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. 2003 Mar;23(5):1775-85.
doi: 10.1128/MCB.23.5.1775-1785.2003.

War1p, a novel transcription factor controlling weak acid stress response in yeast

Angelika Kren  1 , Yasmine M Mamnun, Bettina E Bauer, Christoph Schüller, Hubert Wolfger, Kostas Hatzixanthis, Mehdi Mollapour, Christa Gregori, Peter Piper, Karl Kuchler
Affiliations

War1p, a novel transcription factor controlling weak acid stress response in yeast

Angelika Kren et al. Mol Cell Biol. 2003 Mar.

Abstract

The Saccharomyces cerevisiae ATP-binding cassette (ABC) transporter Pdr12p effluxes weak acids such as sorbate and benzoate, thus mediating stress adaptation. In this study, we identify a novel transcription factor, War1p, as the regulator of this stress adaptation through transcriptional induction of PDR12. Cells lacking War1p are weak acid hypersensitive, since they fail to induce Pdr12p. The nuclear Zn2Cys6 transcriptional regulator War1p forms homodimers and is rapidly phosphorylated upon sorbate stress. The appearance of phosphorylated War1p isoforms coincides with transcriptional activation of PDR12. Promoter deletion analysis identified a novel cis-acting weak acid response element (WARE) in the PDR12 promoter required for PDR12 induction. War1p recognizes and decorates the WARE both in vitro and in vivo, as demonstrated by band shift assays and in vivo footprinting. Importantly, War1p occupies the WARE in the presence and absence of stress, demonstrating constitutive DNA binding in vivo. Our results suggest that weak acid stress triggers phosphorylation and perhaps activation of War1p. In turn, War1p activation is necessary for the induction of PDR12 through a novel signal transduction event that elicits weak organic acid stress adaptation.

