From the Institut Cochin de Génétique
Moléculaire, Département de Génétique,
Développement et Pathologie Moléculaire, INSERM
Unité 257, 24 Rue du Faubourg Saint-Jacques, 75014 Paris, France,
the ¶ GERM-INSERM Unité 435, Université de Rennes 1, Campus Beaulieu, Avenue du Général Leclerc, 35042 Rennes,
France, and the
Laboratoire de Physiologie des Régulations
Cellulaires, UMR 6558, Université de Poitiers, 40 Avenue du
Recteur Pineau, 86022 Poitiers, France
Received for publication, December 27, 2000, and in revised form, February 27, 2001
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ABSTRACT |
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RhoGTPases (Rho, Rac, and Cdc42) are known to
regulate multiple functions, including cell motility, adhesion, and
proliferation; however, the signaling pathways underlying these
pleiotropic effects are far from fully understood. We have recently
described a new RhoGAP (GTPase activating
protein for RhoGTPases) gene, MgcRacGAP, primarily expressed in male germ cells, at the spermatocyte stage. We
report here the isolation, through two-hybrid cloning, of a new partner
of MgcRacGAP, very specifically expressed in the male germ line and
showing structural features of anion transporters. This large protein
(970 amino acids and a predicted size of 109 kDa), we
provisionally designated Tat1 (for testis anion transporter 1), is
closely related to a sulfate permease family comprising three proteins
in human (DRA, Pendrin, and DTD); it is predicted to be an integral
membrane protein with 14 transmembrane helices and intracytoplasmic
NH2 and COOH termini. In situ hybridization studies demonstrate that Tat1 and MgcRacGAP
genes are coexpressed in male germ cells at the spermatocyte stage. On
testis sections, Tat1 protein can be immunodetected in spermatocytes
and spermatids associated with plasma membrane. Two-hybrid and in
vitro binding assays demonstrate that MgcRacGAP stably interacts
through its NH2-terminal domain with the Tat1 COOH-terminal
region. Expression of Tat1 protein in COS7 cells generates a
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene and
chloride-sensitive sulfate transport. Therefore we conclude that Tat1
is a novel sulfate transporter specifically expressed in spermatocytes
and spermatids and interacts with MgcRacGAP in these cells.
These observations raise the possibility of a new regulatory pathway
linking sulfate transport to Rho signaling in male germ cells.
RhoGTPases, which include Rho, Rac, and Cdc42, are signaling
proteins of the Ras superfamily that have been highly
evolutionarily conserved from yeast to mammals. Members of the
Rho family were initially believed to be primarily involved in the
regulation of actin cytoskeleton and to control shape changes in
response to extracellular growth factors. In recent years, they have
been shown, in fibroblasts and other somatic cell types, to regulate a
myriad of other cellular functions, including motility, adhesion, gene
expression, cell cycle progression, and cell division (1). Although
many potential effectors and regulatory proteins of Rho GTPases have
been described, signaling pathways controlling their pleiotropic
functions are far from fully understood (1-4).
We have recently described a new RhoGAP (GTPase
activating protein for RhoGTPases) gene we
called Mgc(Male germ cell)RacGAP, as it is primarily
expressed in male germ cells, at spermatocyte stage (5).
