Roles of Phosphorylation and Dephosphorylation in Phosphatidic Acid Phosphatase Membrane Interaction

Abstract

What molecular machinery underpins the utilization of phosphatidic acid (PA) for homeostasis of membrane phospholipid synthesis and the synthesis of the storage lipid triacylglycerol? PA phosphatase is one of the most important enzymes regulating lipid metabolism due to its function of catalyzing the dephosphorylation of PA to produce diacylglycerol. The importance of this reaction is exemplified by cellular defects and lipid-based diseases associated with the loss of the enzyme. PA phosphatase is controlled by several protein kinases through phosphorylation taking place in cytoplasm; phosphorylation causes retention of the enzyme in this cellular location. Interaction with the endoplasmic reticulum (ER) membrane-associated Nem1-Spo7 protein phosphatase complex followed by its dephosphorylation, causes the enzyme to hop onto the ER membrane where it either binds to a phospholipid molecule or to its substrate PA to catalyze its reaction. PA phosphatase then scoots along the ER membrane towards another PA molecule for another round of catalysis. In this work, the Nem1-Spo7 complex was reconstituted into unilamellar phospholipid vesicles (liposomes) composed of PA and the major ER membrane phospholipids; the complex was examined for its role in regulating phosphorylated PA phosphatase for its membrane interaction and catalytic function. Recombinant PA phosphatase was phosphorylated by Pho85-Pho80 and incubated with the proteoliposomes. The reconstituted protein phosphatase catalyzed the dephosphorylation of PA phosphatase and facilitated its membrane interaction and ability to catalyze the dephosphorylation of PA at the membrane surface.