Shi Wan, Zeng Qin, Running Mark P
Washington University in Saint Louis, St. Louis, MO, USA.
Methods Mol Biol. 2013;1043:147-60. doi: 10.1007/978-1-62703-532-3_16.
Protein prenylation, like other lipid posttranslational modifications of eukaryotic proteins, plays important roles in protein-membrane association and protein-protein interactions. In Arabidopsis, hundreds of proteins involved in a great variety of biological processes are potential prenylation substrates that need to be verified, including heterotrimeric G proteins and most Rop and Rab small GTPases. Also, genetic evidence suggests substrate cross-specificity among different prenyltransferases and/or the existence of unidentified prenylation players. In this chapter we describe a direct and flexible in vitro enzymatic assay designed for testing prenylation activity and substrate specificity in vitro. This protocol takes Arabidopsis Rab-GGT as example and starts with preparation of purified protein components of the reaction, followed by reconstitution of the prenylation reaction in vitro, and autoradiographic detection for qualitative and semiquantitative analysis.
蛋白质异戊二烯化与真核生物蛋白质的其他脂质翻译后修饰一样，在蛋白质与膜的结合以及蛋白质与蛋白质的相互作用中发挥着重要作用。在拟南芥中，参与多种生物学过程的数百种蛋白质是潜在的异戊二烯化底物，有待验证，包括异源三聚体G蛋白以及大多数Rop和Rab小GTP酶。此外，遗传学证据表明不同异戊二烯基转移酶之间存在底物交叉特异性和/或存在尚未鉴定的异戊二烯化作用因子。在本章中，我们描述了一种直接且灵活的体外酶促测定方法，用于测试体外异戊二烯化活性和底物特异性。本方案以拟南芥Rab-GGT为例，首先制备反应所需的纯化蛋白质成分，然后在体外重建异戊二烯化反应，并通过放射自显影检测进行定性和半定量分析。
