8836f6f7c7fb2622e8e1398a0bbb6b12 intjoumolsci_a2020v21n20p7733.pdf d95ffce09bea19d5b5f99185cd4cb7a0c3aedb1b intjoumolsci_a2020v21n20p7733.pdf aa8ed2d28b03f1eef7b11da79ee4782e1c6c8bfb2fdfefb9160a17e61ef65d84 intjoumolsci_a2020v21n20p7733.pdf Title: The N-Terminal Region of Yeast Protein Phosphatase Ppz1 Is a Determinant for Its Toxicity Subject: The Ppz enzymes are Ser/Thr protein phosphatases present only in fungi that are characterized by a highly conserved C-terminal catalytic region, related to PP1c phosphatases, and a more divergent N-terminal extension. In Saccharomyces cerevisiae, Ppz phosphatases are encoded by two paralog genes, PPZ1 and PPZ2. Ppz1 is the most toxic protein when overexpressed in budding yeast, halting cell proliferation, and this effect requires its phosphatase activity. We show here that, in spite of their conserved catalytic domain, Ppz2 was not toxic when tested under the same conditions as Ppz1, albeit Ppz2 levels were somewhat lower. Remarkably, a hybrid protein composed of the N-terminal extension of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1, even if its expression level was comparable to that of Ppz2. Similar amounts of yeast PP1c (Glc7) produced an intermediate effect on growth. Mutation of the Ppz1 myristoylable Gly2 to Ala avoided the localization of the phosphatase at the cell periphery but only slightly attenuated its toxicity. Therefore, the N-terminal extension of Ppz1 plays a key role in defining Ppz1 toxicity. This region is predicted to be intrinsically disordered and contains several putative folding-upon-binding regions which are absent in Ppz2 and might be relevant for toxicity. Keywords: protein phosphatases; cell growth arrest; hybrid proteins; Saccharomyces cerevisiae Author: Carlos Calafí, María López-Malo, Marcel Albacar, Antonio Casamayor and Joaquín Ariño Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Thu Oct 22 04:23:36 2020 CEST ModDate: Thu Oct 22 05:44:13 2020 CEST Custom Metadata: no Metadata Stream: no 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: 3697162 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AHRQMW+VnURWPalladioL Type 1 Custom yes yes no 10 0 EZRNSZ+Pxsy Type 1 Builtin yes yes no 15 0 PVEMZV+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 UYVWRL+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 UDEGHW+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 OFLXAT+Rpxr Type 1 Builtin yes yes 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285 0 MBCDNF+PalatinoLinotype TrueType WinAnsi yes yes no 303 0 MBCEAG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 306 0 MBCEBH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 309 0 MBCELF+TimesNewRoman CID TrueType Identity-H yes yes yes 312 0 TGMEOR+Rpcxr Type 1 Builtin yes yes no 332 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 04:24:45 CEST RepresentationInformation: intjoumolsci_a2020v21n20p7733.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:39:36 CEST Size: 3697162 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 569 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: The N-Terminal Region of Ppz Phosphatases Is Relevant for Toxicity Destination: subsection.2.1 Item: Title: Mutation of Myristoylable Gly2 in Ppz1 only Slightly Attenuates Toxicity Destination: subsection.2.2 Item: Title: Comparison of the Effect of Glc7 and Ppz2 Expression Destination: subsection.2.3 Item: Title: Comparative Analysis of Ppz1 and Ppz2 N-Terminal Regions Destination: subsection.2.4 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Yeast Strains Destination: subsection.4.1 Item: Title: Growth Conditions Destination: subsection.4.2 Item: Title: Plasmid Construction Destination: subsection.4.3 Item: Title: Preparation of Extracts and Immunoblotting Destination: subsection.4.4 Item: Title: In Vivo Fluorescence Microscopy Destination: subsection.4.5 Item: Title: Other Methods Destination: subsection.4.6 Item: Title: References Destination: subsection.4.6 Info: Title: The N-Terminal Region of Yeast Protein Phosphatase Ppz1 Is a Determinant for Its Toxicity Author: Carlos Calafí, María López-Malo, Marcel Albacar, Antonio Casamayor and Joaquín Ariño Subject: The Ppz enzymes are Ser/Thr protein phosphatases present only in fungi that are characterized by a highly conserved C-terminal catalytic region, related to PP1c phosphatases, and a more divergent N-terminal extension. In Saccharomyces cerevisiae, Ppz phosphatases are encoded by two paralog genes, PPZ1 and PPZ2. Ppz1 is the most toxic protein when overexpressed in budding yeast, halting cell proliferation, and this effect requires its phosphatase activity. We show here that, in spite of their conserved catalytic domain, Ppz2 was not toxic when tested under the same conditions as Ppz1, albeit Ppz2 levels were somewhat lower. Remarkably, a hybrid protein composed of the N-terminal extension of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1, even if its expression level was comparable to that of Ppz2. Similar amounts of yeast PP1c (Glc7) produced an intermediate effect on growth. Mutation of the Ppz1 myristoylable Gly2 to Ala avoided the localization of the phosphatase at the cell periphery but only slightly attenuated its toxicity. Therefore, the N-terminal extension of Ppz1 plays a key role in defining Ppz1 toxicity. This region is predicted to be intrinsically disordered and contains several putative folding-upon-binding regions which are absent in Ppz2 and might be relevant for toxicity. 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