MgcRacGAP mRNA was also found in lower amount in many
tissues in human and highly expressed in embryonic cerebral cortex in
mouse (6). MgcRacGAP belongs to the chimaerin family of RhoGAPs,
i.e. comprising a RhoGAP domain in the COOH-terminal half,
and NH2-terminal to this domain, a zinc finger-like motif and a large NH2-terminal extension. We have recently
described a mouse MgcRacGAP cDNA encoding an additional
NH2-terminal sequence of 106 residues resulting in a
~70-kDa protein (6); a human MgcRacGAP cDNA showing a
homologous 5' extension has also been reported (7, 8). Physiological
functions of Chimaerins have been analyzed in details by microinjection
of fibroblasts and neuroblastoma cells with various forms of
n-chimaerin, a brain-specific Rac/Cdc42
GAP1 (9). Whereas the GAP
domain alone inhibited Rac-activated lamellipodia formation,
strikingly, full-length n-chimaerin stimulated formation of the actin-based structures lamellipodia and filopodia. These n-chimaerin-stimulated events required the
non-GAP NH2-terminal half of the protein and were also
dependent on activated Rac and Cdc42(10). These data strongly suggest
that n-chimaerin mediates downstream signaling rather
than down-regulation of Rac and Cdc42. Therefore, while the RhoGAP
domain of MgcRacGAP exhibits GAP activity in vitro toward
Rac and Cdc42 (5), we postulated that MgcRacGAP may also fulfil
RhoGTPase effector functions through its non-GAP regions. A close
homolog of MgcRacGAP is the Drosophila protein Rotund(Rn)RacGAP, the product of a spermatocyte-specific gene essential
for male fertility, as its inactivation leads to male sterility in the
fruit fly (11, 12). Altogether, these data indicate that MgcRacGAP may
operate in a Rho-dependent signaling pathway(s) involved in
the development and/or function of male germ cells.
To elucidate pathways implicating MgcRacGAP and approach their
function, we have searched for partners of the MgcRacGAP
NH2-terminal region, expressed in male germ cells. We
report here the cloning and characterization of an MgcRacGAP
interacting protein specifically expressed in spermatocytes and
spermatids and showing structural and functional features of a sulfate transporter.
Constructs--
cDNAs encoding various domains of MgcRacGAP
and Tat1 proteins were subcloned in pGEX plasmids for GST fusion
protein production, in the pLex plasmid pVJL10 (13) for two-hybrid
assays, and in the expression plasmid pRK5 for transient transfection
experiments. Lex-Mgc(N), Lex-Mgc(GAP), and Lex-MgcRacGAP correspond to
LexA fused to aa 1-180, aa 238-513 of MgcRacGAP, and full-length (aa 1-528) MgcRacGAP, respectively (amino acid numbering refers to MgcRacGAP sequence initially described in Ref. 5); GST-Mgc(N), GST-Mgc(GAP), and GST-MgcRacGAP correspond to the same MgcRacGAP regions fused to GST. GST-Tat1(Cter) corresponds to Tat1 (aa 664-970) fused to GST. pRK5-Tat1 and pRK5myctag-RacL61 express full-length Tat1
and myctagged-RacL61, respectively.
cDNA Cloning--
Using Lex-Mgc(N) as a bait, we have
screened a pACT2 human testis cDNA library
(CLONTECH). Two hybrid procedures for isolation of
specific clones that interact with MgcRacGAP were carried out according
to published methods (13, 14). Full-length Tat1 cDNA was obtained
by screening of Sequence Analysis--
Clones of interest were sequenced using
the Sanger dideoxy termination method adapted to an ABI 373A automated
sequencer. Sequence analysis was performed using computer
facilities provided by Infobiogen. BLAST programs were used to
search for homologies in protein data banks, and the Transmembrane
Protein secondary structure prediction program (TMpred) was used to
elaborate a model of Tat1 topology across plasma membrane.
Northern Blot Analysis--
We probed a multiple human tissue
Northern blot (CLONTECH) with a Tat1
cDNA fragment; a In Situ Hybridization Experiments--
A 1129-base pair
fragment of Tat1 cDNA was subcloned in the Bluescript KS
plasmid allowing both SP6 and T7 transcription; 35S-UTP-labeled antisense or sense cRNA probes were
obtained by linearizing the plasmid, respectively, by
HindIII or BamHI and transcribing with T7 or SP6
RNA polymerase. In the same way, the MgcRacGAP cRNA
antisense probe was generated by linearizing a pExLox plasmid
containing a 1837-base pair cDNA fragment with KpnI and
transcribing with SP6 polymerase as described previously (5). 4-µm
paraffin sections of human adult testis were hybridized overnight at
55 °C, washed as described previously (15), and exposed to
Ilford K5 emulsion for 4 or 5 days. After development of the emulsion,
sections were counterstained with toluidine blue and analyzed.
Cell Isolation--
Sertoli cells were isolated from 20 days
post-partum Harlan Sprague-Dawley rats as described previously
(16). On the second day of culture, the cells were exposed to a
hypotonic treatment to eliminate the contaminating germ cells (17). The
degree of purity of the isolated Sertoli cells was greater than 98%.
The isolation of peritubular cells was carried out during Sertoli cell
preparation as described previously (18). The peritubular cells
obtained were at least 99% pure. Spermatogonia were prepared from
testes of 9 days post-partum male Harlan Sprague-Dawley rats. Seminiferous epithelial cells were dispersed by enzyme treatment and
separated by sedimentation at unit gravity as described previously (19), leading to greater than 90% pure spermatogonia. Pachytene spermatocytes and early spermatids were prepared by centrifugal elutriation with a purity greater than 90% according to a method previously described (20), with the exception that enzymatic dissociation of cells was replaced by a mechanical dispersion. Immediately after isolation, all cell fractions were snap-frozen in
liquid nitrogen and stored at Antibodies--
Antibodies to recombinant Tat1 COOH-terminal
region (aa 664-970) were raised in rabbit and affinity-purified on a
Tat1(aa 664-970)/Affi-Gel column.
Immunolocalization of Tat1 Protein--
Testes of adult rats
were carefully dissected out and immersed in Bouin's fixative solution
for 24 h. Samples were then dehydrated through an increasing
gradient of alcohol and acetone, before being embedded in paraffin wax.
Testis sections (5 µm thick) were microwaved (2 × 3 min in
phosphate-buffered saline) after mounting. Sections were then incubated
with purified rabbit anti-Tat1 antibody, with a working dilution of
1:500. Preimmune serum was also used as a negative control. Complexes
were revealed using a goat anti-rabbit biotinylated antibody (Dako,
Glostrup, France) at a working dilution of 1:500, coupled with a
streptavidin-peroxidase amplification combination. Sections were
counterstained with hematoxylin. After color development, the samples
were mounted with Glycergel (Dako, Glostrup, France)
for microscopic observation.
Analysis of Tat1 Protein by Transient Transfection in HeLa and
COS7 Cells--
Transient transfections (24 or 48 h) of
pRK5-Tat1 and pRK5-myctagged-racL61 expression
vectors were performed in COS7 cells, using the fugene-6 reagent (Roche
Molecular Biochemicals). Cells were washed in phosphate-buffered
saline and then lysed in 50 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100. Lysates were loaded on a 10% SDS-acrylamide gel and
electroblotted onto nitrocellulose. Rabbit anti-Tat1-purified
antibodies were used to detect Tat1 expression in transfected cells.
For glycosylation inhibition experiments, transient transfection of
HeLa cells was performed in a medium containing 10% FCS or 10% FCS
supplemented with N- or O-glycosylation
inhibitors: 25 ng/ml tunicamycin or 2 nM BADG
(benzyl-2-acetamido-2-deoxy- In Vitro Transcription/Translation of Tat1--
1 µg of
Bluescript plasmids containing either Tat1 full-length
cDNA or luciferase cDNA were transcribed and
translated in the presence of 20 µCi of [35S]methionine
in a rabbit reticulocyte lysate (Promega).
In Vitro Binding Assay--
GST-Mgc(N), GST-Mgc(GAP), and
GST-MgcRacGAP fusion proteins (1-3 µg) were coupled to 40 µl of
glutathione-Sepharose beads and incubated with 2.5 µg of Tat1
COOH-terminal fragment (aa 664-970) during 1 h 30 min at
4 °C, in 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100. Beads were sedimented and
washed twice with cold washing buffer (25 mM Hepes, 125 mM NaCl, 1% Triton X-100, 0.1% SDS, protease inhibitors),
once with 1 M NaCl, and once more with cold washing buffer.
