TY - JOUR TI - Identification and characterization of APP1-encoded phosphatidate phosphatase in Saccharomyces cerevisiae DO - https://doi.org/doi:10.7282/T3VM4B0Q PY - 2013 AB - Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomyces cerevisiae, PAP activity is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1Δ dpp1Δ lpp1Δ triple mutant indicated another gene(s) encoding the enzyme. I purified PAP activity from the pah1Δ dpp1Δ lpp1Δ triple mutant by salt-extraction of mitochondria, followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, I found that APP1, a gene whose molecular function has been unknown, confers ~30 % PAP activity of wild type cells. The overexpression of APP1 in the pah1Δ dpp1Δ lpp1Δ mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p that was heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1Δ mutation into the pah1Δ dpp1Δ lpp1Δ mutant resulted in a complete loss of PAP activity, indicating that the enzyme in S. cerevisiae is encoded by APP1, PAH1, DPP1, and LPP1. Protein A-tagged App1p PAP was expressed and purified from S. cerevisiae. The App1p PAP activity followed saturation kinetics with respect to the molar concentration of phosphatidic acid (PA) (Km = 0.5 mM) but followed positive cooperative (Hill number of ~2.3) kinetics with respect to the surface concentration of PA (Km = 2.0 mol %) in Triton X-100/PA mixed micelles. The App1p also exhibited lysoPA phosphatase and diacylglycerol pyrophosphate phosphatase activities. The order of substrate preference was phosphatidic acid> diacylglycerol pyrophosphate > lysoPA. The maximum PAP activity was dependent on Mg2+ ions at pH 7.5 at 30 ⁰C. The activation energy for the reaction was 16.5 kcal/mol, and the enzyme was labile above 40 ⁰C. The App1p PAP activity was inhibited by Ca2+, Mn2+, Zn2+, N-ethylmaleimide, propranolol, nucleotides, diacylglycerol, phosphatidylethanolamine, phosphatidylcholine, and sphinganine, while lysoPA, cardiolipin, phosphatidylglycerol, phosphatidylserine, sphingosine and sphingosine 1-phosphate stimulated the activity. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity. KW - Food Science KW - Phosphatidate phosphatase KW - Saccharomyces cerevisiae LA - eng ER -