From the Centro de Biología Molecular "Severo Ochoa," Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain
Received for publication, July 28, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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Glycine transporter GLYT2 is an axonal
glycoprotein involved in the removal of glycine from the synaptic
cleft. To elucidate the role of the carbohydrate moiety on GLYT2
function, we analyzed the effect of the disruption of the putative
N-glycosylation sites on the transport activity,
intracellular traffic in COS cells, and asymmetrical distribution of
this protein in polarized Madin-Darby canine kidney (MDCK) cells.
Transport activity was reduced by 35-40% after enzymatic
deglycosylation of the transporter reconstituted into liposomes.
Site-directed mutagenesis of the four glycosylation sites (Asn-345,
Asn-355, Asn-360, and Asn-366), located in the large extracellular loop
of GLYT2, produced an inactive protein that was retained in
intracellular compartments when transiently transfected in COS cells or
in nonpolarized MDCK cells. When expressed in polarized MDCK cells,
wild type GLYT2 localizes in the apical surface as assessed by
transport and biotinylation assays. However, a partially unglycosylated
mutant (triple mutant) was distributed in a nonpolarized manner in MDCK
cells. The apical localization of GLYT2 occurred by a glycolipid rafts
independent pathway.
To terminate synaptic transmission in the central nervous system,
neurotransmitters are inactivated by either enzymatic degradation or
active transport into neuronal and/or glial cells (1, 2). The removal
of neurotransmitters is accomplished by transporter proteins located in
the plasma membrane of the presynaptic nerve terminals and surrounding
glial processes. Glycine transporters are encoded by two genes
(glyt1 and glyt2) that produce several alternative isoforms (3-7).
GLYT21 is predominantly
expressed in the spinal cord and the brainstem, associated with the
presynaptic aspect of glycinergic synapses (8-11). Similarly, GLYT1 is
expressed at higher levels in glycinergic areas, but it could also
participate in N-methyl- D-aspartate-mediated glutamatergic neurotransmission (5, 9-14).
GLYT1 and GLYT2 share a common predicted structure comprising 12 putative transmembrane domains and a large hydrophilic loop between
transmembrane domains III and IV which contains several potential
N-linked glycosylation sites. Previous data have shown that
mature GLYT1 and GLYT2 are heavily glycosylated proteins (15, 16).
N-Glycosylation of proteins has been demonstrated to play a
variety of roles including modulation of biological activity,
regulation of intracellular targeting, protein folding, and maintenance
of protein stability. In the case of GLYT1, we have previously shown
that glycosylation is necessary for proper trafficking of the protein,
but it is not indispensable for the transport activity itself (16).
The subcellular localization of transporters seems to be an important
determinant of the impact of transport activity on synaptic function.
In this sense, various transporters show a differential distribution
among membrane domains of the cells where they are expressed. For
instance, the GABA transporter, GABA transporter 1, the transporters
for biogenic amines, and the glycine transporter GLYT2 are concentrated
in presynaptic terminals (17-20). In non-neural cells, the betaine
transporter, a member of the GABA transporter subfamily, is expressed
in the basolateral membrane of epithelial MDCK cells (21). The specific
mechanisms of such a nonuniform distribution have been studied in
experimental systems of polarized cultured cells, especially in the
epithelial MDCK cell line, since somatodendritic neuronal proteins use
to be localized in the basolateral domain, whereas axonal proteins are
in the apical one (22). N-Glycosylation has been shown to be
involved in the apical localization of a number of secretory and
membrane proteins (23), but the molecular mechanisms on apical delivery
are yet unclear.
In the present report we have analyzed the consequences of the
disruption of the potential N-glycosylation sites of GLYT2 on the transport of solute, in transport of the protein to the cell
surface, and in the asymmetrical distribution of GLYT2 in a model of
polarized cells.
Materials--
Dulbecco's modified Eagle's medium and G418
were from Life Technologies, Inc. BHK medium was from Life
Technologies, Inc. Bovine serum albumin was from Sigma.
[2-3H]Glycine (1757.5 GBq/mmol) was from PerkinElmer Life
Sciences. Anti-rabbit fluorescein isothiocyanate-linked was from
Amersham Pharmacia Biotech. Recombinant N-glycosidase F
(PNGaseF) was from New England Biolabs. Transwell inserts were from
Costar. Culture dishes were from Nunc (Denmark). All other reagents
were obtained in the purest form available.
cDNA Constructs and Site-directed Mutagenesis--
All
mutants were performed as described previously (16, 24). Mutant and
wild type cDNAs were cloned in the
HindIII/XbaI or XhoI/XbaI
restriction sites of pcDNA3 for expression in MDCK cells.
