Regulatory aspects of phosphatidic acid biosynthesis in yeast

Phosphatidic acid (PA) holds a pivotal role in cell metabolism, because on one hand it is the key intermediate for the formation of all glycerophospholipids (membrane lipids) and triacylglycerols (storage lipids), and on the other hand involved in cell signaling. Due to these fundamental functions it is important that the cellular PA pool is adjusted to cellular demands. In all eukaryotic organisms enzymes mediating the reactions of PA biosynthesis occur in redundancy. Several differences in the properties of these isoenzymes have been observed. However, our current knowledge about mechanisms regulating the activity of these enzymes, and hence de novo synthesis of PA, is still limited. The aim of the proposed project is to elucidate the regulation of PA biosynthesis by focusing on glycerol- 3-phosphate acyltransferases (GPATs), because these enzymes catalyze the first and rate limiting reaction in PA biosynthesis, and represent the main target of regulation. For these studies we will use as experimental system the budding yeast Saccharomyces cerevisiae, which has been proven to be an invaluable model organism to determine the principles of lipid metabolic processes. In this microorganism two GPATs exist, namely Gat1p (Gpt2p) and Gat2p (Sct1p), which are both target of protein-kinases. By using molecular biological, cell biological and biochemical methods the following questions will be addressed: (i) Is the transcription and/or the phosphorylation of GPATs linked to the cell cycle? (ii) Does phosphorylation of GPATs change their activity, stability and/or subcellular localization? (iii) Is the contribution of GPATs to PA biosynthesis regulated by interaction with other proteins? (iv) Are GPATs regulated by feedback mechanism(s)? And finally, (v) is the dual localization of Gat1p to lipid droplets and the endoplasmic reticulum required to regulate its contribution to PA biosynthesis? Since PA biosynthesis via GPATs is like many cell metabolic processes highly conserved from yeast to human, our results obtained with Saccharomyces cerevisiae will provide the basis for similar studies in higher eukaryotes.

Phosphatidic acid harbors a pivotal role in cell metabolism because it serves as the precursor for all glycerophospholipids (membrane lipids) and triacylglycerols (storage lipids). Moreover, phosphatidic acid is involved in cell signaling. Because of these important roles, the formation of phosphatidic acid has to be strictly adapted to cellular requirements. The first and rate determining reaction of phosphatidic acid biosynthesis is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). This enzyme is also the major point where the regulation of phosphatidic acid biosynthesis occurs. In this project we investigated the regulation of GPATs by using the budding yeast Saccharomyces cerevisiae as our model organism. In yeast two GPATs are expressed which are named Sct1p and Gpt2p. Both enzymes are modified by phosphorylation. We discovered that phosphorylation deficiency of Gpt2p at a highly conserved motif strongly affects triacylglycerol metabolism, but neither alters the glycerophospholipid pattern or glycerophospholipid amount. The respective phosphorylation motif comprises three phosphorylation sites. Phosphorylation deficiency at one or more of these sites strongly increases the enzyme activity of Gpt2p, which leads to a higher triacylglycerol content in the mutant cells compared with control. In the phase of triacylglycerol accumulation both native Gpt2p and the Gpt2p-variant lacking phosphorylation at the conserved motif (Gpt2p-3A) contribute to triacylglycerol formation just differing in extent. However, a striking difference occurs in the phase of triacylglycerol mobilization. Whereas the enzyme activity of native Gpt2p is reduced by phosphorylation which allows efficient triacylglycerol mobilization, phosphorylation deficient Gpt2p-3A remains in the hyperactive state, further contributes to triacylglycerol formation and consequently counteracts net mobilization of triacylglycerols. This effect contributes additionally to the higher triacylglycerol content in the mutant compared with normal cells. Furthermore, we showed that a defect in the phosphorylation of Gpt2p at the conserved motif affects the fatty acid composition of the cell. By extending our studies concerning the physiological meaning of the phosphorylation of Gpt2p to phosphorylation sites different from the ones of the conserved motif we found that phosphorylation/de-phosphorylation additionally affects the protein stability of this GPAT. Collectively, we could show that phosphorylation of the GPAT Gpt2p plays an important role in regulating phosphatidic acid biosynthesis, and changes in the phosphorylation status of this enzyme affect at the protein level the stability of Gpt2p, and at the cellular level the accumulation of triacylglycerols as well as the fatty acid composition.