Phospholipids as cellular mediators in yeast

Diacylglycerol pyrophosphate (DGPP) is a novel phospholipid which contains a pyrophosphate group attached to diacylglycerol. DGPP phosphatase catalyzes the dephosphorylation of the βphosphate of DGPP to yield phosphatidate and PI. DGPP phosphatase was purified 33,333fold from S. cerevisiae by a procedure which included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel blue, hydroxylapatite, and Mono Q. The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa. DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity. The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents. The turnover number (molecular activity) for the enzyme was 5.8 x 103 min^-1 at pH 6.5 and 30 °C. DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (K_m = 0.55 mol %). The K_m value for DGPP was 3-fold greater than its cellular concentration (0.18 mol %). DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction. The role of DGPP phosphatase in phospholipid metabolism and cell signaling will be discussed.