Cloning and characterization of a novel human phosphatidic acid phosphohydrolase

We have identified with a bioinformatics approach a putative phosphatidic acid phosphohydrolase gene using Hidden Markov Models of already known phosphatases. The gene was cloned by PCR from an EST clone and subcloned in an expression vector. The gene was then expressed by transfection in COS‐7 cells and its subcellular localization was analysed by fluorescence immunocytochemistry and by Western Blotting of subcellular fractions. Both approaches showed that the gene protein product was distributed between ER and plasma membrane, with no expression in the cytosol or nucleus. Phosphatidic acid phosphatase assays together with TLC analysis showed that the gene cloned encoded for an enzyme that converted phosphatidic acid to diacylglycerol in the absence of Mg2+. The presence of Mg2+ in the assays eliminated the production of diacylglycerol, suggesting, together with the localization studies, that the enzyme is a type 2 phosphatidic acid phosphohydrolase