"Bound proteins" were recovered by boiling beads in Laemmli sample
buffer and were subjected to a 12% SDS-polyacrylamide gel
electrophoresis and electroblotted onto nitrocellulose. Filters were
incubated with rabbit anti-Tat1 antibodies followed by
peroxidase-labeled swine anti-rabbit antibodies and developed using the
ECL detection system (Amersham Pharmacia Biotech).
Transport Studies--
COS7 cells were transiently transfected
as described above with pRK5 empty vector (mock) or pRK5-expressing
Tat1. Transport activity was assayed by measuring the efflux of sulfate
(35SO4) as described (21, 22) or iodide
(125I) as described previously (23). All experiments were
performed at 37 °C in 12-well plates. Cells were loaded with the
respective radiolabeled anions (PerkinElmer Life Sciences,
Boston, MA) for 30 min in a medium containing: 140 mM NaCl,
5 mM KH2PO4, 1 mM CaCl2, 0.8 mM MgCl2, 5.5 mM glucose, and 10 mM HEPES, pH 7.4. For the
sulfate experiment, the medium also contained
[35S]Na2SO4 (40 µCi/ml)
and 0.3 mM Na2SO4. For iodide
efflux determination, cells were loaded with Na125I (1 µCi/ml) in the presence of 1 µM KI. After incubation,
cells were rinsed three times with cold medium. Efflux was determined every 1 min for 11 min during which the medium was removed at 1-min
intervals to be counted and quickly replaced by 1 ml of the same
medium. For the inhibition assay, 1 mM DIDS
(4,4'-diisothiocyano-2,2'-disulfonic acid stilbene from Sigma) was
added to the efflux buffer. For selectivity study, NaCl was substituted
in the efflux buffer by sodium gluconate. At the end of the efflux, the
medium was recovered, and cells were solubilized in 1 ml of 1 N NaOH. All fractions were counted for radioactivity, and
efflux curves were constructed by plotting the percentage of total
radioactivity released in the medium versus time. Results
are expressed as means ± S.E. of n observations. To
compare sets of data, we used either an analysis of variance or
Student's t test. Differences were considered statistically
significant when p < 0.05. All tests were performed using Prism 3.0 (Graphpad).
In a search for partners of MgcRacGAP in male germ cells, we
screened a two-hybrid library of human testis using as a bait a
non-Rho-binding NH2-terminal region (aa 1-180) of human
MgcRacGAP sequence (5). Among others, a 1.3-kilobase cDNA
was isolated; probing human RNA blots with this fragment revealed a
3.4-kilobase mRNA exclusively expressed in testis (Fig.
1). This 1.3-kilobase cDNA fragment
was subsequently used as a probe to clone the full-length cDNA in human testis cDNA phage libraries. Analysis and
sequencing of overlapping clones predicted an open reading frame of
2910 base pairs encoding a protein of 970 amino acids (109 kDa) of which only the COOH-terminal 300-aa fragment was present in the two-hybrid clone. Searching nucleic sequence data banks with this sequence allowed us to retrieve several human expressed sequence tag sequences showing a perfect match with our cDNA.
Interestingly, all expressed sequence tag sequences found in
data banks originate from testis, male germ cell tumors, or infant
brain libraries, consistent with our finding that among adult human
tissues expression of this gene is restricted to testis. Mouse and rat
sequences encoding very similar proteins are also present in data
banks, suggesting that orthologous genes exist in rodents. A human
genomic fragment encompassing the whole cDNA sequence was also
retrieved in the genomic section (High Throughput Genomic
Sequence) of GenBankTM. Blast search in protein
data banks revealed a clear similarity of the predicted protein with a
family of anion (sulfate) permeases comprising three members in human,
i.e. DRA (24, 25), Pendrin (26), and DTD (27), and the
protein was provisionally designated Tat1, for testis anion transporter
1; Tat1 appears most closely related to Pendrin (26% identity; 47%
similarity) and DRA (28% identity; 46% similarity), which exhibit
transport capacity for chloride/iodide and sulfate/chloride,
respectively (26, 28). Multialignment programs (Fig.
