Membrane targeting of a Rab GTPase that fails to associate with Rab escortprotein (REP) or guanine nucleotide dissociation inhibitor (GDI)

The targeting of various Rab proteins to different subcellular compartments appears to be determined by variable amino acid sequences located upstream from geranylgeranylated cysteine residues in the C-terminal tail. All nasc ent Rab proteins are prenylated by geranylgeranyltransferase II, which reco gnizes the Rab substrate only when it is bound to Rab escort protein (REP), After prenylation, REP remains associated with the modified Rab until it i s delivered to the appropriate subcellular membrane. It remains unclear whe ther docking of the Rab with the correct membrane is solely a function of f eatures contained within the prenylated Rab itself (with REP serving as a " passive" carrier) or whether REP actively participates in the targeting pro cess. To address this issue, we took advantage of a mutation in the alpha2 helix of Rab1B (i.e. Y78D) that abolishes REP and GDI interaction without d isrupting nucleotide binding or hydrolysis. These studies demonstrate that replacing the C-terminal GGCC residues of Rab1B(Y78D) with a CLLL motif per mits this protein to be prenylated by geranylgeranyltransferase I but not I I both in cell-free enzyme assays and in transfected cells. Subcellular fra ctionation and immunofluorescence studies reveal that the prenylated Rab1B( Y78D)CLLL, which remains deficient in REP and GDI association is, nonethele ss, delivered to the Golgi and endoplasmic reticulum (ER) membranes. When t he dominant-negative S22N mutation was inserted into Rab1B-CLLL, the result ing monoprenylated construct suppressed ER --> Golgi protein transport. How ever, when the Y78D mutation was added to the latter construct, its inhibit ory effect on protein trafficking was lost despite the fact that it was loc alized to the ER/Golgi membrane. Therefore, protein interactions mediated b y the alpha2 helical domain of Rab1B(S22N) appear to be essential for its f unctional interaction with components of the ER --> Golgi transport machine ry.