ASSOCIATION OF RAB1B WITH GDP-DISSOCIATION INHIBITOR (GDI) IS REQUIRED FOR RECYCLING BUT NOT INITIAL MEMBRANE TARGETING OF THE RAB PROTEIN

We have identified a Rab1B effector-domain mutant (D44N) that, when ge ranylgeranylated by Rab:geranylgeranyltransferase (GGTase II) in cell- free systems or intact cells, fails to form detectable complexes with GDP . dissociation inhibitors (GDIs). GDI-Rab complexes were collected on anti-FLAG affinity beads after incubating recombinant geranylgeran ylated Rab1B with FLAG epitope-tagged GDI in vitro, or transiently coe xpressing Myc-tagged Rab1B with FLAG-GDI-alpha or FLAG-GDI-2 in human embryonal kidney 293 cells. [H-3]Mevalonate labeling and immunoprecipi tation studies confirmed that the inability of Myc-Rab1B(D44N) to asso ciate with GDI in vivo was not due to failure of the mutant to undergo geranylgeranylation. Immunofluorescence localization and immunoblot a nalysis of subcellular fractions indicated that expressed Myc-Rab1B(D4 4N) efficiently delivered to intracellular membranes in 293 cells. Thi s was confirmed when the fate of the prenylated pool of Rab1B(D44N) in 293 cells was traced by labeling the geranylgeranyl groups attached t o the nascent protein with [H-3]mevalonate. However, in contrast to th e prenylated Rab1B(WT), which was distributed in both the membrane and soluble fractions, the prenylated Rab1B(D44N) was completely absent f rom the cytosol. Overexpression of Myc-Rab1B(D44N) did not impair ER - -> Golgi glycoprotein trafficking in 293 cells, which was assessed by monitoring the Golgi-dependent processing of coexpressed beta-amyloid precursor protein. The current findings suggest that nascent prenylate d Rab1B can be delivered to intracellular membranes in intact cells wi thout forming a stable complex with GDI, but that recycling of prenyla ted Rab1B to the cytosolic compartment is absolutely dependent on GDI interaction.