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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. 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 project was to understand cell biological consequences of dysfunctions in non-polar lipid storage. For this purpose, yeast cells were cultivated on oleic acid which was assumed to provoke lipotoxic stress. We found that under these cultivation conditions TAG synthesis was enhanced creating the major pool for the excess of fatty acids, whereas surprisingly STE synthesis was strongly inhibited. We showed that this effect was not due to decreased expression of ARE2 encoding the major yeast STE synthase at the transcriptional level, but to competitive enzymatic inhibition of Are2p by free oleic acid. As a result, a triple mutant dga1∆lro1∆are1∆ARE2 + grown on oleate did not form substantial amounts of STE and exhibited a growth phenotype similar to the dga1∆lro1∆are1∆are2∆ quadruple mutant which lacks all four acyltransferases involved in neutral lipid synthesis and consequently also lacks lipid particles. Growth of these mutants on oleate was strongly delayed and cell viability was decreased, but rescued by an adaptation process. In these strains, oleate stress caused morphological changes of intracellular membranes, altered phospholipid composition and increased formation of ethyl esters as a possible buffer for fatty acids. 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 a previous study we had demonstrated that squalene accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with TAG and STE. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In a recent study, we investigated the effect of squalene on biophysical properties of lipid particles and membranes and compared these results to artificial membranes. Our experiments showed that squalene lowered the order of STE shells in lipid particles. The majority of squalene, however, was localized to the center of lipid particles where it formed a soft core together with TAG. This view was confirmed with model lipid particles. In biological and artificial membranes fluorescence spectroscopy studies revealed that it is not the absolute squalene level per se, but the squalene to ergosterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membrane there is almost no additive effect of squalene. Our results demonstrated that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties. Pichia pastoris organelles and lipids The yeast Pichia pastoris is an important experimental system for heterologous expression of proteins. Nevertheless, surprisingly little is known about organelles of this microorganism. For this reason, we started a systematic biochemical and cell biological study to establish standardized methods of Pichia pastoris organelle isolation and characterization. Recent work 20
focused on the biochemical characterization of the plasma membrane and secretory organelles from Pichia pastoris. One major aim of this project is the qualitative and quantitative analysis of phospholipids, fatty acids and sterols from organelle membranes when Pichia pastoris is grown on different carbon sources. For this purpose, methods for the isolation of Pichia pastoris organelle fractions were established. Standardized techniques of lipid analysis including lipidome analyses are currently employed to address these problems. Doctoral Theses completed Sona Rajakumari: Role of yeast triacylglycerol lipases in membrane lipid metabolism Previous work from our laboratory had demonstrated that gene products of TGL3, TGL4 and TGL5 encoding the major yeast triacylglycerol (TAG) lipases are located to lipid particles. Deletion of TGL3 and TGL4 resulted in a decreased mobilization of TAG from the lipid particles and a sporulation defect. TAG stored in tgl3∆ and tgl5∆ deletion strains contains slightly increased amounts of C22:0 and C26:0 very long chain fatty acids (VLCFAs) compared to wild type. These VLCFAs are indispensable for sphingolipid biosynthesis and crucial for raft association in yeast. Moreover, deletion of these TAG lipases results in a decreased level of membrane lipid biosynthesis. In this Thesis the role of TAG lipases in membrane lipid metabolism of the yeast Saccharomyces cerevisiae was studied in some detail. First, a metabolic link between TAG lipolysis by TAG lipases and sphingolipid and phospholipid synthesis was shown. Secondly, a dual function of Tgl3p and Tgl5p as lipases and as lysophosphatidylethanolamine or lysophosphatidic acid acyltransferase, respectively, was demonstrated. We also showed that the acyltransferase but not the lipase function of Tgl3p was essential for efficient sporulation. Finally, we also characterized Tgl4p as multifunctional protein exhibiting lipase, phospholipase A2, acyl-CoA dependent LPA acyltransferase and STE hydrolase activity. Altogether, this work shows that catabolic and anabolic activities of TAG lipases play a pivotal role in maintaining lipid homeostasis in the yeast. Karlheinz Grillitsch: Lipid storage and mobilization in the yeast Saccharomyces cerevisiae The yeast Saccharomyces cerevisiae like higher eukaryotic cells (mammals and plants) and Gram-positive bacteria contains a specified organelle for lipid storage, the lipid particle (LP). Unlike other organelles, LP are covered by a phospholipid monolayer that protects its hydrophobic interior formed from densely packed non-polar lipids steryl esters (STE) and triacylglycerols (TAG). Moreover, LP contain a small but specific set of proteins. In this Thesis, storage and mobilization of yeast neutral lipids were studied. First, biochemical properties of the three STE hydolases, Tgl1p, Yeh1p and Yeh2p were investigated. Analysis of enzymatic properties revealed distinct substrate specificities of the three proteins and involvement in sterol homeostasis. Sterol homeostasis is also linked to cell polarity. We showed that two effectors of cell polarity, Ste20p and Cla4p, function as negative modulators of sterol biosynthesis. A major part of this Thesis was devoted to description of the molecular composition of the yeast LP and its modulation upon changes in cultivation conditions. For this purpose, LP from cells grown on either glucose or oleate were analyzed. Strong incorporation of the mono-unsaturated oleic acid into TAG and most phospholipids was observed upon shifting cells to oleate medium. Most notably, the balanced 1:1 ratio of TAG to STE in cells grown on glucose was strongly increased on oleate. Change of the medium also led to changes in the LP protein pattern. This was demonstrated by a combined 21
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
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Page 15 and 16: 2) Jajcanin-Jozic, N., Deller, S.,
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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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