FWF - Pichia Organellen - Characterization of organelles from the yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris is worldwide used as the most important and efficient expression system for heterologous proteins. In addition to this biotechnological application, this yeast has become a suitable model organism for studying protein secretion and the biogenesis of peroxisomes, an organelle which is highly induced when Pichia pastoris is grown on methanol or fatty acids as the only carbon source. Although much progress has been made during the last few years to understand biochemistry and molecular biology of Pichia pastoris, surprisingly little is known about cell biological features of this microorganism. In fact, information in the literature about properties of Pichia pastoris organelles is rare to non-existing. For this reason, we propose in the project presented here a systematic approach to isolate, analyze and characterize major organelles from this microorganism with special emphasis on proteins and lipids of organelle membranes. With increasing specificity we will develop and adapt methods for the isolation of peroxisomes, mitochondria, endoplasmic reticulum fractions, vacuoles, lipid particles, Golgi, plasma membrane and cytosol. This approach is based on the expertise of the applicant's laboratory in cell fractionation and characterization of organelles from baker's yeast Saccharomyces cerevisiae. To characterize the quality of organelles, i.e. to measure enrichment and contamination with other cellular fractions, antibodies against Pichia pastoris organelle markers will be generated and used. Organelles isolated at high purity will be subjected to analysis of proteins and lipids using biochemical methods which are standard in the applicant's laboratory. Fundamental characterization of organelles will be extended to proteome analysis of major subcellular fractions using mass spectrometry. Lipid analysis will include quantification of phospholipids, sterols, neutral lipids, fatty acids and sphingolipids. Moreover, we will establish a collection of organelle specific marker proteins hybridized to green fluorescent proteins (GFP) which will allow visualization of organelles, their structure and their status of development by fluorescence microscopy. Cell structure studies will be supported by electron microscopy performed in cooperation with Prof. Hofer, FELMI, Graz. Altogether, we intend to provide through this project a detailed insight into organelles of this microorganism which will be an important prerequisite to overcome bottlenecks of protein expression at the organelle level. We propose that manipulation of Pichia pastoris organelle and membrane properties will improve formation and yield of heterologously expressed proteins, especially of membrane bound polypeptides.