(Received for publication, June 25, 1996, and in revised form, October 28, 1996)
From the The The plasma membranes of polarized epithelial cells are divided
into apical and basolateral domains that are characterized by distinct
lipid and protein compositions. The generation and maintenance of the
polarized distribution of surface proteins require sorting of newly
synthesized membrane proteins to their appropriate sites of residence.
Thus, although the molecular mechanisms are still poorly understood,
membrane proteins located apically and basolaterally must contain
sorting determinants recognized by the cellular sorting machinery.
So far, the search for apical and basolateral sorting determinants has
been largely focused on monotopic proteins (for review, see Ref. 2).
Two classes of basolateral sorting determinants have been identified:
one related to coated pit localization, and in most cases relying on
the same tyrosine residue required for endocytosis (3-7); the other
unrelated to endocytosis signals (5, 8, 9). Despite their
heterogeneity, all of the identified basolateral sorting determinants
have been found to be located in the cytosolic domains of basolaterally
expressed plasma membrane proteins.
Whereas the identification of two classes of basolateral sorting
signals formally demonstrates that the basolateral pathway is
signal-mediated, the lack of identified apical sorting signals in
transmembrane proteins leaves open the question of whether the apical
pathway occurs by default or by a signal-mediated mechanism. In favor
of a signal-mediated pathway is the observation that glycophosphatidylinositol-anchored proteins are predominantly localized
on the apical surface of MDCK1 cells (10,
11) and that their glycophosphatidylinositol anchors function as apical
sorting signals (12, 13). In addition, N-glycosylation has
been shown to be involved in the apical targeting of a secretory
protein and has been proposed to function as apical sorting signal of
transmembrane proteins as well (14). On the other hand, the removal of
a basolateral sorting signal from a basolaterally targeted protein can
lead to its preferential appearance on the opposite apical domain (5,
7, 8, 15), suggesting that apical sorting could occur by default or
that a hierarchy of signals may occur, with the basolateral signal in
the cytoplasmic domain operating as dominant over the weaker apical
signal located in the lumenal domain (2).
Sorting signals responsible for apical and basolateral delivery of
polytopic proteins have only recently begun to be investigated (16,
17). We wondered if monotopic and polytopic proteins exploit similar
signals and mechanisms to reach their cellular destination. In
particular, we were interested in clarifying whether basolateral
sorting signals of polytopic proteins are located in their cytoplasmic
tails and whether they might cause the basolateral relocation of apical
proteins.
To study sorting determinants of polytopic proteins, we have used as
models two related transporter proteins: the GAT-1 isoform of the
Plasmid Constructions
The original BGT and GAT-1 clones were kindly provided by J. S.
Handler (Johns Hopkins University) and B. Kanner (Hebrew University) and were subcloned in the mammalian expression vector pCB6 (1). Wild
type hNGFR cDNA (23) subcloned into
EcoRI-BamHI restriction sites of pCB6 was kindly
provided by A. Le Bivic (D'Aix-Marseille II University, CNRS UMR
9943). To generate mutants and chimeras, PCR amplifications were
performed, and the PCR products were cloned into pCB6. The absence of
unwanted substitutions in the mutant clones, due to the amplification
processes, was checked by sequencing. The sequences of all primers are
available on request. A representation of the mutants and chimeras is
given in Fig. 1B.
The last COOH-terminal 36 amino
acids of GAT-1 were removed. This mutant was generated by PCR
amplification using GAT-1 cDNA as template. A T was inserted to
change the codon Lys in position 564 of the wild type GAT-1 to a TAA
stop codon. The SmaI-XbaI fragment of pCB6-GAT-1
was replaced with the similarly digested PCR product.
The sequence of the c-Myc epitope tag EQKLISEEDL
was inserted just after the ATG codon corresponding to the first
methionine in BGT cDNA by PCR amplification. The PCR product was
digested with MluI-EcoRI and ligated into
similarly digested pCB6-BGT.
The last COOH-terminal 49 amino acids were removed from the cDNA encoding myc BGT. A fragment
containing an in-frame stop codon was originated by insertion of a T in
position 1814 of BGT in a PCR using BGT as template. The PCR product
was digested with SmaI-ClaI and ligated into
similarly digested pCB6-myc BGT.
The last COOH-terminal 56 amino acids of BGT
were replaced with the analogous 43 amino acids of GAT-1 by two rounds
of sequential PCR amplification. Two separate reactions were carried
out. In the first round, reaction 1, BGT cDNA was used as template
with primers Ia (corresponding to nucleotides 1352-1378 of the BGT sequence) and Ib (containing 21 nucleotides complementary to
oligonucleotide Ic, followed by 21 nucleotides complementary to
nucleotides 1792-1772 of BGT). In reaction 2, GAT-1 was used as
template with primers Ic (corresponding to nucleotides 1860-1880 of
GAT-1) and Id (complementary to bases 1000-983 of the pCB6 cloning
vector, containing the XbaI restriction site). In the second
round of amplification the products of the first round of PCR were used
as template and oligonucleotides Ia and Id as primers. The fusion
product was generated by virtue of the 21-nucleotide overlap between
the fragments generated in the first round. The
AccIII-XbaI digest of this PCR product was ligated into similarly digested pCB6-BGT.
