Neutral lipids in yeast

The present project describes molecular biological, cell biological and biochemical strategies to identify genes and gene products involved in the biosynthesis and metabolism of triacyl-glycerols (TAG) and steryl esters (STE) of the yeast Saccharomyces cerevisiae. This project is aimed at a better understanding of how lipid depots are formed and mobilized from their site of storage, the so-called lipid particles, under conditions of fatty acid and/or sterol shortage. This study will fundamentally contribute to our knowledge of neutral lipid metabolism in yeast. During these investigation we will also address general aspects regarding the physiological role(s) of lipid stores and their formation and degradation. Due to cell biological and biochemical similarities, studies performed with the unicellular eukaryotic model yeast as described in this application will also be relevant for multicellular organisms including man. The following specific aspects will be addressed during this investigation: (i) Identification of new genes encoding enzymes of TAG synthesis; characterization and subcellular localization of the respective gene products; characterization of the coordinate regulation of different pathways of TAG synthesis. (ii) Interplay of lipid particles and endoplasmic reticulum in STE formation with special emphasis on lanosterol transfer between the two compartments; involvement of the protein secretory pathway in STE formation. (iii) Mobilization of TAG and STE from lipid particles and utilization of their components; identification of enzymes (lipases, hydrolases) and of the corresponding genes involved in TAG and STE mobilization; characterization and localization of the respective enzymes; characteri-zation of coordinate regulation of TAG and STE degradation by isoenzymes in different subcellular compartments. (iv) Cell biological consequences of TAG and/or STE depletion or over-production; phenotypic analysis of yeast mutants with single and multiple defect(s) in TAG and STE formation and mobilization. (v)

The aim of this project was to contribute substantially to our understanding of formation, storage and utilization of the neutral lipids, triacylglycerols (TAG) and steryl esters (STE). The model organism baker`s yeast was employed for these studies because general aspects of lipid metabolism in yeast are similar to those in higher eukaryotes including man. Major topics studied and accomplished during this FWF project were: (i) Identification and characterization of novel genes encoding enzymes of TAG synthesis. During these studies we identified the acyl-CoA:diacylglycerol acyltransferase Dga1p which forms a majority of yeast TAG. Specific emphasis was laid on the coordinate synthesis of TAG in different subcellular compartments, the role of different enzymes involved in this process and the biochemical characterization of the respective enzymatic reactions. (ii) Functional characterization of yeast lipid particles where TAG and STE are stored. These studies were focused on the involvement of this compartment in ergosterol biosynthesis with special emphasis on the interplay of lipid particles with the endoplasmic reticulum (ER). The oxidosqualene synthase Erg7p and the 3-keto reductase Erg27p were identified as two lipid particle components interacting during yeast sterol biosynthesis. Flux of sterol intermediates between lipid particles and the ER was studied with special emphasis on the role of Erg11p which catalyzes the reaction following Erg7p is sterol synthesis. Erg11p in contrast to Erg7p was associated with the ER. The biosynthetic pathway of ergosterol therefore requires traffic of sterol intermediates between lipid particles and the ER. (iii) Identification of novel genes and gene products involved in the mobilization process of TAG and STE from lipid particles. In this study, six novel gene products, namely the three TAG lipases Tgl3p, Tgl4p and Tgl5p, and the three STE hydrolases Tgl1p, Yeh1p and Yeh2p, were discovered and characterized. With the exception of Yeh2p, which is a plasma membrane protein, all other polypeptides are localized to lipid particles. Experiments in vivo and in vitro were performed to characterize these enzymes biochemically and cell biologically. (iv) The physiological role of neutral lipids. Using a yeast quadruple mutant lacking all four TAG and STE synthases we demonstrated that proteins typically located to lipid particles in wild type are refined to microsomal fractions of this mutant thus confirming the close relationship between lipid particles and ER.