A genetic screen identifies mutations in the yeast WAR1 gene, linking transcription factor phosphorylation to weak‐acid stress adaptation

Abstract

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.

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

“…2A). The lack of the faster migrating band was already observed for another constitutively active mutant War1-K762N as reported earlier (21). In contrast, the mutants War1-A640T and War1-S703P migrated as double bands with a more pronounced difference when compared with the wild type.…”

“…Notably, as for War1, activation of several zinc cluster factors triggers appearance of phosphorylated forms, and it is not entirely clear whether phosphorylation is the cause or consequence of activation. The rapid weak acid-triggered activation of War1 is accompanied by the concomitant appearance of several phosphorylated War1 isoforms (7,21,22). As reported recently, exposure to the drug fluphenazine causes transient activation of the zinc cluster proteins Pdr1/Pdr3 on a similar time scale (23).…”

“…The previously identified WAR1 gain-of-function mutant exchanges Lys-762 by asparagine (21). The mutant independently isolated in this screen encodes arginine at position 762.…”

“…Regarding War1, a physical interaction with an inducer has not yet been demonstrated. Moreover, genetic evidence for similar upstream regulators is not available (21). Hence, based on the similarity to the above-mentioned family members, a direct regulatory mechanism exerted by weak organic acids seems plausible.…”

“…Cell lysates equivalent to 0.5 A 600 units were resolved by 7.5% SDS-PAGE. War1 and Pdr12 were visualized by immunoblotting using ECL (Amersham Biosciences) and the polyclonal anti-War1 (21) and anti-Pdr12 antisera (4). ␤-Galactosidase assays were carried out in triplicate using total cell-free crude glass bead extracts as described previously (24).…”