ISOLATION AND CHARACTERIZATION OF THE SACCHAROMYCES-CEREVISIAE LPP1 GENE ENCODING A MG2-INDEPENDENT PHOSPHATIDATE PHOSPHATASE()

The DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase enzym e accounts for half of the Mg2+-independent phosphatidate (PA) phospha tase activity in Saccharomyces cerevisiae, The LPP1 (lipid phosphate p hosphatase) gene encodes a protein that contains a novel phosphatase s equence motif found in DGPP phosphatase and in the mouse Mg2+-independ ent PA phosphatase. A genomic copy of the S. cerevisiae LPP1 gene was isolated and was used to construct lpp1 Delta and lpp1 Delta dpp1 Delt a mutants. A multicopy plasmid containing the LPP1 gene directed a 12. 9-fold overexpression of Mg2+-independent PA phosphatase activity in t he S. cerevisiae lpp1 Delta dpp1 Delta double mutant. The heterologous expression of the S. cerevisiae LPP1 gene in Sf-9 insect cells result ed in a 715-fold overexpression of Mg2+-independent PA phosphatase act ivity relative to control insect cells. The Mg2+-independent PA phosph atase activity encoded by the LPP1 gene was associated with the membra ne fraction of the cell. The LPP1 gene product also exhibited lyse-PA phosphatase and DGPP phosphatase activities. The order of substrate pr eference was PA > lyse-PA > DGPP. Like the dpp1 Delta mutant, the lpp1 Delta mutant and the lpp1 Delta dpp1 Delta double mutant were viable and did not exhibit obvious growth defects. Biochemical analyses of lp p1 Delta, dpp1 Delta, and lpp1 Delta dpp1 Delta mutants showed that th e LPP1 and DPP1 gene products encoded nearly all of the Mg2+-independe nt PA phosphatase and lyse-PA phosphatase activities and all of the DG ;PP phosphatase activity in S. cerevisiae. Moreover, the analyses of t he mutants showed that the LPP1 and DPP1 gene products played a role i n the regulation of phospholipid metabolism and the cellular levels of phosphatidylinositol and PA.