The last COOH-terminal 43 amino acids of GAT-1
were replaced with the analogous 56 amino acids of BGT. A fragment
containing the cDNA coding for the COOH-terminal 56 amino acids of
BGT was generated using BGT as template and as upper primer an
oligonucleotide corresponding to nucleotides 1836-1859 of the GAT-1
sequence fused to 1793-1822 of BGT; a T was changed from a C in
position 1801 of the BGT sequence to eliminate the SmaI
site. The lower primer was complementary to the 3 The COOH-terminal 51 amino acids of BGT
were fused to amino acid 278 of p75 hNGFR cDNA to generate a
recombinant receptor with the cytoplasmic tail replaced with that of
BGT. A PCR was carried out using BGT as template. The sequences of the
upper primer contained a PvuII site in its 5 A deletion of amino acids 565-572 (see
Fig. 7A) in the COOH terminus of BGT was introduced by PCR
amplification using BGT as template and an oligonucleotide
corresponding to bases 1799-1813 fused to 1835-1843 of BGT as upper
primer. The lower primer was as for the GBS chimera. The
SmaI-ClaI fragment of pCB6-BGT was replaced with
similarly digested PCR product.
The last COOH-terminal 43 amino acids of
GAT-1 were replaced with the analogous 48 amino acids of BGT Cell Culture and Transfection
MDCK (strain II) cells were grown and transfected with the
calcium phosphate method as described previously (1). Transfected cell
lines were selected by growth in the antibiotic G418 (0.9 mg/ml) (Life
Technologies, Inc.). Expression of recombinant proteins was assayed
initially by GABA uptake and/or by immunofluorescence. At least three
independent clones were analyzed for each recombinant cell line, and
similar polarity ratios were observed. For all polarity studies
transfected and untransfected MDCK cells were grown to confluence for
more than 5 days on 0.4-µm pore size Transwell filter inserts
(Costar).
Antibodies and Immunocytochemistry
To localize wild type and chimeric transporters in both
immunofluorescence and immunoprecipitation experiments the following antibodies were used. GAT-1 and the BGS chimera were localized with
antibody R24, kindly provided by R. Jahn (Yale University). The
antibody was generated against a synthetic peptide corresponding to
amino acids 571-586 in the COOH-terminal domain of GAT-1 (see Fig.
1A). Coupling and immunization were performed as described (1). To localize MDCK cells were fixed in ice-cold methanol and processed for
immunofluorescence as described previously (29). Wild type and chimeric
hNGFR-transfected cells were fixed with freshly made 3%
paraformaldehyde in 125 mM sodium phosphate, pH 7.4, for 15 min, and antibody staining was performed with the same protocol but
without detergent in the reaction buffer. Fluorescein
isothiocyanate-conjugated anti-mouse/rabbit IgG from Jackson
Immunoresearch (West Grove, PA), biotin anti-rabbit/mouse IgG, and
Texas red-conjugated streptavidin (Sigma) were used as secondary
reagents. Confocal images were obtained using a Bio-Rad MRC-1024
confocal microscope. Micrographs were taken using either a Focus
Imagecorder Plus (Focus Graphics Inc.) on Kodak film or a Professional
Color Point 2 dye sublimation printer (Seiko).
Steady-state Cell Surface Biotinylation
Confluent cells grown on 24-mm Transwell filters were starved
for 30 min in Dulbecco's modified Eagle's medium without cysteine and
methionine and metabolically labeled overnight with 0.1 mCi/ml Tran35S-label (ICN Pharmaceuticals, Costa Mesa, CA) (30).
Biotinylation with NHS-ss-biotin (Pierce Chemical Co.) on the apical or
basolateral side was performed according to Sargiacomo et
al. (31). Following biotinylation cells were lysed, and the
protein of interest was incubated with the primary antibodies described
in the preceding section. Following the primary incubation,
anti-rabbit/mouse IgG-conjugated agarose (Sigma) secondary antibodies
were added. Transporters were released from the beads by boiling in 10 µl of 10% SDS, and surface-biotinylated transporters were
reprecipitated with streptavidin beads (Pierce). The beads were then
heated in SDS solubilization buffer, and the released reduced proteins
were alkylated and analyzed on 10% SDS-polyacrylamide gel
electrophoresis (32). Quantitation of the biotinylated proteins was
performed by scanning the developed fluorograph with a LKB Ultroscan XL
laser densitometer.
GABA Influx Assay
GABA influx was performed according to Yamauchi et
al. (22) with modifications (1). Cells were washed twice with
incubation buffer (150 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2,
10 mM HEPES, pH 7.5) and incubated in the same solution
containing [3H]GABA (DuPont NEN). Briefly, for screening
for stable transfectants, cells grown on 24-well plates were incubated
in 0.2 ml of incubation buffer containing 0.5 µCi of
[3H]GABA for 10 or 30 min to measure GAT-1 or BGT
activity, respectively. To study the polarity of expressed transporters
the cells were grown to confluent density for 7 days on 6.5-mm
Transwell filters. [3H]GABA in incubation buffer was
applied either on the apical (100 µl) or basolateral (250 µl) side
at a final concentration of 10 µM for 10 min (for wild
type GAT-1 and related mutants) or 100 µM for 30 min (for
wild type BGT and related mutants). Uptake was terminated by aspirating
the medium, and the cells were washed three times with ice-cold
incubation buffer. After cell solubilization in 0.2 ml of 1% SDS (when
filters were used they were removed from the supports before cell
solubilization) the samples were counted in 5 ml of scintillation
solution (Ultima Gold, Packard).
