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 deve loped 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 isopre noids. Modification of cell-free synthesized Rab5 can be monitored by following the conversion of S-35-labeled peptide to a greater mobility isoform on urea-gradient sodium dodecyl sulfate-polyacrylamide gels. The mobility-shifted isoform also incorporates radiolabel in the prese nce of [H-3]mevalonate or [H-3]geranylgeranyl pyrophosphate, confirmin g post-translational modification with geranylgeranyl. A quantitative assessment of the conversion of mobility-shifted Rab5, promoted by pre nylation, and the amount of incorporated radiolabel from [(3)Hlgeranyl geranyl pyrophosphate was achieved by excising gel slices containing r adiolabeled isoforms and measuring the covalently associated radioacti vity. Using this approach, we have established that 2 moles of geranyl geranyl is attached per mole of Rab5 peptide. A 2:1 molar ratio of ger anylgeranyl:peptide is observed for both Rab5(wt) and a truncation mut ant, Rab5(1-213) containing C-terminal motifs CCXX and XXCC, respectiv ely. When Rab proteins ending in CXC are synthesized and processed in vitro, they also incorporate geranylgeranyl at a 2:1 stoichiometry, al though extended times of incubation are required for full modification . Finally, a C-terminal Rab5 truncation mutant retaining only one cyst eine also becomes modified, although only a minor fraction is fully pr ocessed. This method offers a novel, quantitative approach to investig ate the stoichiometry of post-translational processing of cell-free sy nthesized peptides without the need to purify the native molecules. (C ) 1995 Academic Press,Inc.