2-HYBRID SYSTEM SCREEN WITH THE SMALL GTP-BINDING PROTEIN RAB6 - IDENTIFICATION OF A NOVEL MOUSE GDP DISSOCIATION INHIBITOR ISOFORM AND 2 OTHER POTENTIAL PARTNERS OF RAB6

Rab6 is a small GTP-binding protein that belongs to the Ras superfamil y and is involved in intra-Golgi transport. Using a two-hybrid system screen of a mouse brain cDNA library, we have isolated several clones encoding proteins that interact with Rab6. Approximately 60% of the cl ones identified encoded anew mouse Rab GDP dissociation inhibitor (GDI ) isoform. This GDI isoform is distinct from mouse mGDI-1 and mGDI-2, which have been characterized previously, and most likely represents t he mouse counterpart of the rat Rab GDI beta isoform. In the two-hybri d system, GDI beta interacts with wild-type Rab6 and Rab5, but not wit h a GTP-bound Rab6 mutant, or a Rab6 mutant that cannot be post-transl ationally processed. We further examined whether mouse GDI beta is fun ctional; we show that recombinant mouse GDI beta is able to remove sev eral Rab proteins, including Rab1, Rab2, Rab4, and Rab6, from membrane s. The identification of a third GDI isoform in mouse raised the quest ion whether GDI genes belong to a larger multigenic family. We have sh own, by Southern blot analysis of genomic DNA, that at least five GDI gene copies exist in both the mouse and rat genomes. In our two-hybrid screen, we have also characterized another clone that specifically in teracts with Rab6. This clone was partially sequenced but shows no hom ology to known sequences. Finally, a third clone, interacting with bot h Rab5 and Rab6. also appears to encode a novel protein.