©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (*)

Isabelle Janoueix-Lerosey (1)(§), Florence Jollivet (1), Jacques Camonis (3), Patrice N. Marche (2)(¶), Bruno Goud (1)(**)

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

ABSTRACT
INTRODUCTION
Materials and Methods
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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


INTRODUCTION

Rab proteins are Ras-like GTP-binding proteins involved in the regulation of vesicular transport through the endocytic and secretory pathway(1, 2, 3) . In particular, the Rab6 protein (Rab6p), found associated with medial and trans-Golgi cisternae, regulates intra-Golgi transport events(4, 5) . As for other members of the Ras superfamily, Rab proteins interconvert between a GDP-bound and a GTP-bound forms, but they also switch between a cytosolic and a membrane localization. This cycle is likely to control vesicle targeting and/or fusion at different stages in the endocytic and exocytic pathways.

A model of the Rab functional mechanism has recently been proposed(2) ; in the cytosol, Rab proteins are found as complexes with a GDP dissociation inhibitor (GDI)() protein (6-9). Recent experiments have suggested that the GDI protein could deliver the Rab protein to a specific organelle(10, 11) . The association of the Rab protein with membranes and the subsequent guanine nucleotide exchange may be catalyzed by a GDI displacement factor (GDF) and a guanine nucleotide exchange factor (GEF), respectively(2) . The GTP-bound Rab would then be recruited on nascent transport vesicles and interact with one or more downstream effectors. The final role of Rab proteins may be to catalyze the association of a v-SNARE protein with a t-SNARE protein, providing fidelity in the process of docking and/or fusion of vesicles with their correct acceptor compartment(12) . After fusion, hydrolysis of GTP by the Rab protein, possibly stimulated by a GTPase-activating protein (GAP), would convert it into its GDP-bound conformation. This GDP-bound Rab protein could then be recycled in the cytosol through the action of a GDI protein that is able to extract the GDP-bound Rab proteins from intracellular membranes(9, 10, 11) .

According to this model, several regulators such as GDI, GEF, and GAP proteins, as well as specific downstream effectors, are required to allow the function of Rab proteins. Only some of these regulators have been well characterized. The mammalian protein Mss4 exhibits a guanine nucleotide exchange factor activity toward Rab3A and Rab2, but does not seem to interact with Rab6(13, 14) . Several GAP activities for Rab proteins have been detected biochemically in cell or tissue extracts but only Gyp6, a yeast GAP specific for Ypt6, the yeast Rab6 homolog, have been cloned(15) . Surprisingly, Gyp6 does not show any GAP activity toward Rab6.() The Rabphilin protein, which binds only to the GTP-bound form of Rab3, could constitute a potential effector of this protein(16) .

Rab GDI remain the best characterized Rab accessory proteins. GDI proteins both inhibit GDP release and, as mentioned above, act as chaperones of Rab proteins during their cycling between cytosol and membrane(6, 7, 8, 9, 10, 11) . The first GDI protein (named Rab3A GDI) was originally identified from bovine brain cytosol by its ability to slow the dissociation of GDP from prenylated Rab3A(6) , and its corresponding cDNA was cloned(17) . Rab3A GDI is not, however, a specific regulator of Rab3A and has been shown to interact with many Rab proteins(9) . Recently, cDNAs encoding new GDI isoforms have been characterized in mouse and rat as well as in Drosophila and yeast(18, 19, 20, 21) . It is not known, however, how many GDI isoforms are expressed in these different species or if the different isoforms fulfill specific functions.

As the specific regulators and effectors of Rab6p remain poorly characterized, we have used the yeast two-hybrid system originally described by Fields and Song (22) and modified by Vojtek et al.(23) to search for Rab6 partners. 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 GDI isoform, different from the two GDI proteins recently characterized in mouse, GDI-1, the mouse counterpart of the bovine Rab3A GDI and GDI-2(18) . In the two-hybrid system, this mouse GDI also interacts with Rab5. This suggests that at least three distinct isoforms of GDI may regulate Rab function in mouse. The characterization of a third GDI isoform in mouse prompted us to determine whether the rab GDI genes belong to a larger gene family. To answer this question, we have performed Southern blot analysis of genomic DNA prepared from mice and rats. These experiments indicate that mouse and rat genomes contain at least five rab GDI genes.

