Biosynthesis of Phosphatidic Acid in 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 acyltransferase reactions are involved in conversion of Gro3P to PtdOH, whereas a third reaction 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 studies presented here. Major enzymes catalyzing PtdOH biosynthesis in the yeast have been identified, and the existance of additional PtdOH synthesizing polypeptides has been predicted. At least two isoenzymes per enzymatic step of PtdOH synthesis have to exist. This project is aimed to determine the reason of this redundancy. To meet this goal, we will address questions regarding (i) the interplay of specific isoenzymes during PtdOH biosynthesis, (ii) the formation of specific PtdOH pools, and (iii) the regulation of PtdOH formation. To understand the complexity of PtdOH biosynthesis and the role of this key intermediate in cell metabolism, it will be essential to know the entire set of proteins contributing to its formation. Thus, different screening strategies and bioinformatics, methods of molecular biology, cell biology and biochemistry will be employed for identification and characterization of additional polypeptides governing PtdOH synthesis. The interplay of isoenzymes (organelles) during PtdOH formation and the precise localization of enzymes involved in this process will be determined by fluorescence microscopy (GFP approach) and cell fractionation (Western blot and enzymatic analysis). Phenotypical analyses and lipid profiles of organelles isolated from strains defective in one or more enzymes catalyzing PtdOH formation will reveal whether a preferential interplay of the respective enzymes may lead to the formation of specific PtdOH pools. Determination of gene expression profiles of mutants defective in certain enzymes of PtdOH biosynthesis by DNA microarray experiments will extend our knowledge of PtdOH biosynthesis to the transcriptional level and may also lead to the identification of isoenzymes, suppressors and regulatory proteins involved in PtdOH formation. Detailed analyses of gene expression profiles of enzymes governing PtdOH synthesis will address the regulation of this process. In summary, these investigations of PtdOH biosynthesis will provide a template for similar studies in higher eukaryotes.

Biosynthesis of phosphatidic acid (PA) is essential because this molecule is required for the formation of all glycerophospholipids which are major building blocks of biomembranes, and triacylglycerols serving as storage molecules. Additionally, PA functions in cell signaling. Formation of PA from the precursor glycerol-3-phosphate occurs by two subsequent acylation reactions which are mediated by a glycerol-3-phosphate acyltransferase (GPAT) and a 1-acyl glycerol-3-phosphate acyltransferase (AGPAT), respectively. In all eukaryotes, both GPAT and AGPAT occur in redundancy. The major aim of this project was to elucidate whether these redundant enzymes contribute differentially to lipid/cell metabolism. For these studies the model organism yeast, Saccharomyces cerevisiae, was used. In yeast the first and committed reaction of PA biosynthesis is mediated by two GPAT isoenzymes, Sct1p and Gpt2p. In this study it was observed that overexpression of SCT1 specifically and strongly affected cell growth. Gene expression analyses revealed that upon high level expression of SCT1 the expression level of approximately 20% of all genes were significantly changed. One major group of these genes encoded polypeptides involved in or related to lipid metabolism. By focusing on these genes and gene products an additional AGPAT was identified which plays a major role in PA formation in cells overexpressing SCT1. Since overexpression of SCT1 strongly affected the degree of phosphorylation of the encoded polypeptide, it was hypothesized that the activity of the GPAT is regulated by protein phosphorylation. A major phosphorylation site of Sct1p was identified. Detailed studies revealed that the contribution of Sct1p to lipid/cell metabolism is tightly linked to its degree of phosphorylation.The second GPAT of yeast, Gpt2p, is essential for cell growth in the presence of oleic acid. This growth condition enhances the formation of triacylglycerols, molecules which are deposited in cell compartments known as lipid droplets. Former studies from this laboratory demonstrated that Gpt2p is dually localized to the endoplasmic reticulum and lipid droplets. Most interestingly, we found that in cells cultivated in oleic acid containing medium the dual localization of Gpt2p was lost and Gpt2p exclusively localized to a part of the endoplasmic reticulum which formed crescent structures intimately associated with lipid droplets. Under these conditions the amount of Gpt2p was markedly augmented and the degree of Gpt2p phosphorylation reduced. These results indicate that the contribution of Gpt2p to lipid/cell metabolism is similar to Sct1p regulated by phosphorylation of the protein.Taken together, GPAT isoenzymes indeed contribute in a different way to lipid/cell metabolism, and protein phosphorylation plays a major role in regulating the contribution of these enzymes to lipid/cell metabolism.