Previous results obtained in our laboratory indicate that the
neuronal GAT-1 and the epithelial BGT, despite their high structural (Fig. 1A) and functional similarity, bear opposite apical
and basolateral sorting signals. Indeed, stably transfected MDCK cells localize GAT-1 and BGT apically and basolaterally, as shown previously (1) and in Figs. 2 and 3,
a-f, of this paper. Therefore, we first tested for the
presence of apical and basolateral sorting signals in the COOH-terminal
domain of GAT-1 and BGT, which is a domain with a low degree of
similarity between the two proteins. For this purpose we generated, by
recombinant DNA technology, truncated and chimeric transporters
(schematically represented in Fig. 1 and described under
"Experimental Procedures"), and their sorting behavior was analyzed
in MDCK cells.
The
distribution of the wild type and truncated GAT-1 (
The cellular distribution of wild
type and tail-minus BGT was followed by indirect immunofluorescence
microscopy. To allow the localization of the truncated BGT a c-Myc
epitope was inserted at the amino-terminal domain of the transporter.
In Fig. 3, a-f, epitope-tagged BGT-transfected MDCK cells
were double stained with BGT-KLH (in red) and with mAb 6H
(in green) antibodies. Colocalization of the tagged BGT with
the basolateral marker is revealed by comparison of the confocal
analysis. The yellow/orange color in the merge of vertical and
horizontal sections (e and f) furthermore
demonstrate colocalization of the two antigens. A similar distribution
of the tagged BGT was observed using the antibody against the Myc epitope. No specific staining with the BGT antibody was observed in
untransfected MDCK cells (as shown in b, where nonexpressing cells devoid of staining are observed). This was not surprising in
light of a low endogenous expression of BGT in MDCK cells grown in
isotonic medium, as demonstrated previously (1, 22).
The unchanged basolateral localization of the tagged BGT allowed us to
analyze the sorting behavior of the deleted epitope-tagged BGT
( Consensus sequences for ER retrieval have been identified in the
extremity of cytoplasmic carboxyl termini of ER resident membrane
proteins (34). To exclude that an internal potential ER retrieval motif
was unmasked by the removal of the COOH-terminal 49 amino acids in the
epitope-tagged The ER accumulation of
truncated BGT prevented the determination of a possible involvement of
the cytosolic tail in the specific basolateral localization of the
transporter. Therefore, we generated a GABA transporter chimera
containing the BGT tail (GBS, see Fig. 1). The cellular distribution of
GBS was detected by indirect double immunofluorescence. Confocal images
generated at the horizontal focal plane of the apical surface of
GBS-transfected MDCK cells revealed apical localization of the chimera
(compare Fig. 4, a and b).
However, in contrast to the situation with the wild type GAT-1, the
chimera was also found on the basolateral surface as displayed by the
horizontal section obtained at the basal focal plane, as well as by the
vertical section (compare c and d with e and f).
The apical to basolateral polarity ratio of wild type and mutant
transporters was determined by cell surface biotinylation experiments
and influx studies (Fig. 5).
The steady-state biotinylation experiment (Fig. 5A)
demonstrated that whereas GAT-1 and To perform functional studies, we first measured the kinetic parameters
in the MDCK cell lines expressing wild type and mutated transporters.
Similar apparent GABA affinity constants of about 30 µM
were measured (data not shown), suggesting that GAT mutants retain the
original GAT-1 GABA affinity. This result permitted measurements of
functional activities in the apical or basolateral surface of wild type
and mutants under the same experimental conditions (Fig. 5C
and "Experimental Procedures"). GAT-1 and To investigate whether the identified
basolateral signal of BGT is functional when transferred to a monotopic
protein, we replaced the last COOH-terminal 150 amino acid residues of
p75 hNGFR with the 51 amino acids of the BGT tail (hNGFR-BGT chimera). P75 hNGFR is a type I transmembrane glycoprotein that has been shown
elsewhere (4) to localize apically when transfected in MDCK cells. It
has also been shown that the apical distribution persists after
deletion of the cytoplasmic domain (XI mutant) and that an internal
deletion within the cytosolic domain (PS mutant), which creates a
basolateral sorting signal, is able to redirect the receptor
basolaterally. Because of this sorting behavior, hNGFR appears to be
suitable as polarity reporter protein. Wild type and hNGFR-BGT chimera
were transfected into MDCK cells and their distribution detected by
immunofluorescence and cell surface immunoprecipitation of biotinylated
receptors using mAb ME 20.4. The apical distribution of hNGFR revealed
in vertical and horizontal sections, obtained by confocal analysis, is
shown in Fig. 6, a and b,
respectively. The BGT tail relocated basolaterally the apical hNGFR
(Fig. 6, c and d). These experiments were carried out in nonpermeabilized cells to avoid the interference in surface receptor detection due to the high intracellular expression of both
wild type and chimeric receptor. Cell surface biotinylation experiments
carried out in MDCK-transfected cells confirmed the predominant
basolateral localization of the hNGFR-BGT chimera (Fig.
