The ras-related GTPase rab5 is rate-limiting for homotypic early endosome fusion. We used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. The cDNA sequence encodes a ubiquitous 75-kDa protein with an N-terminal transmembrane domain (TM), a central coiled-coil structure, and a C-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5ipTM(؊)) had a greater affinity in vitro for rab5-guanosine 5-O-2-(thio)diphosphate than for rab5-guanosine 5-3-O-(thio)triphosphate. In transfected HeLa cells, rab5ipTM(؊) was partly cytosolic and localized (by immunofluorescence) with a rab5 mutant believed to be in a GDP conformation (GFP-rab5 G78A ) but not with GFP-rab5 Q79L , a GTPase-deficient mutant. rab5ip with the transmembrane domain (rab5ipTM(؉)) was completely associated with the particulate fraction and localized extensively with GFP-rab5 wt in punctate endosome-like structures. Overexpression of rab5ipTM(؉) using Sindbis virus stimulated the accumulation of fluid-phase horseradish peroxidase by BHK-21 cells, and homotypic endosome fusion in vitro was inhibited by antibody against rab5ip. rab5ipTM(؊) inhibited rab5 wt -stimulated endosome fusion but did not inhibit fusion stimulated by rab5 Q79L . rab5ip represents a novel rab5 interacting protein that may function on endocytic vesicles as a receptor for rab5-GDP and participate in the activation of rab5.

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Tryptic proteolysis of the small GTPases Rab4 and Rab5 is a multi-step, nucleotide-dependent process. Using N-terminal peptide sequencing, matrix-assisted laser desorption ionization-time-of-flight MS and molecular modelling, we identified the three initial sites of proteolysis in Rab5 as Arg-4, Arg-81 and Arg-197. Arg-4 and Arg-81 lie within regions previously implicated in Rab5 endocytic function, and Arg-197 lies in a region involved in membrane targeting. Topologically, Arg-81 lies within the conformationally variable Switch II region shown to be important for protein-protein interactions of other GTPases. Homology modelling studies on Rab5 indicate that the Arg-81 side chain is buried in the Rab5 GTP conformation, but is solvent-accessible in the GDP conformation, explaining the dependence of proteolysis on nucleotides. Peptide mapping of Rab4 was performed to take advantage of additional scissile bonds within Switch II to determine more precisely the limits of the nucleotide-dependent protease-accessible region. The Rab4 cleavage sites corresponded to Arg-81 and Pro-87 of Rab5, and taken together with the finding that Rab5 was not cleaved at Arg-91 this analysis defines an eight-residue surface-exposed conformationally variable region lying in the centre of Switch II. A sequence comparison of Rab proteins shows these eight residues to have a loosely conserved motif that we term Switch II(v) for its relative variability. C-terminal to Switch II(v) is a highly conserved Rab-specific YYRGA motif that we term Switch II(c) for its constant sequence. N-terminal to Switch II(v) is a sequence-invariant G-domain involved in nucleotide binding and hydrolysis. We propose that the Rab Switch II(v) region imparts specificity to nucleotide-dependent protein-protein interactions.