2) demonstrate the presence of conserved blocks among the three proteins and Tat1-specific extra sequences in
amino acids 600-652 and COOH-terminal regions; these
additional sequences account for the longer size ot Tat1 (970 aa) as
compared with DRA (764 aa) and Pendrin (780 aa). Sequence analysis also predicted several N-glycosylation sites in Tat1 sequence; in
particular, there are four putative N-glycosylation sites in
the major predicted external loop (amino acids 570-751) (see Fig.
3).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
gt11 human testis cDNA libraries (CLONTECH).
-actin probe was used as a control.
80 °C until used.
-D-galactopyranoside), respectively. After 24 h, cells were lysed as above, and lysates were submitted to Western blot analysis.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Tissue distribution of Tat1
mRNA. Tat1 and
actin cDNAs were used to
probe multiple human tissue Northern blots
(CLONTECH). Each lane contains 2 µg of
poly(A)+ RNA from the indicated tissues.
View larger version (111K):
[in a new window]
Fig. 2.
Tat1 protein sequence comparison.
Alignment of the full-length proteins Tat1, DRA, and Pendrin.
Identities are shaded black, similarities are shaded
gray, and predicted transmembrane domains in Tat1 are indicated by
dark lines.
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[in a new window]
Fig. 3.
Proposed model for Tat1 protein.
Secondary structure prediction was performed using the "TMpred"
program. N-Linked glycosylation sites are indicated by
G. Asterisks demarcate the region of the Tat1
sequence (aa 664-970) encoded by the initial two-hybrid clone.
As expected from previous studies of DRA and Pendrin (26, 29), the hydrophobicity plot of the Tat1 protein exhibited multiple potential transmembrane helices, and membrane topology prediction programs (TMpred) led to a preferred model with 14 strong transmembrane regions and NH2 and COOH termini inside the cytoplasm (Fig. 3).
Since Tat1 gene appears exclusively expressed in
testis on Northern blot (see Fig. 1), we looked for cell-specific
expression in this organ. In situ hybridization of a
Tat1 cRNA probe to sections of human adult testis showed
that Tat1 expression is (i) detected only in a few tubule
sections and (ii) restricted to germ cells, as the grain density over
somatic cells was not detectably above background (Fig.
4B). Moreover, Tat1
transcript was found primarily, if not exclusively, in spermatocytes.
This pattern is similar to that previously observed for
MgcRacGAP except for a low level expression of
MgcRacGAP in round spermatids (5); consistent with this
finding, in situ hybridization of two serial sections with
Tat1 and MgcRacGAP cRNA probes showed
coexpression of the two genes within the same tubules in the same
groups of germinal cells at the spermatocyte stage (Fig. 4). However,
while all Tat1-positive tubule sections were found
MgcRacGAP-positive as well, MgcRacGAP labeling
was clearly detected in several Tat1-negative tubule sections, suggesting a shorter expression period for the
Tat1 gene.
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Using affinity-purified antibodies directed to the COOH-terminal region
of Tat1 protein (aa 664-970), we did not detect Tat1-specific signals
on Western blots of whole human or rat testis extracts; this could
reflect the facts that (i) the amount of Tat1 protein present in
expressing cells may be quite low, as frequently observed for
membranous ion transporters, and (ii) membranous proteins are usually
very difficult to solubilize from whole testis
extracts.2 Western blot
analysis on enriched populations of germ cells isolated from rat testis
revealed multiple bands, ranging from 60 to 160 kDa and mainly
expressed in spermatocytes and spermatids (Fig. 5); preincubation of antibodies with pure
recombinant Tat1-COOH-terminal fragment resulted in the
disappearance of the 60-160 kDa bands, demonstrating that this pattern
corresponds to Tat1-related proteins (not shown). The complexity of the
pattern of Tat1-related polypeptides probably results from
glycosylation and proteolysis as discussed below. Immunostaining of rat
testis sections using the same antibody also revealed specific
stage-dependent labeling in seminiferous tubules (Fig.