6B).
A comparison of the amino acid
sequences of the cytosolic tails of BGT (dog and human) and GAT-1
reveals a region, proximal to the plasma membrane, which is rich in
positively charged amino acids and conserved in the BGT sequence of dog
and man but not in the GAT-1 sequence (Fig.
7A). Since a similar sequence is a basolateral sequence for the poly IgR (8) we deleted this region in
both the wild type BGT and GBS chimera (BGT and GBS The presence of three distinct domains has made monotopic proteins
useful models with which to identify the sequences responsible for apical and basolateral sorting in polarized cells. Indeed, studies
with truncated and chimeric monotopic proteins have led to the
conclusion that basolateral sorting signals are contained in the
cytosolic domain of basolaterally located proteins whereas apical
sorting signals are probably contained in the lumenal/transmembrane domains of apical proteins (2).
To perform the characteristic transepithelial transport of solutes and
water, polarized epithelia must sort distinct classes of transporter
proteins selectively to their apical or basolateral surfaces. Transport
proteins are polytopic proteins consisting of several cytosolic and
lumenal/transmembrane domains. Their sorting has thus proven difficult
and complex to analyze. In spite of the complexity of the system it is
of particular interest to understand if polytopic proteins follow the
same rules established for monotopic proteins.
So far, the sorting behavior of polytopic proteins has been
investigated principally using as models two proteins belonging to the
E1-E2 class of ion-transporting ATPases, the BGT and GAT-1 are homologous polytopic proteins that exhibit opposite
sorting behavior in both neuronal and epithelial cells (1, 38). We have
used these two proteins as models to compare the sorting mechanisms of
mono- and polytopic proteins. In particular, in this study we have
investigated the role of the cytosolic COOH-terminal domains of GAT-1
and BGT.
Our
data on the truncated GABA transporter ( Because of the structural similarity of BGT with the GAT-1, the ER
localization of We have identified a basolateral sorting signal
contained in the cytosolic tail of BGT. This signal is functional when
transferred on otherwise apically located proteins and presents
similarities with those identified in monotopic proteins, since it is
located in a cytosolic domain. Our data with the hNGFR-BGT and GBS
chimeras demonstrate that this signal can redirect both a monotopic
(the hNGFR) and a polytopic (the GAT-1) protein, normally located on the apical surface, to the basolateral surface of MDCK cells. We have
mapped to an 8-amino acid region the information necessary to localize
basolaterally the GBS chimera. In fact, a deleted GBS chimera that
lacks 8 residues but retains the rest of the BGT tail (GBS Although the basolateral sorting signal contained in the BGT tail which
we identify completely reverses the apical localization of the
monotopic hNGFR (hNGFR-BGT chimera) it only partially relocates the
polytopic GAT-1 (GBS chimera). Since the signal contained in the BGT
tail is dominant on the apical sorting signal of the hNGFR we exclude
the presence in the BGT tail of a weak basolateral signal while we
favor the explanation of both apical and basolateral sorting signals of
comparable strength in the GBS chimera. This explanation implies that
apical sorting signals of this polytopic protein might not be recessive
to basolateral sorting signals, in contrast to the results obtained
with monotopic proteins (2). In a recent report Turner et
al. (17) showed that the basolateral sorting sequence from a
monotopic protein can direct basolaterally a polytopic apical protein
in Caco-2 cells. The authors have epitope tagged the
sodium-dependent glucose cotransporter (SGLT1) with the
sequence containing the residues most relevant to determine basolateral
sorting of the vesicular stomatitis virus G protein. Similarly to our
GBS chimera results, the tagged SGLT1 localized contemporaneously in
both the apical and basolateral surfaces. Thus, our results and those
on the SGLT1 taken together indicate that basolateral sorting signals
can be exchangeable between mono- and polytopic proteins, but they
might behave as dominant only when introduced in monotopic
proteins.
In conclusion, our results suggest that similar mechanisms may underlie
the sorting of polytopic and monotopic proteins in polarized epithelial
cells. There are also, however, important differences, such as in the
apparent strength of apical sorting signals. The future study of the
sorting mechanisms of this type of protein may thus yield novel
information on the genesis of epithelial polarity.
We thank Drs. B. Kanner, J. Handler, A. Le Bivic, R. Jahn, and D. Louvard for providing reagents. We also thank
Dr. F. Clementi and the members of the Borgese/Fornasari laboratories
for helpful suggestions and reading of the manuscript. Special thanks
go to Drs. F. Clementi and N. Borgese for support and encouragement.
Consiglio Nazionale delle Ricerche Cellular
and Molecular Pharmacology Center,
Department of Cellular and Molecular
Physiology, Yale University School of Medicine,
New Haven, Connecticut 06510
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-aminobutyric acid transporter (GAT-1)
isoform of the
-aminobutyric acid and the betaine (BGT) transporters
exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G., and Caplan, M. J. (1994) J. Biol.
