PRENYLATION OF A RAB1B MUTANT WITH ALTERED GTPASE ACTIVITY IS IMPAIRED IN CELL-FREE SYSTEMS BUT NOT IN INTACT MAMMALIAN-CELLS

Previous studies have reached differing conclusions as to whether or n ot guanine-nucleotide-dependent conformational changes affect the abil ity of Rab proteins to undergo post-translational modification by Rab: geranylgeranyltransferase (Rab-GGTase). We now show that the ability of a Rab1B mutant [Q67L (Gln-67 --> Leu)] with reduced intrinsic GTPas e activity to undergo geranylgeranylation in cell-free assays depends on the guanine nucleotide composition of the system. When GTP is the p redominant nucleotide in the assay, Rab1B(Q67L) is a poor substrate. H owever, when GDP is present and GTP is omitted, prenylation of the Q67 L mutant is comparable with that of the wild-type ((WT)) protein. Thes e studies, coupled with the poor prenylation of Rab1B(WT) in the prese nce of the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]trip hosphate, support the notion that Rab-GGTase prefers substrates in the GDP conformation. When the abilities of Rab1B(Q67L) and Rab1B(WT) to undergo prenylation were compared by metabolic labelling of transientl y expressed proteins in cultured human 293 cells, we did not observe a decline in prenylation of the mutant protein as predicted on the basi s of the cell-free assays. Moreover, the Q67L mutant was comparable wi th the wild-type Rab1B in its ability to associate with coexpressed Ra b GDP dissociation inhibitors in 293 cells. These findings raise the p ossibility that unidentified proteins present in intact cells may comp ensate for the reduced intrinsic GTPase activity of the Q67L mutant, a llowing a significant proportion of the nascent Rab1B(Q67L) to assume a GDP conformation. The differential prenylation of Rab1B(Q67L) in cel l-free systems versus intact cells underscores the importance of evalu ating the posttranslational modification of specific Rab mutants in vi vo, where poorly characterized regulatory proteins may have a signific ant effect on GTPase activity or nucleotide exchange rates.