Tài liệu Báo cáo khoa học: A genetic screen identifies mutations in the yeastWAR1 gene, linking transcription factor phosphorylation to weak-acid stress adaptation docx

A genetic screen identifies mutations in the yeast WAR1 gene, linking transcription factor phosphorylation to weak-acid stress adaptation Christa Gregori*, Bettina Bauer*, Chantal Schwartz, Angelika Kren, Christoph SchullerĐ ă and Karl Kuchler Medical University Vienna, Max F Perutz Laboratories, Department of Medical Biochemistry, Campus Vienna Biocenter, Austria Keywords ABC transporter; stress response; weak organic acids; yeast; zinc finger Correspondence K Kuchler, Medical University Vienna, Max F Perutz Laboratories, Department of Medical Biochemistry, Campus Vienna Biocenter, Dr Bohr-Gasse ⁄ 2, A-1030, Vienna, Austria Fax: +43 4277 9618 Tel: +43 4277 61807 E-mail: karl.kuchler@meduniwien.ac.at Present address ´  Universite de Nice-Sophia Antipolis, Inserm, U636, Centre de Biochimie, UFR Sciences, Parc Valrose, Nice, France àInstitute of Biochemistry and Genetics, Department of Clinical and Biological Research (DKBW), Basel, Switzerland §University of Vienna, Max F Perutz Laboratories, Department of Biochemistry & Molecular and Cellular Biology, Campus Vienna Biocenter, Austria Exposure of the yeast Saccharomyces cerevisiae to weak organic acids such as the food preservatives sorbate, benzoate and propionate leads to the pronounced induction of the plasma membrane ATP-binding cassette (ABC) transporter, Pdr12p This protein mediates efflux of weak acid anions, which is essential for stress adaptation Recently, we identified War1p as the dedicated transcriptional regulator required for PDR12 stress induction Here, we report the results from a genetic screen that led to the isolation of two war1 alleles encoding mutant variants, War1-28p and War1-42p, which are unable to support cell growth in the presence of sorbate DNA sequencing revealed that War1-28 encodes a truncated form of the transcriptional regulator, and War1-42 carries three clustered mutations near the C-terminal activation domain Although War1-42 is expressed and properly localized in the nucleus, the War1-42p variant fails to bind the weak-acid-response elements in the PDR12 promoter, as shown by in vivo footprinting Importantly, in contrast with wild-type War1p, War1-42p is also no longer phosphorylated upon weak-acid challenge, demonstrating that phosphorylation of War1p, its activation and DNA binding are tightly linked processes that are essential for adaptation to weak-acid stress * These authors contributed equally to this work (Received 11 January 2007, revised April 2007, accepted 19 April 2007) doi:10.1111/j.1742-4658.2007.05837.x Weak acids have a long history as additives in food preservation In addition to sulfites used in wine making, acetic, sorbic, benzoic and propionic acids are commonly used in the food and beverage industry to prevent spoilage [1,2] In solution, weak acids exist in a dynamic equilibrium between undissociated, uncharged molecules and their anionic form These acids display increased antimicrobial action at low pH, which favors the undissociated state The uncharged molecules can readily diffuse through the plasma Abbreviations GST, glutathione S-transferase; MHR, middle homology region; NLS, nuclear localization signal; PDR, pleiotropic drug resistance; WARE, weak-acid-response element; YPD, yeast peptone ⁄ dextrose 3094 FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gregori et al membrane In the cytoplasm, weak acids encounter a more neutral pH, causing their dissociation into acid anions and protons The protons lead to cytoplasmic acidification, thereby inhibiting important metabolic processes such as glycolysis [3], possibly interfering with active transport and signal transduction [1] Furthermore, sorbate and benzoate may also act as membrane-damaging substances [4] and, at least under aerobic conditions, cause severe oxidative stress [5,6] The antimicrobial action of weak-acid preservatives is usually characterized by extended lag phases and cell stasis, although microbial killing does not occur However, cells can adapt to the presence of weak acid and resume growth In Saccharomyces cerevisiae, this adaptation requires induction of the Pdr12p plasma membrane ATP-binding cassette (ABC) transporter [7] Together with the plasma membrane H+-ATPase, Pma1p, the activity of which is also regulated by weak acid stress [8,9], Pdr12p becomes one of the most abundant surface proteins in stressed cells [7] Whereas Pma1p effluxes protons, Pdr12p mediates cellular extrusion of weak acid anions [7] Notably, other members of the fungal ABC transporter family transport a wide variety of different xenobiotics across the plasma membrane or membranes of subcellular compartments [10,11] Pdr12p is the essential component of this stress response pathway, as cells are hypersensitive to sorbic, benzoic and propionic acid [7] and fail to adapt to such stress conditions in the absence of Pdr12p Moreover, recent data indicate the involvement of Pdr12p in the export of by-products of amino-acid catabolism, as a pdr12D strain displays hypersensitivity to fusel acids derived from leucine, isoleucine, valine, phenylalanine and tryptophan [12] Therefore, Pdr12p is not only required for adaptation to weak-acid stress, but might also efflux weak-acid metabolites Notably, PDR12 is rapidly induced by weak-acid challenge [7], but also in cultures grown with leucine, methionine or phenylalanine as sole nitrogen source [12] A recent study [13] attempted to identify Pdr12p-like proteins in other food spoilage yeasts A sorbic-inducible protein cross-reacting with S cerevisiae Pdr12p antibodies in Saccharomyces bayanus was found In contrast, proteins detectable with the same antibodies in Zygosaccharomyces bailii and Zygosaccharomyces lentus were not up-regulated upon sorbate challenge We are interested in identifying components of the signaling pathway required for this efficient response in S cerevisiae Hence, we pursued two different strategies First, we used a functional genomics approach and screened all putative nonessential transcription factor deletions of the EUROSCARF collection [14] (http:// www.uni-frankfurt.de/fb15/mikro/eroscarf/) for sorbate Yeast weak organic acid stress adaptation hypersensitivity This approach identified the regulator War1p (weak acid resistance) as the main inducer of PDR12 [15] War1p is a nuclear transcription factor, which decorates at least one weak-acid-response element (WARE) in the PDR12 promoter War1p is rapidly phosphorylated upon stress challenge, and phosphorylation is somehow coupled to War1p activation [15] Interestingly, War1p is required for PDR12 up-regulation in response to exogenous weak-acid stress, but it appears also to be involved in the metabolism-derived endogenous fusel acid stress response [12] The War1p protein belongs to the