The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis

Abstract The Ser/Thr protein phosphatase (PPase) Ppz1 is an enzyme related to the ubiquitous type-1 PPases (PP1c) but found only in fungi. It is regulated by an inhibitory subunit, Hal3, which binds to its catalytic domain. Overexpression of Ppz1 is highly toxic for yeast cells, so its de-regulation...

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Autores principales:	Cristina Molero, Carlos Casado, Joaquín Ariño
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7c4ccfbf0c294cab8ab5574d3ef3e0c3
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spelling	oai:doaj.org-article:7c4ccfbf0c294cab8ab5574d3ef3e0c32021-12-02T12:30:36ZThe inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis10.1038/s41598-017-09360-52045-2322https://doaj.org/article/7c4ccfbf0c294cab8ab5574d3ef3e0c32017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-09360-5https://doaj.org/toc/2045-2322Abstract The Ser/Thr protein phosphatase (PPase) Ppz1 is an enzyme related to the ubiquitous type-1 PPases (PP1c) but found only in fungi. It is regulated by an inhibitory subunit, Hal3, which binds to its catalytic domain. Overexpression of Ppz1 is highly toxic for yeast cells, so its de-regulation has been proposed as a target for novel antifungal therapies. While modulation of PP1c by its many regulatory subunits has been extensively characterized, the manner by which Hal3 controls Ppz1 remains unknown. We have used error-prone PCR mutagenesis to construct a library of Ppz1 variants and developed a functional assay to identify mutations affecting the binding or/and the inhibitory capacity of Hal3. We have characterized diverse Ppz1 mutated versions in vivo and in vitro and found that, although they were clearly refractory to Hal3 inhibition, none of them exhibited significant reduction in Hal3 binding. Mapping the mutations strengthened the notion that Hal3 does not interact with Ppz1 through its RVxF-like motif (found in most PP1c regulators). In contrast, the most relevant mutations mapped to a conserved α-helix region used by mammalian Inhibitor-2 to regulate PP1c. Therefore, modulation of PP1c and Ppz1 by their subunits likely differs, but could share some structural features.Cristina MoleroCarlos CasadoJoaquín AriñoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-14 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Cristina Molero Carlos Casado Joaquín Ariño The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis
description	Abstract The Ser/Thr protein phosphatase (PPase) Ppz1 is an enzyme related to the ubiquitous type-1 PPases (PP1c) but found only in fungi. It is regulated by an inhibitory subunit, Hal3, which binds to its catalytic domain. Overexpression of Ppz1 is highly toxic for yeast cells, so its de-regulation has been proposed as a target for novel antifungal therapies. While modulation of PP1c by its many regulatory subunits has been extensively characterized, the manner by which Hal3 controls Ppz1 remains unknown. We have used error-prone PCR mutagenesis to construct a library of Ppz1 variants and developed a functional assay to identify mutations affecting the binding or/and the inhibitory capacity of Hal3. We have characterized diverse Ppz1 mutated versions in vivo and in vitro and found that, although they were clearly refractory to Hal3 inhibition, none of them exhibited significant reduction in Hal3 binding. Mapping the mutations strengthened the notion that Hal3 does not interact with Ppz1 through its RVxF-like motif (found in most PP1c regulators). In contrast, the most relevant mutations mapped to a conserved α-helix region used by mammalian Inhibitor-2 to regulate PP1c. Therefore, modulation of PP1c and Ppz1 by their subunits likely differs, but could share some structural features.
format	article
author	Cristina Molero Carlos Casado Joaquín Ariño
author_facet	Cristina Molero Carlos Casado Joaquín Ariño
author_sort	Cristina Molero
title	The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis
title_short	The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis
title_full	The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis
title_fullStr	The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis
title_full_unstemmed	The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis
title_sort	inhibitory mechanism of hal3 on the yeast ppz1 phosphatase: a mutagenesis analysis
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/7c4ccfbf0c294cab8ab5574d3ef3e0c3
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The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis

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