Weak Organic Acids Trigger Conformational Changes of the Yeast Transcription Factor War1 in Vivo to Elicit Stress Adaptation

Christa Gregori,Christoph Schüller,Ingrid E Frohner,Gustav Ammerer,Karl Kuchler

doi:10.1074/jbc.m803095200

Christa Gregori, Christoph Schüller + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m803095200

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Abstract

The Saccharomyces cerevisiae zinc cluster regulator War1 mediates an essential transcriptional and adaptive response to weak organic acid stress. Here we investigate the mechanism of War1 activation upon weak acid stress. We identified several gain-of-function WAR1 alleles mapping to the central War1 region. These mutations constitutively increase levels of the plasma membrane ABC transporter Pdr12, the main War1 target mediating stress adaptation. Functional analysis of War1 reveals that the central region and its C-terminal activation domain are required for function. Notably, the native DNA-binding and dimerization domains appear dispensable for War1 activity, because they can be replaced by a LexA DNA-binding domain. Chromatin immunoprecipitation demonstrates elevated promoter affinity of activated War1, because its PDR12 promoter association increases upon stress. Hyperactive WAR1 alleles have constitutively high PDR12 promoter association. Furthermore, fluorescence resonance energy transfer of functional CFP-War1-YFP proteins also demonstrates conformational changes of stress-activated War1 in vivo. Our results suggest a mechanism whereby War1 activation is accompanied by conformational changes enhancing promoter association, thus initiating the adaptation process.

Highlights

Weak organic acids, such as potassium sorbate, calcium propionate, and sodium benzoate, are commonly used to preserve foods and beverages [1]
The reporter strain YCG8 (PPDR12-HIS3 war1⌬) transformed with the plasmid pCGWAR1 [21] containing wild type WAR1 was unable to grow on synthetic medium (SC)-His in the presence of 50 mM 3-AT
Addition of 1 mM sorbate to the same plates increased PDR12 promoter-driven HIS3 expression to a level that allowed for growth on this concentration of 3-AT

Summary

Introduction

Weak organic acids, such as potassium sorbate, calcium propionate, and sodium benzoate, are commonly used to preserve foods and beverages [1]. Weak acid regulation of PDR12 occurs at the level of transcription and requires the Zn(II)2Cys zinc cluster transcription factor War, which recognizes and binds response elements within the PDR12 promoter [7]. An orthologue mediating sorbate tolerance was identified in the distantly related human fungal pathogen Candida albicans [8], but the mechanism by which weak organic acids cause War activation is currently an open question. Fungal zinc cluster proteins are involved in diverse cellular processes, including carbon and amino acid metabolism, stress response, mitochondrial signaling, peroxisome biogenesis, and ergosterol biosynthesis [9]. Like War1 [7], many other zinc cluster proteins permanently localize to the nucleus and constitutively bind their target promoters. Several zinc cluster proteins are activating their target genes in response to a signal generated through the interaction with small ligand molecules

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