patch-mating assay. Restrictionendonucleasemappingindicatedthat the isolatedplasmidcontains a genomic DNA insert of approximately 6.2kb.DNA sequencing revealed the presence of an openreadingframe(ORF)of1323aminoacids(Fig. 1C).Com-puter-assisted sequence homology searches of NCBIprotein databases revealed that this ORF codes for anew member of the ABC superfamily of transporters thatis most closely related to  S. cerevisiae   Ste6p (see below).The gene was thus named  HST6  , for homologueof  STE6  .   1998 Blackwell Science Ltd,  Molecular Microbiology  ,  27 , 587–598 588  M. Raymond   et al.  Sequence analysis of   HST6The nucleotide sequence of a 5.0kb  Bgl  II–  Eco  RI fragmentoverlapping the entire  HST6   gene has been determinedand deposited in the GenBank database (accession num-ber U13193). The ATG initiator methionine codon is fol-lowed by a 3969 nucleotide ORF encoding a protein of1323 amino acids with a calculated molecular mass of148620Da. Analysis of the predicted amino acid sequenceof Hst6p indicates that it is formed by two similar halves,each comprising an N-terminal hydrophobic domain withsix predicted transmembrane segments followed by a C-terminal hydrophilic domain with consensus sequencesfor ATP-binding including Walker A, Walker B and ABCsignature motifs (Higgins, 1992; Michaelis and Berkower,1995). These predicted structural features clearly identifyHst6pasamemberoftheABCsuperfamilyoftransportersingeneralandasaclosehomologueofeukaryoticPgpsinparticular. BLAST  and  FASTA  homologysearchesof proteinsequencedatabases with the full-length Hst6p sequence show thatthe highest homology is with  S. cerevisiae   Ste6p (35%sequence identity and 48% similarity; Kuchler  et al  ., 1989;McGrath and Varshavsky, 1989), with the  Schizosaccharo- myces pombe mam1  gene product, which mediates secre-tion of the M-factor pheromone (29% sequence identityand 41% similarity; Christensen  et al  ., 1997) and with themammalian Pgps (approximately 26% sequence identityand 36% similarity, depending on the isoform analysed;Germann, 1996). The regions of highest amino acid iden-tity among these proteins overlap the ATP-binding domainsbut also extend within the TM domains. Short stretches ofcharged amino acids, not present in Ste6p or Pgps, arefound within the ATP binding site of the C-terminal halfof Hst6p, notably a very acidic DNE-rich tract (amino acidpositions 1195–1222). Negatively charged insertions arealso present in the C-terminal ATP-binding domain of the P. falciparum pfmdr1  gene product (Foote  et al  ., 1989).The effect of such charged domains on the activity ofthese transporters with respect to ATP binding and/orhydrolysis is not known.In  S. cerevisiae, STE6   is convergently transcribed atits 3  end with  UBA1 , anessentialgeneencodinga ubiqui-tin-activating enzyme (McGrath and Varshavsky, 1989;McGrath  et al  ., 1991). The two transcription units, sepa-rated by a region of only 188bp between their terminationcodons, map to the left arm of  S. cerevisiae   chromosomeXI (McGrath and Varshavsky, 1989 McGrath  et al  ., 1991).Computer-assisted DNA sequence analysis of  HST6  revealed the presence of an incomplete ORF in the 3  untranslated region of the gene. This ORF, named CAUBA1 , displays 74% amino acid sequence identity(with an additional 12% similarity) with the  S. cerevisiae UBA1  gene. The two genes are also convergently tran-scribed, with their respective termination codons separatedbyaregionof124bp.Thus,the C.albicansHST6/CAUBA1 locuslieswithinaconservedsyntenysegmentbetween C.albicans   chromosome 3 (see Fig. 4) and  S. cerevisiae  chromosome XI. The high degree of conservation betweenthe  STE6/UBA1  and the  HST6/CAUBA1  loci strongly sug-gests that  C. albicans HST6   is indeed the  S. cerevisiae STE6   equivalent and that both genes arise from thesame ancestor. Quantitative determination of mating efficiency  We have used a quantitative mating experiment to com-pare the efficiency of Ste6p and Hst6p with respect toa-factor export (Table 1). JPY201 cells were transformed   1998 Blackwell Science Ltd,  Molecular Microbiology  ,  27 , 587–598 Fig. 1.  