Measurement of Rab35 activity with the GTP-Rab35 trapper RBD35.
Measurement of Rab35 activity with the GTP-Rab35 trapper RBD35.
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DOI：
10.1007/978-1-4939-2569-8_18
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发表时间：
2015-01-01
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影响因子：
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通讯作者：
Fukuda, Mitsunori
中科院分区：
文献类型：
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作者：
Kobayashi, Hotaka;Etoh, Kan;Fukuda, Mitsunori
小GTP酶Rab 35通过在GTP结合的活性形式和GDP结合的非活性形式之间循环，作为膜运输的分子开关，特别是用于内吞再循环。虽然Rab 35已被证明可以调节各种细胞过程，包括胞质分裂，细胞迁移和神经突生长，但其在这些过程中的确切作用尚未完全了解。由于可用于测量Rab 35活性的分子工具对于确定Rab 35介导膜运输的机制是有用的，我们最近使用RUSC 2的含有RUN结构域的区域来开发活性Rab 35捕获器，我们将其命名为RBD 35（Rab 35特异性Rab-binding domain specific）。由于RBD 35特异性地与Rab 35的GTP结合活性形式相互作用，并且不与在人类和小鼠中鉴定的任何其他59种Rab蛋白相互作用，因此RBD 35是用于通过下拉测定测量活性Rab 35的水平和用于通过过表达抑制Rab 35的功能的有用工具。在本章中，我们描述了用RBD 35分析Rab 35的测定方法。
Small GTPase Rab35 functions as a molecular switch for membrane trafficking, specifically for endocytic recycling, by cycling between a GTP-bound active form and a GDP-bound inactive form. Although Rab35 has been shown to regulate various cellular processes, including cytokinesis, cell migration, and neurite outgrowth, its precise roles in these processes are not fully understood. Since a molecular tool that could be used to measure Rab35 activity would be useful for identifying the mechanisms by which Rab35 mediates membrane trafficking, we recently used a RUN domain-containing region of RUSC2 to develop an active Rab35 trapper, and we named it RBD35 (Rab-binding domain specific for Rab35). Because RBD35 specifically interacts with the GTP-bound active form of Rab35 and does not interact with any of the other 59 Rab proteins identified in humans and mice, RBD35 is a useful tool for measuring the level of active Rab35 by pull-down assays and for inhibiting the function of Rab35 by overexpression. In this chapter, we describe the assay procedures for analyzing Rab35 with RBD35.