Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana
Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana
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DOI：
10.1074/mcp.m300030-mcp200
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发表时间：
2003-05-01
影响因子：
7
通讯作者：
Rolland, N
中科院分区：
文献类型：
--
作者：
Ferro, M;Salvi, D;Rolland, N
叶绿体的发育及其功能在植物细胞内的整合依赖于位于其限制性被膜内的复杂生化机制的存在。为了提供最详尽的叶绿体被膜蛋白库的视图，我们分析了这个膜系统使用蛋白质组学。为此，我们首先开发了一种从拟南芥叶绿体制备高纯度包膜膜的方法。然后，我们使用不同的方法提取包膜蛋白，即氯仿/甲醇提取和碱或盐水处理，以检索尽可能多的蛋白质，从最疏水的到最不疏水的。然后对每个包膜膜亚组分进行液相色谱串联质谱分析，鉴定出100多种蛋白质。已知约80%的已鉴定蛋白质位于或很可能位于叶绿体被膜中。两个磷酸盐转运蛋白的包膜中的本地化的验证证明了需要一种策略的组合，以执行最详尽的鉴定真正的叶绿体包膜蛋白。有趣的是，一些鉴定的蛋白质被发现是N（α）-乙酰化的，这表明相应成熟蛋白质的N末端的准确位置。关于功能，超过50%的已鉴定蛋白质具有已知或非常可能与叶绿体被膜相关的功能。这些蛋白质a）参与离子和代谢物运输，B）蛋白质输入机制的组分，和c）参与叶绿体脂质代谢。一些可溶性蛋白质，如蛋白酶，参与碳代谢的蛋白质，或参与氧化应激反应的蛋白质，与包膜有关。我们发现的蛋白质中有近三分之一没有已知的功能。目前的工作有助于在分子水平上理解叶绿体被膜的代谢，并提供了一个新的概述叶绿体被膜的生化机制。
The development of chloroplasts and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting envelope membranes. To provide the most exhaustive view of the protein repertoire of chloroplast envelope membranes, we analyzed this membrane system using proteomics. To this purpose, we first developed a procedure to prepare highly purified envelope membranes from Arabidopsis chloroplasts. We then extracted envelope proteins using different methods, i.e. chloroform/methanol extraction and alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to least hydrophobic ones. Liquid chromatography tandem mass spectrometry analyses were then performed on each envelope membrane subfraction, leading to the identification of more than 100 proteins. About 80% of the identified proteins are known to be, or are very likely, located in the chloroplast envelope. The validation of localization in the envelope of two phosphate transporters exemplifies the need for a combination of strategies to perform the most exhaustive identification of genuine chloroplast envelope proteins. Interestingly, some of the identified proteins are found to be N(alpha)-acetylated, which indicates the accurate location of the N terminus of the corresponding mature protein. With regard to function, more than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are a) involved in ion and metabolite transport, b) components of the protein import machinery, and c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism, or proteins involved in responses to oxidative stress, were associated with envelope membranes. Almost one-third of the proteins we identified have no known function. The present work helps understanding chloroplast envelope metabolism at the molecular level and provides a new overview of the biochemical machinery of the chloroplast envelope membranes.