Modification of Rab5 with a Photoactivatable Analog of Geranylgeranyl Diphosphate

• Published in: The Journal of biological chemistry Vol. 276; no. 44; pp. 40727 - 40733
• Main Authors: Quellhorst, George J., Allen, Charles M., Wessling-Resnick, Marianne
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.11.2001; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Diazonium Compounds - chemistry; Diazonium Compounds - metabolism; Electrophoresis, Polyacrylamide Gel; Membrane Proteins - metabolism; Photoaffinity Labels - chemistry; Photoaffinity Labels - metabolism; Photochemistry; Protein Prenylation; rab5 GTP-Binding Proteins - chemistry; rab5 GTP-Binding Proteins - metabolism; Rabbits; Ultraviolet Rays
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M104398200

A photoprobe analog of geranylgeranyl diphosphate (2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate or DATFP-FPP) inhibits mevalonate-dependent prenylation ofin vitro translated Rab5 in rabbit reticulocyte lysate, suggesting that it competes for lipid binding to the Rab geranylgeranyl transferase. Modification of Rab5 with DATFP-FPP, demonstrated by gel mobility shift and Triton X-114 phase separation experiments, confirms that the enzyme uses the analog as a substrate. The sedimentation of DATFP-modified Rab5 as a larger mass complex on sucrose density gradients indicates that it binds to other factors in rabbit reticulocyte lysate. Most importantly, DATFP-Rab5 cross-links to these soluble factors upon exposure to UV light. Immunoprecipitation with antibodies raised against proteins known to interact with Rab5 reveals that the cross-linked complexes contain Rab escort protein and GDI-1. DATFP-Rab5 also associates with membranes in a guanosine-5′-O-(3-thiotriphosphate)-stimulated manner. However, although prenylated Rab5 can be cross-linked to two unknown membrane-associated factors by the chemical cross-linker disuccinimidyl suberate, these proteins fail to be UV cross-linked to membrane-bound DATFP-Rab5. These results strongly suggest that membrane-associated factors bind Rab5 through protein-protein interactions rather than protein-prenyl interactions. The modification of Rab5 with DATFP-FPP establishes a novel photoaffinity technique for the characterization of prenyl-binding sites.