Analysis of the stoichiometry of Rab protein prenylation

Rab5 is a Ras-related GTP-binding protein that is post-translationally prenylated with the 20-carbon isoprenoid geranylgeranyl. We have developed a method to determine the stoichiometry of prenylation of Rab5, and Rab family members in general, based on the cell-free translation of these peptides in the presence or the absence of appropriate isoprenoids. Modification of cell-free synthesized Rab5 can be monitored by following the conversion of 35S-labeled peptide to a greater mobility isoform on urea-gradient sodium dodecyl sulfate-polyacrylamide gels. The mobility-shifted isoform also incorporates radiolabel in the presence of [3H]mevalonate or [3H]geranylgeranyl pyrophosphate, confirming post-translational modification with geranylgeranyl. A quantitative assessment of the conversion of mobility-shifted Rab5, promoted by prenylation, and the amount of incorporated radiolabel from [3H]geranylgeranyl pyrophosphate was achieved by excising gel slices containing radiolabeled isoforms and measuring the covalently associated radioactivity. Using this approach, we have established that 2 moles of geranylgeranyl is attached per mole of Rab5 peptide. A 2:1 molar ratio of geranylgeranyl:peptide is observed for both Rab5wt and a truncation mutant, Rab5(1-213), containing C-terminal motifs CCXX and XXCC, respectively. When Rab proteins ending in CXC are synthesized and processed in vitro, they also incorporate geranylgeranyl at a 2:1 stoichiometry, although extended times of incubation are required for full modification. Finally, a C-terminal Rab5 truncation mutant retaining only one cysteine also becomes modified, although only a minor fraction is fully processed. This method offers a novel, quantitative approach to investigate the stoichiometry of post-translational processing of cell-free synthesized peptides without the need to purify the native molecules.