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
Plant Biochemistry and Biophysics
Final Report Abstract
The project deals with two aspects of lipid and membrane functions in the yeast Pichia pastoris. This yeast and many other fungi contain two types of membrane-bound glycosphingolipids, glucosylceramides and glycosyl inositolphosphorylceramides which show structural differences in their lipid backbone as well as in their sugar headgroup. These structural features are probably essential for their different biological functions in separate domains of the plasma membrane of the yeast cells. In the project we used a series of Pichia mutants with different impairments in sphingolipid biosynthesis to analyse their sphingolipid composition by mass spectrometry methods. In addition, the enzymatic properties of a key enzyme of the pathway, the ceramide synthase Bar1, were determined. We found two checkpoints of glycosphingolipid biosynthesis which are responsible for the formation of the two structurally distinct glycosphingolipids. These findings are valuable for future investigations of their biological functions. Pichia pastoris contains a second glycolipid, ergosteryl β-glucoside which is involved in the functions of peroxisomes. When the cells grow on methanol as the sole carbon source, peroxisomes proliferate to support the utilization of methanol. Switching the carbon source to glucose results in the degradation of the peroxisomes by an autophagic mechanism called micropexophagy. We used mutants of Pichia expressing different cyan fluorescent protein-labelled and enzymatically active or inactive variants of the sterol β-glucosyltransferase. These cells were investigated by fluorescence microscopy, lipid analysis and enzymatic assays representing peroxisome proliferation and degradation, respectively. Surprisingly, we found that the lipid ergosteryl -glucoside is not only essential for the formation of a membrane system required for peroxisome degradation, but seems also to be involved in the proliferation of peroxisomes. This novel hypothesis, developed at the late phase of the project, requires further investigations prior to publication. In the course of this project, we identified two novel sterol glycosyltransferases of the pathogenic bacteria Borrelia burgdorferi and B. hermsii.
Publications
- (2010). The functions of steryl glycosides come to those who wait: Recent advances in plants, fungi, bacteria and animals. Prog Lipid Res. 49: 262-288
Grille S, Zaslawski A, Thiele S, Plat J, Warnecke D
- (2011). Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris. J Biol Chem. 286:1 1401-14. Epub 2011 Feb 8
Temes P, Wobbe T, Schwarz M, Albrecht S, Feussner K, Riezman I, Cregg JM, Heinz E, Riezman H, Feussner I, Warnecke D
