Modification of Rab5 with a Photoactivatable Analog of
Geranylgeranyl Diphosphate*
George J.
Quellhorst Jr.
§,
Charles M.
Allen¶, and
Marianne
Wessling-Resnick
From the Department of Nutrition, Harvard School of
Public Health, Boston, Massachusetts 02115 and the ¶ Department of
Biochemistry and Molecular Biology, J. Hillis Miller Health Center,
University of Florida, Gainesville, Florida 32610
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.
*
This work was supported in part by Research Grants CB-15 (to
M. W. R.) and F93UF-2 (to C. M. A.) from the
American Cancer Society and by funds from the Cancer Center of the
University of Florida (to C. M. A.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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