Chem. 269, 4668-4674). We have investigated the presence of
sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric
transporters. Whereas truncated GAT-1 (
C-GAT) maintained the
original functional activity and apical localization, either the
removal (
C-myc BGT) or the substitution (BGS chimera) of the
cytosolic tail of BGT generated proteins that accumulated in the
endoplasmic reticulum. Moreover, we have found that the cytosolic tail
of BGT redirected apical proteins, the polytopic GAT-1 (GBS chimera)
and the monotopic human nerve growth factor receptor, to the
basolateral surface. These results suggest the presence of basolateral
sorting information in the cytosolic tail of BGT. We have further shown
that information necessary for the exit of BGT from the endoplasmic
reticulum and for the basolateral localization of the GBS chimera is
contained in a short segment, rich in basic residues, within the
cytosolic tail of BGT.
-aminobutyric acid (GABA) transporter (18) and the dog betaine
transporter (BGT) (19). These proteins contain 12 putative hydrophobic
transmembrane
-helices, with both amino and carboxyl termini
predicted to face the cytoplasm (see Fig.
1A). GAT-1 and BGT are members of the same
sodium- and chloride-dependent neurotransmitter transporter
gene family, share high structural (60% of identity at the amino acid
level, see Fig. 1A) and functional (they both transport
GABA) similarities, and both exert their specific function in polarized
cells (20). Whereas GAT-1 is expressed predominantly on axonal
processes of GABA-ergic neurons (1, 21) where it mediates GABA
reuptake, BGT is accumulated on the basolateral domain of renal
epithelial cells exposed to hyperosmotic conditions (22). We have shown
previously that GAT-1 and BGT localize to opposite plasma membrane
domains when transfected in MDCK cells (1). Thus, these two homologous
proteins provide a good system for the investigation of apical and
basolateral sorting determinants in polytopic proteins. Our results
show that, as is true for monotopic basolateral proteins, BGT contains
a basolateral sorting determinant in a cytosolic domain. In addition,
they suggest the presence of strong apical determinants in GAT-1.
Fig. 1.
Structure of GAT-1 and BGT and representation
of mutants and chimeras used in this study with their localization in
transfected MDCK cells. Panel A, predicted transmembrane
topology of betaine and GABA transporters. Filled circles
represent amino acids that are identical in BGT and GAT-1. Positions of
synthetic peptides used to raise the indicated antibodies are shown.
Panel B, wild type and truncated transporters, as well as
the chimeras constructed in this study are shown schematically, and
their localization in transfected MDCK cells is indicated. The
contributions of BGT (filled bars), GAT-1 (hatched
bars), and hNGFR (dotted bars) cDNAs are indicated
in each chimera. The location of the Myc tag is indicated by the
white bar at the NH2 terminus of BGT. The Myc sequence in myc BGT and C-myc BGT was inserted immediately after the
AUG start codon. The last 49 amino acids at the COOH terminus of BGT
were deleted in the
C-myc BGT construct. The COOH termini of GAT-1
and BGT were exchanged from the first amino acid after transmembrane 12 in the BGS and GBS chimeras.
C-GAT was constructed by removal of the
last 36 amino acids of GAT-1. The cytosolic tail of hNGFR was replaced
by 51 amino acids of the cytosolic tail of BGT. The vertical
dashed double line indicates the 12th transmembrane domain of the
transporters and the unique transmembrane domain of hNGFR. * The apical
localizations of hNGFR and
C-hNGFR have been established in previous
studies by Le Bivic et al. (4).
[View Larger Version of this Image (32K GIF file)]
C-GAT (Tail-minus GAT-1)
C-myc BGT (Tail-minus BGT)
-noncoding region of
BGT and contained a ClaI restriction site. The PCR fragment
was digested with SmaI and ClaI restriction
enzymes and ligated to similarly digested pCB6-GAT-1.
extremity. The
PCR fragment was digested with PvuII and ClaI and
ligated into similarly digested pCB6-hNGFR.
565-572
Fig. 7.
Distribution of BGT 565-572 and GBS
565-572 expressed in MDCK cells. Panel A, comparison
between the cytosolic tails of canine and human BGT and of rat GAT-1.
The region containing amino acids 565-572 of BGT, deleted from BGT and
GBS cDNAs, is indicated. Panel B, immunofluorescence
analysis of MDCK transfected with BGT
565-572 (a) or
GBS
565-572 (b and c), stained with antibodies BGT-KLH (in red) and mAb 6H against the sodium
pump (in green). Merging of horizontal (a and
c) or vertical (b) confocal sections is shown. In
a the enlarged shape of the cells is due to the overnight
treatment with sodium butyrate. Bar, 15 µm. Panel C, quantification of apical (empty columns) and
basolateral (filled columns) transport activity assayed by
[3H]GABA uptake. Values are presented as the percent of
total cell surface transporter activity and represent means ± S.E. of five independent GBS
565-572 expressing cell lines
performed in duplicate. The comparison of the apical to basolateral
ratio between GBS
565-572, wild type GAT-1, and GBS constructs
together with the immunofluorescence experiments indicates that a
basolateral signal is contained in the 8-amino acid deleted region
within the cytosolic tail of BGT. Moreover, this region contains
information necessary for the ER exit of BGT.
