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Identification of a novel phosphatidate phosphatase gene PAH2 from Saccharomyces cerevisiae
Author(s) -
Chae Minjung,
Carman George M.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.787.2
Subject(s) - phosphatase , phosphatidate , biochemistry , diacylglycerol kinase , dusp6 , dephosphorylation , saccharomyces cerevisiae , mutant , alkaline phosphatase , gene , microbiology and biotechnology , protein phosphatase 1 , enzyme , biology , protein phosphatase 2 , chemistry , protein kinase c
Phosphatidate (PA) phosphatase catalyzes the dephosphorylation of PA to yield diacylglycerol (DAG) and Pi. In the yeast Sacchromyces cerevisiae , the enzyme activity is encoded by the PAH1 , DPP1 , and LPP1 genes. However, cells lacking these genes still exhibit PA phosphatase activity indicating the presence of another gene(s) encoding the enzyme. In this study, we purified this activity with an aim to identify the unknown gene(s) for PA phosphatase. We purified PA phosphatase activity from membranes of the pah1 Δ dpp1 Δ lpp1 Δ triple mutant by a nine‐step purification scheme, which included chromatography with phosphocellulose, Affi‐gel blue, phenyl Sepharose, Mono Q, and Superdex 200. The purification scheme resulted in an 8,300‐fold enrichment of PA phosphatase activity. Mass spectrometry analysis of proteins from SDS‐polyacrylamide gels revealed multiple proteins that contained putative phosphatase motifs. The analysis of PA phosphatase activity in available mutants lacking genes encoding proteins with the phosphatase motifs led to the identification of a novel PA phosphatase gene that we named PAH2 . Supported by NIH grant GM‐28140.