The major sorting station of biosynthetic material destined for the cell surface or secretion is the trans Golgi Network, TGN. This organelle sorts proteins and lipids into vesicular transport carriers that are targeted via different pathways to distinct membrane compartments of the cell. The molecular principles that operate in cargo sorting at the TGN are still not very well understood. Especially, we know very little about the sorting of lipids. It was postulated that a sorting mechanism based on clustering of lipid rafts, dynamic membrane domains enriched in sphingolipids and sterols, could be an important part of the picture. My thesis study dealt with the elucidation of the molecular sorting principles at the TGN and their exploitation for cell surface polarity in the yeast Saccharomyces cerevisiae. To this end, we conducted a genome wide screen that identified yeast mutants defective in cell surface delivery of the model cargo protein FusMid-GFP. The most striking result of this screen was that mutant strains with defects in ergosterol (the major yeast sterol) and sphingolipid biosynthesis lost sorting competence. To elucidate a direct role for sphingolipids and ergosterol in cargo sorting and secretion we sought to characterize the lipid composition of secretory vesicles. Hence, we established a vesicle purification protocol based on an immunoisolation strategy. Additionally, in collaboration with the group of A. Shevchenko, we developed a mass spectrometry methodology that allows the comprehensive and quantitative lipid analysis of subcellular organelles. Preliminary results corroborate our genetic evidence. The data show that the vesicles are enriched in sphingolipids and decreased in phosphatidylcholine indicating a role for raft clustering in cargo sorting at the TGN. The studies of cell polarity during yeast mating also unraveled a role for raft clustering. We could identify that the lipid bilayer at the tip of the mating projection was more ordered than at the plasma membrane enclosing the cell body and that this was dependent on sphingolipid synthesis. The results of my thesis suggest that in the yeast Saccharomyces cerevisiae fundamental cell biological processes such as cargo sorting and vesicle formation at the TGN as well as cell surface polarity during mating employ raft clustering mechanisms.

The major sorting station of biosynthetic material destined for the cell surface or secretion is the trans Golgi Network, TGN. This organelle sorts proteins and lipids into vesicular transport carriers that are targeted via different pathways to distinct membrane compartments of the cell. The molecular principles that operate in cargo sorting at the TGN are still not very well understood. Especially, we know very little about the sorting of lipids. It was postulated that a sorting mechanism based on clustering of lipid rafts, dynamic membrane domains enriched in sphingolipids and sterols, could be an important part of the picture. My thesis study dealt with the elucidation of the molecular sorting principles at the TGN and their exploitation for cell surface polarity in the yeast Saccharomyces cerevisiae. To this end, we conducted a genome wide screen that identified yeast mutants defective in cell surface delivery of the model cargo protein FusMid-GFP. The most striking result of this screen was that mutant strains with defects in ergosterol (the major yeast sterol) and sphingolipid biosynthesis lost sorting competence. To elucidate a direct role for sphingolipids and ergosterol in cargo sorting and secretion we sought to characterize the lipid composition of secretory vesicles. Hence, we established a vesicle purification protocol based on an immunoisolation strategy. Additionally, in collaboration with the group of A. Shevchenko, we developed a mass spectrometry methodology that allows the comprehensive and quantitative lipid analysis of subcellular organelles. Preliminary results corroborate our genetic evidence. The data show that the vesicles are enriched in sphingolipids and decreased in phosphatidylcholine indicating a role for raft clustering in cargo sorting at the TGN. The studies of cell polarity during yeast mating also unraveled a role for raft clustering. We could identify that the lipid bilayer at the tip of the mating projection was more ordered than at the plasma membrane enclosing the cell body and that this was dependent on sphingolipid synthesis. The results of my thesis suggest that in the yeast Saccharomyces cerevisiae fundamental cell biological processes such as cargo sorting and vesicle formation at the TGN as well as cell surface polarity during mating employ raft clustering mechanisms.

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