Characterization of peroxisomes from the methylotrophic yeast Pichia pastoris.
Tamara Wriessnegger (1), Georg Guebitz (2), Erich Leitner (3), Elisabeth Ingolic (4), Guenther Daum (1)
(1) Institute of Biochemistry, Graz University of Technology, Petersgasse 12/II, Graz, 8010, Austria; (2) Institute of Environmental Biotechnology; Graz University of Technology, Austria; (3) Institute of Food Chemistry and Food Technology; Graz University of Technology, Austria; (4) Research Institute for Electron Microscopy; Graz University of Technology, Austria
The methylotrophic yeast Pichia pastoris is widely used as an efficient expression system for heterologous proteins. This yeast has also become a suitable model organism for studying peroxisome biogenesis due to the fact that growth on methanol or oleate strongly induces proliferation of this organelle. Nevertheless, surprisingly little is known about biochemical and cell biological properties of peroxisomes from Pichia pastoris. For this reason, we aimed to isolate highly purified peroxisomes from Pichia pastoris cells grown on methanol or oleate media and characterize this organelle with special emphasis on its lipid and protein composition. Electron microscopy revealed huge clustered peroxisomes when cells were grown on methanol. Under these conditions alcohol oxidase becomes the predominant protein in peroxisomes. Cultivation on oleate changes the peroxisomal polypeptide pattern, and enzymes involved in fatty acid beta-oxidation are expressed as documented by mass spectrometric analysis. Major phospholipids of peroxisomal membranes are phosphatidylcholine and phosphatidylethanolamine, and major fatty acids are oleic acid and stearic acid. Ergosterol is the most abundant sterol of Pichia pastoris peroxisomes, and sterol precursors were detected only at minor amounts. This work is the starting point of a systematic approach to isolate and characterize subcellular fractions of Pichia pastoris to obtain fundamental knowledge about the cell biology of this versatile yeast.