Michaud Morgane
Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, USA.
Laboratoire de Physiologie Cellulaire et Végétale, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Commissariat à l'Energie Atomique et aux Energies Alternatives, CEA Grenoble, UMR5168, Université Grenoble Alpes, Grenoble, France.
Methods Mol Biol. 2018;1829:173-188. doi: 10.1007/978-1-4939-8654-5_12.
Plastids are organelles playing fundamental roles in different cellular processes such as energy metabolism or lipid biosynthesis. To fulfill their biogenesis and their function in the cell, plastids have to communicate with other cellular compartments. This communication can be mediated by the establishment of direct contact sites between plastids envelop and other organelles. These contacts are dynamic structures that are modified in response to stress. As example, during phosphate (Pi) starvation, the number of contact sites between plastids and mitochondria significantly increases. In this situation, these contacts play an important role in the transfer of galactoglycerolipids from plastids to mitochondria. Recently, Pi starvation stress was used to identify key proteins involved in the traffic of galactoglycerolipids from plastids to mitochondria in Arabidopsis thaliana. A mitochondrial lipoprotein complex called MTL (mitochondrial transmembrane lipoprotein complex) was identified. This complex contains mitochondrial proteins but also proteins located in the plastid envelope, suggesting its presence at the plastid-mitochondria junction. This chapter describes the protocol to isolate the MTL complex by clear-native polyacrylamide gel electrophoresis (CN-PAGE) from the mitochondrial fraction of Arabidopsis cell cultures and the methods to study different features of this complex.
质体是在能量代谢或脂质生物合成等不同细胞过程中发挥基础作用的细胞器。为了完成其生物发生及其在细胞中的功能，质体必须与其他细胞区室进行通讯。这种通讯可以通过在质体包膜与其他细胞器之间建立直接接触位点来介导。这些接触是动态结构，会因应激而发生改变。例如，在磷酸盐（Pi）饥饿期间，质体与线粒体之间的接触位点数量会显著增加。在这种情况下，这些接触在半乳糖甘油脂从质体向线粒体的转移中发挥重要作用。最近，Pi饥饿胁迫被用于鉴定拟南芥中参与半乳糖甘油脂从质体向线粒体运输的关键蛋白。一种名为MTL（线粒体跨膜脂蛋白复合物）的线粒体脂蛋白复合物被鉴定出来。该复合物不仅包含线粒体蛋白，还包含位于质体包膜中的蛋白，这表明它存在于质体 - 线粒体交界处。本章描述了通过清晰天然聚丙烯酰胺凝胶电泳（CN - PAGE）从拟南芥细胞培养物的线粒体部分分离MTL复合物的方案以及研究该复合物不同特性的方法。