[View Larger Version of this Image (56K GIF file)]
565-572
565-572 by PCR amplification using as upper primer an oligonucleotide
corresponding to nucleotides 1836-1859 of the GAT-1 sequence fused to
1793-1849 of the BGT sequence in which nucleotides 1810-1835 were
removed and GBS as template. The lower primer was as for the GBS
chimera. The SmaI-ClaI fragment of pCB6-GBS was
replaced with the similarly digested PCR product.
C-GAT we used a rabbit polyclonal serum (GAT-KLH) (keyhole limpet hemocyanin) produced against a synthetic peptide comprising amino acids 189-205 of GAT-1 (in the second extracellular loop, see Fig. 1A). BGT and GBS chimera localization was
revealed with an antibody (BGT-KLH) raised against the synthetic
peptide comprising amino acids 563-591 in the COOH terminus of BGT
(see Fig. 1A). A cysteine followed by a glycine were added
at the amino termini of the GAT-KLH and BGT-KLH synthetic peptides to
facilitate coupling to KLH. Haptenization to KLH and injection were as
described (24, 25). C-Myc epitope-tagged wild type and truncated BGT were localized with clone 9 E10 monoclonal antibody (Oncogene Science).
The Na,K-ATPase
1 subunit was localized using monoclonal antibody
6H. Production and characterization of the 6H antibody are described
elsewhere (26). The wild type hNGFR and the chimera hNGFR-BGT were
localized using ME 20.4 (kindly provided by A. Le Bivic; 27). An
anti-rat ER antibody (28), a gift of D. Louvard, was used in double
labeling experiments.
Fig. 2.
Immunofluorescence analysis of the surface
distribution of GAT-1 and C-GAT in transfected MDCK cells.
GAT-1 (panels a-f) and
C-GAT (panels g-l)
transfected MDCK cells grown to confluence on Transwell filters were
fixed with ice-cold methanol and double stained with anti-GAT-1 R24
(panels a and b) or GAT-KLH (panels g
and h) antibodies (in red) and with mAb 6H
against the sodium pump (panels c, d,
i, and j) (in green). Confocal
immunofluorescence micrographs of vertical (panels a,
c, e, g, i, and
k) or horizontal (panels b, d,
f, h, j, and l) focal
planes are shown. Note that horizontal sections were taken at a focal
plane which include portions of both the apical and basolateral
surfaces. A merge of the two patterns (panels e,
f, k, and l) clearly shows a lack of
colocalization of GAT-1 and
C-GAT with the basolateral marker sodium
pump. Bar, 15 µm.
[View Larger Version of this Image (99K GIF file)]
Fig. 3.
Immunofluorescence analysis of the cellular
distribution of wild type, truncated, and chimeric BGT in transfected
MDCK cells. Confluent MDCK cells transfected with Myc-tagged BGT (panels a-f), Myc-tagged C-BGT (panels g-i),
or BGS chimera (panels j-l) were stained with the polyclonal
BGT-KLH raised against BGT (panels a and b),
anti-Myc epitope mAb 9E10 (panel g), or polyclonal R24
against the cytosolic tail of GAT-1 (panel j) antibodies (in red), and double stained with anti-sodium pump mAb 6H
(panels c, d, and k) or polyclonal
anti-ER (panel h) antibodies (in green). Confocal
immunofluorescence micrographs of vertical (panels a and
c) or horizontal (panels b, d,
g, h, j, and k) focal
planes and merging of the two staining patterns are shown (panels
e, f, i, l).
Yellow/orange colors indicate colocalization. The addition of the c-Myc epitope in the NH2 terminus of BGT does not
interfere with the basolateral localization of the transporter, whereas removal or substitution of the cytosolic tail of BGT generates proteins
unable to reach the cell surface. Bar, 15 µm.
[View Larger Version of this Image (131K GIF file)]
C-GAT) stably
expressed in MDCK cells was investigated by double immunostaining with
the antipeptide antibodies raised against GAT-1 (Fig. 2, in
red) and the basolateral marker
1 subunit of the sodium
pump (Fig. 2, in green). Horizontal (XY section) and
vertical (XZ cross-section) images were obtained by confocal laser
scanning analysis. A punctate pattern, typical of apical microvillar
staining, was revealed by the GAT-1 antibodies in horizontal sections
both in MDCK cells expressing the wild type (b and
f) and the truncated transporters (h and
l). The predominant apical localization of GAT-1 and
C-GAT is clearly identifiable in vertical sections (compare Fig. 2, a and g with c and i,
respectively). Virtually no yellow color, indicating colocalization
with the basolateral marker, was revealed by merging of the confocal
images. These data show that the removal of the last 36 amino acids in
the wild type GAT-1 does not affect the apical localization of the
protein. Together with the unaltered sorting behavior, the deleted
GAT-1 transporter retained its functional surface activity, as shown by
the GABA uptake assay (see Fig. 5C).
Fig. 5.
Quantitation by surface biotinylation and
GABA uptake assay of cell surface distribution of wild type and mutated
GABA and betaine transporters expressed in MDCK cells. Panel
A, steady-state biotinylation experiments were carried out in
confluent transfected cells grown on Transwell filters. Cells were
metabolically labeled overnight with [35S]methionine and
cysteine. Surface-expressed transporters were biotinylated from the
apical (A) or basolateral (B) side. After cell
lysis, the proteins were immunoprecipitated and then reprecipitated with streptavidin beads. The precipitates were analyzed on 10% SDS-polyacrylamide gel electrophoresis and visualized by fluorography. Panel B, quantification of apical and basolateral surface
expression of transporters assayed by cell surface biotinylation.