Functional complementation of  S. cerevisiae ste6    by the C. albicans HST6   gene. JPY201 transformed with plasmidYEp352-STE6, YEp352-HST6 or YEp352 were tested for theproduction of extracellular a-factor by patch-mating and halobioassays.A. Patch-mating assay. Transformants were grown on an SD-uraplate and replicated onto YPD on a lawn of DC17 cells. Mating wasallowed for 12h at 30  C. Cells were replicated onto a MM plate andgrown for a further 2 days at 30  C to assess diploid formation.B. Halo assay. Transformants were grown to stationary phase inSD-ura medium. Cells (10 6 ) were spotted directly onto a lawn ofstrain M323-2A. The plate was photographed after incubation at30  C for 24h.C. A partial restriction map of the genomic DNA insert of plasmidYEp352-HST6 is shown. The endonuclease restriction sites are asfollows: B,  Bam  HI; Bg,  Bgl  II; E,  Eco  RI; H,  Hin  dIII; K,  Kpn  I; N, Nhe  I; Sa,  Sac  I; Sp,  Spe  I. Parentheses indicate that the  Bam  HIcloning site was destroyed by the cloning process. The scale (1kb)is shown below. DNA sequence analysis of the  Bgl  II–  Eco  RI 5.0kbfragment overlined by the arrow indicates that the genomic DNAinsert contains an ORF of 1323 amino acids, with the direction oftranscription shown from left to right. A Ste6p/Pgp homologue in   Candida albicans 589  witheither the S.cerevisiaeSTE6   or the C.albicansHST6  genes carried on a high-copy-number (YEp352) or a low-copy-number (pRS314) plasmid. Cultures of transformantswere mated with the tester strain DC17, and diploid for-mation was assessed on selective medium. When over-expressed from a high-copy-number vector,  HST6   wasfound to support mating at significant levels, with an effi-ciency approximately 6% that of  STE6   (Table 1). Whenexpressed from a low-copy-number vector,  HST6   wasalso able to suppress the mating defect of the  ste6    cells,althoughwithlowerefficiency,0.1%thatof STE6   expressedfrom the same vector (Table 1). The 60-fold difference in HST6   activity observed between the high- and low-copy-number vectors correlates well with the copy number oftheseplasmidsinyeastcells(Rose etal  ., 1990).Themea-sureddifferenceinmatingefficienciesbetween HST6  -and STE6  -expressing cells could reflect the differential abilityof the two transporters to export a-factor, a possible con-sequence of the evolutionary distance between the  C.albicans   and the  S. cerevisiae   genes. This differencecould also result from a low level of  HST6   expression in S. cerevisiae   cells used as a heterologous expressionsystem. Cell type-specific expression of   HST6  in   S. cerevisiae S. cerevisiae   has two haploid mating types,  MAT  a and MAT   , which mate to form a  MAT  a/    diploid cell. Thespecific expression of particular sets of genes ( MAT  a-specific,  MAT   -specific and haploid-specific genes) inthese different cell types is determined by a combinationof positiveand negativetranscriptionalregulatory proteins(Herskowitz  et al  ., 1992).  