Integral membrane proteins of the chloroplast envelope:: Identification and subcellular localization of new transporters
Integral membrane proteins of the chloroplast envelope:: Identification and subcellular localization of new transporters
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DOI：
10.1073/pnas.172390399
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发表时间：
2002-08-20
影响因子：
11.1
通讯作者：
Rolland, N
中科院分区：
文献类型：
--
作者：
Ferro, M;Salvi, D;Rolland, N
一个双膜系统，或包膜，包围着质体。由于植物细胞内叶绿体代谢的整合，被膜是许多特异性运输活动的场所。然而，只有少数蛋白质参与的过程中的运输通过叶绿体被膜已被确定在分子水平上。为了发现新的包膜转运蛋白，我们开发了一种亚细胞蛋白质组学方法，旨在鉴定最疏水的包膜蛋白。该策略结合了使用高度纯化和表征的膜组分，用有机溶剂提取疏水蛋白，SDS/PAGE分离和串联质谱分析。为了处理大量的MS/MS数据，开发了一个基于BLAST的程序，用于蛋白质、表达序列标签和植物基因组数据库的搜索。在54个已鉴定的蛋白中，有27个是新的包膜蛋白，其中大多数带有多个α-螺旋跨膜区，很可能是包膜转运蛋白。目前的蛋白质组学研究还使我们能够确定已知的和新发现的推定的包膜内膜转运蛋白之间的共同特征。这些特征被用来挖掘拟南芥全基因组，并允许我们建立一个虚拟的质体包膜整合蛋白数据库。总而言之，蛋白质组学和计算机模拟方法确定了50多个候选人尚未以前未知的质体包膜转运蛋白。这些蛋白质的可预测功能开辟了研究领域，可能会导致更好地了解叶绿体代谢。目前的亚细胞蛋白质组学的方法是服从其他细胞内膜系统的疏水核心的分析。
A two-membrane system, or envelope, surrounds plastids. Because of the integration of chloroplast metabolism within the plant cell, the envelope is the site of many specific transport activities. However, only a few proteins involved in the processes of transport across the chloroplast envelope have been identified already at the molecular level. To discover new envelope transporters, we developed a subcellular proteomic approach, which is aimed to identify the most hydrophobic envelope proteins. This strategy combined the use of highly purified and characterized membrane fractions, extraction of the hydrophobic proteins with organic solvents, SDS/PAGE separation, and tandem mass spectrometry analysis. To process the large amount of MS/MS data, a BLAST-based program was developed for searching in protein, expressed sequence tag, and genomic plant databases. Among the 54 identified proteins, 27 were new envelope proteins, with most of them bearing multiple a-helical transmembrane regions and being very likely envelope transporters. The present proteomic study also allowed us to identify common features among the known and newly identified putative envelope inner membrane transporters. These features were used to mine the complete Arabidopsis genome and allowed us to establish a virtual plastid envelope integral protein database. Altogether, both proteomic and in silico approaches identified more than 50 candidates for the as yet previously uncharacterized plastid envelope transporters. The predictable function of some of these proteins opens up areas of investigation that may lead to a better understanding of the chloroplast metabolism. The present subcellular proteomic approach is amenable to the analysis of the hydrophobic core of other intracellular membrane systems.