fbf1e879f94c59ff0c99d9cdb215ac2b ijms_24_05548.pdf 9a8c1e9d6ad86483b5597b880cff1ab21c6c7878 ijms_24_05548.pdf c102e849f6afbfd5b78e97d30e2064bc6277730a77bec3dbabc6b5688aee8026 ijms_24_05548.pdf Title: The ENA1 Na+-ATPase Gene Is Regulated by the SPS Sensing Pathway and the Stp1/Stp2 Transcription Factors Subject: The Saccharomyces cerevisiae ENA1 gene, encoding a Na+-ATPase, responds transcriptionally to the alkalinization of the medium by means of a network of signals that involves the Rim101, the Snf1 and PKA kinases, and the calcineurin/Crz1 pathways. We show here that the ENA1 promoter also contains a consensus sequence, located at nt -553/-544, for the Stp1/2 transcription factors, the downstream components of the amino acid sensing SPS pathway. Mutation of this sequence or deletion of either STP1 or STP2 decreases the activity of a reporter containing this region in response to alkalinization as well as to changes in the amino acid composition in the medium. Expression driven from the entire ENA1 promoter was affected with similar potency by the deletion of PTR3, SSY5, or simultaneous deletion of STP1 and STP2 when cells were exposed to alkaline pH or moderate salt stress. However, it was not altered by the deletion of SSY1, encoding the amino acid sensor. In fact, functional mapping of the ENA1 promoter reveals a region spanning from nt -742 to -577 that enhances transcription, specifically in the absence of Ssy1. We also found that the basal and alkaline pH-induced expression from the HXT2, TRX2, and, particularly, SIT1 promoters was notably decreased in an stp1 stp2 deletion mutant, whereas the PHO84 and PHO89 gene reporters were unaffected. Our findings add a further layer of complexity to the regulation of ENA1 and suggest that the SPS pathway might participate in the regulation of a subset of alkali-inducible genes. Keywords: transcriptional regulation; environmental alkalinization; stress response; Saccharomyces cerevisiae Author: Abdelghani Zekhnini, Marcel Albacar, Antonio Casamayor and Joaquín Ariño Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Mar 14 10:47:49 2023 CET ModDate: Tue Mar 14 10:53:14 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 8127064 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WLENKM+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 MWHIBA+VnURWPalladioL Type 1 Custom yes yes no 15 0 SNPJTP+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 HEIKNL+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 VRPOZE+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 KMPOEG+URWPalladioL-BoldItal Type 1 Custom yes yes no 32 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 36 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 54 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 57 0 KSGHMQ+PazoMath Type 1 Builtin yes yes no 94 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 109 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes no 188 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-17 02:31:30 CEST RepresentationInformation: ijms_24_05548.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-05-10 01:34:23 CEST Size: 8127064 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 428 FreeObjects: 17 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: A Short Region of the ENA1 Promoter Containing the Mig1/2 Consensus Sequence Unexpectedly Increases the Activity of a Hybrid Promoter Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: The Putative Stp1/2 Site Present in the ENA1 Promoter Is Relevant for Transcriptional Induction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: The Stp1/2 Site in the ENA1 Promoter Responds to the Activation of the SPS Pathway Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Non-Homogenous Impact of Mutations in the SPS Pathway on the Expression Driven from the Entire ENA1 Promoter Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Functional Mapping of the Alkaline pH Response of the ENA1 Promoter and Its Relationship with the SPS Pathway Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Participation of Stp1/2 in the Expression of Other Genes Responsive to Alkaline pH Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Children: Item: Title: Yeast Strains and Culture Media Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Recombinant DNA Techniques and Reporter Construction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Fluorimetric Determination of yeGFP Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Flow Cytometry Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: -Galactosidase Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Info: Title: The ENA1 Na+-ATPase Gene Is Regulated by the SPS Sensing Pathway and the Stp1/Stp2 Transcription Factors Author: Abdelghani Zekhnini, Marcel Albacar, Antonio Casamayor and Joaquín Ariño Subject: The Saccharomyces cerevisiae ENA1 gene, encoding a Na+-ATPase, responds transcriptionally to the alkalinization of the medium by means of a network of signals that involves the Rim101, the Snf1 and PKA kinases, and the calcineurin/Crz1 pathways. We show here that the ENA1 promoter also contains a consensus sequence, located at nt -553/-544, for the Stp1/2 transcription factors, the downstream components of the amino acid sensing SPS pathway. Mutation of this sequence or deletion of either STP1 or STP2 decreases the activity of a reporter containing this region in response to alkalinization as well as to changes in the amino acid composition in the medium. Expression driven from the entire ENA1 promoter was affected with similar potency by the deletion of PTR3, SSY5, or simultaneous deletion of STP1 and STP2 when cells were exposed to alkaline pH or moderate salt stress. However, it was not altered by the deletion of SSY1, encoding the amino acid sensor. In fact, functional mapping of the ENA1 promoter reveals a region spanning from nt -742 to -577 that enhances transcription, specifically in the absence of Ssy1. We also found that the basal and alkaline pH-induced expression from the HXT2, TRX2, and, particularly, SIT1 promoters was notably decreased in an stp1 stp2 deletion mutant, whereas the PHO84 and PHO89 gene reporters were unaffected. Our findings add a further layer of complexity to the regulation of ENA1 and suggest that the SPS pathway might participate in the regulation of a subset of alkali-inducible genes. 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