Densitometric quantitation of the biotinylation experiment shown in
panel A is presented as the percent of total cell surface
(apical + basolateral) biotinylated transporters. Panel C,
quantification of apical and basolateral transporter activity assayed
by [3H]GABA uptake. [3H]GABA influx
measurements were carried out on cells grown confluent in Transwell
filters at a final [3H]GABA concentration of 10 µM for wild type and GAT-1 mutants and of 100 µM for BGT-related transporters. Labeled GABA was applied from the apical or basolateral surface and the total activity measured
was: ~35 (GAT-1), 16 (C-GAT), 20 (GBS), 7 (BGT), and 5.5 (myc BGT)
pmol/min/well. Values are presented as the percent of total cell
surface transporter activity and represent means ± S.E. of at
least five independent experiments performed in duplicate. Empty and filled columns indicate apical and
basolateral surfaces, respectively. Bars indicate S.E.
[View Larger Version of this Image (24K GIF file)]
C-myc BGT). Immunolocalization experiments carried out in
C-myc
BGT-transfected MDCK cells showed an intracellular distribution of the
tailless BGT typical of ER localization (Fig. 3g). No
staining was observed in untransfected MDCK cells with the antibody
raised against the Myc epitope (data not shown). Double staining with an antibody raised against an ER marker (28) confirmed the ER localization of the truncated BGT (revealed by the yellow staining, i). The inability of the truncated transporter to reach the
cell surface was further confirmed by the [3H]GABA uptake
transport assay. No surface transport activity was observed, even after
enhancing the expression of exogenous proteins by overnight treatment
with 10 mM sodium butyrate (33) (data not shown).
C-myc BGT, the BGT tail was replaced by the GAT-1
tail. The inability of the GAT-1 tail to restore the surface expression
of BGT was revealed by confocal microscope analysis of transfected MDCK
cells (j-l) and by [3H]GABA transport
experiments (data not shown). These data suggest that information
necessary for the BGT exit from the ER is contained in the cytosolic
tail of BGT.
Fig. 4.
Immunofluorescence analysis of the surface
distribution of the GBS chimera in transfected MDCK cells. Cells
grown to confluent density on Transwell filters after ice-cold methanol fixation were double stained with polyclonal BGT-KLH (panels
a, c, and d) and mAb 6H against the sodium
pump (panels b, e, and f) antibodies.
Confocal immunofluorescence micrographs of horizontal sections through
the apical (panels a and b) and the basal
(panels c and e) surface of the monolayer, and
vertical focal planes (panels d and f) are shown.
The apical and basolateral localization of the GBS chimera demonstrates
the presence in the BGT tail of a basolateral sorting signal.
Bar, 15 µm.
[View Larger Version of this Image (111K GIF file)]
C-GAT are available to cell
surface biotinylation predominantly from the apical side, BGT is
predominantly biotinylated when the NHS-biotin was added to the
basolateral surface. In agreement with the results obtained by
immunofluorescence experiments the GBS chimera was almost equally
biotinylatable from both sides.
C-GAT transfected-MDCK cells mediate a higher GABA influx at the apical versus the
basolateral surface, whereas higher GABA influx was observed at the
basolateral surface with BGT and myc BGT- transfected MDCK cells.
Comparable apical and basolateral GABA influx levels were determined in
GBS expressing MDCK cells. A comparison of the results obtained by biotinylation and GABA influx assays is presented in Fig. 5,
B and C, respectively. The predominant apical
localization of
C-GAT and the equal distribution in the apical and
basolateral plasma membranes of GBS were confirmed by both methods.
Thus, these data suggest the presence of a basolateral sorting signal
in the cytosolic tail of BGT. However, this signal is not capable of
completely reversing the polarity of GAT-1.
Fig. 6.
Surface distribution of the wild type hNGFR
and hNGFR-BGT chimera. Panel A, indirect immunofluorescence
analysis. Confluent monolayers expressing wild type hNGFR (a
and b) or chimeric receptor hNGFR-BGT (c and
d) were grown on Transwell filters and fixed with 3%
paraformaldehyde. The cells were then incubated in the absence of
detergent with mouse anti-hNGFR (ME 20.4) antibody. Vertical
(a and c) and horizontal (b and
d) sections were taken by confocal microscopy.
Bar, 10 µm. Panel B, cell surface biotinylation was performed as described in the legend for Fig. 5A, and
anti-hNGFR mAb 20.4 antibody was used to immunoprecipitate the wild
type and chimeric receptor. Densitometric quantitation of the cell surface biotinylation experiments indicated that the BGT tail relocated
basolaterally 75% of the hNGFR. The position corresponding to the
apparent molecular weight of the hNGFR is indicated on the
left by an arrowhead.
