Substrate Selectivities and Lipid Modulation of Plant Phospholipase Dα, -β, and -γ

Abstract

Three classes of phospholipase D (PLD), designated PLDα, -β, and -γ, have been cloned from plants, but their substrate selectivities have not been established. Using active PLDs expressed from their cDNAs inEscherichia coli,we compared the hydrolytic activities of these three PLDs toward various phospholipids and the influence of substrate composition on their substrate selectivities. When single-class phospholipid vesicles of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4,5-bisphosphate (PIP2),N-acylphosphatidylethanolamine (NAPE), and cardiolipin (CL) were examined, PLDα hydrolyzed PC, PE, and PG but PLDβ and -γ showed no activity toward any of these lipids. When PIP2was included in mixed vesicles with the phospholipids above, PLDα showed the same PC-, PE-, and PG-hydrolyzing ability, whereas PLDβ and -γ were able to hydrolyze both PE and PS. When both PE and PIP2were included in substrate vesicles, PLDβ and PLDγ hydrolyzed PC, PG, and NAPE, showing that both PE and PIP2are required for PC, PG, and NAPE hydrolysis by PLDβ and -γ. The PE activation of PLDβ and -γ required lipid vesicles made of mostly PE, suggesting that PE may affect the substrate presentation rather than serve as a cofactor of these PLDs. Under equivalent reaction conditions, PLDβ displayed a similar preference for PC and NAPE, whereas PLDγ preferred NAPE to PC by nearly three times. None of the three PLDs used PI, CL, or PIP2as substrates. These results have identified PS- and NAPE-hydrolyzing PLDs and have indicated an important role for lipid composition in regulating the substrate selectivity of PLDβ and -γ.