Graz University of Technology Austria Institute of Biochemistry ...

grown on glucose or on oleic acid. This approach identified a number of lipid particle proteins
that were already known but also some novel polypeptide candidates. We also realized
through this approach that there were some differences in the lipid particle proteome from
cells grown on glucose or oleic acid. Finally, mass spectrometric analyses revealed marked
differences in the lipidome of lipid particles from cells grown on the two different carbon
sources. This study sets the stage for further investigation of protein-lipid interaction on the
surface of lipid particles and provides basic evidence for the coordinated biosynthesis of lipid
and protein components from lipid particles.
Another important aspect of this project was to understand cell biological consequences of
dysfunctions in non-polar lipid storage. For this purpose, yeast cells were cultivated on oleic
acid which was assumed to provoke lipotoxic stress. We found that under these cultivation
conditions TAG synthesis was enhanced creating the major pool for the excess of fatty acids,
whereas surprisingly STE synthesis was strongly inhibited. We showed that this effect was
not due to decreased expression of ARE2 encoding the major yeast STE synthase at the
transcriptional level, but to competitive enzymatic inhibition of Are2p by free oleic acid. As a
result, a triple mutant dga1∆lro1∆are1∆ARE2 + grown on oleate did not form substantial
amounts of STE and exhibited a growth phenotype similar to the dga1∆lro1∆are1∆are2∆
quadruple mutant which lacks all four acyltransferases involved in neutral lipid synthesis and
consequently also lacks lipid particles. Growth of these mutants on oleate was strongly
delayed and cell viability was decreased, but rescued by an adaptation process. In these
strains, oleate stress caused morphological changes of intracellular membranes, altered
phospholipid composition and increased formation of ethyl esters as a possible buffer for fatty
acids.
Another potential non-polar storage lipid is squalene. This component belongs to the group of
isoprenoids and is precursor for the synthesis of sterols, steroids and ubiquinons. In a previous
study we had demonstrated that squalene accumulates in yeast strains bearing a deletion of the
HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets
together with TAG and STE. In mutants lacking the ability to form lipid particles, however,
substantial amounts of squalene accumulate in organelle membranes. In a recent study, we
investigated the effect of squalene on biophysical properties of lipid particles and membranes
and compared these results to artificial membranes. Our experiments showed that squalene
lowered the order of STE shells in lipid particles. The majority of squalene, however, was
localized to the center of lipid particles where it formed a soft core together with TAG. This
view was confirmed with model lipid particles. In biological and artificial membranes
fluorescence spectroscopy studies revealed that it is not the absolute squalene level per se, but
the squalene to ergosterol ratio which mainly affects membrane fluidity/rigidity. In a fluid
membrane environment squalene induces rigidity of the membrane, whereas in rigid
membrane there is almost no additive effect of squalene. Our results demonstrated that
squalene (i) can be well accommodated in yeast lipid particles and organelle membranes
without causing deleterious effects; and (ii) although not being a typical membrane lipid may
be regarded as a mild modulator of biophysical membrane properties.
Pichia pastoris organelles and lipids
The yeast Pichia pastoris is an important experimental system for heterologous expression of
proteins. Nevertheless, surprisingly little is known about organelles of this microorganism.
For this reason, we started a systematic biochemical and cell biological study to establish
standardized methods of Pichia pastoris organelle isolation and characterization. Recent work
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