Department of Natural Sciences, International Christian University, Tokyo, Japan.
Institute for Global Leadership, Ochanomizu University, Tokyo, Japan.
Methods Mol Biol. 2020;2177:183-197. doi: 10.1007/978-1-0716-0767-1_15.
RAB GTPases regulate membrane traffic by interacting with effector proteins in the GTP-bound active form. RAB GTPases are highly conserved in a broad range of eukaryotic organisms, while land plants and some green algal species possess a plant-specific RAB5 group. A plant-specific RAB5 in Arabidopsis called ARA6 was shown to regulate a characteristic trafficking route, and participate in abiotic and biotic stress responses. The identification of ARA6 effectors is a powerful strategy to get insights into the molecular basis of ARA6 functions. Recently, we identified an ARA6 effector, PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2), and characterized its functions by biochemical means. PUF2 was hardly expressed as a recombinant protein in the bacterial system, but we solved this problem by optimizing the codon usage of PUF2 CDS to suite for expression in Escherichia coli. Here, we present the protocol we employed to purify PUF2 protein, and to test its nucleotide state-specific interaction with ARA6 by in vitro pull-down assay. This approach would be extended to analyze the molecular functions of other effector proteins of RAB GTPases.
RAB GTPases 通过与 GTP 结合的活性形式中的效应蛋白相互作用来调节膜运输。RAB GTPases 在广泛的真核生物中高度保守，而陆地植物和一些绿藻物种则具有植物特异性的 RAB5 组。拟南芥中一种称为 ARA6 的植物特异性 RAB5 被证明可以调节一种特征性的运输途径，并参与非生物和生物胁迫反应。鉴定 ARA6 的效应物是深入了解 ARA6 功能的分子基础的有效策略。最近，我们鉴定了一种 ARA6 的效应物，即 PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2)，并通过生化手段对其功能进行了表征。PUF2 作为重组蛋白在细菌系统中几乎不表达，但我们通过优化 PUF2 CDS 的密码子使用来适应大肠杆菌中的表达，解决了这个问题。在这里，我们介绍了我们用于纯化 PUF2 蛋白的方案，并通过体外下拉测定测试了其与 ARA6 的核苷酸状态特异性相互作用。这种方法将扩展到分析 RAB GTPases 的其他效应蛋白的分子功能。