[View Larger Version of this Image (67K GIF file)]
565-572, respectively), and their cellular distribution was analyzed by indirect
immunofluorescence microscopy and [3H]GABA influx
studies. Confocal images revealed an intracellular accumulation of the
BGT
565-572 construct (Fig. 7B), and influx studies
confirmed the absence of surface localization of the mutant transporter
(data not shown). In contrast to the results obtained with the BGT
565-572, the deleted GBS chimera (GBS
565-572) was able to reach
the cell surface and, more interestingly, the deletion restored the
original apical localization of GAT-1 (Fig. 7B). Influx
experiments performed on several GBS
565-572 MDCK-transfected cell
lines confirmed the predominant apical localization of the deleted GBS
chimera (see in Fig. 7C the comparison of the average uptake
of several clones expressing the deleted GBS chimera with GAT and GBS).
Thus, our data indicate that information necessary for both the BGT
export from the ER and for the basolateral localization of the GBS
chimera is contained within amino acids 565-572 of BGT.
1 subunits of the Na,K-
and the H,K-ATPases. From these studies, an apical sorting signal was
found to be contained within the amino-terminal half of the H,K-ATPase
(16). More recently this signal has been located to 8 amino acids in
the 4th transmembrane domain.2 From these
results, it would appear that the apical signals of polytopic proteins
might be located in the lumenal/transmembrane domains just as they are
in monotopic proteins. A recent report indicates that the basolateral
localization of the Na,K-ATPase, rather than being mediated by a
basolateral sorting signal, might derive both from its exclusion from
the apical pathway and from a cytoskeleton-mediated retention mechanism
operating selectively on the basolateral plasma membrane (35).
Moreover, in contrast with the behavior documented for several membrane
proteins, which maintain a polarized distribution in both MDCK cells
and hippocampal neurons (36, 37), two isoforms of the Na,K-ATPase with
exclusive basolateral localization in MDCK cells are distributed
homogeneously in neuronal (hippocampal) cells (26). Our observations of
a nonpolarized neuronal expression versus the exclusive
epithelial basolateral localization of the sodium pump taken together
with the report of Mays et al. (35) might lead to the
conclusion that polytopic proteins make use of different sorting
mechanisms than monotopic proteins to localize basolaterally.
C-GAT) demonstrate that the
cytoplasmic COOH-terminal domain of GAT-1 is not necessary either for
its apical sorting or for its functional activity. The latter
observation is in agreement with a previous in vitro study
of Mabjeesh and Kanner (39). The authors showed that a proteolytic
fragment of purified GAT-1 lacking the cytosolic and most likely the
last transmembrane domain still exhibits transport activity upon
reconstitution in liposomes. Our results demonstrate that 36 amino
acids at the carboxyl terminus of GAT-1 are not required for GABA
transport and, furthermore, to localize the transporter on the apical
plasma membrane of MDCK cells.
C-myc BGT was not expected. Moreover, both a short
deletion in the BGT tail (BGT
565-572) as well as the tail
replacement with that of GAT-1 (BGS chimera) generates transporters
that fail to reach the cell surface. Many natural and artificial
mutations have been observed to result in ER retention and degradation,
apparently because they affect protein folding or oligomerization (40).
So far, studies on the oligomerization of GABA and betaine transporters
have not been carried out, but an oligomeric structure has been
inferred for several carrier systems, and an oligomeric structure has
been documented in the case of the glucose transporter (41) and Na/H
exchanger (42). Our data suggest a direct involvement of an 8-amino
acid region in the COOH terminus of BGT in the folding/oligomerization
process of the betaine transporter. Further investigations are required to clarify the cellular process in which this sequence is involved. However, the present work provides clear evidence for a different role
of the COOH-terminal domains of these two homologous proteins.
565-572) was sorted apically. The 8-amino acid region is rich in basic
residues and, interestingly, basic residues have been shown to play a
role in the basolateral sorting of monotopic proteins (4, 5, 43). In
case of the poly IgR, basolateral sorting seems to depend, as shown by
alanine scanning mutagenesis, on a 3-basic residue segment. Further
analysis is required to determine the contribution of the positively
charged residues for the basolateral sorting of GBS and BGT.
Unfortunately, the inability of both the tail-minus and the
565-572
BGT to move out of the ER prevented us from directly testing the role
of the entire cytosolic tail and of the 8-amino acid region in the
basolateral sorting of the transporter. Most of the basolateral sorting
signals so far identified rely on a tyrosine residue (2). Since the tyrosine residue in the canine BGT tail is not conserved in the human
BGT homolog (44), a prevalent role of the tyrosine residue in the
basolateral sorting of BGT seems unlikely, although it remains to be
investigated.
*
This work was supported in part by Consiglio Nazionale delle
Ricerche Grant 94.00377.CT14.115.28212B/0004 (to G. P.).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.
**
To whom correspondence should be addressed: CNR Cellular and
Molecular Pharmacology Center, Via Vanvitelli, 32, 20129 Milano, Italy.
Tel.: 39-2-701-46358; Fax: 39-2-749-0574; E-mail: Grazia{at}Farma1.csfic.mi.cnr.it.
1
The abbreviations used are: MDCK, Madin-Darby
canine kidney; GABA, -aminobutyric acid; GAT-1, GABA transporter;
BGT, betaine transporter; hNGFR, human nerve growth factor receptor;
PCR, polymerase chain reaction; KLH, keyhole limpet hemocyanin; ER,
endoplasmic reticulum; mAb, monoclonal antibody.
2
M. J. Caplan, unpublished data.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.