Steryl ester hydrolases of the yeast

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.

The two major storage lipids in the yeast Saccharomyces cerevisiae are the nonpolar lipids triacylglycerol and steryl esters. Both lipid species are synthesized in the endoplasmic reticulum. Dga1p and Lro1p are responsible for the formation of triacylglycerols and Are1p and Are2p catalyze the synthesis of steryl esters. The latter enzymes also contribute to the formation of triacylglycerols, although to a minor amount. Once steryl esters and triacylglycerols are built up, they can be stored in an organelle-like structure termed lipid droplet. In times of starvation or growth, these nonpolar lipids serve as important building blocks for membrane lipid formation and as energy pool. Therefore steryl esters and triacylglycerols need to be mobilized by hydrolytic enzymes. Whereas triacylglycerols are hydrolyzed by triacylglycerol lipases, our focus was set on the three steryl ester hydrolases Tgl1p, Yeh1p and Yeh2p. Tgl1p and Yeh1p are known to be lipid droplet resident enzymes, in contrast of Yeh2p which is located at the cell periphery. The overall aim of this work was to shed light on regulatory aspects of steryl ester metabolism. On the one hand, we investigated the steryl ester synthases Are1p and Are2p under conditions of deprived steryl ester mobilization. We clearly demonstrated a feedback regulation on the two steryl ester forming enzymes. Although gene expression and protein levels of the two acyltransferases were not affected in a strain lacking all three steryl ester hydrolases, the in vitro activity of Are1p and Are2p and the in vivo incorporation of radio-labelled fatty acids into steryl esters was significantly reduced under the tested conditions. On the other hand, we were curious about the fate of the three steryl ester hydrolases in the absence or presence of nonpolar lipids. For these studies we used strains either lacking steryl esters, triacylglycerols or lipid droplets at all. Based on the performed experiments we showed that Tgl1p and Yeh1p are retained to the endoplasmic reticulum solely in the strain lacking lipid droplets. As a consequence of this relocalization, the enzymes lost their hydrolytic activity and were highly unstable. Yeh2p was confirmed to be a protein localized at the plasma membrane, even in a strain lacking lipid droplets. Furthermore, Yeh2p is subject to a posttranslational modification, namely phosphorylation. In summary, results shown in this project are one step forward to understand how steryl ester metabolism is regulated in the yeast S. cerevisiae.