Rabs regulate the important cellular process of vesicular transport. The importance of the prenyl modification of Rab proteins and how other proteins and membranes interact with this modification remains unclear. This proposal specifically aims to define the kinetic parameters of GDI- mediated Rab5 retrieval from membranes and to investigate the effects of synthetic Rab5 peptides on those parameters. The assay monitors the release of Rab5, by quantitative Western analysis, into the supernatant after pelleting the membrane source by centrifugation. The proposal outlines the determination of the cytosol, energy, and nucleotide requirements for the reaction as well as the activity's dependence on GDI, Rab5, and peptide concentrations. This proposal also specifically aims to determine how a photoreactive prenyl pyrophosphate analog affects the in vitro prenylation of Rab5. An in vitro system for the prenylation or Rab5 is available in the sponsoring lab. An in vitro system for the prenylation of Rab5 is available in the sponsoring lab. This proposal outlines how to test whether the analog competitively inhibits the reaction and whether it modifies the beta subunit of the Rab prenyl transferase enzyme. The proposal outlines how to test whether the analog competitively inhibits the reaction and whether it modifies the beta subunit of the Rab prenyl transferase enzyme. The proposal also describes how to create a photoreactive Rab5 probe for other proteins that interact with the Rab5 and for amino acid residues of GDI that lie in close proximity to the lipid modification. The results of these studies should provide important clues as to how prenyl modifications mediate protein-protein interactions.
| Quellhorst Jr, G J; Allen, C M; Wessling-Resnick, M (2001) Modification of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate. J Biol Chem 276:40727-33 |
