Staff Members of the Institute of Biochemistry, TU Graz http://www ...

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
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