Abstract
Unlike most other organisms, the essential five-step coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the genes encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remain unidentified. Sequence homology analyses suggest three candidates—Ykl088w, Hal3 and Vhs3—as putative PPCDC enzymes in Saccharomyces cerevisiae. Notably, Hal3 and Vhs3 have been characterized as negative regulatory subunits of the Ppz1 protein phosphatase. Here we show that YKL088w does not encode a third Ppz1 regulatory subunit, and that the essential roles of Ykl088w and the Hal3 and Vhs3 pair are complementary, cannot be interchanged and can be attributed to PPCDC-related functions. We demonstrate that while known eukaryotic PPCDCs are homotrimers, the active yeast enzyme is a heterotrimer that consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues. Our results unveil Hal3 and Vhs3 as moonlighting proteins involved in both CoA biosynthesis and protein phosphatase regulation.
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Acknowledgements
Thanks are given to B. Weiss (Emory University School of Medicine) for E. coli strain BW369, to A. Osterman (Burnham Institute for Medical Research) for the human PPCDC cDNA and to R. Serrano (Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas) and P. Sanz (Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas) for diverse constructs and plasmids. We thank H. Ceulemans, E. Simón, A. Barceló and A. Vivó for their contribution in different phases of the project. The excellent technical assistance of A. Vilalta, M.J. Álvarez and M. Robledo is acknowledged. This work was supported by grants BFU2005-06388-C4-04-BMC and BFU2008-04188-C03-01 to J.A. (Ministerio de Educación y Ciencia, Spain and Fondo Europeo de Desarrollo Regional) and grant FA2007041600013 to E.S. (National Research Foundation, South Africa). J.A. is recipient of an “Ajut de Suport a les Activitats dels Grups de Recerca” (2009SGR-1091, Generalitat de Catalunya). A.G. was recipient of a fellowship from the Spanish Ministry of Education and Science. J.A. and E.S. are recipients of a Spain/South Africa Research Cooperation grant (HS2007-0022) from the National Research Foundation (South Africa) and the Ministerio de Educación y Ciencia (Spain). J.A. dedicates this paper to the memory of his father.
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A.R. performed genetic, phenotypic and biochemical experiments. A.G. performed genetic experiments, prepared recombinant proteins and carried out binding and cross-linking experiments and in vitro activity measurements. I.M. and R.S. performed genetic experiments. J.A.A. prepared recombinant proteins and performed in vitro activity measurements. E.S. and J.A. directed the research and co-wrote the paper.
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Ruiz, A., González, A., Muñoz, I. et al. Moonlighting proteins Hal3 and Vhs3 form a heteromeric PPCDC with Ykl088w in yeast CoA biosynthesis. Nat Chem Biol 5, 920–928 (2009). https://doi.org/10.1038/nchembio.243
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DOI: https://doi.org/10.1038/nchembio.243
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