STE6   is a  MAT  a-specific gene;its expression is repressed in  MAT    and  MAT  a/    cells bythe  MAT   2 product of the mating type locus (Wilson andHerskowitz, 1984; 1986). On the other hand, the expres-sion of haploid-specific genes in diploid  MAT  a/    cells isrepressed by a transcriptional complex formed by the MAT  a1 and  MAT   2 proteins (Herskowitz  et al  ., 1992).This repression is mediated by a 20bp DNA sequence,which consists of two conserved symmetrical half-sitesseparated by eight less conserved AT-rich basepairsand has been found in the promoter region of severalhaploid-specific genes in  S. cerevisiae   (consensus: 5  -TCRTGTNNWNANNTACATCA) (Goutte and Johnson,1988). Examination of the promoter region of  HST6  revealed the presence of a potential  MAT  a1- MAT   2binding sequence (5  -TCATGTTAAAAAGTACATCT; posi-tions ¹ 211to ¹ 191withrespecttothetranslationinitiationcodon). Interestingly, a similar sequence has also beenfound in the promoter region of another  C. albicans   gene, CAG1 , encoding a homologue of a G-protein   -subunitthat functions in the  S. cerevisiae   mating signal transduc-tion pathway (Sadhu  et al  ., 1992).In order to examine the level and cell type specificity of HST6   expression in  S. cerevisiae   cells, isogenic haploid MAT  a, haploid  MAT    and diploid  MAT  a/    strains weretransformed with plasmids pRS314, pRS314-STE6 orpRS314-HST6, and the level of  HST6   and  STE6   expres-sion was determined in individual transformants (Fig. 2).As expected,  STE6   expression was  MAT  a specific, withhigh levels of expression from the chromosomal copy ofthe  STE6   gene detectable in  MAT  a cells transformedwith any of the three plasmids (Fig. 2, STE6 panel, lanes1–3) but undetectable in any of the  MAT    and  MAT  a/   transformants (Fig. 2, STE6 panel, lanes 4–9). The plas-mid-borne copy of the  STE6   gene was also regulated in a MAT  a-specific manner, giving rise to detectable  STE6  expression in  MAT  a cells, as judged from the strongersignal intensity in pRS314-STE6 compared with pRS314transformants (Fig. 2, STE6 panel, compare lanes 2 and1) and undetectable  STE6   expression in  MAT  a and MAT  a/    cells (Fig. 2, STE6 panel, lanes 5 and 8). Inte-restingly, we found that  HST6   is expressed at high levelsin  MAT  a and  MAT    cells (Fig. 2, HST6 panel, lanes 3and 6 respectively) but is not expressed in diploid  MAT  a/   cells (Fig. 2, HST6 panel, lane 9), suggesting that the MAT  a1–  MAT   2 consensus sequence present in the HST6   promoter is probably functional in  S. cerevisiae  .Taken together, these results indicate that (i)  HST6   expres-sionishaploidspecificin S.cerevisiae  ,unlike STE6   expres-sion, which is  MAT  a specific; and (ii)  HST6   and  STE6   areexpressed at roughly the same level in  MAT  a cells, sug-gesting that a difference in transcriptional levels cannotaccount for the 1000-fold difference in mating activityobserved between the two genes (Table 1). Immunodetection of Hst6p  We haveusedanepitope-taggingapproachtoanalysethesize and abundance of Hst6p expressed in  S. cerevisiae  cells. To this end, an antigenic determinant from the c-mycproto-oncogene (EQKLISEEDL) (Evan  et al  ., 1985), for   1998 Blackwell Science Ltd,  Molecular Microbiology  ,  27 , 587–598 Table 1.  Quantitative determination of mating efficiency.Mating efficiency a VectorInsert YEp352 pRS314STE6 1.2 (100) 1.0 (100)HST6 6.7 × 10 ¹ 2 (5.6) 1.0 × 10 ¹ 3 (0.1)None 1.2 × 10 ¹ 6 (0.0001) 2.0 × 10 ¹ 6 (0.0002) a.  Mating efficiency of JPY201 cells carrying the different plasmidslisted was determinedas describedin  Experimentalprocedures  . Mat-ing efficiencies are expressed as absolute values or as percentagesrelative to the values of  STE6   transformants set at 100% (paren-theses). The reported values are the mean of two independentexperiments performed in duplicate. 