fungal-specific Zn(II)2Cys6 zinc finger family of transcriptional regulators with some 54 other putative members in S cerevisiae [16] These are implicated in various important cellular processes, including amino-acid [17] and galactose [18] metabolism, nitrogen source utilization [16], peroxisomal proliferation [19,20], respiration [21,22] and even pleiotropic drug resistance (PDR) [11] For example, Pdr1p and Pdr3p are key players in yeast PDR development, because they control ABC drug efflux pumps such as Pdr5p [23,24], Snq2p [25,26] and Yor1p [27], all of which are involved in PDR [10,11] Most regulators harbor a binuclear DNA-binding zinc cluster at the N-terminus, whereas the acidic activation domain is usually present at the C-terminus The middle homology region (MHR) bridging the DNA-binding and the activation domain may control the activity or specificity of the transcription factor, as deletions or mutations in this region often result in constitutive activity [22,28–30] Notably, a WAR1 orthologue has been identified in the human fungal pathogen Candida albicans [31] Consistent with its role in S cerevisiae, this WAR1 is also required for sorbate tolerance Moreover, our group recently identified the Candida glabrata homologue of War1p (C Gregori and K Kuchler, unpublished work) Preliminary experiments show that it is also required for a response to weak organic acids in the human fungal pathogen C glabrata, demonstrating the evolutionary conservation of this weak-acid stress in the fungal kingdom (C Gregori and K Kuchler, unpublished work) Secondly, we applied a classical genetic screen using a PDR12prom-lacZ reporter gene to identify components of the weak-acid response pathway Here, we report the results of the genetic approach, which leads to the isolation of two war1 mutant alleles that are unable to drive Pdr12p induction Remarkably, the genetic screen identified mutations in the WAR1 gene only, indicating that weak-acid stress response requires two major components, a dedicated stress regulator and the Pdr12p efflux pump The defective War1-42p mutant is no longer phosphorylated upon stress and FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS 3095 Yeast weak organic acid stress adaptation C Gregori et al unable to bind to cis-acting WARE motifs, suggesting that activation of War1p or its binding to WARE is tightly linked to its post-translational modification A WT - war1-28 + - + war1-42 - + pdr12Δ - + sorbate Results Pdr12p Isolation of sorbate-sensitive mutant strains control To identify components of the stress response pathway that mediates induction of the Pdr12p efflux pump, we set up a classical mutagenesis screen For the isolation of mutant cells that fail to induce PDR12 upon weak acid challenge, we constructed a reporter strain carrying the lacZ gene driven by the PDR12 promoter integrated into the ura3 loci of two different genetic backgrounds, creating the strains YCS12ZI and YAK3 These strains were grown to the exponential growth phase, plated and irradiated with UV light to randomly introduce mutations After a 2-day incubation, colonies were replica-plated on plates containing mm sorbate and the dye X-Gal to induce the PDR12 promoter and to visualize LacZ expression In a first round of screening, we obtained 111 white colonies (62 for YAK3 and 49 for YCS12Z-I) To determine if the white color resulted from a lack of PDR12 promoter induction, and thus no expression of lacZ, these colonies were re-screened for their Pdr12p protein concentrations by immunoblotting Although several mutants showed reduced Pdr12p concentrations under stress conditions (data not shown), only two mutant strains, 42 and 28, lacked detectable Pdr12p induction upon sorbate stress (Fig 1A) Both mutants were back-crossed with the wild-type several times to clean up the genetic background and determine whether the phenotype was caused by mutations in a single gene As tetrad analysis revealed a : cosegregation of sorbate sensitivity with the inability of lacZ induction, both mutations must reside in a single gene (data not shown) Growth-inhibition assays (Fig 1B) showed that mutants 28 and 42 grew at a sorbate concentration of up to 0.25 mm The pdr12D control strain was viable, but exhibited reduced growth on 0.5 mm sorbate plates, and failed to grow on mm sorbate, whereas the wild-type control even grew at concentrations above mm Therefore, we isolated two yeast mutants with defects in a single gene representing at least one component of the weak-acid response machinery that acts through Pdr12p induction to trigger adaptation Identification of mutated genes In addition to the classical genetic approach, we recently pursued a functional genomics approach to 3096 B WT war1-28 YPD pH 4.5 control war1-42 pdr12Δ 0.25 mM 0.5 mM mM + Sorbate Fig war1 mutants are sorbate-sensitive and fail to induce Pdr12p upon sorbate stress (A) The strains W303-1A (WT), YAK4 (War1-28), YCS42-D4 (War1-42) and control strain YBB14 (pdr12D) were grown in YPD to an A600 of  The cultures were split and one half was stressed with mM sorbate for h Cell extracts equivalent to 0.5 A600 were separated by SDS ⁄ PAGE (7% gel), and the immunoblots were decorated with polyclonal anti-Pdr12p serum A cross-reaction to the antibodies served as loading control (B) The strains W303-1A (WT), YAK4 (War1-28), YCS42-D4 (War142) and YBB14 (pdr12D) were grown in YPD to an A600 of  Then the A600 was adjusted to 0.2, and the cells were spotted along with three : 10 serial dilutions on YPD, pH 4.5, containing the indicated sorbate concentrations Growth was monitored after a 48-h incubation at 30 °C identify regulators of weak organic acid resistance Making use of the EUROSCARF haploid deletion strain collection of S cerevisiae [14] (EUROSCARF, Germany; http//http://www.uni-frankfurt.de/fb15/ mikro/euroscarf/), we tested all viable transcription factor deletion strains for their ability to grow in the presence of sorbic acid This approach identified the transcription factor essential for Pdr12p induction and hence weak-acid resistance, War1p [15] To determine if the mutants isolated in the classical genetic screen are allelic to WAR1, appropriate selection markers were integrated and the strains subjected to complementation analysis Figure shows the growth phenotypes of the resulting diploid strains on yeast peptone ⁄ dextrose (YPD), pH 4.5, with different sorbate FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gregori et al Yeast weak organic acid stress adaptation WT/WT WT/war1Δ WT/28 WT/42 war1Δ/28 war1Δ/42 28/42 pdr12Δ YPD pH 4.5 control 0.5 mM mM + Sorbate Fig The sorbate-sensitive mutants carry loss-of-function alleles of WAR1 The strains W303-D (WT ⁄ WT), YBB21 (WT ⁄ war1D), YBB24 (WT ⁄ 28), YBB22 (WT ⁄ 42), YBB25 (war1D ⁄ 28), YBB26 (war1D ⁄ 42), YBB23 (28 ⁄ 42) and YBB14 (pdr12D) were grown to an A600 of 1, diluted to A600 of 0.2 and spotted on to YPD, pH 4.5, agar