Final Activity Report Summary - PTDINS BIOSYNTHESIS (Regulation of mammalian phosphatidylinositol biosynthesis) Phosphatidate phosphatases, PAPs, are key enzymes in lipid biosynthesis and signaling. Functionally, PAP1 enzymes participate in de-novo phospholipid biosynthesis, whereas PAP2 enzymes have an established role in lipid signalling. To identify PAP2 enzymes that were potentially involved in de-novo phospholipid synthesis we first screened the human genome for PAP2 enzymes and after identifying all family members, we screened these for exposure of their predicted active site residues to the cytosolic side of membranes. These criteria identified two related enzymes, PAP2F and PAP2G, which show differential tissue and subcellular distribution, as well as novel yet differential roles in lipid metabolism. Specifically, we found that myc/His-PAP2F, but not myc/His-PAP2G, is a potent Mg2+-independent type II PAP which exhibited surface dilution kinetics. Subcellular fractionation detection showed that both PAPs were associated with membranes, while immunofluorescent imaging revealed an exclusive ER distribution. Most importantly, PAP2F accelerated the synthesis of phosphatidylcholine and caused accumulation of triacylglycerol while decreased phosphatidylinositol. On the contrary, myc/His-PAP2G had no effect on lipid metabolism. Coexpression of CTP:phosphocholine cytidylyltransferase-a with PAP2F enhanced the effect of PAP2F on phosphatidylcholine levels, yet attenuated its effect on triacylglycerol. Taken together, our studies provide the first evidence that, in addition to PAP1, there is a eukaryotic, ER-resident PAP2 enzyme, PAP2F, which regulates de-novo biosynthesis of phospholipids and triacylglycerols.