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.
Sanford
J C
JC
Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Yu
J
J
Pan
J Y
JY
Wessling-Resnick
M
M
D000818:Animals; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics; D017368:Protein Prenylation; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
D020730:Guanine Nucleotide Dissociation Inhibitors; D046911:Macromolecular Substances; D016376:Oligonucleotides, Antisense; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D001710:Biotin; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins