Tài liệu Báo cáo khoa học: A genetic screen identifies mutations in the yeastWAR1 gene, linking

A genetic screen identifies mutations in the yeast WAR1

gene, linking transcription factor phosphorylation

to weak-acid stress adaptation

Christa Gregori*, Bettina Bauer*, Chantal Schwartz, Angelika Kren, Christoph Schu¨ ller§

and Karl Kuchler

Medical University Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, Campus Vienna Biocenter, Austria

Weak acids have a long history as additives in food

preservation. In addition to sulfites used in wine mak￾ing, acetic, sorbic, benzoic and propionic acids are

commonly used in the food and beverage industry

to prevent spoilage [1,2]. In solution, weak acids exist

in a dynamic equilibrium between undissociated,

uncharged molecules and their anionic form. These

acids display increased antimicrobial action at low pH,

which favors the undissociated state. The uncharged

molecules can readily diffuse through the plasma

Keywords

ABC transporter; stress response; weak

organic acids; yeast; zinc finger

Correspondence

K. Kuchler, Medical University Vienna, Max

F. Perutz Laboratories, Department of

Medical Biochemistry, Campus Vienna

Biocenter, Dr Bohr-Gasse 9 ⁄ 2, A-1030,

Vienna, Austria

Fax: +43 1 4277 9618

Tel: +43 1 4277 61807

E-mail: [email protected]

Present address

Universite´ de Nice-Sophia Antipolis,

Inserm, U636, Centre de Biochimie, UFR

Sciences, Parc Valrose, Nice, France

Institute of Biochemistry and Genetics,

Department of Clinical and Biological

Research (DKBW), Basel, Switzerland

§University of Vienna, Max F. Perutz

Laboratories, Department of Biochemistry &

Molecular and Cellular Biology, Campus

Vienna Biocenter, Austria

*

These authors contributed equally to this

work

(Received 11 January 2007, revised 4 April

2007, accepted 19 April 2007)

doi:10.1111/j.1742-4658.2007.05837.x

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 ani￾ons, 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 sorb￾ate. 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.

Abbreviations

GST, glutathione S-transferase; MHR, middle homology region; NLS, nuclear localization signal; PDR, pleiotropic drug resistance; WARE,

weak-acid-response element; YPD, yeast peptone ⁄ dextrose.

3094 FEBS Journal 274 (2007) 3094–3107 ª 2007 The Authors Journal compilation ª 2007 FEBS