ISOLATION AND CHARACTERIZATION OF THE SACCHAROMYCES-CEREVISIAE DPP1 GENE ENCODING DIACYLGLYCEROL PYROPHOSPHATE PHOSPHATASE

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel li pid signaling pathway, DGPP phosphatase (DGPP phosphohydrolase) is a m embrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which c atalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol, A mino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phospha tase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity i n S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DG PP phosphatase activity over that expressed in wild-type S. cerevisiae . DGPP phosphatase possesses a Mg2+-independent PA phosphatase activit y, and its expression correlated with the overexpression of DGPP phosp hatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembra ne-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. A dpp1 Delta mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1 Delta mutant was viable and did not exhibit any obvious growth de fects. The mutant was devoid of DGPP phosphatase activity and accumula ted (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene wa s not responsible for all of the Mg2+-independent PA phosphatase activ ity in S. cerevisiae.