Modification of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate

A photoprobe analog of geranylgeranyl diphosphate (2-diazo-3,3,3-trifluorop ropionyloxy-farnesyl diphosphate or DATFP-FPP) inhibits mevalonate-dependen t prenylation of in vitro translated Rab5 in rabbit reticulocyte lysate, su ggesting that it competes for lipid binding to the Rab geranylgeranyl trans ferase. Modification of Rab5 with DATFP-FPP, demonstrated by gel mobility s hift and Triton X-114 phase separation experiments, confirms that the enzym e uses the analog as a substrate. The sedimentation of DATFP-modified Rab5 as a larger mass complex on sucrose density gradients indicates that it bin ds to other factors in rabbit reticulocyte lysate. Most importantly, DATFP- Rab5 cross-links to these soluble factors upon exposure to UV light. Immuno precipitation with antibodies raised against proteins known to interact wit h Rab5 reveals that the crosslinked complexes contain Rab escort protein an d GDI-1. DATFP-Rab5 also associates with membranes in a guanosine-5'-O-(3-t hiotriphosphate)-stimulated manner. However, although prenylated Rab5 can b e crosslinked to two unknown membrane-associated factors by the chemical cr oss-linker disuccinimidyl suberate, these proteins fail to be W cross-linke d to membrane-bound DATFP-Rab5. These results strongly suggest that membran e-associated factors bind Rab5 through protein-protein interactions rather than protein-prenyl interactions. The modification of Rab5 with DATFP-FPP e stablishes a novel photoaffinity technique for the characterization of pren yl-binding sites.