Site-directed mutagenesis was performed using a modification of the
method of Higuchi (25) as described (24). Mutants and wild type
cDNAs were subcloned downstream from the cytomegalovirus promoter
of pcDNA3 mammalian expression vector (Invitrogen). To disrupt the
glycosylation sequences (NX(S/T)), codons coding for Asn
were mutagenized to Asp. Glycosylation sites were eliminated sequentially, starting with single mutations as follows: N345D, N355D,
N360D, and N366D. Mutants devoid of two, three, and four glycosylation
sites were generated, using as a template DNA sequences mutated at one,
two, or three glycosylation sites, respectively. All mutations were
confirmed by DNA sequencing.
Transient Transfection in COS Cells--
COS-7 cells (American
Type Culture Collection) were grown at 37 °C and 5% CO2
in high glucose Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml
streptomycin sulfate. Transient expression in COS cells, was carried
out using LipofectAMINE Plus (Life Technologies, Inc.) following the
procedure indicated by the supplier. Cells were incubated for
48 h at 37 °C and then used to assay transport activity,
surface expression, and immunofluorescence.
Transfection and Stable Expression of MDCK Cells--
The
parental MDCK type II cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum and 2 mM L-glutamine at 37 °C, 7%
CO2. MDCK cells were transfected by electroporation with
cDNAs encoding the transporters cloned into the expression vector
pcDNA3 that carries the neomycin resistance gene for selection.
After selection with 0.6 g/liter G418 for 12-15 days, single colonies
were isolated with cloning cylinders and tested for transporter
expression by immunocytochemistry. Clones stably transfected were then
assayed for transport activity and maintained in DMEM without glycine,
supplemented with 10% fetal calf serum, 2 mM
L-glutamine, and 0.6 g/liter G418.
Glycine Transport Assay--
Transport assays in transfected
COS-7 cells were performed at 37 °C in HEPES-buffered saline (150 mM NaCl, 10 mM HEPES-Tris, pH 7.4, 1 mM Ca2Cl, 2.5 mM KCl, 2.5 mM MgSO4, 10 mM glucose) as described previously (26). Cells were incubated for the indicated times
in 0.3 ml of an uptake solution that contained an isotopic dilution of
3H-labeled glycine in the former solution yielding a 10 µM final glycine concentration (or the desired
concentration). Kinetic analysis was performed by varying glycine
concentration in the uptake medium between 10 µM and 1 mM. Transport into reconstituted liposomes was measured at
room temperature using an inwardly directed NaCl gradient in the
presence of a negative membrane potential, as described (27, 28).
Nonspecific transport was defined as the glycine transport shown by
mock-transfected cells or proteoliposomes obtained from
mock-transfected cells.
Vectorial Glycine Uptake Assay--
Parental and transfected
MDCK cells were plated at 50% confluence on Transwell tissue culture
inserts (6.5 mm diameter, 0.4 µm filter pore size, Costar Co.,
Cambridge, MA) and grown for 5-7 days. Glycine uptake was performed at
room temperature according to a modification of the method of Yamauchi
et al. (29). After one wash of each chamber with complete
phosphate-buffered saline (PBS/Ca/Mg: 137 mM NaCl, 2.7 mM KCl, 0.89 mM CaCl2, 0.49 mM MgCl2, 4.3 mM
Na2HPO4·7H2O, 1.4 mM
KH2PO4, pH 7.3) and another wash with uptake
buffer (150 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 10 mM D(+)-glucose, 10 mM HEPES-Tris,
pH 7.4), the cells were incubated with 0.2 µM
[3H]glycine in uptake buffer either in the upper or in
the lower chamber for 10 min. At the end of the incubation, the cells
were washed with ice-cold PBS/Ca/Mg three times from the side they were
incubated with the 3H-labeled substrate and once from the
opposite side. The cells were dried and lysed in 0.2 M
NaOH. Aliquots were taken and counted in scintillation fluid, and
protein concentrations were determined with the Bio-Rad protein assay.
Basal glycine uptake was defined as glycine transport shown by
nontransfected MDCK cells.
Solubilization and Reconstitution Procedure--
For each
reconstitution experiment, transfected cells from one dish (10-cm
diameter) were used. Cells were scraped, collected by centrifugation,
and adjusted at a protein concentration of 5-10 mg/ml with
phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4·7H2O, 1.4 mM
KH2PO4, pH 7.3). Solubilization of cells was
performed using sodium cholate at a 1:1 detergent/protein ratio. After
10 min on ice, solubilized proteins were reconstituted with
asolectin/brain lipids (28), and proteoliposomes were kept on ice until used.
