Klug Lisa, Tarazona Pablo, Gruber Clemens, Grillitsch Karlheinz, Gasser Brigitte, Trötzmüller Martin, Köfeler Harald, Leitner Erich, Feussner Ivo, Mattanovich Diethard, Altmann Friedrich, Daum Günther
Biochim Biophys Acta. 2014 Feb;1841(2):215-26. doi: 10.1016/j.bbalip.2013.11.005.
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.
甲醇营养型酵母毕赤酵母是生物技术领域中用于生产异源蛋白的一种常用酵母表达系统。有趣的是，迄今为止，在这一过程中发挥重要作用的细胞器尚未得到充分研究。因此，我们启动了一项系统研究，旨在从毕赤酵母中分离并鉴定细胞器。在本研究中，我们展示了一种高纯度分离微粒体膜的方法。这些样本代表内质网（ER）组分，并对其进行了脂质和蛋白质的分子分析。细胞器脂质组学包括对甘油磷脂、脂肪酸、固醇和鞘脂的详细分析。通过质谱分析的微粒体蛋白质组鉴定出了其他细胞类型（尤其是酿酒酵母）中已知的内质网典型蛋白质，同时也鉴定出了一些未归类的基因产物。毕赤酵母微粒体的脂质组和蛋白质组分析是更好地理解该细胞器功能以及为生物技术应用对该细胞器进行改造的前提条件。
