In Vitro Prenylation Assay of Arabidopsis Proteins

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.