Lipases of the yeast S. cerevisiae

All types of eukaryotes including yeast contain lipid depots. Depending on the cell type, storage lipids are in most cases triacylglycerols (TAG) and steryl esters (STE). These storage lipids accumulate in a specific compartment called lipid particle or lipid droplet. TAG and STE serve as a source of energy, but also as building blocks (fatty acids, sterols) for the formation of biological membranes and bioactive metabolites. Therefore, formation, storage and mobilization of non-polar lipids (TAG and STE) are important for various cell biological processes. The present proposal will focus on TAG storage and mobilization in the yeast Saccharomyces cerevisiae thereby continuing studies performed during the previous FWF Project 18857. Yeast has proven a highly suitable cellular model for studies of lipid metabolism and membrane biology. The specific goal of this project will be investigating biochemical and cell biological properties of the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. As shown most recently in our lab these three enzymes, which are located to the surface of lipid particles/droplets, do not only hydrolyze TAG with different specificity, but also fulfill biosynthetic function(s) by catalyzing lysophospholipid acyltransferase reactions. As result of these dual and independent activities, TAG lipases need to get efficient access to their substrates which are TAG molecules in the core of the lipid particle/droplet; and contribute to lipid anabolism by channeling fatty acids set free to the formation of complex lipid products. Whereas basic biochemical knowledge about Tgl3p, Tgl4p and Tgl5p is available, little is known about molecular structure and function of these proteins, their topology in lipid particles, and their physical and functional interaction with partner components. Therefore, aims of this project will be to (i) perform topology studies with TAG lipases in their membrane environment with emphasis on the orientation of active sites (lipase, acyltransferase) of the enzymes; (ii) address regulatory aspects of TAG lipases with emphasis on the link to TAG formation and utilization under different cultivation conditions; (iii) investigate physical interaction of TAG lipases with potential partner proteins, which might act as regulators or adaptors; and (iv) isolate yeast TAG lipases and/or fragments of these enzymes with focus on Tgl3p, the major yeast TAG lipase, for protein structure analysis. Methods and tools required for these studies are either well established in our lab or covered by collaborations with scientific partners. Data available through previous projects and recent investigations will be the basis for detailed studies suggested in this proposal.

All types of eukaryotes including yeast contain lipid depots. Depending on the cell type, storage lipids are in most cases triacylglycerols (TAG) and steryl esters (SE). These storage lipids accumulate in a specific compartment called lipid particle or lipid droplet. TAG and SE serve as a source of energy, but also as a depot of building blocks (fatty acids, sterols) for the formation of biological membranes. Therefore, synthesis, storage and mobilization of non-polar lipids (TAG and SE) are important for various cell biological processes. The present proposal will focus on TAG storage and mobilization in the yeast Saccharomyces cerevisiae thereby continuing studies performed during a previous FWF project. Yeast has proven a highly suitable cellular model for studies of lipid metabolism and membrane biology. The specific goal of this project was to study biochemical and cell biological properties of the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Three lines of investigation were in the focus of this project. (i) A major goal of our studies was to address regulatory aspects of TAG lipases with emphasis on the link between TAG formation and utilization. (ii) A further important aspect which we studied was topology of TAG lipases, especially of Tgl3p, in their membrane environment with emphasis on the orientation of active sites of the enzymes. (iii) Finally, we wished to identify and characterize novel TAG lipases from the yeast. Among several candidates Ayr1p was studied in more detail. Methods required for these studies, e.g., microbiology of the yeast, handling of organelles, and analysis of lipids and proteins, were standard in our lab and supplemented by collaborations with national and international scientific partners.