Synthese von Ester- und Etherlipiden durch die GPAT/AGPAT-Proteinfamilie und Lipin in Dictyostelium

The synthesis of glycerolipids starts with the acylation of lipid backbones. Two structurally different glycerolipids emerge in the cell: ether-lipids and ester-lipids. In the case of ether-lipids the first fatty acid is substituted with a fatty alcohol inside the peroxisome. By the characterization of the protein FARAT in this thesis, a key enzyme in etherlipid-synthesis was identified for D. discoideum. FARAT shows a peroxisomal localization and is in principle able to do two jobs. Firstly, it initiates the peroxisomal lipid synthesis by the acylation of dihydroxyacetone phosphate. And secondly, it is able to form fatty alcohols needed for the ether-lipid synthesis. Gpat is a protein that is responsible for initiating exclusively the ester-lipid synthesis at the endoplasmic reticulum (ER). In combination with FARAT it was used to examine the interaction of ester- and ether-lipid-synthesis in D. discoideum. The present data shows that exclusively ether-lipids derive from the peroxisome and that increasing the peroxisomal lipid synthesis results in the accumulation of lysophospholipids. Without FARAT the cell loses the ability to synthesize ether-lipids and also to phagocyte particles. In contrast, ether-lipids and maybe also lysophospholipids have a generally positive effect on the phagocytosis of yeast particles. The second step in glycerolipid synthesis is the addition of another fatty acid to the backbone. This step is committed by acylglycerol-3-phosphate acyltransferases (AGPATs). AgpD is a putative AGPAT in D. discoideum and was therefore characterized. It localizes on the ER and the corresponding knockout surprisingly shows an increase in the neutral lipid content. Additionally, it was analyzed whether redundancy occurs in the AGPAT protein family in D. discoideum. Therefore, multiple gene knockouts were generated but even the triple knockout of agpA/agpB/agpC shows only a marginal reduction in glycerolipid synthesis. No additional information was gained by the analysis of strains overexpressing already characterized, putative AGPAT isoforms. Consequently, it is not yet clear which protein mediates the second step in glycerolipid synthesis. The overall data suggests that there are uncharacterized proteins that are able to catalyze the second step in glycerolipid synthesis, too. The next step in neutral lipid synthesis is the dephosphorylation of phosphatidic acid (PA), committed by PA phosphatases. The characterization of the cytoplasmic protein LPIN2 shows that it is indispensable for neutral lipid synthesis and also for lipid droplet appearance. Both properties match the predicted function as a PA phosphatase. Further, the knockout of LPIN2 causes a reduced growth rate in liquid media and on a bacterial lawn, too. The overall phospholipid content of the knockout is not dramatically affected even though a significant reduction for one lipid, presumably cardiolipin, is detected.