Biosynthesis of Phosphatidic Acid in the Yeast

In eukaryotic cells, the biosynthesis of phosphatidic acid (PtdOH) can be accomplished by two pathways, namely (i) the glycerol-3-phosphate (Gro3P) and (ii) the dihydroxyacetonephosphate (GrnP) pathway named after the respective precursors utilized. Two acyltransferases catalyze the conversion of Gro3P to PtdOH, whereas a third enzyme is required for the GrnP pathway to convert 1-acyl-GrnP to 1 acyl-Gro3P. In all types of eukaryotic cells, enzymes for the formation of PtdOH occur in redundancy. This is also true for the yeast Saccharomyces cerevisiae, which will be used as a model for the studies presented here. So far, only two of these enzymes were identified at the molecular level. The aim of the project presented in this proposal will be identification and characterization of additional yeast genes and gene products involved in PtdOH formation. Methods of bioinformatics, molecular biology, cell biology and biochemistry will be employed for these investigation. Localization of the respective enzymes will be studied to address the question as to the interplay of organelles during PtdOH synthesis, the redundancy of these proteins and the possible compatibility of isoenzymes. Subcellular distribution of these proteins will be shown by fluorescence microscopy (green fluorescent protein approach or immunological methods) and cell fractionation techniques (Western blot and enzymatic analysis). Species analysis of PtdOH formed in wild-type or in strains deleted of candidate ORFs will demonstrate the contribution of each specific enzyme to the total amount of cellular PtdOH synthesized. This analysis will also demonstrate whether different pools of PtdOH, probably in different subcellular compartments, exist for the formation of glycerophospholipids and triacylglycerols. Taken together, this study will contribute to our understanding of the cell biological role of PtdOH in yeast, which may also be correlated to the function of this important key intermediate of glycerolipid biosynthesis in higher eukaryotic systems.

In eukaryotic cells, the biosynthesis of phosphatidic acid (PtdOH) can be accomplished by two pathways, namely (i) the glycerol-3-phosphate (Gro3P) and (ii) the dihydroxyacetonephosphate (GrnP) pathway named after the respective precursors utilized. Two acyltransferases, Gat1p and Gat2p, catalyze the first acylation step in the formation of PtdOH via the Gro3P pathway in the yeast Saccharomyces cerevisiae. During my Firnberg project, the subcellular localization of these two proteins was determined by enzymatic measurements, tagging with GFP (green fluorescent protein) and microscopic inspections, and by Western blot analysis. These methods demonstrated that Gat1p is dually localized to lipid particles and the endoplasmic reticulum (ER), whereas Gat2p is found only in the latter compartment. Previous results from our laboratory suggested the existence of at least another yeast acyltransferase with minor activity. A double deletion mutant gat1 gat2, however, is not viable. Thus, the activity of additional acyltransferases may not be sufficient to complement for a defect in Gat1p and Gat2p. Future experiments employing molecular biological screenings will address this question. Besides acyltransferases, lipid particles of the yeast harbor a number of additional enzymes which are involved in lipid metabolism. Among these proteins a putative triacylglycerol lipase was identified. This is the first enzyme of this kind that was characterized by function in the yeast. Moreover, several enzymes involved in sterol metabolism such as Erg7p are localized to lipid particles. The interplay of lipid particles with the ER was shown through localization studies of Erg11p and Erg27p. Erg11p, which catalyzes the metabolic step subsequent to Erg7, and Erg27p, which appears to be a regulator of Erg7p localization, were mainly found in the ER. Thus, both compartments interact during synthesis of yeast sterols. The latter study was performed in cooperation with Dr. G. Balliano (Turino, Italy) and Dr. M. Bard (Indianapolis, USA).