Glycosidase Treatment--
Wild type GLYT2 protein transiently
transfected in COS cells was solubilized and reconstituted into
liposomes as described above. Proteoliposomes were treated with or
without PNGaseF (150 units/µg protein) in the presence of protease
inhibitors (4 µM pepstatin A and 0.4 mM
phenylmethylsulfonyl fluoride) for 3 h at 37 °C with gentle
agitation. After treatment, proteoliposomes were washed and immediately
used for glycine transport assays or for SDS-PAGE and immunoblotting.
Cell Surface Biotinylation--
MDCK cells were plated at 50%
confluence on 0.4-µm pore size, 25-mm Transwell cell culture filter
inserts for 6-well plates and grown for 7 days. After 3 washes with 2 ml of ice-cold PBS/Ca/Mg, cell surface proteins of either apical or
basolateral domains of cell plasma membrane were biotinylated by
exposing that side of the cell monolayer to 1 ml of sulfosuccinimidyl
2-(biotinamido)ethyl-1,3'-dithiopropionate (Sulfo-NHS-SS-Biotin)
(Pierce) (1 mg/ml in PBS/Ca/Mg) for 20 min on ice. Cells were then
washed with 2 ml of PBS/Ca/Mg plus 100 mM lysine for 20 min
to quench the reagent. After three more washes with PBS/Ca/Mg, filters
were excised, and cells were lysed with 1 ml of lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM
HEPES-Tris, 0.25% deoxycholate, 1% Triton X-100, 0.1% SDS, pH 7.4)
for 30 min. The cell lysate was clarified by sedimentation at
14,000 × g for 10 min. COS cell proteins were labeled
following the same procedure except they were plated on regular culture
dishes. Biotinylated proteins were recovered by adding
streptavidin-agarose beads to a portion of the cell lysate followed by
incubation for 2 h with gentle agitation at room temperature.
Beads were pellet by centrifugation. After three washes with 1 ml of
lysis buffer, proteins bound to the beads were eluted in 2× Laemmli
sample buffer, separated by SDS electrophoresis, and transferred to
nitrocellulose filters. The filters were probed with antibodies
recognizing GLYT2 (10, 11) as described previously (10). Bands were
visualized with the ECL detection method (Amersham Pharmacia Biotech)
and quantified by densitometry (Molecular Dynamics ImageQuant version
3.0) by using film exposures that were in the linear range.
Isolation of Low Density Membrane Domains--
The procedure
used to prepare Triton X-100-insoluble membranes by centrifugation to
equilibrium in sucrose density gradients was essentially as described
by Brown and Rose (30). Cells grown to confluency in four 100-mm dishes
were rinsed with PBS and lysed for 20 min in 1 ml of 150 mM
NaCl, 25 mM Tris-Cl, pH 7.5, 5 mM EDTA, 1%
Triton X-100 at 4 °C. The lysate was scraped from the dishes with a
rubber policeman, and the dishes were rinsed with 1 ml of the same
buffer at 4 °C, and the lysate was homogenized by passing the sample
through a 22-gauge needle. The lysate was brought to 40% sucrose in a
final volume of 4 ml and placed at the bottom of an 8-ml 5-30% linear
sucrose gradient. Samples were centrifuged for 18 h at 39,000 rpm
at 4 °C in a Beckman SW 41 rotor. Fractions of 1 ml were harvested
from the bottom of the tube, and aliquots were subjected to immunoblot
analysis. MAL proteolipid was detected by the dot-blot technique by
spotting 100 µg of protein onto a nitrocellulose filter. The filter
was probed with a highly specific antibody for MAL kindly provided by
Dr. M. A. Alonso (Centro de Biología Molecular) and
previously characterized (31).
Protein Concentration--
Protein concentration was determined
by the method of Bradford (32).
