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However, mechanisms involved in these translocation processes are still unclear. To address these questions, we established in vitro and in vivo assays for studying phospholipid supply to peroxisomal membranes. We constructed a strain which lacks the gene product of OPI3, the major PE methyltransferase localized to the ER, and bears an Opi3p-GFP hybrid with an SKL targeting sequence that directs the enzyme to peroxisomes. In this “reporter mutant”, the only site of phosphatidylcholine (PC) formation via methylation of PE are the peroxisomes, and the appearance of PC becomes an indicator and measure for PE translocation from the different sites of synthesis to peroxisomes. Currently permeabilized cells of the “reporter mutant” are used to characterize the PE transport to peroxisomes in some detail. This system in combination with mutations in the different PE biosynthetic pathways will allow us to investigate the different mechanisms of PE translocation between organelles involved in aminoglycerophospholipid biosynthesis. Most recently, we also studied the link between PE metabolism and neutral lipid storage. For this purpose we analyzed lipids from strains bearing defects in PE synthesis. These analyses showed that a mutant bearing a defect in the CDP-ethanolamine pathway had a decreased level of triacylglycerols (TAG). In cki1∆dpl1∆eki1∆ mutants bearing defects in the CDPethanolamine pathway both the cellular and the microsomal levels of PE were markedly decreased, whereas in other mutants of PE biosynthetic routes depletion of this aminoglycerophospholipid in microsomes was less pronounced. This observation is important because the TAG synthesisizing enzyme Lro1p similar to the enzymes of the CDPethanolamine pathway is a component of the ER. We conclude from these results that in cki1∆dpl1∆eki1∆ insufficient local supply of PE to Lro1p was a major reason for the strongly reduced TAG level. Neutral lipid storage in lipid particles and mobilization Yeast cells have the capacity to store neutral lipids TAG and STE (steryl esters) in subcellular structures named lipid particles/droplets. Upon requirement, TAG and STE can be mobilized and serve as building blocks for membrane biosynthesis. In a long-standing project of our laboratory, we investigate the characterization of enzymatic steps which lead to the mobilization of TAG and STE depots. A major focus of our neutral lipid project was the biochemical characterization of the three yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast TAG lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out biochemical experiments with wild type and deletion strains bearing defects in Tgl3p, Tgl4p and Tgl5p. We demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. The pattern of phosphatidylcholine (PC), PE and PS molecular species was altered in tgl deletion strains underlining the important role of TAG turnover in maintenance of the pool size and remodelling of complex membrane lipids. This study shed new light on the physiological role of TAG lipases in yeast and in general. 18
Even more surprising evidence was obtained when the enzymology of the three TAG lipases Tgl3p, Tgl4p and Tgl5p was studied in some detail. Motif search analysis indicated that Tgl3p and Tgl5p did not only contain the TAG lipase but also an acyltransferase motif. Interestingly, lipid analysis revealed that deletion of TGL3 resulted in a decrease and overexpression of TGL3 in an increase of glycerophospholipids. Similar results were obtained with TGL5. Therefore, we tested purified Tgl3p and Tgl5p for acyltransferase activity. Indeed, both enzymes did not only exhibit lipase activity but also catalyzed acylation of lysophosphatidylethanolamine and lysophosphatidic acid, respectively. Experiments using variants of Tgl3p created by site-directed mutagenesis clearly demonstrated that the two enzymatic activities act independently of each other. These results demonstrated that yeast Tgl3p and Tgl5p play a dual role in lipid metabolism contributing to both anabolic and catabolic processes. P-Lipase 28 GGGTFG 29 Acyltransferase 48 HIIACQ 49 1aa 282 Patatin domain 48 3 910aa 31 GSSAG 31 Lipase Domains of Tgl3p (from Rajakumari and Daum, 2010, J. Biol. Chem. 285, 15769-15776) In another study, we demonstrated that the yeast TAG lipase Tgl4p, the functional ortholog of adipose TAG lipase (ATGL), catalyzes multiple functions in lipid metabolism. An extended domain and motif search analysis revealed that Tgl4p bears not only a lipase consensus domain but also a conserved motif for calcium independent phospholipases A2 (PLA2). We showed that Tgl4p exhibits TAG lipase, STE hydrolase and PLA2 activities, but also catalyzes acyl-CoA dependent acylation of lysophosphatidic acid (LPA) to phosphatidic acid. Heterologous overexpression of Tgl4p in Pichia pastoris increased total phospholipid and specifically phosphatidic acid synthesis. Moreover, deletion of TGL4 in Saccharomyces cerevisiae showed an altered pattern of PC and phosphatidic acid molecular species. Altogether, our data suggested that yeast Tgl4p functions as hydrolytic enzyme in lipid degradation, but also contributes to fatty acid channelling and phospholipid remodelling. Several years ago we identified through a mass spectrometric approach for the first time the major lipid particle proteins of Saccharomyces cerevisiae. This approach was a milestone in the field because it identified a number of new gene products and their function and provided valuable hints for processes associated with lipid particles. Recently, a more precise lipid particle proteome analysis was initiated in collaboration with M. Karas from the Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University, Frankfurt, Germany. This proteome study was combined with a lipidomics investigation that was performed in collaboration with H. Köfeler from the Center for Medical Research, Medical University of Graz, Austria. In this study, we compared lipid particle components from cells which were 19
Page 1 and 2: Graz University of Technology Austr
Page 3 and 4: Brief History of the Institute of B
Page 5 and 6: Highlights of 2010 In 2010, Peter M
Page 7 and 8: Biochemistry group Group leader: Pe
Page 9 and 10: plant genes that apparently encode
Page 11 and 12: unusual amino acids, i.e. hydroxypy
Page 13 and 14: words, the hydroxyl group attacks t
Page 15 and 16: 2) Jajcanin-Jozic, N., Deller, S.,
Page 17: most promising candidates of this s
Page 21 and 22: focused on the biochemical characte
Page 23 and 24: Research projects FWF P21429: Phosp
Page 25 and 26: characteristics of these two TAG sy
Page 27 and 28: Biophysical chemistry group Group l
Page 29 and 30: 2.1. Fluorescent suicide inhibitors
Page 31 and 32: International cooperations: T. Hian
Page 33 and 34: Chemistry of Functional Foods Group
Page 35 and 36: structural characterization of TAGs
Page 37 and 38: Lectures and Laboratory Courses Akt

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