Abstract #71

The PDR12-inducing transcription factor War1p undergoes stress-induced conformational changes on the promoter to elicit weak organic acid stress adaptation in yeast. Christa Gregori¹, Ingrid Frohner¹, Bettina Bauer¹, Dorothea Anrather², Gustav Ammerer², Karl Kuchler¹. 1) Medical University Vienna, Max F. Perutz Laboratories, Department Medical Biochemistry, Dr. Bohr-Gasse 9/2, Vienna, A-1030 Austria; 2) University of Vienna, Max F. Perutz Laboratories, Department of Biochemistry & Molecular Cell Biology, Dr. Bohr-Gasse 9/5, Vienna, A-1030 Austria.
   Exposure of the yeast Saccharomyces cerevisiae to weak organic acids such as sorbate or benzoate leads to the pronounced induction of the plasma membrane ABC transporter Pdr12p. Induction of PDR12 requires the transcription factor War1p, a Zn(II)2Cys6 zinc finger transcription factor. We proposed earlier that War1p receives and transmits signals through a novel signaling mechanism dedicated to stress adaptation. However, the upstream sensor, components transducing the stress signal and the mechanism of War1p function or activation remained unknown. In this work, we investigated a loss-of-function mutant of War1p that fails to regulate PDR12, showing that War1p phosphorylation is tightly linked to its activity. We also show that the so-called middle homology region of War1p plays an important role in regulating weak acid response, since deletion of parts as well as mutations in the MHR lead to constitutive War1p hyperactivity. Moreover, semi-quantitative phosphopeptide mass spectrometry on purified War1p demonstrate that weak acids cause a bulk hyper-phosphorylation of already pre-phosphorylated War1p, thereby activating War1p on the PDR12 promoter. Importantly, single cell FRET analysis of functional YFP-War1p-CFP fusion proteins indicates extensive conformational changes of War1p following its stress-induced activation. Using a ChIP approach, we provide strong evidence that phosphorylated War1p binds much tighter to the cis-acting WARE element in the PDR12 promotor. Hence, the higher affinity binding of activated War1p might facilitate recruitment of other co-activators or the common transcriptional mediator complex, thereby causing increased PDR12 expression, which is essential for weak acid stress adaptation.

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