plates containing the indicated sorbate concentrations along with three : 10 serial dilutions Colony growth was inspected after 48 h at 30 °C concentrations Mutants 28 and 42, as well as the war1D deletion, when in combination with a wild-type gene, displayed similar growth to the diploid wild-type strain Thus, the mutant alleles, 28 and 42, are recessive for sorbate growth, and a heterozygous wildtype ⁄ war1D strain did not show any haplo-insufficiency phenotypes (Fig 2) However, when mutants 28 and 42 were crossed with the war1D strain, the diploid strains remained hypersensitive to sorbate, and displayed the same growth behavior as the pdr12D control strain These data suggest that the mutants isolated in the UV mutagenesis screen were allelic to WAR1 Thus, the mutant alleles were named War1-28 and War1-42, respectively Interestingly, diploid War128 ⁄ War1-42 cells were more resistant than war1D ⁄ mutant diploids, suggesting a possible cross-complementation of mutant alleles, and implying that War1p acts as a dimer [15] Identification of the mutations in war1 To identify the actual mutations leading to the loss-offunction phenotypes, the defective war1 alleles were amplified by PCR from genomic DNA obtained from the mutant strains and subjected to DNA sequencing Sequencing of both DNA strands of War1-28 identified an A to T mutation at position 1286, and a change of C to T at position 1288, the latter introducing a translational stop codon (Fig 3) At the aminoacid level, these mutations resulted in a N429I residue exchange, and the nonsense mutation leads to truncated War1-28p protein (Fig 3A) For the mutant War1-42 allele, four clustered mutations were found: deletion of A2286, T2287 and T2288, and the G2291T transversion These four mutations caused three amino acid changes, namely K762N, F763M and the R764D deletion The rest of the protein remained unchanged As depicted in the cartoon (Fig 3A), War1p is representative of the binuclear Zn(II)2Cys6 transcription factor family, all members of which contain a DNAbinding zinc finger at their N-terminus (amino acids 75–111), followed by two predicted nuclear localization signals (NLS amino acids 106–123 and 286–303), and a coiled-coil domain mediating protein–protein interactions The putative transcriptional activation domain is located near the C-terminus, residues 911–937 Hence, a loss-of-function phenotype of War1-28 may easily be explained by the absence of the activation domain, whereas the effect of the mutations in War1-42 on War1p function is not immediately obvious Characterization of War1-42p and its post-translational modification To determine if the mutant proteins are properly expressed, we epitope-tagged both War1-28p and War1-42p at the C-terminus by genomic integration of a triple 3HA epitope, creating the strains YBB30 and YBB31, respectively Immunoblotting of protein extracts from exponentially growing cultures revealed that both mutant proteins displayed a mobility corresponding to their predicted molecular mass (Fig 3B) However, when compared with the wild-type, the steady-state concentrations of mutant War1-42p-3HA appeared to be markedly reduced Notably, the concentrations of the truncated War1-28p-3HA appeared slightly increased, implying that the stability of the protein is affected by the different mutations To address this point, we performed cycloheximide chase experiments The strains, YAK111, YBB30 and YBB31, were grown in YPD to an A600 of  1; then cycloheximide was added to block protein synthesis, and samples were collected at the indicated time points for immunoblotting (Fig 3C) The results show that the wild-type protein was quite stable, with a half-life of 100–120 Likewise, War1-28p-3HA was detectable throughout the whole chase period (Fig 3C) In contrast, War1-42p-3HA displayed a much faster proteolytic turnover, as it was already below the detection limit 40 after cycloheximide addition (Fig 3C) Thus, the low steady-state concentrations of War142p-3HA may be explained by its reduced stability Notably, sorbate failed to influence War1p stability, as the half-life was unchanged under stress (data not shown) FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS 3097 Yeast weak organic acid stress adaptation C Gregori et al Stress-induced phosphorylation is absent in War1-42p Whereas for War1-28p the inability to induce PDR12 transcription was attributable to the lack of the activation domain, the explanation was not obvious for War1-42p Notably, we have previously shown that PDR12 induction coincides with War1p phosphorylation [15] Therefore, we determined the post-translational modification status of War1-42p-3HA under both stressed and nonstressed conditions using immunoblotting (Fig 3D) The cultures were grown to an A600 of  1, split, and one half was treated with mm sorbate After 30 min, cells were harvested, and protein extracts prepared and subjected to immunoblotting As A Functional analysis of single-residue changes, K762N, F763M and R764D war1-42 K762N F763M R764Δ war1-28 N429I STOP AD Zn W T -3 H A 42 H -3 28 W T- 3H A B A NLS NLS War1p-3HA War1-28p-3HA cross reaction loading control C 20 40 60 80 100 120 CHX War1p-3HA War1-28p-3HA 3H A W T A H -3 TW 42 W T TW 42 -3 H 3H A A War1-42p-3HA D War1p-3HA unstressed 3098 + mM sorbate 30 reported previously [15], wild-type War1p migrated as a double band in unstressed cells and shifted to slower mobility forms upon sorbate addition (Fig 3D) In contrast, no mobility shift was detectable for War142p-3HA, as it migrated as a single band under both stressed and nonstressed conditions (Fig 3D) Therefore, the post-translational modification pattern of War1p, which is intimately linked to PDR12 stress induction, is absent in the War1-42p-3HA mutant, indicating that phosphorylation may be an essential step in War1p activation Remarkably, War1-42p-3HA from unstressed cells exhibited a faster mobility on SDS ⁄ polyacrylamide gels than authentic War1p (Fig 3D), suggesting that the basal modification in the absence of stress was also affected in War1-42p-3HA The War1-42 allele contains a cluster of four mutations, leading to three residue changes To address which mutation alone or in combination with another one causes the phenotype, we constructed the CENbased plasmids pCGWAR1-K762N, pCGWAR1F763M and pCGWAR1-R764D carrying the single mutations, respectively Each of the three plasmids expressed a mutated version War1p with only one of three residue changes of War1-42p To determine the phosphorylation status of War1p-K762N, War1p- Fig Organization, expression, stability and modification of War1p variants (A) The cartoon depicts the localization of mutations in the WAR1 gene abrogating its function as a specific Pdr12p regulator Zn, zinc finger; AD, activation domain Cartoon not drawn to scale (B) Expression and stability of the wild-type and mutant War1p variants Cultures of the strains YAK111 (WT-3HA), YBB31 (28-3HA), YBB30 (42-3HA) and YPH499 (WT) were grown in YPD to an A600 of  and harvested Yeast crude protein extracts equivalent to A600 were separated by PAGE (10% gel) and analyzed by immunoblotting using the 12CA5 HA antibody Cross-reactions to the HA antibody served as a loading control (C) The strains YAK111 (War1p-3HA), YBB31 (War1-28p-3HA) and YBB30 (War1-42p-3HA) were grown in YPD to an A600 of  1, then cycloheximide (CHX) was added to a final concentration of 0.1 mgỈmL)1, and samples were taken at the indicated time points Extracts (0.5 A600 for War1p-3HA and War1-28p-3HA, 1.5 A600 for War1-42p-3HA) were fractionated by SDS ⁄ PAGE (10% gel), followed by immunodetection of the War1p-3HA and variants by monoclonal 12CA5 HA antibody (D) The strains YAK111 (WT-3HA), YBB30 (42-3HA) and YPH499 (WT) were grown in YPD to an A600 of  1, then the cultures were split, and one half was treated with mM sorbate for 30 Crude cell extracts (equivalent to 1.5 A600 for 42-HA and 0.5 A600 for WT-HA and WT) were separated by SDS ⁄ PAGE (7% gel) and immunodetected using the monoclonal 12CA5 HA antibody FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gregori et al A WT - K762N F763M + - + - + R764Δ - mM sorbate + War1p Pdr12p Pdr5p B w ar K7 1Δ F7 2N R7 3M 64 Δ F763M and War1p-R764D, as well as their ability to induce Pdr12p expression, we performed immunoblotting under stressed and nonstressed conditions (Fig 4A) Cultures of the war1D strain YAK120, harboring pCGWAR1-K762N, pCGWAR1-F763M, pCGWAR1-R764D or the control plasmid expressing wild-type WAR1, were grown to an A600 of  The cultures were split in half; one half was treated with mm sorbate for 30 min, and the other left unstressed Cells were then harvested, and protein extracts were prepared and subjected to immunoblotting Whereas War1p-F763M behaved as the wild-type War1p under unstressed conditions, War1p-K762N showed a slightly different modification pattern in unstressed cells In contrast with wild-type War1p, which migrated as a double band under nonstressed conditions, the lowermigrating band was hardly detectable in War1p-K762N (Fig 4A) However, sorbate shifted both War1pK762N and War1p-F763M to a slower mobility, as was also observed for the wild-type control In contrast, War1p-R764D remained unmodified in response to sorbate stress The mobility shift in response to stress is tightly linked to PDR12 induction, which was absent in the strain expressing War1p-R764D (Fig 4A) In contrast, strains expressing the mutants War1pK762N and War1p-F763M showed greatly increased Pdr12p concentrations in both the absence and presence of weak-acid stress Addition of sorbate did not further increase Pdr12p expression levels (Fig 4A), demonstrating that the War1p-K762N and War1pF763M single mutants are gain-of-function variants However, their hyperactivity is suppressed by the presence of the additional R764D deletion in War1-42p Furthermore, we tested the strains expressing the mutant War1p variants for their ability to grow on YPD, pH 4.5, in the presence or absence of different sorbate concentrations (Fig 4B) Consistent with the immunoblotting data, the War1p-R764D strain was as hypersensitive to sorbate as the war1D control because of the inability to induce Pdr12p, whereas War1pK762N and War1p-F763M showed normal growth when compared with wild-type cells (Fig 4B) Notably, the hyperactivity of War1p-K762N and War1pF763M variants failed to cause hyper-resistance to weak organic acids (data not shown) Because War1-42p displayed reduced protein stability (Fig 3C), cycloheximide chase experiments were also performed with the War1p-R764D single mutant exactly as described above for War1-42p (Fig 3C) Detection of the different War1p variants demonstrated a markedly reduced stability of War1p-R764D compared with wild-type, although protein concentrations did not decrease as fast as for War1-42p Yeast weak organic acid stress adaptation T W YPD pH 4.5 + 0.5 mM sorbate + mM sorbate C 20 40 60 80 100 120 + mM sorbate CHX War1p War1p-R764Δ War1-42p Fig War1p-764D causes sorbate hypersensitivity and fails to induce Pdr12p (A) Cultures of YAK120 cells were transformed with pCGWAR1, pCGWAR1-K762N, pCGWAR1-F763M or pCGWAR1R764D and grown in YPD to an A600 of  1; cultures were split in half, and one half was treated with mM sorbate for 30 min, while the other remained untreated Crude cell extracts (0.5 A600) were separated by SDS ⁄ PAGE (7% gel), and immunodetected using polyclonal antibodies against War1p, Pdr12p and Pdr5p (B) YAK120 was transformed with pCGWAR1, pCGWAR1-K762N, pCGWAR1F763M or pCGWAR1-R764D and grown to an A600 of 1, diluted to A600 ¼ 0.2, 0.02 and 0.002; culture aliquots were spotted on to YPD, pH 4.5, agar plates containing sorbate concentrations as indicated Colony growth was inspected after 48 h at 30 °C (C) The strain YAK120 transformed with pCGWAR1 or pCGWAR1-R764D and strain YCS42-D4 (War1-42p) were grown in YPD to an A600 of  Cycloheximide (CHX) was added at a final concentration of 0.1 mgỈmL)1; samples were taken at the indicated time points Cell-free extracts (0.5 A600 for War1p, 1.0 A600 for War1-42p and War1-R764Dp) were separated by SDS ⁄ PAGE (7% gel) and transferred to nitrocellulose membranes War1p and variants were detected by immunoblotting using polyclonal antibodies to War1p FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS 3099 Yeast weak organic acid stress adaptation C Gregori et al S 3100 42 C S N war1Δ C S N C War1p Swi6p Hxk1p W T B We have previously demonstrated that War1p is a nuclear protein [15] Although the mutational changes in War1-42p left the DNA-binding domain and both NLS unaffected, we wanted to test whether War1-42p is also properly localized to the nucleus Hence, we carried out fractionation experiments using purified nuclear fractions from various strains Subcellular fractions were isolated by following a gentle cell lysis procedure to preserve nuclear integrity, and subjected to immunoblotting using polyclonal antibodies specific for War1p, the nuclear marker protein Swi6p and the cytoplasmic hexokinase Hxk1p (Fig 5A) As shown in Fig 5A, War1-42p, like the wild-type control War1p, localized to the nucleus in the steady state As expected, Hxk1p was predominantly found in the soluble cytoplasmic fraction The signal for Hxk1p in the nuclear fraction is due to normal unavoidable contamination of the nuclear fraction with cytosolic proteins However, the nuclear marker, Swi6p, entirely cofractionated with both War1-42p and wild-type War1p (Fig 5A), demonstrating that the normal nuclear localization of War1-42p is unaffected by the mutations No immunoreactive material was detectable in war1D cells, confirming the specificity of the polyclonal anti-War1p serum Notably, the polyclonal antibodies also detected a War1p degradation product (Fig 5A), which was not recognized by the monoclonal HA antibody (Fig 3) Hence, the lack of