Nucleotide Dependence of Rab Geranylgeranylation

Abstract

Geranylgeranylation of Rab GTPases is an essential post-translational modification that enables Rabs to as- sociate with intracellular membranes where they regu- late exocytic and endocytic pathways. Geranylgeranyla- tion is initiated by formation of a stable complex between newly synthesized Rab proteins and Rab escort protein (REP). The complex is recognized by Rab gera- nylgeranyl (GG) transferase, which transfers two GG groups to Rabs. The geranylgeranylated Rabs regulate vesicular movement by oscillating between an inactive GDP-bound form and an active GTP-bound form. In this study, I show that the kinetics of geranylgeranylation is influenced by the nucleotide status of nascent Rab. GDP-bound Rab is geranylgeranylated with 10–50-fold higher affinity than GTP-bound Rab (or GTP analog- bound Rab), as indicated by the apparent Km of the reaction. In vitro REP?Rab binding assays demonstrate that REP forms a stable complex only with the GDP- bound form of Rab but not the GTP-bound form, suggest- ing that the apparent Km effect in the prenylation reac- tion is due to a discrimination between the two different nucleotide-bound forms of Rab by REP. Inasmuch as Rabs are likely GTP-bound after synthesis andREPdoes not possess GTPase-activating protein activity, these re- sults raise the possibility that a Rab GTPase-activating protein enhances the REP?Rab interaction prior to prenylation.