The transcriptional response of the yeast Na(+)-ATPase ENA1 gene to alkaline stress involves three main signaling pathways.
Adaptive response of the yeast Saccharomyces cerevisiae to environmental alkalinization results in remodeling of gene expression. A key target is the gene ENA1, encoding a Na(+)-ATPase, whose induction by alkaline pH has been shown to involve calcineurin and the Rim101/Nrg1 pathway. Previous functional analysis of the ENA1 promoter revealed a calcineurin-independent pH responsive region (ARR2, 83 nucleotides). We restrict here this response to a small (42 nucleotides) ARR2 5.-region, named MCIR (minimum calcineurin independent response), which contains a MIG element, able to bind Mig1,2 repressors. High pH-induced response driven from this region was largely abolished in snf1 cells and moderately reduced in a rim101 strain. Cells lacking Mig1 or Mig2 repressors had a near wild type response, but the double mutant presented a high level of expression upon alkaline stress. Deletion of NRG1 (but not of NRG2) resulted in increased expression. Induction from the MCIR region was marginal in a quadruple mutant lacking Nrg1,2 and Mig1,2 repressors. In vitro band shift experiments demonstrated binding of Nrg1 to the 5. end of the ARR2 region. Furthermore, we show that Nrg1 binds in vivo around the MCIR region under standard growth conditions, and that binding is largely abolished after high pH stress. Therefore, the calcineurin-independent response of the ENA1 gene is under the regulation of Rim101 (through Nrg1) and Snf1 (through Nrg1 and Mig2). Accordingly, induction by alkaline stress of the entire ENA1 promoter in a snf1 rim101 mutant in the presence of the calcineurin inhibitor FK506 is completely abolished. Thus, the transcriptional response to alkaline stress of the ENA1 gene integrates three different signaling pathways.
Platara
Maria
M
Department of Bioquímica i Biologia Molecular, Universitat Autónoma de Barcelona, Bellaterra 08193, Barcelona.
Ruiz
Amparo
A
Serrano
Raquel
R
Palomino
Aarón
A
Moreno
Fernando
F
Ariño
Joaquín
J
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D019703:Calcineurin / Q000378:metabolism; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D002874:Chromosome Mapping; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase; D016559:Tacrolimus / Q000494:pharmacology; D014158:Transcription, Genetic
D027682:Cation Transport Proteins; C086375:ENA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019703:Calcineurin; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D016559:Tacrolimus