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the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Moreover, activity and expression levels of fatty acid elongases, Elo1p and Elo2p, were enhanced as a consequence of reduced TAG lipolysis emphasizing the link to sphingolipid formation. Finally, the pattern of phosphatidylcholine (PC), PE and PS molecular species was altered in tgl deletion strain 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. Figure 2: Domains of Tgl3p (from Rajakumari et al., 2010, in press) 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 (Figure 2). 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. We also showed that Tgl3p is important for efficient sporulation of yeast cells, but rather through its acyltransferase than lipase activity. These results demonstrated that yeast Tgl3p and Tgl5p play a dual role in lipid metabolism contributing to both anabolic and catabolic processes. 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 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. We realized, however, that this study was 18
incomplete and did not provide the complete picture of the yeast lipid particle proteome. For this reason, 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 grown on glucose or on oleic acid. This approach identified a number of lipid particle proteins that were already known but also some novel polypeptide candidates. Detailed investigations of these proteins are currently in progress. We also realized through this approach that there were some differences in the lipid particle proteome from cells grown on glucose or oleic acid. Finally, mass spectrometric analyses revealed marked differences in the lipidome of lipid particles from cells grown on the two different carbon sources. This study sets the stage for further investigation of protein-lipid interaction on the surface of lipid particles and provides basic evidence for the coordinated biosynthesis of lipid and protein components from lipid particles. Another important aspect of this study was the physiological relevance of TAG and STE depot formation. Therefore, attempts were made to understand cell biological consequences of dysfunctions in neutral lipid storage. Previously, it has been reported that yeast cells lacking neutral lipid depots appear to become apoptotic. More recently, we and others were able to demonstrate that lipid depot formation plays an important role especially when yeast cells are stressed in the presence of exogenous fatty acids. However, it appears that an adaptation to this stress situation occurs which helps to overcome the deleterious effect of this lipotoxic stress. Changes in the lipid pattern caused by cultivation of yeast cells on oleic acid are currently investigated in some detail. In a previous study we had observed that variation of the yeast lipid particle composition by deletion of either TAG or STE synthesizing enzymes led to changes in the protein pattern on the lipid particle surface. In particular we realized that Erg1p, which is present at large amounts on lipid particles of wild type cells, loses stability in strains missing STE. During an ongoing project we performed stability and topology studies with different variants and constructs of Erg1p to elucidate embedding of this protein in the lipid particle surface membrane. This problem is related to the general question how lipid particle proteins are anchored in the monolayer membrane of the particle and/or interact with the hydrophobic core of the compartment. Although the evidence obtained so far is only preliminary we believe that the lipid status of lipid particles has a direct influence on the protein equipment of this compartment. This link may be related to the biogenesis of lipid particles. Another potential non-polar storage lipid is squalene. This component belongs to the group of isoprenoids and is precursor for the synthesis of sterols, steroids and ubiquinons. In the yeast, the amount of squalene can be increased by varying culture conditions or by genetic manipulation. As an example, in strains deleted of HEM1 squalene accumulates and is stored mainly in lipid particles. Interestingly, a heme deficient dga1∆lro1∆are1∆are2∆ quadruple mutant (Qhem1∆) which is devoid of the classical storage lipids, TAG and SE, accumulates substantial amounts of squalene in cellular membranes, especially in microsomes and the plasma membrane. The fact that Qhem1∆ does not form lipid particles suggests that biogenesis of these storage particles cannot be initiated by squalene which is synthesized in the ER similar to TAG and SE. This result also indicates that squalene accumulation, at least under the conditions tested, is not lipotoxic. Finally, our experiments demonstrate that squalene incorporated into organelle membranes does not compromise cellular function. 19
Page 1 and 2: Staff Members of the Institute of B
Page 3 and 4: the future direction of research. G
Page 5 and 6: 2009 in Graz under the chairmanship
Page 7 and 8: The BBE-catalysed oxidative carbon-
Page 9 and 10: Nikkomycin biosynthesis Nikkomycins
Page 11 and 12: HO R 1 H R 2 HSO 4 - Inversion H R
Page 13 and 14: substrate to product, was reduced e
Page 15 and 16: Cell Biology Group Group leader: G
Page 17: Organelle association and membrane
Page 21 and 22: Diploma Thesis completed Sabine Zit
Page 23 and 24: Publications 1. Wagner, A., Grillit
Page 25 and 26: characteristics of these two TAG sy
Page 27 and 28: Biophysical chemistry group Group l
Page 29 and 30: esterases are important biocatalyst
Page 31 and 32: Doctoral Theses completed: Maria Mo
Page 33 and 34: measurement of hydrolytic activitie
Page 35 and 36: carotenoids, while 110 o C, 120 o C
Page 37 and 38: Lectures and Laboratory Courses Anl

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