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Storage of non-polar lipids in lipid droplets and mobilization Yeast cells like most other cell types have the capacity to store non-polar lipids. In the case of Saccharomyces cerevisiae triacylglycerols (TG) and steryl esters (SE) are the predominant lipid storage molecules which accumulate in subcellular structures named lipid particles/droplets. Upon requirement, TG and SE 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 formation and mobilization of TG and SE depots. Life cycle of yeast neutral lipids: Genes involved in non-polar lipid formation and degradation. Previous studies in our laboratory had identified Tgl3p, Tgl4p and Tgl5p as major yeast TG lipases. In addition, however, a number of candidate gene products with potential lipase/esterase activity were also identified. One of these polypeptides is Ldh1p, a lipid droplet associated hydrolytic enzyme. In collaboration with the laboratory of R. Erdmann, Bochum, Germany, we demonstrated that recombinant Ldh1p exerts esterase and TG lipase activities. The enzyme activity was abolished upon mutation of the conserved GXSXG-type lipase motif of the protein. Saccharomyces cerevisiae deleted of LDH1 was characterized by the appearance of giant lipid droplets and accumulation of non-polar lipids and phospholipids in LDs, indicative of a role of Ldh1p in maintaining lipid homeostasis. A major focus of our non-polar lipid project was the biochemical characterization of the three yeast TG lipases, Tgl3p, Tgl4p and Tgl5p. Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast TG lipase resulted in decreased mobilization of TG, 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 TG fraction. Surprising evidence was obtained when the enzymology of the three TG lipases Tgl3p, Tgl4p 18
and Tgl5p was studied. Motif search analysis indicated that Tgl3p, Tgl4p and Tgl5p do not only contain the TG lipase but also an acyltransferase motif. Indeed, all three enzymes exhibit lipase activity but also catalyze acylation of lysophosphatidylethanolamine and lysophosphatidic acid, respectively. Experiments using variants of lipases demonstrated that the two enzymatic activities act independently of each other. These results demonstrated that yeast lipases may play a dual role in lipid metabolism contributing to both anabolic and catabolic processes. This finding led us to investigate more deeply the biochemistry of yeast lipases. Currently, molecular properties of these enzymes including their topology on lipid droplets and the functional link between non-polar lipid formation and degradation are studied. Several years ago we identified through a mass spectrometric approach for the first time the major lipid droplet proteins from 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/droplets. Recently, a more precise yeast lipid droplet proteome analysis was initiated in collaboration with M. Karas, Frankfurt, Germany. This proteome study was combined with a lipidome investigation performed in collaboration with H. Köfeler, Medical University of Graz, Austria. In this study, we compared lipid droplet components from cells grown on glucose or oleate. This approach identified a number of lipid droplet 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 droplet proteome from cells grown on glucose or oleate. 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 investigations of protein-lipid interaction on the surface of lipid droplets and provides fundamental evidence for the coordinated biosynthesis of lipid and protein components during biogenesis of this compartment. Identification of novel lipid droplet proteins from cell grown on glucose (YPD) or oleate (YPO). Another 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 TG and SE. In mutants lacking the ability to form lipid droplets, however, substantial amounts of squalene accumulate in organelle membranes. In a recent study, we 19
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