Schikowsky Christine, Thal Beate, Braun Hans-Peter, Eubel Holger
Institute of Plant Genetics, Plant Proteomics, Leibniz University Hannover, Herrenhäuser Str. 2, 30419, Hannover, Germany.
Methods Mol Biol. 2018;1696:163-183. doi: 10.1007/978-1-4939-7411-5_11.
Containing plastids and vacuoles in addition to those organelles also found in other (heterotrophic) cells, the plant cell displays an extraordinary level of compartmentalization, largely obtained by the utilization of membranes. These membranes not only confine reaction spaces but must also facilitate cross-talk between organelles and other cell compartments. They also host important components of the plant energy metabolism, i.e., the electron transport chains of mitochondria and chloroplasts. Characterization of the proteomes of these membranes requires isolation of pure and intact organelles from plant tissues followed by subsequent purification of their respective membranes. Membrane fractions are then amenable for further analyses using gel electrophoresis procedures or gel-free proteomic approaches. Here, we describe the preparation of intact mitochondria from Arabidopsis thaliana cell-culture, the isolation of outer and inner mitochondrial membranes and downstream proteomic applications for analyzing their membrane protein content.
除了在其他（异养）细胞中也能找到的那些细胞器外，植物细胞还含有质体和液泡，其呈现出非凡的区室化水平，这在很大程度上是通过膜的利用实现的。这些膜不仅界定了反应空间，还必须促进细胞器与其他细胞区室之间的相互作用。它们还承载着植物能量代谢的重要组成部分，即线粒体和叶绿体的电子传递链。对这些膜的蛋白质组进行表征需要从植物组织中分离出纯净且完整的细胞器，随后对其各自的膜进行纯化。然后，膜组分适合使用凝胶电泳程序或无凝胶蛋白质组学方法进行进一步分析。在这里，我们描述了从拟南芥细胞培养物中制备完整线粒体、分离线粒体外膜和内膜以及用于分析其膜蛋白含量的下游蛋白质组学应用。