We have also identified in our two-hybrid screen two other potential partners of Rab6; one only interacts with Rab6, and the other one forms complexes in the yeast with either Rab5 or Rab6. Characterization of the interaction between these clones and various Rab6 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 therefore represent novel potential Rab partners.


Materials and Methods

Strains and Media

The genotype of the Saccharomyces cerevisiae reporter strain L40 is MATatrp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ(23) . Yeast strains were grown at 30 °C in rich medium (1% yeast extract, 2% Bacto-Peptone, 2% glucose) or in synthetic minimal medium with appropriate supplements(24) .

Plasmids

The pLexA-Rab6wt, pLexA-Rab6Asn and pLexA-Rab6C plasmids were constructed by inserting the EcoRI/PstI fragments of the pGEM-Rab6wt, pGEM-Rab6Asn, and pGEM-Rab6C plasmids(5) , respectively, into the pBTM116 plasmid (a kind gift of A. Vojtek)(23) . The resulting plasmids express Rab6 as a fusion protein to the DNA binding domain of LexA with a short linker between the LexBD and the Rab6 initiator methionine, consisting of the following amino acids: EFRSGRSSSST. The Val and Ile mutations were generated by oligonucleotide-directed mutagenesis on M13mp10 vector of Rab6 cDNA using following primers: 5`-TCCAACGCTTACCTCCCCCAGG-3` (Val, provided by Gress Kadaré and Jean de Gunzburg) and 5`-AGCAAGATCTGTTTTAATTCCTACTAGCATGAT-3` (Ile). After sequencing, the mutated cDNAs were inserted into pGEM-Rab6 and the pLexA-Rab6Val and pLexA-Rab6Ile were derived from these plasmids as described above. The pLexA-Rab6 Leu plasmid was constructed by PCR from pGEM-Rab6Leu(5) using the following forward and reverse oligonucleotide primers: 5`-GGCCGGATCCGGAATGTCCACGGGCGGAGACTTC-3` and 5`-GGCCGTCGACACATTAGCAGGAACAGCCTCCTTC-3`. Standard PCR conditions were used. The PCR product was digested with BamHI and SalI and then inserted into pBTM116. The sequence of the PCR product was verified by dideoxy sequencing. The resulting plasmid expresses Rab6Leu as a fusion protein to the LexBD with a glycine between the LexBD and the Rab6 initiator methionine. pLexA-lamin (23) and pLexA-Rab5 wt (kindly provided by H. Stenmark and M. Zerial, EMBL, Heidelberg, Germany) express human lamin C (amino acids 66-230) and Rab5wt protein as fusions to the DNA-binding domain of LexA, respectively. A cDNA library from mouse BALB/c brain poly(A) RNA was constructed in fusion with GAL4AD, in pGAD1318 (25) using the Stratagene cDNA synthesis kit. pGAD-SNIF4 is a generous gift of Linda Van Aelst (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Two-hybrid Screen

The yeast reporter strain L40, which contains the reporter genes LacZ and HIS3 downstream of the binding sequences for LexA, was sequentially transformed with the pLexA-Rab6Val plasmid and with a mouse brain cDNA library using the lithium acetate method (24) and subsequently treated as described(23) . Double transformants were plated to synthetic medium lacking histidine, leucine, tryptophan, uracil, and lysine. The plates were incubated at 30 °C for 3 days. His colonies were patched on selective plates and assayed for -galactosidase activity by a filter assay(26) . Plasmid DNA was prepared from colonies displaying a His/LacZ phenotype and used to retransform the L40 strain containing the LexA-Rab6Val hybrid; this step is necessary to correlate the His/LacZ phenotype with a single library plasmid. Indeed, several different library plasmids are often (approximately 30% of the cases) recovered from a single yeast colony and retransformation of the reporter strain often gives His/LacZ colonies and His/LacZ colonies. pGAD1318 library plasmids were then rescued from His/LacZ colonies and tested for specificity by cotransformation into L40 with pLexA-Rab6wt, pLexA-Rab5wt, or pLexA-lamin.

The cDNA inserts from specific clones were sequenced using the Sanger dideoxy-termination method. The sequence of one clone (clone A) containing the full-length cDNA encoding mouse GDI was determined from both strands either directly with oligonucleotide primers or after progressive deletions in the coding sequence with an ALF automated sequencer (Pharmacia Biotech Inc.). Sequences were analyzed with the GCG software package(27) , and protein sequence comparisons were carried out with CLUSTAL V software(28) .

