PRENYLATION OF RAB8 GTPASE BY TYPE-I AND TYPE-II GERANYLGERANYL TRANSFERASES

Rab GTPases are post-translationally modified by addition of geranylge ranyl moieties to carboxyl-terminal cysteine residues. For Rab protein s ending with xxCC xCxC and CCxx motifs this modification is catalysed by geranylgeranyltransferase type II (GGTaseII), and is entirely depe ndent on the Rab substrate being bound to Rab escort protein (REP), Se veral Rab proteins contain carboxyl-terminal CaaL prenylation motifs t ypical of members of the Rho family, which are modified in a REP-indep endent manner by geranylgeranyltransferase type I (GGTaseI). The prese nt studies show that one such Rab protein (Rab8), which ends with a CV LL motif, is uniquely able to serve as a substrate for either REP/GGTa seII or GGTaseI in cell-free assays. The modification of Rab8 by GGTas eI did not require REP, indicating that a REP-induced conformational c hange is not essential for exposure of the Rab carboxyl-terminal cyste ine prenylation site. To determine whether one enzyme plays a predomin ant role in Rab8 prenylation in vivo, the incorporation of [H-3]mevalo nate into Rab8 was measured in human embryonal kidney 293 cells under conditions where the activity of GGTaseI, but not GGTaseII, was blocke d by the peptidomimetic inhibitor GGTI-298. The GGTaseI inhibitor did not prevent prenylation of either overexpressed Myc-tagged Rab8 or end ogenous Rab8, whereas prenylation of a known GGTaseI substrate with th e same carboxyl-terminal motif, Cdc42Hs, was completely blocked. To ru le out the possibility that the apparent prenylation of Rab8 by GGTase II occurs only when GGTaseI activity is eliminated, metabolic labellin g studies were carried out in the absence of the GGTaseI inhibitor, us ing a REP-binding-deficient Rab8 construct (Y78D) that cannot serve as a substrate for GGTaseII, but is indistinguishable from wild-type Rab 8 as a substrate for GGTaseI. Prenylation of the Y78D mutant was reduc ed by 60-70% in intact cells, consistent with the conclusion that the majority of Rab8 is prenylated by the REP/GGTaseII system in vivo.