Mutations at the hydrophobic core affect Hal3 trimer stability, reducing its Ppz1 inhibitory capacity but not its PPCDC moonlighting function
Santolaria Bello, Carlos (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Velázquez, Diego (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Strauss, Erick (Stellenbosch University. Department of Biochemistry)
Ariño Carmona, Joaquín (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)

Data:	2018
Resum:	S. cerevisiae Hal3 (ScHal3) is a moonlighting protein that, is in its monomeric state, regulates the Ser/Thr protein phosphatase Ppz1, but also joins ScCab3 (and in some instances the Hal3 paralog Vhs3) to form an unusual heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. PPCDC is required for CoA biosynthesis and in most eukaryotes is a homotrimeric complex with three identical catalytic sites at the trimer interfaces. However, in S. cerevisiae the heterotrimeric arrangement results in a single functional catalytic center. Importantly, the specific structural determinants that direct Hal3's oligomeric state and those required for Ppz1 inhibition remain largely unknown. We mutagenized residues in the predicted hydrophobic core of ScHal3 (L403-L405) and the plant Arabidopsis thaliana Hal3 (AtHal3, G115-L117) oligomers and characterized their properties as PPCDC components and, for ScHal3, also as Ppz1 inhibitor. We found that in AtHal3 these changes do not affect trimerization or PPCDC function. Similarly, mutation of ScHal3 L403 has no effect. In contrast, ScHal3 L405E fails to form homotrimers, but retains the capacity to bind Cab3-explaining its ability to rescue a hal3 vhs3 synthetically lethal mutation. Remarkably, the L405E mutation decreases Hal3's ability to interact with and to inhibit Ppz1, confirming the importance of the oligomer/monomer equilibrium in Hal3's Ppz1 regulating function.
Ajuts:	Ministerio de Ciencia e Innovación BFU2017-82574-P Ministerio de Economía y Competitividad BFU2014-54591-C2-1-P
Drets:	Aquest 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.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Arabidopsis Proteins ; Carboxy-Lyases ; Cell Cycle Proteins ; Hydrophobic and Hydrophilic Interactions ; Models, Molecular ; Mutagenesis, Site-Directed ; Phosphoprotein Phosphatases ; Protein Binding ; Protein Multimerization ; Recombinant Proteins ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae Proteins ; Synthetic Lethal Mutations
Publicat a:	Scientific reports, Vol. 8 (2018) , art. 14701, ISSN 2045-2322

DOI: 10.1038/s41598-018-32979-x
PMID: 30279472

12 p, 2.4 MB

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