Purification and identification of novel Rab effectors using affinity chromatography
Purification and identification of novel Rab effectors using affinity chromatography
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DOI：
10.1006/meth.2000.0953
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发表时间：
2000-04-01
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影响因子：
--
通讯作者：
Zerial, M
中科院分区：
文献类型：
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作者：
Christoforidis, S;Zerial, M
Rab GTP酶是真核细胞胞内转运机制的核心调控元件。为了调节囊泡对接和融合以及细胞器动力学，Rab蛋白在GTP结合的活性状态下与效应分子相互作用。因此，Rab效应物的鉴定对于了解细胞内转运的机制至关重要。在这里，我们描述了我们开发的实验系统，用于生物化学纯化和鉴定小GTPase Rab 5的效应物。该方法，这是基于亲和层析程序，结果在大规模纯化的Rab效应的量足以为他们的鉴定，通过微测序技术和他们的功能特性。在Rab5的情况下，该程序允许全面分析该GTdR的下游效应子和调节子。我们希望这种策略提供膜转运的分子机制的基本见解，但也适用于其他几个GTP酶依赖的生物功能。(C)北京大学出版社.
Rab GTPases are central regulatory elements of the intracellular transport machinery of eukaryotic cells. To regulate vesicle docking and fusion as well as organelle dynamics Rab proteins interact with effector molecules in the GTP-bound active state. The identification of Rab effecters is, therefore, of primary importance for the mechanistic understanding of intracellular transport. Here we describe the experimental system we have developed to biochemically purify and identify effecters of the small GTPase Rab5. The method, which is based on an affinity chromatography procedure, results in the large-scale purification of Rab effectors in amounts sufficient for both their identification by microsequencing techniques and their functional characterization. In the case of Rab5, the procedure allows a comprehensive analysis of the downstream effecters and regulators of this GTPase. We expect this strategy to provide fundamental insights into the molecular mechanism of membrane transport but also to be applicable to several other GTPase-dependent biological functions. (C) 2000 Academic Press.