Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions.
Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions.
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DOI：
10.1007/978-1-61779-234-2_18
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发表时间：
2011
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影响因子：
--
通讯作者：
Schnell, Danny J
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文献类型：
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作者：
Inoue, Hitoshi;Wang, Fei;Inaba, Takehito;Schnell, Danny J
大多数叶绿体蛋白在细胞质中作为带n端可切割转运肽的前蛋白合成，并通过TOC-TIC转座系统导入细胞器。导入涉及一套复杂的识别和膜易位步骤，以确保多肽在双膜叶绿体包膜上的保真度和单向运输。为了理解进口的机制，必须定义每个步骤的分子相互作用和能量学。在这里，我们描述了通过操纵叶绿体的能量状态来捕获进口过程中的中间体的方法，并使用两种不同的化学交联方法来检查介导进口过程的分子相互作用，并评估转座子的组装状态。这些方法可用于鉴定蛋白质-蛋白质序列相互作用，从而剖析蛋白质输入叶绿体过程中输入组分的途径和作用。
Most chloroplast proteins are synthesized in the cytosol as preproteins with N-terminal cleavable transit peptides and are imported into the organelle through the TOC–TIC translocon system. Import involves a complex set of recognition and membrane translocation steps that ensure the fidelity and unidirectional transport of the polypeptide across the double-membrane chloroplast envelope. To understand the mechanism of import, the molecular interactions and energetics of each step must be defined. Here, we describe the methods for capturing intermediates in the import process through the manipulation of the energy state of chloroplasts, and the use of two different chemical cross-linking approaches to examine the molecular interactions that mediate the import process and to assess the assembly state of the translocons. These approaches can be employed to identify sequential protein–protein interactions, and thereby dissect the pathway and roles of import components during protein import into chloroplasts.