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Molecular Biology Group Group leader: Karin Athenstaedt Postdoctoral Fellow: Andreas Beranek Acyltransferases catalyzing triacylglycerol synthesis in the oleaginous yeast Yarrowia lipolytica The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to accumulate huge amounts of triacylglycerols (TAG). Along with other neutral lipids, TAG are stored in socalled lipid particles, cell compartments with a rather simple structure. A neutral lipid core mainly formed of TAG and steryl esters is surrounded by a phospholipid monolayer with few proteins embedded. By growing Yarrowia lipolytica cells in media containing, e.g., industrial fats or glycerol as a carbon source, the amount of TAG can be increased up to 40% of cell dry weight. This ability leads to its application in biotechnological processes such as single cell oil production or production of nutrients enriched in essential fatty acids which can serve as nutritional complements. However, Yarrowia lipolytica may also serve as a model organism to study lipid turnover in adipocytes, since not only the ability to store excessive amounts of TAG in lipid particles but also the composition of this cell compartment resembles adipocytes of higher eukaryotes. Despite these potentials of Yarrowia lipolytica information about TAG (lipid) metabolism in this yeast is rather limited. Thus, we started to investigate the proteome of Yarrowia lipolytica for proteins involved in TAG synthesis. Homology searches with TAG synthases of other eukaryotes as queries highlighted two candidate gene-products of the oleaginous yeast potentially catalyzing the formation of TAG. A decreased amount of TAG in mutant cells defective in these candidate genes already pinpointed to a function of these polypeptides in TAG formation. To investigate whether these candidate genes encode true TAG synthases these genes were heterologously expressed in cells of a Saccharomyces cerevisiae mutant defective in neutral lipid synthesis and as a consequence lacking lipid particles (Fig. 1). A B C D Figure 1: Restoration of lipid particle formation upon heterologous expression of Yarrowia lipolytica TAG synthase candidates in mutant cells of the budding yeast Saccharomyces cerevisiae lacking lipid particles. In contrast to the negative control (B; mutant + empty plasmid), lipid particles are formed in mutant cells transformed with plasmids bearing either of the respective candidate genes of Yarrowia lipolytica (C, D). Panel A shows lipid particle formation in a wild-type cell of Saccharomyces cerevisiae. Lipid particles are indicated by arrows. Size bar: 10 µm. Fluorescent microscopic inspection and lipid analyses of the respective mutants clearly demonstrated that these Yarrowia lipolytica genes encode true TAG synthases. The 24
characteristics of these two TAG synthases of the oleaginous yeast are currently under investigation. Biosynthesis of phosphatidic acid in yeast Phosphatidic acid is of utmost importance, since it is the key intermediate for the formation of all glycerolipids, namely glycerophospholipids (membrane lipids) and triacylglycerols (storage lipids). Furthermore, this lipid functions in cell signaling. In all eukaryotic organisms enzymes catalyzing phosphatidic acid biosynthesis occur in redundancy. One reason for the existence of isoenzymes may be the formation of different phosphatidic acid pools supplying different pathways. In this project this hypothesis is tested in the model organism yeast Saccharomyces cerevisiae, which contains two glycerol-3-phosphate acyltransferases, Gat1p and Gat2p, and two 1-acyl glycerol-3-phosphate acyltransferases, Slc1p and Lpt1p. Glycerol- 3-phosphate acyltransferases catalyze the first and rate limiting step in de novo synthesis of phosphatidic acid via the glycerol-3-phosphate pathway, namely the acylation of glycerol-3- phosphate yielding 1-acyl glycerol-3-phosphate (lyso-phosphatidic acid). A subsequent acylation converts lyso-phosphatidic acid to phosphatidic acid and is catalyzed by a 1-acyl glycerol-3-phosphate acyltransferase. The precise localization of glycerol-3-phosphate acyltransferases tagged with green fluorescent protein (GFP) has been determined by fluorescence microscopy and Western blot analyses with highly purified cell fractions of the respective mutants. Whereas Gat1p is associated with lipid particles and the endoplasmic reticulum, Gat2p is exclusively localized to the latter compartment in wild-type background (Fig. 2). A B LP LP Wild-type + GFP-Gat1p Wild-type + GFP-Gat2p Figure 2: Fluorescence microscopy of Gat1p and Gat2p, respectively, tagged with green fluorescent protein (GFP) in wild-type background. A: GFP-Gat1p localizes to the endoplasmic reticulum and lipid particles (indicated by arrows). In contrast, GFP-Gat2p is only present in the endoplasmic reticulum (B). Most interestingly, the absence of either glycerol-3-phosphate acyltransferase results in a different distribution pattern of its counterpart, and in growth defects. Whether these phenotypes are caused by alterations in the lipid pattern of the respective mutants is currently under investigation. International cooperations T. Chardot, Institut National de la Recherche Agronomique, Institut Jean-Pierre Bourgin, UMR1318, Versailles, France J.-M. Nicaud, Institut National de la Recherche Agronomique, Laboratoire de Microbiologie et Génétique Moléculaire, UMR1319, Jouy-en-Josas, France 25
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 and 18: most promising candidates of this s
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Page 21 and 22: focused on the biochemical characte
Page 23: Research projects FWF P21429: Phosp
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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