From the
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
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.
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)(
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.(
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.
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
50 µg of membranes prepared
from HeLa cells were incubated without or with 30 µg of purified
His-tagged mouse GDI
Approximately 40
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
As
mentioned above, 170 clones among the 261
His
To
determine whether mouse GDI
One can exclude the possibility
that the absence of interaction between GDI
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
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
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
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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.
ABSTRACT
INTRODUCTION
Materials and Methods
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)
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) .
)
The Rabphilin
protein, which binds only to the GTP-bound form of Rab3, could
constitute a potential effector of this protein(16) .
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-Rab6
C plasmids were constructed by inserting the EcoRI/PstI fragments of the pGEM-Rab6wt,
pGEM-Rab6Asn
, and pGEM-Rab6
C 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.
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.
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.
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.
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.
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.
/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.
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 Rab6
C 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) .
and
Rab6Leu
, Rab6Ile
, or Rab6
C 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.
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.
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
, Rab6
C, 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 Rab6
C (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) .
; 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
/EMBL Data Bank with accession number(s) L36414, L40894,
and L40934.
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.