GLYT2 is an axonal glycoprotein involved in the re-uptake of the
neurotransmitter glycine in the glycinergic synapses. Based on its
deduced amino acid sequence, the protein has four consensus sites
(NX(T/S)) for N-glycosylation in the second
extracellular loop (Asn-345, Asn-355, Asn-360, and Asn-366) as depicted
in Fig. 1. Glycosylation has been shown
to play a functional role in other members of the sodium- and
chloride-dependent neurotransmitter transporter family (24,
33). To address the role of the carbohydrate moiety on different
functional aspects of GLYT2, we disrupted the glycosylation consensus
sequences by site-directed mutagenesis replacing the asparagine
residues with aspartate. Then we analyzed the effects of these
mutations on transport activity, on transport of the protein from the
intracellular compartments to the cell surface, and on asymmetrical
distribution of the protein in the surface of polarized cells. First,
we produced a series of mutants by progressive removal of the potential
glycosylation sites. We prepared four single mutants (N345D, N355D,
N360D, and N366D), a double mutant (N355D/N360D), a triple mutant
(N345D/N355D/N360D), and the quadruple mutant
(N345D/N355D/N360D/N366D). Wild type or mutant forms of GLYT2 were then
transiently transfected into COS cells, and their transport activities
were assayed. As shown in Fig. 2, mutants
deficient in one glycosylation site sustained glycine transport
activities similar to those displayed by wild type transporter,
indicating that no single N-linked carbohydrate chain is
specifically required for the GLYT2 function. The double and triple
mutants retained 74 and 58% of wild type glycine transport activity,
respectively. When all four potential glycosylation sites were mutated,
a complete lost of activity was observed. These results indicate that
the effects of disrupting N-glycosylation sites on GLYT2
activity are cumulative. The progressive decrease in transport activity
was not due to a change in the affinity of the transporter for the
substrate, as deduced from kinetic analysis of the triple mutant. The
calculated Km for glycine was nearly identical in
wild type and triple mutant of GLYT2 (102 ± 4 and 93 ± 3 µM, respectively). However, there were decreases in the
Vmax values (3.3 ± 0.5 and 2.0 ± 0.2 nmol of glycine/mg of protein/6 min, respectively).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Location of the potential
N-linked glycosylation sites in the scheme of GLYT2
structure. Potential sites for N-glycosylation
(NX(S/T)) are represented as . Arrowheads and
sugar chain symbols point out to residues that were not
mutated and mutated, respectively.
View larger version (18K):
[in a new window]
Fig. 2.
Transport activity of
N-glycosylation-deficient mutants. Each of the
seven asparagine replacement mutants were assayed for transport
activity 48 h after transfection in COS-7 cells as described under
"Experimental Procedures." Activities are shown as a percentage of
GLYT2 transport activity that was 1.2 ± 0.3 nmol glycine/mg of
protein/6 min. Error bars represent S.E. of three
determinations.
To determine whether the impaired transport of the mutants was due to a
poor expression or to a deficient transport of the protein to the
plasma membrane, we performed cell surface biotinylation experiments
followed by immunodetection (Fig. 3).
Plasma membrane proteins were labeled with the membrane-impermeant
reagent Sulfo-NHS-SS-Biotin. The biotinylated proteins were
precipitated with immobilized streptavidin and immunodetected by
Western blot using a previously characterized specific antibody against
the NH2-terminal domain of GLYT2 (10, 11). In the total
cell lysate the antibody recognized two protein bands either for the
wild type or for the mutants, except in the case of the quadruple
mutant that was expressed as a single band (Fig. 3A). The
upper band was very diffuse, a characteristic of heavily glycosylated
proteins. In the triple mutant this smeared band was very faint and
could be barely detected with longer exposures of the film. Only the
upper bands were biotinylated when exposed to the biotin reagent (Fig.
3B), indicating that these forms of the protein correspond
to extracellularly accessible, and therefore plasma membrane inserted,
mature transporters. However, the lower bands were not biotinylated as
expected for partially processed intracellular transporters. The
electrophoretic mobility of the lower band slightly increased with
every single mutation and increased stepwise as more mutations were
progressively introduced. The size of the lower bands ranged from
around 71 kDa in the wild type to 63 kDa in the quadruple mutant. This
indicates that the four consensus N-glycosylation sites
located in the large hydrophilic loop of GLYT2 are in fact glycosylated
and that the location of this loop is consistent with GLYT2 predicted
topology.
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We quantified the relative amounts of the mutants on the plasma membrane compared with their total amount in cell extracts by a densitometric analysis considering as 100% the fraction of total wild type protein that reached the membrane (Fig. 3C). For the mutants, a parallelism between the difficulty to arrive to the cell surface and the decrease in transport activity was observed. No significant differences were detected in cell and membrane expression of all single and double mutants as compared with the wild type. However, mutants devoid of three or four N-glycosylation sites showed low expression levels and difficulties reaching the plasma membrane. The quadruple mutant was not detected in the surface, which is consistent with the absence of transport activity shown when this protein is expressed in COS cells (Fig. 2).
The subcellular localization of mutants was additionally examined by immunocytochemistry on transfected COS cells. Whereas the plasma membrane of wild type transfected cells was clearly labeled, a poor presence or total absence of labeling was observed at the surface of cells expressing the triple and quadruple mutants, respectively.2 Immunofluorescence together with biotinylation experiments led us to conclude that, although each carbohydrate chain considered individually is not specifically essential for GLYT2 function, the progressive removal of carbohydrate chains produced an accumulative decrease in surface expression and, consequently, in transport activity of the glycine transporter. Similar results have been described for the norepinephrine transporter (33) and GLYT1 (24).