function in War1-42p is probably a consequence of impaired activation or direct binding to the WARE rather than aberrant cellular localization War1-42 strains cannot tolerate sorbate, suggesting that they may lack the capacity for proper modification of War1p under stress conditions To exclude this possibility, we introduced War1-42p into a wild-type background to obtain strain YBB32 With this strain carrying one wild-type and one mutated allele of WAR1, we repeated the stress experiments described above and checked for the mobility of War1-42p by immunoblot analysis Even in the presence of wild-type War1p and a normal stress response, War1-42p remains unmodified (data not shown), suggesting that the mutations prevent normal modification of the transcription factor rather than indirect effects, which N ar w -42 ar 1Δ War1-42p localizes to the nucleus but is unable to bind to the WARE in vivo WT A w (Fig 4C) This may indicate that the cluster of three residue changes in War1-42p, as well as single residue changes, even if two of them lead to a gain-of-function, change War1p folding, thereby destabilizing War1p more than the loss of a single amino acid as in War1-R764Dp * 1+2 1+3 2+3 * WT war1-42 war1Δ Fig Nuclear War1-42p is unable to decorate the PDR12 promoter in vivo (A) Subcellular fractions were prepared from wild-type cells (WT), War1-42 mutants (42) and cells lacking War1p (war1D) as described in Experimental procedures About A600 equivalents were subjected to immunoblotting using polyclonal antibodies against War1p, Swi6p and Hxk1p S, Total input; N, nuclear pellet; C, cytoplasmic fraction (B) YPH499 wild-type (WT), YAK110 (war1D) and YCS42-D4 (War1-42) cells were grown to the early exponential growth phase and treated with dimethyl sulfoxide to methylate DNA For in vivo footprinting, chromosomal DNA was prepared and used as a template for primer extension with a labeled oligonucleotide primer corresponding to )497 to )472 of the PDR12 promoter The reaction mixture was resolved through a sequencing gel, exposed to a phosphoimaging screen, and signals were quantified Intensities of traces are compared in the indicated combinations An asterisk marks a protected G ()631) in the War1-42 allele Differences are indicated by bars for deprotected G residues ()643, )642, )617, )618) and aligned with the sequence of the region FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gregori et al would be a consequence of the impaired growth of War1-42 mutants under stress Because nuclear localization of War1-42p was unaffected, we determined whether War1-42p still binds to the WARE in the PDR12 promoter To clarify this, we performed in vivo footprinting experiments in cells expressing War1-42p The strains YPH499 (wild-type), YAK110 (war1D) and YCS42-D4 (War1-42) were cultivated to the exponential growth phase and then treated with dimethyl sulfoxide to methylate guanines and to some extent adenines in the genomic DNA Chromosomal DNA was isolated, and the methylation status was determined by primer extension analysis (Fig 5B) Comparison of the methylation patterns of the different strains revealed that the mutant War1-42 and war1D exhibit almost identical patterns (Fig 5B, 2+3) In contrast, when both were compared with the wild-type (Fig 5B, 1+2 and 1+3), deprotection at the same nucleotides (black bars) was observed These results clearly indicate that War1-42p is unable to decorate the WARE in the PDR12 promoter in vivo, nicely explaining the loss-of-function phenotype and the sorbate hypersensitivity of War1-42 mutant cells Taken together, our genetic screen identified two mutations in the putative MHR region of the War1p transcriptional regulator, suggesting that the MHR is essential for War1p function Mutations in the MHR may cause structural changes that impair post-translational phosphorylation, affecting DNA binding of War1p or the recruitment of other as yet unknown coregulators Discussion We are interested in dissecting the response pathway necessary for cellular adaptation to stress from weak organic acids in the yeast, S cerevisiae Using a functional genomic approach, we have identified the War1p regulator as the dedicated transcription factor required for Pdr12p induction following weak-acid stress exposure [15] In this study, we report the isolation and characterization of two loss-of-function war1 alleles that give rise to War1p variants that are unable to mediate Pdr12p induction in response to sorbate stress We exploited a classical genetic screen, taking advantage of a lacZ reporter driven by the PDR12 promoter, which otherwise controls expression of the Pdr12p weak-acid anion-efflux pump [7] After UV mutagenesis, we screened more than 10 genome equivalents of mutant colonies for their capacity in PDR12 induction We expected to isolate mutants in membrane sensors, signaling components such as kinases, phosphatases and perhaps transcriptional Yeast weak organic acid stress adaptation regulators However, most remarkably, only two yeast mutants were isolated in which sorbate-mediated induction of Pdr12p was completely abolished (Fig 1A) The weak-acid hypersensitivity of both mutant strains was attributed to mutations in the WAR1 gene (Fig 3) DNA sequencing identified the mutations in the nonfunctional war1 alleles Whereas War1-28 encoded a truncated regulator which was due to a stop codon, War1-42 carried three residue changes close to the C-terminus Hence, the genetic approach yielded only mutant variants of the War1p regulator, suggesting that War1p is the major and perhaps only stress regulator of Pdr12p The genetic approach confirms our strategy of using functional genomics which led to the identification of War1p Nevertheless the genetics data allow several interpretations about the function of War1p Firstly, none of the signaling components except War1p, including the War1p kinase(s), appear to be essential, perhaps because of redundant functions in the pathway Secondly, Pdr12p and War1p represent the key elements of the response pathway and there are no other essential components Thirdly, we cannot entirely exclude the possibility that other mutants have been missed by the low amount of sorbate used for the PDR12 promoter induction screen because of weak-acid hypersensitivity However, as Pdr12p is the major determinant of weakacid resistance, and the main target of War1p [32], we reason that defects in genes encoding components acting upstream of War1p should not display higher sorbate sensitivities than nonfunctional War1p variants themselves The sorbate hypersensitivity phenotype of War1-28 cells can easily be explained, because this allele carries a nonsense mutation leading to a truncated War1-28p (Fig 3A) Hence War1-28p lacks the C-terminal activation domain, which is necessary for transcriptional activation of the target genes by other Zn(II)2Cys6 transcription factors [33] Notably, the truncated War1-28p