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Abstract
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.
Highlights
These GTPases act as regulators of the assembly and disassembly of protein complexes involved in the docking and fusion of vesicles [10]
Our results demonstrate that the only GDP dissociation inhibitor (GDI) isoform present in reticulocyte lysate, a 45-kDa GDI2-like species, forms a complex with nascent prenylated biotin-Rab5
Disposed Rab proteins are known to complex with GDIs [23, 28]
Summary
These GTPases act as regulators of the assembly and disassembly of protein complexes involved in the docking and fusion of vesicles [10]. Alexandrov et al [19] have demonstrated that Rab can be modified in a reconstitution assay using purified prenylation reaction components and that Rab remains associated with REP after modification to be delivered to membranes in the absence of other cytosolic factors These observations help to explain why the in vitro prenylation reaction is limited in the absence of detergents, which help to dissociate the Rab-REP complex; delivery and release of Rab proteins to membranes subsequent to prenyl transfer would help to recycle REP and to recover catalytic activity of geranylgeranyl transferase II. Otherwise, this enzyme appears to act in a stoichiometric fashion in vitro [18]. In contrast to the findings of Alexandrov et al [19], stable complexes between
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