Release of Rab Proteins from Membranes

The entire mouse GDI protein was expressed as a His-tagged protein by using the pET-15b vector (Novagen) and purified on Ni-agarose (Qiagen) according to the manufacturer's instructions.

50 µg of membranes prepared from HeLa cells were incubated without or with 30 µg of purified His-tagged mouse GDI as described previously(29) . The reaction mixtures were centrifuged at 4 °C for 15 min at 150,000 g in a TL100 Beckman centrifuge; pellet (membrane fraction) and supernatant (soluble fraction) were then analyzed for Rab1, Rab2, Rab4, or Rab6 content by SDS-polyacrylamide gel electrophoresis and Western blotting with affinity-purified anti-Rab antibodies.

Southern Blot Analysis

General procedures and buffers used for the preparation of high molecular weight DNA, electrophoresis, blotting, and hybridization were previously described(30) . 10 µg of DNA samples were digested to completion by BamHI, EcoRI, and XbaI restriction endonucleases. After electrophoresis on a 0.7% agarose gel, DNA was transferred on a Nylon membrane (Qiabrane, Qiagen, Germany) and then hybridized with P probes at 2-4 10 cpm/ml labeled using Ready to Go kit (Pharmacia). Filters were washed, the final wash stringency was adjusted with either 0.2 SSC, 45 °C, or 0.1 SSC, 65 °C. Same filters were used in turn with the different probes after removing hybridization by treatment with 0.4 M NaOH, 45 °C, 30 min. The probes were derived from the mouse GDI 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 mGDI for P1 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.


RESULTS AND DISCUSSION

Interaction Screening of a Mouse Brain cDNA Library with Rab6

In order to identify partners of Rab6p, we performed interaction screening using the two-hybrid system in yeast. The yeast reporter strain L40 containing the Rab6Val protein fused to the LexA binding domain (LexBD) was transformed with a library of mouse brain cDNA fragments inserted 3` to the GAL4 activation domain (GAL4AD), in pGAD1318 plasmid. In this assay, the formation of a complex between Rab6Val fused to LexBD and a protein fused to GAL4AD confers histidine auxotrophy and -galactosidase activity.

Approximately 40 10 yeast transformants were screened. 350 colonies were found to grow on histidine-free plates, and, among them, 261 displayed -galactosidase activity. Plasmid DNA was prepared from these 261 His/LacZ colonies. First, the L40 strain containing pLexA-Rab6Val was retransformed with 40 selected library plasmids in order to correlate the His/LacZ phenotype with a unique library plasmid (see ``Materials and Methods''). PCR reactions were then performed to determine the size of the inserts contained in the plasmids yielding a His/LacZ phenotype; 36 plasmids contained a 2-kb insert, and 4 contained a 3-kb insert. These two species of clones were then tested for specificity by co-transformation into the L40 strain with plasmids directing the expression of Rab6 wt, Rab5 wt, or lamin fused to LexBD. One clone (clone A) gave a positive signal in combination with either Rab6, or Rab5 fused to LexBD; the other one (clone B) appeared to be specific for Rab6 as it yielded growth on histidine-free plates only when co-expressed with the Rab6 fusion protein. It appeared from the analysis of these first 40 clones that some of them were highly redundant. To reduce the number of remaining clones to be analyzed by the above procedure, the DNA from the 261 originally positive clones were hybridized with probes corresponding to clones A and B. 170 clones gave a positive signal when hybridized with probe A, and 61 were positive with probe B. 231 redundant clones were thus eliminated. The 30 remaining clones were then analyzed for specificity, and a new cDNA sequence (clone C) was identified as a potential partner of Rab5 and Rab6 proteins. The present screening therefore led to the isolation of two clones encoding proteins that interact with Rab6 and Rab5, but not with lamin (clones A and C), and one that specifically interacts with Rab6 (clone B). They were further characterized for interaction with various Rab6 mutants and their DNA sequence determined.

Clone A Encodes Mouse GDI

Clone A, that interacts with Rab6p and Rab5p contains a 2-kb cDNA insert with a 1335-bp open reading frame. The nucleotide sequence of clone A share 75% identity with mGDI-1 (partial sequence) and 88% identity with mGDI-2, two GDI isoforms recently characterized in mouse(18) . Since these three genes have been cloned from the same mouse strain (BALB/c), differences between sequences could not be attributed to divergence between species. Clone A therefore represents a new mouse GDI isoform.