protein, although expressed at higher levels than the wild-type, does not interfere with the function of authentic War1p in diploid cells (Fig 2), which might be a direct consequence of impaired dimer formation of War1p or a lack of DNA binding Indeed, in the case of War1-42p, in vivo footprinting data (Fig 5) indicate an inability to bind to the WARE, which is normally decorated by wild-type War1p in the presence or absence of the stress agent [15] Thus, the lack of PDR12 stress induction by War1-42p is perhaps due to its inability to bind to the promoter WARE of its target gene Alternatively, the mutations may also reduce the binding affinity of the War1-42p, FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS 3101 Yeast weak organic acid stress adaptation C Gregori et al thereby causing impaired assembly of the active transcription complex A lack of DNA binding by War1-42p may be explained in several ways First, immunoblotting cycloheximide chase experiments indicated that War142p shows lower steady-state protein concentrations and decreased stability compared with the wild-type War1p (Fig 4) Hence, the amount of protein might simply be too low to allow binding to the target DNA Secondly, the mutations may reduce affinity for WARE binding or prevent the formation of War1p dimers, which appears necessary for War1p function [15] Thirdly, reduced protein concentrations may still allow WARE binding, but other determinants are preventing the DNA recognition Although band shift experiments using glutathione S-transferase (GST)War1p suggested that WARE binding does not require additional factors or modifications [15], this may not entirely reflect the situation in vivo As shown for Gal4p, the prototype zinc cluster transcription factor, its DNA-binding properties can be different in vitro and in vivo and may involve sequences in the regulatory domains distinct from the zinc finger or even additional factors [34] Therefore, the war1 mutations identified perhaps destroy structural features necessary for the interaction with accessory proteins involved in target site recognition, WARE binding or post-translational modification of War1p Genetic separation of the clustered mutations in War1-42p revealed that R764D displays a complete loss-of-function phenotype In sharp contrast, however, the residue changes, K762N and F763M, lead to constitutive War1p hyperactivity (Fig 4A) Strikingly, War1p-R764D is no longer phosphorylated in response to sorbate challenge Conversely, War1p-K762N and War1p-F763M are phosphorylated upon stress, although they are already active in the absence of sorbate stress (Fig 4A) Therefore, in constitutively active War1p variants, sorbate stress is tightly linked to phosphorylation, even if the protein is already activated War1-42p is functionally inactive and also not phosphorylated in response to weak-acid treatment (Fig 3D) Hence, the R764D mutation can be considered dominant for War1p loss-of-function when present in combination with the hyper-activating mutations, K762N and F763M The basal modification status of War1-42p is different from wild-type War1p, as they display distinct mobilities on immunoblotting DNA binding in vivo may well require basal post-translational modifications, as present in wild-type War1p but absent in the mutant variant (Fig 3D) These modifications either directly influence the DNA-binding capability or are 3102 necessary for the interaction with another as yet unknown cofactor that would facilitate binding to the PDR12 promoter The fact that loss-of- function mutations reside outside the zinc finger or the NLS suggests an altered conformation or structure This is consistent with the apparent absence of stress-induced phosphorylation in War1p-R764D and its reduced protein stability Thus, only massive folding changes can explain the inactivity of War1p-R764D, which may hinder phosphorylation Mutations may also affect the structure of confined domains such as the MHR rather than the whole tertiary structure Hence, the lack of phosphorylation is most likely a consequence of massively altered War1p conformation rather than altered structure of the kinase targets themselves Further, the residue changes in War1-42p not involve serine, threonine, tyrosine or histidine (Fig 3A) In any case, the nonphosphorylated war1 alleles are nonfunctional, indicating that certain post-translational modifications are essential for War1p to induce transcription of PDR12 upon stress From the homologies between zinc finger regulators, a defined MHR [33] is not immediately apparent in War1p However, it seems plausible that the stretch carrying the residue changes is functionally similar to the MHR Because the War1p-K762N and War1pF763M mutants in this stretch are constitutively active, the MHR of War1p is likely to play a major role in the regulation of its transcriptional activity, as well as in the specificity of target site recognition If the regulator is present in limiting amounts, a reduced or altered specificity because of lack of a functional MHR will remove the protein from its binding sites in vivo [33] However, we wish to provide another explanation for the loss-of-function in War1-42p The drastic decrease in the total amount of the transcriptional regulator in combination with a reduction in sequence recognition specificity may account for the observed lack of WARE binding by War1-42p Defective nuclear localization can be excluded, because the NLSs are not affected by the mutations, and because the mutant proteins display proper nuclear localization Another possibility is that War1-42p binds to WARE with much lower affinity, insufficient to be detected by in vivo footprinting (Fig 5B) Thus, more information about the structure and potential interaction partners of War1p is required For instance, the War1-42 mutant can be used as a tool to identify intragenic suppressor mutations Furthermore, high-copy or second-site suppressors may lead to the identification of unknown War1p-interacting partners This seems to be a feasible and promising approach FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gregori et al Yeast weak organic acid stress adaptation considering the suppression of the constitutive activity of K762N and F763M by an additional R764D deletion Finally, the War1-42 mutant allele should be useful as a tool for learning more about the molecular structure, as well as the protein–protein and protein– DNA interactions, of this binuclear zinc transcriptional regulator The polyclonal antibodies to War1p should be useful in identifying the upstream components of the response pathway, including War1p-specific kinases and phosphatases implicated in the modulation of War1p activity during adaptation to stress induced by weak organic acids Experimental procedures Yeast strains, growth conditions, and growth inhibition assays Rich medium (YPD) and synthetic medium were prepared essentially as