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

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 DNAbinding 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.Weak organic acids, such as potassium sorbate, calcium propionate, and sodium benzoate, are commonly used to preserve foods and beverages (1). In Saccharomyces cerevisiae, uncharged weak acids diffuse across the plasma membrane and dissociate inside the cell, causing intracellular acidification, free radical production, and oxidative stress (1), as well as inhibition of several metabolic processes (2). The yeast ABC transporter Pdr12 confers resistance to monocarboxylic acids with chain lengths up to C 7 (3, 4). Only organic acids but not other stresses cause a dramatic induction of PDR12 transcription and a concomitant increase of Pdr12 (4, 5). A physiological role of Pdr12 is suggested from its proposed ability to extrude amino acid catabolism by-products (6). Weak acid regulation of PDR12 occurs at the level of transcription and requires the Zn(II) 2 Cys 6 zinc cluster transcription factor War1, which recognizes and binds response elements within the PDR12 promoter (7). War1 is functionally conserved among several yeast species, including fungal pathogens. 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 War1 activation is currently an open question.In S. cerevisiae, some 55 members of the binuclear zinc cluster regulator family exist (for a review see Ref. 9). Most exhibit a similar molecular architecture and domain organization. The N-terminal DNA-binding domain is followed by a coiled-coil dimerization domain, by an extended region of limited homology referred to as the "middle homology region" (MHR), 3 and finally by a short acidic stretch encompassing the C-terminal activation domain (10). Fungal zinc cluster pro...

Sorbic acid stress activates the Candida glabrata high osmolarity glycerol MAP kinase pathway

“…Attempts to assign a regulatory function to weak acid induced changes of the War1 phosphorylation status, which results in a robust migration difference, were unsuccessful (Frohner et al, 2010; Mollapour and Piper, 2012). It has been suggested that War1 becomes directly activated by weak acids triggering conformational changes (Gregori et al, 2008). In addition, adaptation to weak acids also requires Haa1, a further weak acid inducible transcription factor S. cerevisiae (Fernandes et al, 2005).…”

Adaptive Response and Tolerance to Weak Acids inSaccharomyces cerevisiae: A Genome-Wide View

“…1.63 Preservation of cheeses, jams, and fish products Gregori et al, 2008;Abbot et al, 2007;Makrantoni et al, 2007Schuller et al, 2004Mollapour et al, 2004;Mollapour et al, 2006Hatzixhantis et al, 2003Kren et al, 2003;Pearce et al, 2001;Piper et al, 1999;Holyoak et al, 1996;Holyoak et al, 1999 Lactic acid 3.86…”

“…Preservation of bakery and fresh dairy products Mira et al, 2009;Gregori et al, 2008;Mollapour and Piper, 2007;Abbot et al, 2007;Fernandes et al, 2003;Simõ es et al, 2006;Piper et al, 1998, Kren et al, 2003 Octanoic acid 4.89…”

Role of the DHH1 Gene in the Regulation of Monocarboxylic Acids Transporters Expression in Saccharomyces cerevisiae

“…For instance, the expression of the transcription factor encoding gene WAR1 (involved in weak acid resistance) and of its main target gene PDR12 decreased in the dhh1 mutant. Noteworthy, the level of expression of DHH1 increased in a WAR1 gain of function mutant [87]. Similarly, Dhh1 apparently controls the level of expression of several proteins regulating mRNA stability, including for instance PUF2 and PUF3 .…”