f3ce95cb1ee43b8a0112fa2dcafa7de4 pmc_34946993.pdf 1d2db8e18fa3b4e1015ea6daabfe26f2d326ef10 pmc_34946993.pdf 42b623064fc0fe75ace7e7b0b2f4547806a0897b640d41c2420dde1541c13008 pmc_34946993.pdf Title: The Toxic Effects of Ppz1 Overexpression Involve Nha1-Mediated Deregulation of K+ and H+ Homeostasis Subject: The alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K+ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na+, K+/H+ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H+ and efflux of K+, which is detrimental for growth. Keywords: Ppz1 phosphatase; cation homeostasis; Nha1; intracellular pH; K+ transport; Saccharomyces cerevisiae Author: Marcel Albacar, Lenka Sacka, Carlos Calafí, Diego Velázquez, Antonio Casamayor, Joaquín Ariño and Olga Zimmermannova Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Nov 29 03:29:42 2021 CET ModDate: Mon Nov 29 03:47:33 2021 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: 3376140 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SSNIUI+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 AHRQMW+VnURWPalladioL Type 1 Custom yes yes no 15 0 GAYHJY+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 VGVITQ+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 FUTQYW+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 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yes no 242 0 ICHJOO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 245 0 ICHJOK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 265 0 ICHJOL+PalatinoLinotype CID TrueType Identity-H yes yes yes 268 0 ICHJOM+PalatinoLinotype TrueType WinAnsi yes yes no 274 0 ICHJOO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 277 0 ICHJOK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 287 0 ICHJOL+PalatinoLinotype CID TrueType Identity-H yes yes yes 290 0 ICHJOM+PalatinoLinotype TrueType WinAnsi yes yes no 296 0 ICHJOO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 299 0 ICHJOK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 312 0 ICHJOL+PalatinoLinotype CID TrueType Identity-H yes yes yes 315 0 ICHJOM+PalatinoLinotype TrueType WinAnsi yes yes no 321 0 ICHJOO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 324 0 ICHPLN+Arial CID TrueType Identity-H yes yes yes 327 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-22 02:25:06 CET RepresentationInformation: pmc_34946993.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-01-10 07:56:09 CET Size: 3376140 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 594 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Yeast Strains and Culture Media Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Generation of Nha1 Variants Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Media Acidification Tests Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Preparation of Protein Extracts and Inmmunoblot Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Intracellular pH Measurements Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Potassium Content and Efflux Measurements Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Fluorescence Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Statistics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Children: Item: Title: The Toxic Effects of Ppz1 Overexpression Are Supressed by Deletion of the NHA1 Antiporter Gene but Not by Potassium Supplementation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Cells Overexpressing Ppz1 Show Noticiable Loss of Intracellular Potassium Levels and Increased Intracellular Acidification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Functionally Impaired Versions of Nha1 Do Not Counteract the Effect of Nha1 Loss in Ppz1-Overexpressing Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Overexpression of Ppz1 Limits the Capacity for Acidification of the Medium Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Info: Title: The Toxic Effects of Ppz1 Overexpression Involve Nha1-Mediated Deregulation of K+ and H+ Homeostasis Author: Marcel Albacar, Lenka Sacka, Carlos Calafí, Diego Velázquez, Antonio Casamayor, Joaquín Ariño and Olga Zimmermannova Subject: The alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K+ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na+, K+/H+ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H+ and efflux of K+, which is detrimental for growth. 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