described elsewhere [35] Unless otherwise indicated, all yeast strains were grown routinely at 30 °C S cerevisiae strains used in this study are listed in Table To determine weak-acid susceptibility, exponentially growing cultures were adjusted to A600 of 0.2 and diluted : 10, : 100 and : 1000 Equal volumes of these serial dilutions were spotted on to YPD, pH 4.5, plates containing the indicated sorbate concentrations exactly as previously described [15] Gene disruptions and strain constructions The deletion of WAR1 was performed by a PCR-based method using the disruption cassette of the plasmid pFA6aHIS3MX6 [36] The PDR12 gene was disrupted with a hisG-URA3-hisG cassette from the plasmid pYM63 [7] For epitope tagging of the wild-type or mutant versions of WAR1, the triple HA tag or GFP tag was amplified from plasmids pFA6a-3HA-KANMX6 or pFA6a-3HA-HIS3MX6 [37] using appropriate primers, followed by integration at the genomic locus For the PDR12prom-lacZ reporter construct used in the UV-mutagenesis screen, we amplified the PDR12 promoter by PCR, introducing an EcoRV site at position )1168 and a HindIII site at position +8 The EcoRV–HindIII fragment was then cloned into the vector YIp357 [38], yielding plasmid pCS12Z-I The correct sequence of the insert was verified by DNA sequencing To construct the strain carrying lacZ under the control of the PDR12 promoter, plasmid Table Yeast strains used in this study Strains Genotype Source W303–1B MATa ura3-1 leu2-3112 his3-11,15 trp1-1 ade2-1 can1-100 (W303-1A - MATa, W303-D - MATa ⁄ a) MATa ura3-52 leu2-D1 his3-D200 trp1-D1 ade2-10oc lys2-801a MATa ura3-52::pCS12ZI URA3 (isogenic to YPH499) MATa ura3-1::pCS12ZI URA3 (isogenic to W303-1B) MATa ura3-1::pCS12ZI URA3 LEU2 (isogenic to W303-1B) MATa ura3-1::pCS12ZI URA3 LEU2 War1-28 (isogenic to W303-1B) MATa war1D::HIS3MX6 (isogenic to YPH499) MATa WAR1-3HA KANMX6 (isogenic to YPH499) MATa war1D::HIS3MX6 (isogenic to W303-1A) MATa ura3-52::pCS12ZI URA3 War1-42 (isogenic to YPH499) MATa pdr12::hisG-URA3-hisG (isogenic to W303-1A) MATa ⁄ a LEU2 war1D::HIS3MX6 (isogenic to W303-D) MATa ⁄ a ura3-1 leu2-3112 trp1-1 ade2-1 can1-100 ura3-52::pCS12ZI URA3 leu2-D1 his3-D200 trp1-D1 ade2-10oc lys2-801a War1-42 MATa ⁄ a ura3-1::pCS12ZI URA3 LEU2 his3-11,15 trp1-1 ade2-1 can1-100 War1-28 ura3-52::pCS12ZI URA3 leu2-D HIS3 trp1-D1 ade2-10oc lys2-801a War1-42 MATa ⁄ a ura3-1::pCS12ZI URA3 LEU2 HIS3 trp1-1 ade2-1 can1-100 War1-28 (isogenic to W303-D) MATa ⁄ a ura3-1::pCS12ZI URA3 LEU2 his3-11,15 trp1-1 ade2-1 can1-100 War1-28 war1D::HIS3MX6 (isogenic to W303-D) MATa ⁄ a ura3-1 leu2-3112 his3-11,15 trp1-1 ade2-1 can1-100 war1D::HIS3MX6 ura3-52::pCS12ZI URA3 leu2-D1 his3-D200 trp1-D1 ade2-10oc lys2-801a War1-42 MATa ura3-52::pCS12ZI URA3 War1-42-3HA HIS3MX6 (isogenic to YPH499) MATa ura3-1::pCS12ZI URA3 LEU2 War1-28-3HA HIS3MX6 (isogenic to W303-1B) MATa ⁄ a ura3-1 LEU2 his3-11,15 trp1-1 ade2-1 can1-100 ura3-52::pCS12ZI URA3 leu2-D1 his3-D200 trp1-D1 ade2-10oc lys2-801a War1-42-3-HAHIS3MX6 [45] YPH499 YCS12ZI YAK2 YAK3 YAK4 YAK110 YAK111 YAK120 YCS42-D4 YBB14 YBB20 YBB22 YBB23 YBB24 YBB25 YBB26 YBB30 YBB31 YBB32 FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS [39] This [15] [15] [15] This [15] [15] This This This This study study study study study study This study This study This study This study This study This study This study 3103 Yeast weak organic acid stress adaptation C Gregori et al pCS12Z-I was linearized with StuI and integrated into the ura3-52 locus of YPH499 or ura3-1 of W303-1B, creating the strains YCS12Z-I and YAK2, respectively Correct genomic integration was confirmed by PCR, and lacZ functionality was tested by b-galactosidase assays For backcrossing of war1 mutants after the screening, as well as complementation analysis, additional markers were integrated into the strains (Table 1), and diploids were selected by double selection using appropriate auxotrophic markers UV mutagenesis For mutagenesis by UV irradiation, the strains YCS12Z-I or YAK3 were grown to the exponential growth phase, diluted, and plated on YPD at about 1000 cells ⁄ plate Then, the cells were treated in a UV Stratalinker 2400 (Stratagene, La Jolla, CA) with a dose allowing for 80% survival of cells After days incubation at 30 °C, the colonies were replicaplated on X-Gal (5-chloro-4-bromo-3-indolyl-b-d -galactoside) plates containing mm sorbate and grown for another days For YAK3, about 80 000 independent yeast colonies, and for YCS12Z-I about 50 000 colonies, were screened for the loss of sorbate-mediated lacZ induction White colonies were re-streaked to single colonies on X-Gal plates containing mm sorbate for easier inspection of color development All colonies that remained white were re-screened for Pdr12p concentrations by immunoblotting using polyclonal antibodies to Pdr12p [7] Isolated mutant cells were back-crossed at least three times to clean up the genetic background and to verify : segregation of sorbate sensitivity Site-directed mutagenesis and plasmid construction To generate the single-residue changes K762N, F763M and R764D, a PCR fragment containing 170 bp of the WAR1 promoter and 2568 bp of the WAR1 ORF was ligated to the vector pGEMT-easy (Promega, Mannheim, Germany) resulting in the plasmid pIF1 This plasmid served as a template for site-directed mutagenesis reactions using the QuickChange Site-Directed Mutagenesis kit (Stratagene) Site-directed mutagenesis was carried out exactly as recom- mended by the manufacturer using the customized oligonucleotide primers listed in Table Mutations in the WAR1 sequence are indicated in bold italic letters in the primer sequence The plasmids obtained were named pIF1-762, pIF1-763 and pIF-764, and successful mutagenesis was verified by DNA sequencing Fragments containing the indicated mutations were cloned into a yeast vector as follows A 4.32-kb PCR fragment containing the entire WAR1 ORF, as well as kb of the 5¢-region and 0.45 kb of the 3¢-region were isolated by PCR using genomic DNA from W303-1A After digestion with SalI and SacI, the PCR fragment was cloned into the corresponding sites of pRS315 [39], resulting in the plasmid pCGWAR1 The plasmid was sequenced to exclude PCR errors Plasmids pIF1-762, pIF1-763 and pIF-764 were digested with NsiI, and the resulting 2440-bp fragments containing the desired WAR1 mutations were used to replace the corresponding wild-type fragment in pCGWAR1, yielding the plasmids pCGWAR1-K762N, pCGWAR1-F763M and pCGWAR1-R764D Production of rabbit polyclonal antibodies and immunoblotting Polyclonal antibodies to War1p were raised in rabbits against a GST-War1p fusion protein containing 294 amino acids of the C-terminal part of War1p (amino acids 650– 944) fused in-frame to the C-terminus of GST The gene fusion was constructed as follows A 900-bp fragment of WAR1 was generated by PCR, using the customized primers WAR1-GSTs (5¢-AAGAATTCTCCATGGGGGAAAT GTCGCATACCATA-3¢) and