The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase

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...

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Autores: Molero Merinero, Cristina, Casado Vázquez, Carlos, Ariño Carmona, Joaquín|||0000-0002-6774-2987
Formato: artículo
Fecha de publicación:2017
País:España
Recursos:Universitat Autònoma de Barcelona
Repositorio:Dipòsit Digital de Documents de la UAB
Idioma:inglés
OAI Identifier:oai:ddd.uab.cat:205909
Acesso em linha:https://ddd.uab.cat/record/205909
https://dx.doi.org/urn:doi:10.1038/s41598-017-09360-5
Access Level:acceso abierto
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spelling The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphataseA mutagenesis analysisMolero Merinero, CristinaCasado Vázquez, CarlosAriño Carmona, Joaquín|||0000-0002-6774-2987The 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 afecting 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 signifcant 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 difers, but could share some structural features. 22017-01-0120172017-01-01Articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://ddd.uab.cat/record/205909https://dx.doi.org/urn:doi:10.1038/s41598-017-09360-5reponame:Dipòsit Digital de Documents de la UABinstname:Universitat Autònoma de BarcelonaInglésengopen accesshttp://purl.org/coar/access_right/c_abf2Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:ddd.uab.cat:2059092026-06-06T12:50:31Z
dc.title.none.fl_str_mv The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase
A mutagenesis analysis
title The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase
spellingShingle The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase
Molero Merinero, Cristina
title_short The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase
title_full The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase
title_fullStr The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase
title_full_unstemmed The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase
title_sort The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase
dc.creator.none.fl_str_mv Molero Merinero, Cristina
Casado Vázquez, Carlos
Ariño Carmona, Joaquín|||0000-0002-6774-2987
author Molero Merinero, Cristina
author_facet Molero Merinero, Cristina
Casado Vázquez, Carlos
Ariño Carmona, Joaquín|||0000-0002-6774-2987
author_role author
author2 Casado Vázquez, Carlos
Ariño Carmona, Joaquín|||0000-0002-6774-2987
author2_role author
author
description 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 afecting 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 signifcant 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 difers, but could share some structural features.
publishDate 2017
dc.date.none.fl_str_mv 2
2017-01-01
2017
2017-01-01
dc.type.none.fl_str_mv Article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
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dc.identifier.none.fl_str_mv https://ddd.uab.cat/record/205909
https://dx.doi.org/urn:doi:10.1038/s41598-017-09360-5
url https://ddd.uab.cat/record/205909
https://dx.doi.org/urn:doi:10.1038/s41598-017-09360-5
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
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eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Dipòsit Digital de Documents de la UAB
instname:Universitat Autònoma de Barcelona
instname_str Universitat Autònoma de Barcelona
reponame_str Dipòsit Digital de Documents de la UAB
collection Dipòsit Digital de Documents de la UAB
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