At the protein level, clone A (445 amino acids with a calculated mass of 50,543 kDa) shares respectively 84.5% and 95.3% identity with mouse GDI-1 and GDI-2. Interestingly, it presents 98% identity with the rat Rab GDI isoform(21) . Moreover, the 5`-noncoding region of mouse GDI is distinct from that of mouse GDI-2, but very similar to that of rat Rab GDI(21) . For these reasons, clone A most likely corresponds to the mouse counterpart of rat GDI and was thus named mouse GDI. Fig. 1shows an alignment of the three mouse GDI isoforms. Differences in the sequences are distributed almost randomly along the complete coding region; however, two blocks are extremely well conserved between residues 200 and 250 and between residues 300 and 350.


Figure 1: Alignments of deduced amino acid (single-letter code) sequences of mouse GDI, GDI-1, and GDI-2. Identical residues are denoted by colons, and differences are indicated by the corresponding residue. Dashes mark the missing first 124 amino acids of GDI-1.



It was not clear previously whether GDI and GDI-2 were the same or different proteins. Our findings show that mouse GDI and GDI-2 are encoded by different genes. Therefore, at least three different Rab GDI isoforms, mGDI-1, mGDI-2, and mGDI, exist in mouse.

As mentioned above, 170 clones among the 261 His/LacZ clones were positive when hybridized with a probe corresponding to mouse GDI. As the different GDI cDNAs share a high level of identity and could potentially cross-hybridize in our assay, we have randomly selected 10 clones among the 170 positives and sequenced them partially; all of them were strictly identical to mouse GDI. Several reasons could explain why we only picked up mouse GDI in our two-hybrid screen. Mouse GDI-2 contains an in-frame terminator upstream its ATG initiation codon(18) ; therefore, a complete cDNA inserted 3` to the GAL4 activation domain would never allow the synthesis of a fusion protein between GAL4AD and GDI-2. Mouse GDI-1 could be poorly represented in the library; alternatively, it may also contain a terminator upstream from its initiating Met codon.

To determine whether mouse GDI is active, we have expressed it as an His-tagged protein in Escherichia coli and tested its ability to remove different Rab proteins from membranes. We show that purified mGDI is able to extract Rab1A, Rab2, Rab4A, and Rab6 from membranes prepared from HeLa cells (Fig. 2). Moreover, in this study we found that mGDI also interacts with Rab5, since the GAL4AD-GDI hybrid confers the ability to grow in the absence of histidine, when expressed with a LexBD-Rab5wt hybrid, but not with a LexBD-lamin hybrid (Fig. 3). Rat GDI has been shown to inhibit GDP release from both Rab3A and Rab11(21) . We also recently found that GDI from Chinese hamster ovary cells forms in vivo complexes with several proteins of the Rab family including Rab1A, Rab2, Rab4A, and Rab6(29) . Altogether, these results indicate that GDI can interact with many, if not all, Rab proteins.


Figure 2: Recombinant His-tagged GDI releases membrane-bound Rab1, Rab2, Rab4, and Rab6. Membranes prepared from HeLa cells were incubated with or without recombinant GDI as described under ``Materials and Methods.'' The reaction mixtures were centrifuged, and the membrane (pellet, p) and soluble (s) fractions were analyzed by immunoblotting with affinity-purified polyclonal antibodies directed against Rab1, Rab2, Rab4, and Rab6.




Figure 3: Interaction of wild-type and mutant Rab6 proteins with mouse GDI in the yeast two-hybrid system. The S. cerevisiae reporter strain L40 was cotransformed with pairs of the indicated proteins fused to LexBD and to GAL4AD. Transformants were plated on synthetic medium without (left) or with (right) histidine. Plates were then incubated at 30 °C for 2 days. The LexBD-Rab6wt hybrid in the presence of a GAL4AD-SNIF4 fusion protein (SNIF4 being an extraneous target, 22) or the GAL4AD-GDI in the presence of a LexBD-lamin hybrid do not permit growth in the absence of histidine, showing that histidine auxotrophy is the result of interaction between Rab6 and GDI. Each patch represents an independent transformant.



