Projekt A) Identification of the functions of ergosteryl-ß-glucoside in the degradation of peroxisomes in the yeast. Projekt D) Dissecting glucosylceramide biosynthesis in the yeast Pichia pastoris

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Projekt A)Autophagy is a cellular, degenerative pathway which is conserved in eukaryotes. The yeast Pichia pastoris can degrade its peroxisomes by a specific autophagic process called micropexophagy. We showed that P. pastoris mutants which are either deficient in ergosteryl-β-glucoside biosynthesis or in the localization of the ergosterol-β-glucosyltransferase are impaired in micropexophagy. The aim of the project is to reveal whether the ergosterol-β-glucosyltransferase activity and its lipid product, ergosteryl-β-glucoside, are essential for micropexophagy or the protein itself. It might be that the protein supports micropexophagy independently of its ergosterol-β-glucosyltransferase activity e.g. by protein-protein interactions. Therefore, we expressed variants of the ergosterol-β-glucosyltransferase with single amino acid alterations within the catalytic domain in P. pastoris. These cells will be examined for ergosteryl-β-glucoside biosynthesis and micropexophagy. Since preliminary results suggest that the glycolipid is required for micropexophagy, it is interesting to determine the structural details of the ergosteryl-β-glucoside which are required to fulfill its function. Therefore, we will replace the native ergosteryl-β-glucoside with structurally similar glycolipids and determine whether they support proper micropexophagy. To reach this goal, sterol glycosyltransferases of different sugar specificity will be expressed in P. pastoris.Projekt D)Sphingolipids are ubiquitous membrane lipids present in all eukaryotic and some prokaryotic organisms. Most fungi contain two different types of glycosylated sphingolipids with a distinct ceramide backbone: Glucosylceramide (GlcCer) and glycosyl inositol phosphorylceramide (GIPC). Our longterm goal is to establish the yeast Pichia pastoris as a model system to investigate the spatial separation and intracellular trafficking of sphingolipids, the formation of lipid domains and the biological functions of these structurally different types of glycosphingolipids. We started our work by the identification and characterization of six genes and their corresponding enzymes which are involved in the introduction of functional groups into the ceramide backbone, such as desaturases, hydroxylases, methyltransferases and glycosyltransferases. Subsequently, knock-out mutants of P. pastoris, each impaired in the function of one of these genes, were generated. In order to achieve a complete description of GlcCer biosynthesis in P. pastoris, the free ceramides present in these mutant strains will now be analyzed by LC/MS. In addition, the so far hypothetical GIPCs from this yeast will be purified and identified by mass spectrometrical methods.