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inactivation of SWS (swiss cheese), the homolog of NTE in the fruit fly Drosophila melanogaster, leads to a progressive degeneration of the nervous system, which can be observed as big “holes” in the brain of the fly. Heterologous expression of the murine NTE can completely substitute SWS in Drosophila, demonstrating the functional conservation of this protein. A homolog of NTE is also present in yeast Saccharomyces cerevisiae encoded by the NTE1. In contrast to higher eukaryotes, deletion of this protein in the model organism yeast does not result in a phenotype under several standard conditions tested. However, due to the presence of this polypeptide in a single cell organism, NTE must be involved in a more general context as in development, neural survival and brain integrity. All efforts to identify the biological function, substrate, and interaction partners of NTE in higher eukaryotes have failed so far. In contrast, Nte1p of the yeast has been reported to exhibit phospholipase B activity, thus indicating a similar function of the respective counterparts of higher eukaryotes. In this project, we are investigating whether Nte1p of Saccharomyces cerevisiae is a true member of the NTE protein family and thus suitable as a model for unravelling the biological function of this protein family. In vitro, the functional conservation between NTE of higher eukaryotes and Nte1p of the yeast has already been demonstrated. But is this also the case in vivo For answering this question, I expressed NTE1 of the yeast heterologously in the fruit fly Drosophila melanogaster. A functional compensations would be indicated by reversion of the phenotype of an sws-mutant fly, i.e., observable as a decrease in the size and / or number of “holes” in the brain of Drosophila. Several different mutant lines were constructed expressing the yeast NTE1 to different extent and in different tissues. Subsequently, the brains of these mutants were analyzed by fluorescence microscopy for the presence of “holes”. Any result from full reversion of the phenotype to no effect has been considered, however, the phenotype was even worse in the transfectants showing a higher number of “holes”. In addition, the life span of the sws-mutants expressing NTE1 of yeast was significantly reduced compared to control flies. This result indicates that there is no functional conservation among the NTE protein family in vivo. However, my searches for an explanation what caused this rather striking effect provided some evidence that Nte1p interacts with enzymes of the lipid metabolism and forms a protein complex with at least one more polypeptide. Preliminary results indicated that this is also true for Sws of the fruit fly. Furthermore, mutations in certain lipid synthesizing pathways led to smaller and a fewer number of holes in the brain of a sws-mutant fly, thus indicating that the phenotype of the sws-mutant is caused by major changes in the lipid pattern. Analysis of the lipid pattern of the respective mutant flies will reveal which lipid parameters are required for normal development of the nervous system, and will provide valuable information for potential targets to compensate effects caused by neurons in metabolic disorders (e.g., diabetes), toxic states and advanced age. This work was performed at OHSU (Oregon Health and Sciences University, Portland, Oregon) during my post doctoral studies in 2007. Lipid metabolism of the yeast Yarrowia lipolytica The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols. For this reason, this microorganism has become attractive for industrial applications such as production of single cell oil. In a former project, we investigated lipid particles of the oleaginous yeast with respect to their lipid and protein composition. Analysis of the lipid particle proteome identified major lipid particle 24
proteins. The “standard” proteome of lipid particles isolated from glucose grown cells was complemented by several others when cells were grown on oleic acid. Homology searches using the ORFs coding for lipid particle polypeptides of Yarrowia lipolytica as a query revealed that most lipid particle proteins are counterparts of proteins involved in lipid metabolism in Saccharomyces cerevisiae. However, since the function of these proteins was only deduced upon homology, we are currently investigating the “true” function of these proteins with a special emphasis on those involved in triacylglycerol synthesis. Figure 2: Accumulation of neutral lipids in lipid particles of Yarrowia lipolytica wildtype cell either grown on oleic acid (left) or glucose (right). Neutral lipids are stained with the fluorescent lipophilic dye NileRed®. Special Activities Habilitation for the subject Biochemistry Postdoctoral studies at the Oregon Health and Sciences University (OHSU), Portland, Oregon, USA International Cooperations T. Chardot and J.-M. Nicaud, Institute National de la Recherche Agronomique, UMR Chimie Biologique, Thiverval Grignon, France D. Kretzschmar, Oregon Health and Sciences University, Portland, Oregon, USA J. M. Thevelein, Centre for Food and Microbial Technology, Department of Microbial and Molecular Systems, Katholeke Universiteit Leuven, Heverlee, Belgium Publications 1) Daum, G., Wagner, A., Czabany, T. and Athenstaedt, K. Dynamics of neutral lipid storage and mobilization in the yeast Biochimie 89 (2007) 243-248 2) Czabany, T., Athenstaedt, K. and Daum, G. Synthesis, storage and degradation of neutral lipids in yeast Biochim. Biophys. Acta 1771 (2007) 299-309 3) Daum, G, Wagner, A., Czabany, T, Grillitsch, K. and Athenstaedt, K. Lipid storage and mobilization pathways in yeast Novartis Foundation Symposium No.286 (2007), 142-154 25
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