We took advantage of the two-hybrid system to further characterize the interaction between Rab6p and mouse GDI. For this purpose, several Rab6 mutants altered in their GDP/GTP binding properties and GTP hydrolysis, or in their processing, were fused to LexBD. All hybrids were expressed at approximately the same level in the transformed yeasts, as determined by Western blot analysis (data not shown). As shown in Fig. 3, mutations in Rab6 such as Ile(31) or Leu(5, 32) that are thought to lock the protein in its active GTP-bound conformation, completely abolish interaction with GDI. This is in good agreement with previous finding showing that GDI proteins specifically interact with the GDP-bound form of the Rab proteins(7) . Interestingly, interaction between Rab6 and GDI was enhanced by a valine 22 mutation in Rab6 (Fig. 3, lane5), suggesting that this mutation rather favors the GDP-bound form of the protein over the GTP-bound form. This is similar to the effect of an equivalent mutation in Rab3A(33) , but opposite to the case of Ras p21 in which the corresponding Val mutation locks the protein in its active GTP-bound form(34) . More surprisingly, GDI seems not to be able to interact with a Rab6 protein bearing an Asn mutation (Fig. 3, lane10), which should favor the GDP-bound conformation of the protein(35, 36, 37) . One possibility is that this mutation decreases the affinity of the protein for GDI so that the resulting interaction is too weak to be detected in the two-hybrid system. Finally, a Rab6C mutant, deleted for the last three amino acids at the carboxyl end and which therefore cannot be isoprenylated, is no longer capable of interacting with GDI (Fig. 3, lane9). This is consistent with the fact that GDI can only interact with isoprenylated forms of Rab proteins(38, 39) .

One can exclude the possibility that the absence of interaction between GDI and Rab6Leu, Rab6Ile, or Rab6C could be due to an instability or improper folding of these mutants, since they are able to form complexes in vivo with other partners, with the same efficiency as the Rab6 wild-type protein (see below). The same holds true for Rab6Asn, since this mutant was found to interact with cDNA inserts in another screen.() Therefore, the above results indicate that mouse GDI has similar properties to Rab3AGDI with respect to its interaction with Rab proteins. They also illustrate that the two-hybrid system can be a powerful assay to study the interaction between a Rab protein and a GDI protein.

Mouse Genome Contains at Least Five Distinct rab GDI Genes

The existence of three isoforms of GDI in mouse prompted us to determine the number of GDI genes in mouse genome. To address this issue, genomic DNA were analyzed by Southern blots and hybridized with a probe corresponding to the most conserved region among the three mouse GDI (P1 probe, corresponding to residues 255-356 of the GDI protein). Four fragments hybridized to BALB/c mouse DNA following EcoRI or BamHI restriction endonuclease digests (Fig. 4, lanes1 and 2) and five using XbaI restriction endonuclease digest (Fig. 4, lane3). Similar results were obtained with another mouse strain, C57Bl/6, with EcoRI and BamHI (data not shown). However, a polymorphism can be revealed with XbaI enzyme (Fig. 4, lane4) as the fragments revealed in C57BL/6 were different in size to those observed in BALB/c. When the same mGDI probe was used with rat (Lewis strain) genomic DNA, 5 fragments were also detected in XbaI digest (Fig. 4, lane5). Similar results were obtained with the Lou rat strain (data not shown). Taken together, these data support the hypothesis that four or five GDI gene copies are present in mouse and rat genomes.


Figure 4: Southern blots of restriction endonuclease digested genomic DNA from mouse and rat. Genomic DNA prepared from mouse BALB/c and C57BL/6 strains and from rat was digested with the indicated restriction endonucleases. Electrophoresis and transfer were performed as described under ``Materials and Methods.'' The blot was then hybridized with the P1 probe corresponding to the 763-1064 region of the mouse GDI cDNA. The sizes (in kb) of HindIII fragments of phage are indicated at the left. Lanes 1-3 correspond to mouse BALB/c DNA digested by EcoRI, BamHI, and XbaI restriction enzymes, respectively; lane4, mouse C57Bl/6; lane5, rat DNA digested both with XbaI restriction enzyme. The stringency of the final wash was 0.2 SSC, 45 °C.