590  M. Raymond   et al.  which a monoclonal antibody is commercially available(mAb 9E10), was introduced in frame in the coding regionof  HST6   between amino acid positions 1314 and 1315,using oligonucleotide-mediated site-directed mutagenesis.A plasmid carrying this tagged version of Hst6p (YEp352-HST6[myc]) was introduced into JPY201 cells, along withplasmidsYEp352andYEp352-HST6asnegativecontrols.For the purposes of comparison, a plasmid carrying amyc-tagged STE6 (YEp352-STE6[myc]) was also usedto transform JPY201 cells. This tagged version of Ste6pcontains the c-myc epitope inserted between amino acids1280 and 1281 and is readily detected by mAb 9E10 inimmunoprecipitation experiments (M. Raymond and D. Y.Thomas, unpublished). The epitope-tagged Ste6p andHst6p derivatives were found to function normally  in vivo  as determined by complementation of the  ste6    mutationby mating assay (data not shown).JPY201 cells transformed with plasmids YEp352,YEp352-HST6, YEp352-HST6[myc] and YEp352-STE6[myc] were metabolically labelled with [ 35 S]-Cys and[ 35 S]-Met. Cellular extracts were prepared and subjectedtoimmunoprecipitation with mAb 9E10. In cells transformedwithplasmidYEp352-HST6[myc]andYEp352-STE6[myc](Fig. 3, lanes 3 and 4 respectively), the antibody recog-nizes two high-molecular-weight proteins absent from cellstransformed with plasmids YEp352 or YEp352-HST6 (Fig.3, lanes 1 and 2 respectively), confirming that these pro-teins are indeed Hst6p and Ste6p. The abundance ofHst6p in JPY201 cells is about two to three times lowerthan the level of Ste6p in the same cells (compare lanes3 and 4), explaining in part the difference in activityobservedbetweenthetwoproteinswithrespecttoa-factortransport (Fig. 1B and Table 1). Hst6p was found tomigrate faster than Ste6p, an unexpected result given thatthe calculated molecular mass of Hst6p predicted from itsgene sequence (149kDa) is greater than that of Ste6p(145kDa) (Kuchler  et al  ., 1989). The reason for this differ-ence is not known. It has been reported that treatment of MAT  a cells with   -factor leads to increased levels ofSte6p, through transcriptional induction of the  STE6   gene(Kuchler  et al  ., 1993). In order to test if Hst6p expressionwas also responsive to pheromone treatment in  S. cere- visiae  ,JPY201cellstransformedwithYEp352-HST6[myc]and YEp352-STE6[myc] were radiolabelled in parallel inthe presence of   -factor at 5  M. As expected, treatmentof JPY201 cells carrying plasmid YEp352-STE6[myc] with  -factor increased the expression of Ste6p (Fig. 3, com-pare lanes 4 and 6). However, we found that the level ofHst6p was not significantly and reproducibly increased   1998 Blackwell Science Ltd,  Molecular Microbiology  ,  27 , 587–598 Fig. 2.  Cell type-specific transcription of  HST6   in  S. cerevisiae  .Total RNA was isolated from  S. cerevisiae   haploid  MAT  a(W303-1A), haploid  MAT    (W303-1B) and diploid  MAT  a/    (W303)cells carrying either plasmid pRS314 (lanes 1, 4 and 7), pRS314-STE6 (lanes 2, 5 and 8) or pRS314-HST6 (lanes 3, 6 and 9). RNAsamples (20  g) were separated in duplicate by electrophoresison a 1% agarose gel and transferred to nylon membranes. Themembranes were probed with an  HST6   (top) or an  STE6   (bottom)radiolabelled fragment, washed and exposed for 6h with twointensifying screens. The membranes were then reprobed with aradiolabelled fragment from the  LEU2   gene to monitor RNAloading and transfer, washed and exposed for 20h with twointensifying screens. The probes are indicated on the left of thefigure. Fig. 3.  Detection of epitope-tagged Hst6 protein byimmunoprecipitation. JPY201 cells carrying either plasmid YEp352(lane 1), YEp352-HST6 (lane 2), YEp352-HST6[myc] (lanes 3 and5) or YEp352-STE6[myc] (lanes 4 and 6) were metabolicallylabelled with [ 35 S]-Met and [ 35 S]-Cys in the absence (lanes 1–4) orthe presence (lanes 5 and 6) of 5  M   -factor. Cellular extractswere prepared and subjected to immunoprecipitation with anti-mycmonoclonal antibody 9E10. Immune complexes were recovered,separated by SDS–PAGE on a 7.5% gel and analysed byfluorography. Molecular mass standards shown on the right aremyosin (200kDa) and phosphorylase  b   (97kDa). The Hst6 proteinis indicated by the arrow. A Ste6p/Pgp homologue in   Candida albicans 591
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