Membrane Phospholipid Composition Governs Pah1 Phosphatidate Phosphatase Activity

In yeast and higher eukaryotes, all membrane phospholipids and the storage lipid triacylglycerol, are derived from phosphatidate (PA), which is also implicated as an activator of cell growth and proliferation, vesicular trafficking, secretion, and endocytosis. PA also regulates expression of lipid synthesis genes via the Henry (Opi1/Ino2‐Ino4) regulatory circuit, where the key step is the PA‐mediated translocation of the Opi1 repressor between the nuclear/ER membrane and the nucleus. Data indicate that several enzymes play important roles in the PA‐mediated regulation of lipid synthesis that occurs at the nuclear/ER membrane. Of the enzymes that metabolize PA, the PA phosphatase enzyme, which is encoded by the PAH1 gene, has emerged as key regulator of PA homeostasis. Indeed, cells lacking this enzyme exhibit several phenotypes that include elevated levels of PA, derepression of phospholipid synthesis genes, increased synthesis of membrane phospholipids, and the abnormal expansion of the nuclear/ER membrane. These mutant cells also exhibit a drastic reduction in TAG and lipid droplets. The PA phosphatase, which catalyzes the conversion of PA to diacylglycerol, must translocate from the cytoplasm to the ER membrane where its substrate PA resides to function in lipid synthesis. Thus, studies to examine mechanisms for the enzyme reaction at the membrane surface are warranted. In this work, we implemented defined unilamellar vesicles with a diameter of 100 nm composed of the major phospholipids within the ER membrane. Vesicles composed of phosphatidylcholine (PC) and 10 mol% PA supported PA phosphatase activity. The addition of phosphatidylethanolamine (PE) or phosphatidylinositol (PI) stimulated the activity, but phosphatidylserine (PS) had little effect. In more complex vesicle compositions, the maximum activity was observed with vesicles containing PC/PE/PS/PA or PC/PE/PI/PA. Yet, the activity was reduced when vesicles contained PC/PE/PI/PS/PA. The PA phosphatase activity was dependent on vesicle number as well as the surface concentration of PA within the vesicles. This vesicle system is being used to assess the roles of phosphorylation/dephosphorylation of Pah1 on membrane interaction and PA phosphatase activity. Support or Funding Information Supported by NIH grant GM028140