To examine further the gene complexity of the GDI family, the same blots were hybridized with two probes (P2 and P3 probes corresponding to regions 186-284 and 341-428 of mGDI protein, respectively) derived from regions that display a greater level of divergence among mouse GDI. The summary of the hybridization patterns obtained with BALB/c mouse is presented in . Following EcoRI digestion, 16.5-, 11.8-, and 1.5-kb fragments hybridized with the three probes. The fact that fragments of the same size are detected with all the probes strongly suggested that a complete mGDI is included. Alternatively, one cannot formally exclude the possibility of comigration of two independent DNA fragments bearing different parts of the gene. This later explanation is unlikely, since the same observation can be made with BamHI, for 25-, 22-, and 4.1-kb fragments and with XbaI, for 12.6-, 10.9-, and 3.0-kb fragments. In all digestions, a fragment, the 14.3-kb EcoRI, the 3.6-kb BamHI, or the 4.9-kb XbaI, was revealed with P1 and P3 probes, but not with the most 5` part P2 probe. This could be due to polymorphism in the region covered by the P2 probe precluding the labeling of these fragments. When the blots were washed at a higher stringency, the hybridization of these fragments was lost with the P1 probe, arguing that the mouse GDI gene present in these fragments is divergent from the mGDI gene used to probe them. Finally, one additional XbaI fragment hybridized with P1 probe (9.8 kb) and with P2 probe (6.2 kb). This argues that at least one GDI gene copy was not detected in the EcoRI and BamHI digests, either because one of the hybridizing fragments contains two linked genes or because two genes are born on a comigrating DNA fragment. The fact that the P1 and P2 probes did not hybridize to identical fragments can be explained by the presence of an XbaI site that would split the GDI gene in two parts, each of them hybridizing with one of the probes, respectively. As none of cDNA sequences from the known mGDI contains a XbaI site, this copy might correspond to a new mGDI gene, or the XbaI site could be located in a noncoding region such as a putative intron sequence; these two hypotheses are not exclusive. Finally, none of these additional XbaI fragments were detected with the P3 probe, nor were any new fragments detected, suggesting that this mGDI gene is divergent from the mGDI in the 3` region.

It is unlikely that genes encoding REP-1 (40) and REP-2 (41) (for Rab escort proteins) that show some similarity with GDI proteins in two domains could hybridize with the GDI probes used for the Southern blot analysis. Indeed, the first homology domain is not covered by any of the probes, and in the second one, which is contained in the P2 probe, the similarity between GDI and REP is only approximately 50% on a segment of 135 bp, whereas the rest of the probe does not match with REP nucleotide sequence.

In conclusion, one can evaluate to at least five the number of mouse GDI genes. Three of them are probably well conserved with the mGDI gene, whereas one may diverge in the 5` region, and the other in the 3` region of the gene.

Clones B and C Encode Two New Potential Partners of Rab6

Clone B, containing a 3-kb insert, interacts in the two-hybrid system with Rab6p, but not with Rab5p or lamin (Fig. 5). This clone was partially sequenced (558 bp). Its 5` region presents 86% identity with an expressed sequenced tag (264 bp sequenced) isolated from a directionally cloned human infant brain cDNA library (EST07229, Homosapiens cDNA clone HIBBS52, 5` end)(42) . Since some bases were not determined in the human sequence, the identity between the mouse and the human cDNAs could reach 90%. Clone B is therefore probably the mouse counterpart of the human HIBBS52 clone. The nucleotide sequence of clone B contains an open reading frame, in frame with the GAL4AD. However, the deduced amino acid sequence (186 amino acids, see Fig. 6) shows no homology with any sequence of the GenBank data base; thus, clone B represents a new protein.


Figure 5: Clone B specifically interacts with Rab6 in the yeast two-hybrid system. The L40 reporter strain was transformed in order to coexpress the indicated hybrid proteins. The figure shows growth of the transformants on synthetic medium without (left) or with (right) histidine. Growth in the absence of histidine indicates the interaction between hybrid proteins. Each patch represents an independent transformant.




Figure 6: Nucleotide and deduced amino acid sequences of clone B. The partial nucleic acid sequence of clone B was translated in frame with the upstream GAL4AD coding sequence, yielding a 186-amino acid sequence (linker sequence is in italics). Nucleotide residues are numbered on the top; amino acid residues are numbered on the bottom.