WAR1-GSTas (5¢-CTGCA GTCAAATGTCGACATTCATGAAAAGGTCTGTCC-3¢), as described elsewhere [40] The PCR product was digested with EcoRI and SalI and cloned into the corresponding sites of pGEX-5X-1 (Amersham Biosciences, Piscataway, NJ) The resulting plasmid, pCG1950, allowed expression of the C-terminal 294 amino acids of WAR1 fused in-frame to the C-terminus of GST Escherichia coli strain DH5a harboring pCG1950 was grown on 37 °C to an A600 of 0.5 Expression of the GST-War1p fusion protein was induced at 30 °C for h by adding isopropyl b-d-thiogalactopyranoside to a final concentration of 0.2 mm The Table Oligonucleotides for site-directed mutagenesis of WAR1 Bases leading to residue changes in mutagenic oligonucleotides are given in bold italic letters Name Oligonucleotide sequence Source K762Ns K762Nas F763Ms F763Mas R764Ds R764Das 5¢-CCCTTCAACAACTCTCTTTACAAC TTTAGGTATGTTATTGCG-3¢ 5¢-CGCAATAACATACCTAAAGTT GTAAAGAGAGTTGTTGAAGGG-3¢ 5¢-CTTCAACAACTCTCTTTACAAAATG AGGTATGTTATTGCGTTATTTTG-3¢ 5¢-CAAAATAACGCAATAACATACCTCAT TTTGTAAAGAGAGTTGTTGAAG-3¢ 5¢-CCCTTCAACAACTCTCTTTACAAATTTTATGTTATTGCGTTATTTTGTC-3¢ 5¢-GACAAAATAACGCAATAACATAAAATTTGTAAAGAGAGTTGTTGAAGGG-3¢ This This This This This This 3104 study study study study study study FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS C Gregori et al GST-War1p fusion protein was purified as described elsewhere [15], except that the binding to the glutathione–Sepharose beads (Amersham) was performed at °C for 16 h and elution was carried out with 0.05% SDS Removal of SDS and concentration of the GST-War1p fusion protein was carried out in a Centricon YM-10 centrifugal filter device (Millipore, Billerica, MA) Antiserum to the purified GST-War1p fusion protein was raised in rabbits using a standard immunization regimen as described elsewhere [41] Preparation of yeast cell extracts and immunoblotting were performed exactly as described previously [41] To determine Pdr12p induction, strains were grown in YPD to an A600 of  The cultures were split, and one half was stressed with mm potassium sorbate for h, and the other left untreated as a control Cell-free extracts equivalent to 0.5 A600)2 A600 were separated by SDS ⁄ PAGE (7% gel), followed by immunoblotting using polyclonal antibodies to Pdr12p [7], War1p, Swi6p (a gift from K Nasmyth, IMP, Vienna, Austria) and Hxk1p (Biotrend, Koln, Germany) The protein concentrations of wild-type ă and mutant War1p variants were analyzed by immunoblotting of extracts from exponentially growing yeast cultures using the monoclonal HA antibody, 12CA5, or polyclonal War1p antibodies using the ECL chemiluminescence detection and conditions as suggested by the manufacturer (Amersham Biosciences) Subcellular fractionation and analysis of protein modification and stability Subcellular fractionation experiments were performed essentially as previously described [42] Briefly, cells grown in 500 mL YPD to an A600 of 1.0 were harvested, washed and pretreated with 2-mercaptoethanol [43] For spheroblasting, cells were resuspended in mL S-buffer (1.0 m sorbitol, 25 mm KH2PO4 pH 6.5, 0.4 mm CaCl2), Zymolyase 100.000 was added (25 mL)1), and cells were incubated with gentle shaking at 30 °C for h Spheroblasts were centrifuged at 3600 g for 10 and washed once with S-buffer An aliquot was mixed with sample buffer for direct lysis (total input) Spheroblasts were lysed by adding volumes of N-buffer (18% Ficoll, 20 mm KH2PO4 pH 6.5, 0.5 mm CaCl2, mm phenylmethanesulfonyl fluoride) and vigorous vortex-mixing After centrifugation at 2.500 g, the supernatant was re-centrifuged at 20 000 g for 30 The supernatant representing the cytoplasmic fraction was carefully removed; aliquots of the cytoplasmic fraction and the pellet with the nuclear fraction were mixed with sample buffer and separated by SDS ⁄ PAGE (7% gel) To check if the mutant War1p variants are post-translationally modified upon stress, cultures were grown to an A600 of  1, and split in half; one half was treated with mm potassium sorbate for 30 the other remained untreated Cell lysates from cultures with an A600 of 0.5 and 1.5 for War1-42p, were separated by SDS ⁄ PAGE (7% Yeast weak organic acid stress adaptation gel), followed by immunoblotting using the monoclonal HA antibody, 12CA5, to visualize War1-42p Polyclonal War1p antibodies were used to detect War1p-K762N, War1p-F763M and War1p-R764D Cycloheximide-chase experiments were performed to analyze changes in protein stability in the War1p mutants Cultures were grown to an A600 of  1, and cycloheximide was added at a final concentration of 0.1 mgỈmL)1 Equal amounts of cells were harvested at the indicated time points, and cell extracts analyzed by immunoblotting as described above In vivo footprinting experiments and DNA sequencing In vivo footprints were performed exactly as described previously [44] Cells were grown in 500 mL YPD to the early exponential growth phase, concentrated in 10 mL YPD, and treated with lL dimethyl sulfoxide The reaction was stopped after min, and chromosomal DNA prepared Primer extension was carried out with a 32P-labeled oligonucleotide corresponding to the residues )497 to )472 of the PDR12 promoter, resolved through a 8% sequencing gel, exposed to a phosphoimager screen, and quantified Traces were captured using ImageQuant software, converted into vector graphs, and aligned For identification of the mutations in war1 alleles, the WAR1 gene was amplified from genomic DNA To eliminate the danger of PCR-derived mutations, the pool of PCR fragments was subjected directly to DNA sequencing using the BigDye Terminator Cycle Sequencing Kit version 3.0 according to the instructions of the manufacturer and the ABI PRISM Sequencing System 310 (Applied Biosystems, Foster City, CA) Acknowledgements We thank Manuela Schutzer-Muhlbauer and all laboră ă atory members for critical reading of the manuscript and helpful discussions Peter Piper and Mehdi Mollapour are acknowledged for sharing unpublished information and their long-standing collaboration We appreciate the gifts of polyclonal anti-Swi6p and antiHxk1p sera from Kim Nasmyth and Rudolf Schweyen, respectively This work was supported by a grant from the FWF (Austrian Science Foundation Project P-15934-B12) to K.K References Lambert RJ & Stratford M (1999) Weak-acid preservatives: modelling microbial inhibition and response J Appl Microbiol 86, 157–164 Chichester DF & Tanner FW (1972) Antimicrobial food additives In Handbook of Food Additives (Furia TE, ed.), pp 115–184 CRC Press, Cleveland, OH FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS 3105 Yeast weak organic acid stress adaptation C Gregori et al Krebs HA, Wiggins D, Stubbs M, Sols A & Bedoya F (1983) Studies on the mechanism of the antifungal action of benzoate Biochem J 214, 657–663 Stratford M & Anslow PA (1998) Evidence that sorbic acid does not inhibit yeast as a classic ‘weak 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