Modification of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate.
A photoprobe analog of geranylgeranyl diphosphate (2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate or DATFP-FPP) inhibits mevalonate-dependent prenylation of in 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.
Quellhorst
G J
GJ
Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, USA. gquellho@hsph.harvard.edu
Allen
C M
CM
Wessling-Resnick
M
M
D000818:Animals; D003979:Diazonium Compounds / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000378:metabolism; D010777:Photochemistry; D017368:Protein Prenylation; D011817:Rabbits; D014466:Ultraviolet Rays; D020696:rab5 GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism
C095155:2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate; D003979:Diazonium Compounds; D008565:Membrane Proteins; D019920:Photoaffinity Labels; D020696:rab5 GTP-Binding Proteins