DETERMINATION OF STRUCTURAL REQUIREMENTS FOR THE INTERACTION OF RAB6 WITH RABGDI AND RAB GERANYLGERANYLTRANSFERASE

The importance of geranylgeranylation to the interaction of Rab protei ns with RabGDI was investigated with a set of Rab6 mutants post-transl ationally modified by all known C-terminal lipid combinations. Rab6 pr oteins geranylgeranylated on CXC or CC motifs were found to be signifi cantly better substrates for membrane extraction by RabGDI than either Rab6 proteins geranylgeranylated on CAAL motifs or Rab6 proteins that were farnesylated and palmitoylated. The methylation status of the CX C motif did not significantly affect interaction of wild type Rab6 wit h RabGDI. Rab6 protein sequences required for RabGDI interaction were then identified. Consistent with the significant homology between RabG DI and the Rab escort protein, a subunit of Rab geranylgeranyltransfer ase (RabGGTase), we show that there is an overlap between RabG motifs required for RabGDI binding and RabGGTase processing. The effector dom ain, loop3/beta 3 and the hypervariable region of RabG are all require d for RabGDI binding, whereas loop3/beta 3 and the hypervariable regio n but not the effector domain are required for efficient processing of RabG by RabGGTase. Interestingly, however, loop3/beta 3 of RabG when introduced into H-Ras is sufficient to allow some in vivo processing o f a C-terminal CSC motif.