We then examined the interaction of this new protein with various Rab6 mutants using the two-hybrid system. Clone B gave a positive signal (growth on plates lacking histidine) when co-expressed with Rab6wt, Rab6Leu, Rab6Ile, Rab6C, or Rab6Val and no signal with Rab6Asn (see Fig. 5). However, when measured through galactosidase activity, the interaction between clone B and Rab6Val was much weaker compared to that observed with Rab6wt, Rab6Leu, or Rab6C (data not shown). Therefore, since this partner seems to prefer the GTP-bound form of Rab6 and does not require a post-translational modified Rab6 to interact with, it could represent a GAP protein or an effector of Rab6p. However, clone B does not display any homology with Gyp6, a GAP for Ypt6, the yeast homolog of Rab6 nor with Rabphilin, a putative target of Rab3A(15, 16) . Clone B has also no homology with Rabin3, a recently identified partner of Rab3 with unknown function(43) .

Clone C, containing a 0.8-kb insert, appears to be a potential regulator of several Rab proteins rather than a specific partner of Rab6p, since it is able to form complexes in the L40 strain with either Rab6p or Rab5p, but not with lamin (Fig. 7). Moreover, it shows the same behavior with the different Rab6 mutants tested compared to GDI; indeed, this clone only interacts with Rab6wt and Rab6Val (see Fig. 7). This clone was also partially sequenced; the nucleotide sequence contains an open reading frame, in frame with the GAL4AD. However, neither the nucleotide sequence (308 bp), nor the deduced amino acid sequence (102 amino acids, see Fig. 8) presents similarity with any sequence of the GenBank data base. Nevertheless, it should be pointed out that a long hydrophobic domain is found between amino acids 77 and 102 of clone C. This domain could act as a membrane anchoring region. As this new protein seems to interact preferentially with the GDP-bound form of Rab6, and also form a complex with Rab5, it could represent an exchange factor of Rab6 and Rab5 proteins or an hypothetical GDI displacement factor. Indeed, according to the Rab functional mechanism, GDF and GEF are required to allow the association of the Rab proteins with membranes. Until now, the only characterized exchange factor has been the mammalian protein Mss4, which is active toward a subset of Rab proteins that does not include Rab6 and Rab5(14) . However, clone C does not display any homology with Mss4, and additional experiments will be required to determine whether clone C act as an exchange factor on a subset of Rab proteins.


Figure 7: Interaction of Rab6 and Rab5 proteins with clone C using the yeast two-hybrid system. The L40 strain expressing the pairs of indicated hybrid proteins was analyzed for histidine auxotrophy. Transformants were plated on synthetic medium without (left) or with (right) histidine. Growth in the absence of histidine indicates the interaction between hybrid proteins. Each patch represents an independent transformant.




Figure 8: Nucleotide and deduced amino acid sequences of clone C. The partial nucleic acid sequence of clone C was translated in frame with the upstream GAL4AD coding sequence, yielding a 102-amino acid sequence (linker sequence is in italics). Nucleotide residues are numbered on the top; amino acid residues are numbered on the bottom.



In conclusion, we have characterized in this study several clones encoding proteins that interact with Rab6p. Among them, we have identified a new mouse GDI isoform, mouse GDI, and have shown that this protein is functional. These results therefore indicate that at least three GDI isoforms exist in mice. One can not exclude that additional GDI isoforms may exist; indeed, Southern blot analysis revealed that mouse genome contain at least five GDI genes. It remains to be determined whether more than the three isoforms now characterized are expressed. This study led also to the identification of two other potential partners of Rab6p, one being fully specific for Rab6p and the other being able to interact also with Rab5p. As these clones exhibit no homology with known sequences, they appear to represent new proteins.

  
Table: DNA fragments of BALB/c mouse hybridizing with mGDI probes



FOOTNOTES

*
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.

§
Supported by a fellowship from the Fondation pour la Recherche Médicale.

Investigator of the INSERM.

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

The abbreviations used are: GDI, GDP dissociation inhibitor; LexBD, LexA binding domain; GAL4AD, GAL4 activation domain; -gal, -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.

P. Mollat and B. Goud, unpublished results.

I. Janoueix-Lerosey, F. Jollivet, and B. Goud, unpublished results.


ACKNOWLEDGEMENTS

We thank Anne Vojtek for providing the pBTM116 plasmid and the L40 yeast strain, Marino Zerial and Harald Stenmark for the pLexA Rab5 construct, and Linda Van Aelst for the pGAD1318 plasmid. We acknowledge the expert technical assistance of Carine Liebe-Gris and Olivier Gorgette in the Southern analysis. We are indebted to Professor Gérard Buttin for constant support.


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