Membrane interactions of yeast phosphatidate phosphatase

The yeast PAH1‐encoded phosphatidate (PA) phosphatase (PAP) catalyzes the formation of diacylglycerol (DAG) from PA. The DAG generated in the reaction is used for the synthesis of triacylglycerol and for the synthesis of phospholipids via the Kennedy pathway. While PAP is primarily associated with the cytosolic fraction of the cell, the enzyme must associate with the membrane where its substrate PA resides to be physiologically active. In this work, we examined the association of PAP with phospholipid vesicles using an assay based on fluorescence emission. The interaction of PAP with phospholipid vesicles resulted in an increase in tryptophan fluorescence intensity as well as a shift from 350 to 343 nm in wavelength of the maximum emission. For wild type PAP, there was a dose‐dependent increase in fluorescence by the addition of vesicles. The dephosphorylation of PAP significantly enhanced the fluorescence increase, indicating an increase in phospholipid vesicle interaction. The interaction of PAP with the phospholipid vesicles was confirmed by immunoblot analysis using anti‐PAP antibodies. The association of PAP with lipid membranes was also dependent on the amphipathic helix found at the N‐terminus of the enzyme. Loss of the amphipathic helix or mutations within the helix prevented membrane association. The helix‐dependent association with vesicles was governed by the phosphorylation state of the enzyme.