Sterol esters (SE) together with triacylglycerols (TG) are the major storage lipids in most eukaryotic cells. Formation of these molecules and degradation/mobilization are important processes for the overall lipid homeostasis in the cell. While storage of TG and SE in lipid droplets, synthesis and lipolysis of TG as well as formation of SE have been studied in some detail, little evidence about SE hydrolysis is available. The goal of the project presented here will be to close this gap and to generate data on the biochemistry, molecular biology and cell biology of SE hydrolases using the well-established model cell, the yeast Saccharomyces cerevisiae, as an experimental system. In the yeast, three major SE hydrolytic enzymes (sterol esterases) have been identified, namely the gene products of YEH1, YEH2 and TGL1. To shed more light on these three yeast proteins, enzymatic properties, subcellular localization and topology of the enzymes, and regulatory aspects affecting both enzymatic properties and subcellular localization of the enzymes will be studied. When revisiting the enzymatic properties of Yeh1p, Yeh2p and Tgl1p, much emphasis will be placed on second or side activities of these enzymes, e.g. phospholipase or acyltransferase activities, as shown recently for other lipid hydrolytic enzymes. Subcellular localization of Yeh1p and Tgl1p in lipid droplets, and of Yeh2p on the cell periphery was shown before, but details such a dual localization, e.g. in lipid droplets and the endoplasmic reticulum, need to be tested. Also shift of the enzymes under certain conditions between organelles will be an important issue. These investigations will lead us to a closer study of possible targeting domains and membrane anchor(s), but also of active sites, substrate binding sites and regulatory domains of the enzymes. Studies of the membrane topology/orientation of the yeast SE hydrolases in the different subcellular membrane compartments will help to elucidate their enzymological and cellular properties. Finally, regulatory aspects of yeast SE hydrolases will be addressed at an individual but also at a genome wide level. The effects of compromised SE synthesis on expression, subcellular localization and activity of the YEH1, YEH2 and TGL1 gene products will be studied. Vice versa, the effect of SE hydrolase depletion on expression and activity of SE synthesizing enzymes will be investigated. Feedback control of SE hydrolysis to ergosterol and fatty acid synthesis enzymes will be included in these investigations. In summary, our studies will provide information about important links between gene expression, protein formation, enzymatic properties, subcellular localization and cellular function of yeast SE hydrolases which have not been addressed before. The expected results will broaden our general knowledge of lipid homeostasis not only in the yeast but may also become relevant for other cell types.
|Effective start/end date||1/09/14 → 31/08/17|