The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris
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- 作者：Lisa Klug ; Pablo Tarazona ; Clemens Gruber ; Karlheinz Grillitsch ; Brigitte Gasser ; Martin Tr枚tzm眉ller ; Harald K枚feler ; Erich Leitner ; Ivo Feussner ; Diethard Mattanovich ; Friedrich Altmann ; G眉nther Daum
- 关键词：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
- 刊名：Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids
- 出版年：February, 2014
- 年：2014
- 卷：1841
- 期：2
- 页码：215-226
- 全文大小：1127 K
文摘
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.