Staff Members of the Institute of Biochemistry, TU - Institut für ...

Diploma Thesis completed
Sabine Zitzenbacher: Characterization of organelles from the yeast Pichia pastoris
Characterizations of cell organelles from organisms start with a successful isolation of the
organelle of interest. Isolation protocols are available, but they usually have to be optimized.
A problem of each organelle preparation is cross-contamination with other cellular organelles
in the isolated organelle fraction. Here we tested the quality of preparation protocols for
mitochondria and microsomes, plasma membrane and vacuoles, used for the strain Pichia
pastoris. Isolated fractions were analysed by Western blot analysis and functional enzyme
assays. Results of isolated mitochondrial and microsomal fractions showed a clear enrichment
of mitochondria as well as microsomes. In the mitochondrial fraction a minor ER
contamination was observed, whereas the microsomes fractions were free from crosscontamination.
Analysis of the isolated plasma membrane fraction showed enrichment of
plasma membrane without any cross contamination with other organelles. Both preparation
protocols have proven to be well established for Pichia pastoris. The last protocol
investigated showed an enrichment of vacuoles in the isolated fraction, but the vacuolar
fraction also showed a high contamination with mitochondria. Thus, the protocol has to be
modified to get an isolated vacuolar fraction without any or with only little crosscontamination.
In the past, different cell organelles, such as peroxisomes, mitochondria,
microsomes and plasma membrane from Pichia pastoris were characterized. The missing
organelle is the Golgi apparatus. Here we established an isolation protocol for Golgi isolation
from Pichia pastoris. The obtained Golgi fraction was analysed by Western Blot and
enzymatic assays. Applying this new preparation protocol we showed an enrichment of Golgi
in the isolated fraction largely free of cross-contamination with ER.
Doctoral Thesis completed
Tibor Czabany: Cell biology of neutral lipid synthesis in yeast Saccharomyces cerevisiae
Previously, four acyltransferases involved in the synthesis of neutral lipids (NL),
triacylglycerols (TAG) and steryl esters (SE), have been identified. Dga1p and Lro1p esterify
DAG to produce TAG, and Are1p and Are2p are responsible for SE synthesis. After the NL
have been synthesized they are assembled in specialized organelles called lipid particles (LP).
The hydrophobic core of LP containing TAG and SE is separated from the hydrophilic
cytosol by a phospholipid monolayer. The present study is focused on elucidation of the
detailed structure using a set of triple mutants (TM) which contain only one of the four
acyltransferases active. Using these mutants we were able to investigate the influence of
individual enzymes on LP composition and structure. It was demonstrated that the four
acyltransferases act independently of each other and are able to synthesize individually a
sufficient amount of neutral lipids for LP biogenesis. Furthermore, TM and the quadruple
mutant, lacking all four acyltransferases, were also used for studies of NL synthesis when
fatty acids were supplied as the only carbon source. Under these conditions, fatty acids enter
the cell and accumulate at high amounts which leads to disturbed lipid homeostasis. Changes
in lipid metabolism caused by the lack of individual acyltransferase are described, especially
the influence of excess fatty acids on neutral lipid synthesis. It was shown that yeast cells
grown on oleic acid upregulated TAG synthesis and down regulated esterification of sterols.
Detailed analysis of SE synthase revealed that the down regulation of this activity was not by
transcriptional control but rather by direct inhibition of the enzyme in its membrane
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