PRENYLATION OF RAB5 IS DEPENDENT ON GUANINE-NUCLEOTIDE-BINDING

Rab5 is a small molecular weight GTP-binding protein that functions in endocytic vesicle traffic. Like other Ras-related proteins, Rab5 is p renylated on C-terminal cysteine residues, although it lacks the typic al C-terminal CAAX motif (where A is any aliphatic amino acid and X is any amino acid) to direct this post-translational modification. We ha ve investigated structural requirements for the in vitro geranylgerany lation of Rab5. Rab5N133I, a point mutant that has impaired ability to bind GTP or GDP, undergoes modification to a limited extent and at a severely reduced rate when compared to cognate Rab5. A second point mu tant, Rab5Q79L, can be processed to approximately the same extent as w ild-type albeit at a reduced rate. Since the latter mutation results i n defective GTPase activity, these combined observations indicate that guanine nucleotide binding plays an important role in the geranylgera nylation reaction and suggest that the GDP-bound form of Rab5 is the p referred conformation for interaction with Rab prenyltransferase. This idea is supported by the finding that non-hydrolyzable GTP analogs in hibit Rab5 prenylation, while in vitro processing of both H-ras and th e gamma2 subunit of regulatory G proteins is unaffected at concentrati ons of guanosine 5'-O-(thiotriphosphate) (GTP-gammaS) up to 400 muM. M oreover, a truncation mutant lacking the C-terminal cysteines, Rab5(1- 211), serves as an inhibitor of Rab5wt geranylgeranylation when ligand ed with GDP but not GTPgammaS. Thus, the recognition of Rab5 as a subs trate by Rab prenyltransferase involves structural elements exclusive of the C terminus and dependent upon the GDP-binding conformation of t he protein.