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

Pichia pastoris organelles
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
focused on the biochemical characterization of mitochondrial membranes and the plasma
membrane from Pichia pastoris grown on different carbon sources. The major aim of this
project was the qualitative and quantitative analysis of phospholipids, fatty acids and sterols
from both types of membranes, when Pichia pastoris was grown on different carbon sources.
We also started to study selected lipid synthesizing enzymes from Pichia pastoris with
emphasis on the PE biosynthetic machinery. We identified two Pichia pastoris PS
decarboxylases (PSD) encoded by genes homologous to PSD1 and PSD2 from
Saccharomyces cerevisiae (Figure 3). Using Pichia pastoris psd1 and psd2 mutants we
investigated the role of these two gene products in PE synthesis and the effect of their
deletions on cell growth. Mitochondrial Psd1p is the major enzyme for PE synthesis in Pichia
pastoris. Deletion of PSD1 caused a loss of PSD activity in mitochondria and a severe growth
defect on minimal media which was partially cured by supplementation with ethanolamine.
This result indicated that the CDP-ethanolamine but not the Psd2p pathway provided
sufficient PE for cell viability in a psd1 background. Under these conditions, however,
cellular and mitochondrial PE levels were dramatically reduced. Fatty acid analysis from wild
type, psd1 and psd2 demonstrated the selectivity of both PSDs in vivo for the synthesis of
unsaturated PE species. Short chain saturated (16:0) PE species were preferentially imported
into mitochondria irrespective of psd1 or psd2 deletions. Thus, biosynthesis and subcellular
distribution contribute to PE homeostasis in mitochondria of Pichia pastoris.
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Figure 3:
Phylogenetic tree of PSD sequences from different organisms. The tree was constructed using
the neighbor joining (NJ) method of the CLUSTALW program.
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