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

iosynthesis in a type of feedback regulation. Conclusively, SE storage and mobilization
although being dispensable for yeast viability under laboratory conditions contribute
markedly to sterol homeostasis in this microorganism.
Previous work from our laboratory had demonstrated that deletion of TGL3, the
major yeast TAG lipase located to lipid particles, resulted in a decreased mobilization of
triacylglycerols (TAG) from the lipid particles. TAG stored in a tgl3 mutant contained
slightly increased amounts of C22:0 and C26:0 very long chain fatty acids (VLCFAs).
These VLCFAs are indispensable for sphingolipid biosynthesis and crucial for raft
association in the yeast. To understand the possible role of TAG lipolysis in
sphingolipid metabolism, we carried out radiolabeling experiments with sphingolipid
precursors in wild type and tgl mutants. These results indicated that the mutants have a
significantly reduced rate of sphingolipid biosynthesis and an increased rate of turnover
compared to wild type. Competition experiments with labeled palmitic acid (C16:0) and
non-radiolabeled cerotic acid (C26:0) demonstrated that the C26:0 fatty acid competed
with C16:0 fatty acid labeling of sphingolipids. Thus, we hypothesize that stored TAG
can be a fatty acid donor for sphingolipid formation and TAG lipases contribute to this
pathway of the yeast.
Another aspect addressed in our laboratory is characterization of yeast TAG
lipases with special emphasis on the topology of these proteins on the surface of lipid
particles where these proteins occur. This study is paralleled by topology studies of
other proteins located to the lipid particle surface, especially Erg1p, a squalene
epoxidase, which has been studied in our laboratory before. Preliminary studies
addressing accumulation of squalene in the yeast under certain conditions revealed that
this component is localized to different subcellular sites depending on the cell biological
and metabolic status of the yeast.
Characterization of organelles from the yeast Pichia pastoris
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. The expertise of our laboratory in cell fractionation and organelle
isolation from the yeast Saccharomyces cerevisiae has been developed through a
number of investigations in the past. Some of the methods applied for Saccharomyces
cerevisiae can be used for Pichia pastoris cell fractionation, whereas others cannot or
need to be adapted.
Recent work focused on the biochemical characterization of mitochondrial
membranes from Pichia pastoris grown on different carbon sources (Figure 4). The
major aims of this project were (1) qualitative and quantitative analysis of
phospholipids, fatty acids and sterols from mitochondrial membranes; and (2) analysis
of mitochondrial subfractions from Pichia pastoris grown on different carbon sources.
More recently, experiments were started to study selected lipid synthesizing enzymes
from Pichia pastoris with emphasis on the phosphatidylethanolamine biosynthetic
machinery. Preliminary data are a first step towards the understanding of the Pichia
pastoris lipidome which will provide initial insight into the variety of membrane
components of this yeast.
19