Semi-synthetic Rab proteins as tools for studying intermolecular interactions

Rab GTPases play a key role in the regulation of membrane traffic. Posttran slational geranylgeranylation is critical for their biological activity and is conferred by a Rab geranylgeranyl transferase (RabGGTase). To study the interactions between Rab proteins and RabGGTase, we used in vitro ligation methodology to generate a fluorescent semi-synthetic Rab7 protein. The obt ained protein was functionally active and was used to demonstrate a micromo lar affinity interaction of Rab7 with the RabGGTase in the absence of Rab e scort protein (REP). This finding is consistent with an earlier proposed mo del according to which RabGGTase possesses two independent weak binding sit es for REP and Rab proteins. (C) 2000 Federation of European Biochemical So cieties.