The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris |
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| Authors: | Lisa Klug,Pablo Tarazona,Clemens Gruber,Karlheinz Grillitsch,Brigitte Gasser,Martin Trö tzmü ller,Harald Kö feler,Erich Leitner,Ivo Feussner,Diethard Mattanovich,Friedrich Altmann,Gü nther Daum |
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| Affiliation: | 1. Institute of Biochemistry, Graz University of Technology, Austria;2. Department for Plant Biochemistry, Albrecht-von-Haller Institute for Plant Sciences, Georg-August-University, Goettingen, Germany;3. Austrian Centre of Industrial Biotechnology, Vienna, Austria;4. Department of Chemistry, BOKU University of Natural Resources and Life Sciences Vienna, Austria;5. Austrian Centre of Industrial Biotechnology, Graz, Austria;6. Department of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Austria;g Core Facility for Mass Spectrometry, Center for Medical Research, Medical University of Graz, Austria;h Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Austria |
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| Abstract: | The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications. |
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| Keywords: | ER, endoplasmic reticulum M30, 30,000 g microsomes M40, 40,000 g microsomes BM, bulk membranes PMSF, phenylmethylsulfonyl fluoride SDS, Sodium dodecyl sulfate DTT, dithiothreitol Cer, ceramide HexCer, hexosyl ceramide LCB, long chain base MS, mass spectrometry cww, cell wet weight Td, doubling time |
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