GDP dissociation inhibitor serves as a cytosolic acceptor for newly synthesized and prenylated Rab5.

In vitro synthesis and post-translational prenylation of Rab5 is accomplished using reticulocyte lysate supplemented with prenyl precursors (Sanford, J. C., Pan, Y., and Wessling-Resnick, M. (1993) J. Biol. Chem. 268, 23773-23776). When Rab5 is translated in the presence of biotin-lysine-tRNA, it incorporates biotin-lysine into its peptide backbone and is efficiently prenylated; since this modification is dependent on guanine nucleotide binding, biotin-Rab5's functional integrity must be maintained. Prenylated biotin-Rab5 associates with a 45-kDa reticulocyte GDP dissociation inhibitor (GDI), sedimenting as a approximately 70-kDa particle on 5-20% sucrose density gradients. The GDI-Rab5 complex can be captured using streptavidin-linked agarose beads. Only Rab5 peptides that are substrates for prenylation are found to cosediment with the lysate GDI on sucrose gradients. Post-translational association of Rab5 and GDI is a novel finding, since previous reports suggested Rab5 remains associated with Rab escort protein (REP) after prenylation (Alexandrov, K., Horiuchi, H., Steele-Mortimer, O., Seabra, M. C., and Zerial, M. (1994) EMBO J. 13, 5262-5273). Since post-translational prenylation is catalytically mediated by REP, our study suggests that a complex between Rab5 and this factor is transient in nature. Thus, newly synthesized and prenylated Rab5 is most likely escorted to its target membrane by a GDI acceptor molecule. Biotin-Rab5 provides a novel tool for future efforts to capture and characterize additional accessory factors required for Rab protein function in vesicle transport.