Abstract
Rab6 is a small GTP-binding protein that belongs to the Ras superfamily 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 clones identified encoded a new 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 the mouse counterpart of the rat Rab GDI beta isoform. In the two-hybrid system, GDI beta interacts with wild-type Rab6 and Rab5, but not with a GTP-bound Rab6 mutant, or a Rab6 mutant that cannot be post-translationally processed. We further examined whether mouse GDI beta is functional; we show that recombinant mouse GDI beta is able to remove several Rab proteins, including Rab1, Rab2, Rab4, and Rab6, from membranes. The identification of a third GDI isoform in mouse raised the question whether GDI genes belong to a larger multigenic family. We have shown, 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 interacts with Rab6. This clone was partially sequenced but shows no homology to known sequences. Finally, a third clone, interacting with both Rab5 and Rab6, also appears to encode a novel protein.
Highlights
In our two-hybrid screen, we have characterized another clone that interacts with Rab6
We have identified from a mouse brain cDNA library several clones encoding proteins that interact with Rab6; among them, we have isolated a new mouse GDP dissociation inhibitor (GDI) isoforrn, different from the two GDI proteins recently characterized in mouse, GDI-1, the mouse counterpart of the bovine Rab3A GDI and GDI-2 [18]
The probes were derived from the mouse GDIf3 cDNA by PCR amplification with primers 5'-ATTGAAGAAATCATTGTG-3' and 5'-GTTGTACTTACAATGGCA-3', spanning bases 763-1064, which correspond to residue 255 and 356 in mGDIf3 for PI probe; with primers 5'GCCATTCTCTTGCACTGT-3' and 5' -GGGATCACAGATGAGCTG-3', spanning bases 557-832, which correspond to residue 186 and 284 for P2 probe; and with primers 5 '-ACATAATGTGGCAGCACA-3, and 5'CTCAGATCCTGTCATCCT-3', spanning from bases 1020-1284, which correspond to residue 341 and 428 for P3 probe
Summary
Vol 270, No 24, Issue of June 16, pp, 14801-14808, 1995 Printed in U.S.A. IDENTIFICATION OF A NOVEL MOUSE GDP DISSOCIATION INHIBITOR ISOFORM AND TWO OTHER POTENTIAL PARTNERS OF Rab6*. Hydrolysis of GTP by the Rab protein, possibly stimulated by a GTPase-activating protein (GAP), would convert it into its GDP-bound conformation This GDPbound Rab protein could be recycled in the cytosol through the action of a GDI protein that is able to extract the GDPbound Rab proteins from intracellular membranes [9,10,11]. Characterization of the interaction between these clones and various Rab mutants altered in their GDP/GTP binding properties, or in their processing, was used to define their possible relationship with Rab6 These clones were partially sequenced but show no homology with known sequences; they represent novel potential Rab partners
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