Two-hybrid System Screen with the Small GTP-binding Protein Rab6. IDENTIFICATION OF A NOVEL MOUSE GDP DISSOCIATION INHIBITOR ISOFORM AND TWO OTHER POTENTIAL PARTNERS OF Rab6

doi:10.1074/jbc.270.24.14801

Two-hybrid System Screen with the Small GTP-binding Protein Rab6. IDENTIFICATION OF A NOVEL MOUSE GDP DISSOCIATION INHIBITOR ISOFORM AND TWO OTHER POTENTIAL PARTNERS OF Rab6 (*)

From the (1) Unité de Génétique Somatique URA CNRS 361,
(2)Unité d'Immunochimie Analytique URA CNRS 359, Département d'Immunologie, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15 and
(3)INSERM U248, Faculté de Médecine Lariboisière-Saint Louis, 10 avenue de Verdun, 75010 Paris, France

** To whom correpondence should be addressed. Tel.: 33-1-45-68-85-68; Fax: 33-1-40-61-31-71; E-mail: bgoud{at}pasteur.fr.

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 GDIβ isoform. In the two-hybrid system, GDIβ 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 GDIβ is functional; we show that recombinant mouse GDIβ 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.

Footnotes

↵^§ Supported by a fellowship from the Fondation pour la Recherche Médicale.
↵^¶ Investigator of the INSERM.
↵* This study was supported in part by Grant RG-380/92 from the Human Frontier Science Program (to B. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L36414, L40894, and L40934.
↵¹ The abbreviations used are:

GDI

GDP dissociation inhibitor

LexBD

LexA binding domain

GAL4AD

GAL4 activation domain

β-gal

β-galactosidase

GAP

GTPase-activating protein

PCR

polymerase chain reaction

GDF

GDI displacement factor

GEF

guanine nucleotide exchange factor

bp

base pair(s)

kb

kilobase pair(s)

wt

wild type.
↵²P. Mollat and B. Goud, unpublished results.
↵³I. Janoueix-Lerosey, F. Jollivet, and B. Goud, unpublished results.