6A). In the tubules, a strong
labeling was observed in germ cells from spermatocytes to elongated
spermatids (Fig. 6B). Immunoreactivity is clearly localized
at the cell periphery of young spermatocytes, pachytene spermatocytes,
and early and elongating spermatids, strongly suggesting a membranous
expression of Tat1 (Fig. 6, A and B). A more
diffuse cytoplasmic immunolabeling is still present in elongated
spermatids at the very last steps of spermiogenesis (Fig.
6B). Altogether, data from Northern blot, in situ
hybridization, and immunodetection experiments clearly demonstrate that
the Tat1 gene and Tat1 protein are exclusively expressed in
spermatocytes and spermatids.
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To further characterize Tat1 protein, we expressed Tat1
cDNA in cultured cells. Transfection of Tat1 constructs
in COS7 (or HeLa cells) resulted in a high level of protein expression
as evidenced by the strong specific immunofluorescence labeling of transfected cells (not shown). Immunoblots of lysates from COS7 cells
overexpressing Tat1 protein show a heavily labeled diffuse band with a
high molecular mass (>200 kDa) and a series of minor bands ranging from 110 to 50 kDa molecular mass (Fig.
7A). These bands are
completely absent in a lysate from COS7 cells transfected with an
irrelevant expression plasmid (pRK5-racL61) and thus must correspond to specific Tat1 expression. This pattern is
reminiscent of the one reported for the intestine-specific anion
transporter DRA (29, 30) and suggests that the major slow migrating
form of Tat1 could result from glycosylation of the protein, as usually found in integral membrane proteins. The 110-kDa band fits with the
expected size of the protein as deduced from open reading frame
translation (109 kDa) and might represent a minor fraction of non
post-translationnally modified protein. The exact nature of the
additional multiple sized proteins detected in COS7 lysates is not
known; as mentioned previously for DRA, this complexity could result
from a combination of NH2-terminal truncation and glycosylation (30). In vitro transcription/translation
experiments using Tat1 cDNA as a template also resulted
in a complex pattern of protein synthesized (Fig. 7B); this includes
high molecular mass possibly glycosylated forms, 110-kDa and
shorter bands, which could correspond to the use of multiple initiation
sites.
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Treatment of Tat1-expressing HeLa cells with tunicamycin, an inhibitor of N-glycosylation, resulted in the complete disappearance of the diffuse high molecular mass species (Fig. 7C); by contrast, O-glycosylation inhibitor BADG had no effects. This result clearly demonstrates that the bulk of Tat1 protein present in those cells is N-glycosylated. Altogether our data strongly suggest that within the different systems analyzed (HeLa human cells, COS7 simian cells, rat germ cells, and rabbit reticulocyte lysate) Tat1 protein is synthesized as a high molecular mass species corresponding to various degrees of glycosylation; this probably reflects species and/or tissue specificity of glycosyltransferase (31) and glycosidase (32) involved in N-glycan biosynthesis.
Tat1/MgcRacGAP interaction was analyzed in two-hybrid experiments and
in vitro binding assays using recombinant proteins and anti-Tat1C-ter antibodies. In the yeast two-hybrid assay, Tat1 COOH-terminal region strongly interacts with the Mgc-N region (aa
1-180) but not with the GAP domain (Fig.
8A). In vitro,
GST-Mgc-N fusion protein actually binds to the carboxyl-terminal domain of Tat1. The Tat1 COOH-terminal region also interacts with the full-length MgcRacGAP protein, but not with the GAP domain (Fig. 8B, lanes 2-4). Taken together, these results
demonstrate that MgcRacGAP interacts through its
NH2-terminal region with the cytoplasmic COOH-terminal
domain of Tat1.
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In a first approach to Tat1 function, COS7 cells expressing Tat1 were
assayed for anion transport. As shown in Fig.
9A, sulfate efflux was
markedly enhanced in Tat1-expressing cells as compared with mock
transfected cells. When extracellular chloride was replaced by
gluconate, the Tat1-mediated sulfate transport was markedly reduced
showing a dependence upon extracellular Cl (Fig.
