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.
|Effective start/end date||1/02/09 → 31/01/12|