Weak organic acid stress triggers hyperphosphorylation of the yeast zinc-finger transcription factor War1 and dampens stress adaptation.
Exposure of Saccharomyces cerevisiae to weak organic acids such as sorbate, propionate, or benzoate rapidly induces the plasma membrane ABC transporter Pdr12, requiring the Zn(II)(2)Cys(6) zinc-finger transcription factor War1. Weak acid stress rapidly triggers War1 phosphorylation but its role for War1 function is not clear yet. Here, we provide new insights into sorbate-induced phosphorylation of War1. A War1 zinc-finger mutant is still hyperphosphorylated in response to sorbate stress, indicating that War1 phosphorylation occurs independently of DNA recruitment. To map and identify phosphoresidues, War1 purified from stressed and unstressed cells was subjected to semiquantitative phosphopeptide mass spectrometry analysis. Remarkably, we show that weak acid stress causes a dramatic hyperphosphorylation of several already prephosphorylated residues. WAR1 alleles harboring combinations of mutations identified phosphoresidues were generated, some of which display altered gel mobility. Certain mutational combinations almost completely abolish stress-induced gel-shift, suggesting alternative phosphorylation. Surprisingly, PDR12 expression levels are similar in these mutants, demonstrating that War1 phosphorylation is not required for PDR12 induction. Strikingly, absence of hyperphosphorylation in response to stress leads to a faster stress adaptation, suggesting that phosphorylation might play a role in stabilizing War1 activity on the promoter elements, hence changing the dynamics and kinetics of the stress response.
Frohner
Ingrid E
IE
Medical University Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, Vienna, Austria.
Gregori
Christa
C
Anrather
Dorothea
D
Roitinger
Elisabeth
E
Schüller
Christoph
C
Ammerer
Gustav
G
Kuchler
Karl
K
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000143:Acids / Q000494:pharmacology*; D000222:Adaptation, Physiological / Q000187:drug effects*; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological / Q000187:drug effects*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000143:Acids; C472673:Pdr12 protein, S cerevisiae; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C472674:War1 protein, S cerevisiae