9B). In addition, Tat1-mediated sulfate efflux was inhibited by the anion exchanger inhibitor DIDS as illustrated in Fig.
9C. By contrast, the iodide transport (Fig. 9D)
was not modified by Tat1 expression. Together, these data support the
idea that Tat1 is a bona fide anion transporter displaying
sulfate transport at least partly through a sulfate/chloride exchange
mechanism (21, 22).
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As mentioned earlier (see Figs. 2 and 3), the Tat1 COOH-terminal region has no counterpart in DRA, Pendrin, and DTD and is predicted to be located in cytoplasm; this suggests that MgcRacGAP binding to the Tat1 COOH-terminal region could provide a specific regulatory mechanism of Tat1 transport function and that Rho GTPases might participate in the regulation of sulfate transport in male germ cells.
Although we have no clue as to the role of sulfate transport in male germ cells, two lines of evidence support the hypothesis that the MgcRacGAP/Tat1 pathway could fulfil important functions. 1) Recently, the orthologous gene of MgcRacGAP in Caenorhabditis elegans, CYK-4, and human MgcRacGAP have been shown to play an essential role in the process of cell division (8, 33). More specifically, those proteins have been shown to associate with mitotic spindle and to be required for cytokinesis. Interestingly, throughout spermatogenesis, both mitotic and meiotic cell divisions are characterized by incomplete cytokinesis resulting in the persistence of intercytoplasmic bridges (34). While incomplete cytokinesis during mitotic divisions may be related to the absence of MgcRacGAP in spermatogonia (5),2 we speculate that, in spermatocytes, where MgcRacGAP and Tat1 are coexpressed, Tat1/MgcRacGAP interaction may interfere with MgcRacGAP function and eventually preclude complete cytokinesis during meiotic divisions.
2)Tat1 belongs to a family of anion transporters comprising three human
proteins, i.e. DRA, DTD, and Pendrin. Strikingly, mutations
in all three corresponding genes Dra, DTD, and
PDS are responsible for human diseases, i.e.
congenital chloride diarrhea (28), diastrophic dysplasia (27, 35, 36),
and goiter associated with congenital deafness known as Pendred
syndrome (26, 37), respectively. This implies that each of these genes
plays a specific and essential role in affected tissues. In agreement
with this view, Dra and PDS show very specific
expression in intestine epithelium and thyroid, respectively. As
Tat1 gene expression appears restricted to the male germ
line at the spermatocyte stage, we speculate that Tat1 may fulfil
critical functions in these cells and that mutations in the
Tat1 gene may result in impaired spermatogenesis in human.
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ACKNOWLEDGEMENTS |
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We are grateful to F. Letourneur, N. Lebrun, and C. Jougla for excellent technical assistance. We thank Drs. P. Camparo and M. Arborio for providing human testis sections and P. Chaffey for advice in protein analysis.
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FOOTNOTES |
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* This work was supported by INSERM and by grants from the Ligue Nationale Contre le Cancer and the Association pour la Recherche sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of fellowships from the Ministère de l'Éducation Nationale, de la Recherche et de la Technologie and from the Fondation pour la Recherche Médicale.
** To whom correspondence should be addressed: Institut Cochin de Génétique Moléculaire, Dépt. de Génétique, Développement et Pathologie Moléculaire, INSERM Unité 257, 24 Rue du Faubourg Saint-Jacques, 75014 Paris, France. Tel.: 33-1-44-41-24-70; Fax: 33-1-44-41-24-62; E-mail: gacon@cochin.inserm.fr.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M011740200
2 A. Touré, L. Morin, C. Pineau, and G. Gacon, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
GAP, GTPase
activating protein;
GST, glutathione S-transferase;
aa, amino acid(s);
FCS, fetal calf serum;
BADG, benzyl-2-acetamido-2-deoxy--D-galactopyranoside;
DIDS, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene.
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