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Figures

FIG. 1.
FIG. 1.
Deletion of WAR1 abolishes stress-induced Pdr12p expression. (A) YAK3 cells were grown on YPD and stressed with 10 mM sorbate or left untreated. β-Galactosidase activity in three independent samples was determined. (B) BY4741 and the single-deletion strains were grown in YPD to an OD600 of 1.0 and spotted onto pH 4.5 YPD plates containing various concentrations of sorbate. Cell growth was inspected after a 48-h incubation. WT, wild type. (C) BY4741 and Δyml076c (Δwar1) cells were grown on YPD and treated with 8 mM sorbate for the indicated times. RNA was isolated and blotted and PDR12, WAR1, and ACT1 mRNAs were detected by Northern analysis.
FIG. 2.
FIG. 2.
War1p mediates resistance to several weak organic acids. (A) BY4741 (wild type [WT]) and Δyml076c (Δwar1) cells transformed with pYA313WAR1 and the empty vector control pRS313 were grown in SC to an OD600 of 1.0 and spotted onto pH 4.5 SC plates containing sorbate. Cell growth was inspected after a 48-h incubation. (B) W303-1A (WT), YAK120 (Δwar1), YBB14 (Δpdr12), and YBB15 (Δwar1 Δpdr12) cells growing exponentially in YPD were spotted onto pH 4.5 YPD plates containing the indicated amounts of sorbate. Colonies were inspected after a 48-h incubation. (C) FY1679-28c (WT) and YAK100 (Δwar1) transformed with YEp352 or pYA352WAR1 were grown to an OD600 of 1.0 and spotted on pH 4.5 YPD plates containing weak acids as indicated.
FIG. 3.
FIG. 3.
War1p localizes to the nucleus and is able to form homodimers. (A) YAK122 cells expressing a War1p-GFP fusion were grown in YPD to the exponential growth phase and stressed with 8 mM sorbate for 2 h (d to f) or left untreated (a to c). Living cells were treated with DAPI for 15 min to stain nuclear DNA and inspected in a Zeiss Axiovert 10 fluorescence microscope. (a and d) Fluorescein isothiocyanate filter, War1-GFP; (b and e) DAPI staining; (c and f) Nomarski image.
FIG. 4.
FIG. 4.
War1p recognizes a cis-acting WARE in the PDR12 promoter. (A) FY1679-28c transformed with plasmids carrying the indicated fragments of the PDR12 promoter fused to lacZ were grown to the logarithmic growth phase, shifted to pH 4.5 YPD, and treated with 1 mM sorbate. β-Galactosidase assays were performed in triplicate in the absence (−) and presence (+) of sorbate, and activity is given in Miller units. (B) FY1679-28c (wild type [WT]) and YAK100 (Δwar1) cells transformed with pRW95-3 and pRW95-W1 were grown in SC medium to the logarithmic growth phase, shifted to pH 6.8 YPD, and stressed with 8 mM sorbate. β-Galactosidase activity was assayed, and activities are given in Miller units. Three independent experiments were performed with each construct, and standard deviations are given in parentheses.
FIG. 5.
FIG. 5.
War1p binds to the WARE in the PDR12 promoter. (A) For mobility shift experiments, the 32P-end-labeled WARE probe (bp −655 to −604 of the PDR12 promoter) was incubated with GST-War1p alone (lanes 2 and 3) or in the presence of a specific (S; lanes 5 and 6) or unspecific (US; lanes 6 and 7) competitor probe for 30 min. WARE alone (lane 1) or extracts from uninduced E. coli (lanes 8 and 9) served as controls. Complexes were separated by native gel electrophoresis and detected by autoradiography. (B) For in vivo footprint experiments, FY1679-28c (lanes 1 and 2) or YAK100 (Δwar1; lanes 3 and 4) cells were grown to the logarithmic growth phase and stressed with 8 mM sorbate for 10 min (+; lanes 1 and 3) or left unstressed (−; lanes 2 and 4). Cells were treated with DMS to methylate DNA. Primer extension was carried out on both strands (left, antisense; right, sense) to detect DNA methylation. Products were separated by native acrylamide gel electrophoresis and autoradiographed. Graphs reflect intensities of the bands (lane 1, solid line; lanes 2 and 3, dashed line). A, B, and C, sequences in the WARE. Boxed nucleotides show changes in methylation intensity corresponding to protection or deprotection.
FIG. 6.
FIG. 6.
War1p is reversibly phosphorylated during weak acid stress. (A) Exponentially growing YAK101 (WAR1-HA) cells were treated with 8 mM sorbate for the time periods indicated. War1p-HA and Pdr12p were immunodetected in cell extracts with monoclonal anti-HA antibody 12CA5 and polyclonal anti-Pdr12p antibodies, respectively. (B) YAK101 cells were stressed with 8 mM sorbate for 1 h in YPD or left unstressed. Protein extracts were incubated with phosphatase (CIP) for 1 h at 37°C, mock treated, or treated with CIP in the presence of orthophosphate as an inhibitor. (C) YAK101 cells were grown to an OD600 of 1.0; then, 8 mM sorbate was added for the time periods indicated (sorbate). Sorbate was removed by spinning out cells and resuspending them in fresh prewarmed YPD medium (YPD). Samples were taken, and protein extracts were analyzed by immunoblotting with anti-HA antibody 12CA5 and anti-Pdr12p antibodies.
FIG. 7.
FIG. 7.
Model for War1p-mediated weak acid stress adaptation. Uncharged weak acids enter the cell and dissociate in the cytoplasm, generating a lower intracellular pH. The resulting protons are extruded by Pma1p, and acid anions are effluxed by induced Pdr12p in cells adapting to weak acid stress. We propose that acid anions directly or indirectly activate War1p, which is constitutively bound to the WARE in the PDR12 promoter. War1p is rapidly phosphorylated upon weak acid stress and thus activated to induce PDR12, leading to a massive upregulation of Pdr12p in the plasma membrane.
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