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 important 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 shown through a number of investigations in the past.
Recently, our studies were focused on Pichia pastoris mitochondria. Appropriate growth conditions were chosen to induce proliferation of this organelle. A method originally developed for Saccharomyces cerevisiae was adapted to isolate inner and outer membranes of Pichia pastoris mitochondria. Proteins and lipids of total mitochondria and submitochondrial membranes were identified and quantified. Other Pichia pastoris organelles currently under investigation are the plasma membrane and the Golgi.
Figure 3: Phylogenetic tree of PSD sequences from different organisms. The tree was constructed using the neighbor joining (NJ) method of the CLUSTALW program
We also started to study selected lipid synthesizing enzymes from Pichia pastoris with emphasis on the phosphatidylethanolamine (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. Such manipulations may provide a useful tool to adapt membranes from Pichia pastoris for biotechnological purposes.