To determine whether the inactivation and intracellular retention of
the transporter produced by the total disruption of
N-glycosylation sites could be explained by misfolding of
the protein, the quadruple mutant and the wild type form of GLYT2 were
expressed in COS cells, solubilized with detergent, and reconstituted
into liposomes. Fig. 4A shows
that whereas the reconstituted wild type was fully active, the
quadruple mutant was completely inactive after reconstitution. Moreover, when the wild type was deglycosylated by treatment with PNGaseF after being reconstituted into liposomes, the transporter reduced the transport activity by 35-40%. The removal of
N-linked sugar chains by PNGaseF produced a shift in the
electrophoretic mobility of GLYT2 protein as shown on Western blot
analysis (Fig. 4B, lanes 1 and 2). The
size of the band (63 kDa) corresponding to the nonglycosylated protein
(Fig. 4B, lane 2) agrees with that of the
quadruple mutant (Fig. 4B, lane 3), indicating
that there are no additional sites for N-glycosylation on
GLYT2. This rules out the use of the N-glycosylation
consensus sites located at the extracellular loop VI and those of the
amino terminus of the transporter (Fig. 1).
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These results indicated that the loss of carbohydrates renders a GLYT2 transporter that is in an inactive conformation, probably misfolded, and that is unable to reach the cell surface. The importance of glycosylation for a proper surface delivery has been previously stated for other transporters (24, 33-37) and for various plasma membrane proteins (38). Moreover, the reduction in the wild type GLYT2 activity after enzymatic deglycosylation indicates that the oligosaccharide chains stabilize the optimal active conformation of the transporter once it has been synthesized. Interestingly, these results contrast with those reported for GLYT1, norepinephrine transporter, and serotonin transporter where surface trafficking but no transport activity impaired in the nonglycosylated mutant (24, 33, 34) but are consistent with results obtained with a glycine transporter purified from pig spinal cord (15).
GLYT2 is expressed mainly in axons and terminals of neurons in the
spinal cord and other glycinergic areas but is absent in most of
dendrites and cell bodies. A number of transporters of this gene family
also show an asymmetrical distribution on the surface of cells where
they are expressed, and thus, it is supposed that the specific
subcellular distribution of transporters must be essential to
accomplish their biological functions. To study the molecular mechanism
involved in asymmetrical distribution of neuronal proteins, a
frequently used experimental model is found in the epithelial MDCK cell
line. We recently developed MDCK cell lines constitutively expressing
GLYT2 in which most of the wild type transporters were localized in the
apical domain of polarized cells (39). Because glycosylation is known
to be an apical determinant for a number of secreted and membrane
proteins (40), we decided to analyze the distribution of deglycosylated GLYT2 mutants in this experimental system. However, in transiently transfected MDCK cells, like in COS cells, the quadruple mutant remained accumulated in intracellular compartments (not shown), and
thus, this mutant could no be further analyzed. Again like in COS
cells, the triple mutant was partially active, and thus we generated
stable cell lines with this construct. The distribution of the protein
between the apical and basolateral domains was analyzed by vectorial
transport assays and by biotinylation experiments (Fig.
5). In agreement with our previous
observations, 80 ± 8% of the transport activity determined in
MDCK cells stably transfected with wild type GLYT2 was located in the
apical side (Fig. 5A), and most of the protein (89 ± 6%) was labeled when the Sulfo-NHS-SS-Biotin reagent was added
from the apical side (Fig. 5B). However, in MDCK cells
transfected with the triple GLYT2 mutant, the transport activity and
the labeled protein were detected at about the same levels at both cell
surfaces (60 ± 3% apical in transport assays and 55 ± 7%
apical in biotinylation experiments) (Fig. 5, A and B). The high intracellular retention of this mutant
precluded the production of reliable confocal microscopy images.
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These results clearly indicate that full glycosylation is not only necessary for proper conformation and cell surface delivery but also for asymmetrical distribution in polarized cells, a property that must be physiologically relevant for neuronal proteins that need to be asymmetrically distributed. The mechanisms for apical sorting are diverse, and signals have been found in the extracellular (41), in the transmembrane (42), and in the intracellular protein domains (43, 44). The carbohydrate moiety is involved in apical localization of other proteins (45), but this has not been reported for neurotransmitter transporters. In fact, the mechanism for apical localization of the GABA transporter GABA transporter 3 in MDCK cells has been investigated, and it was shown to depend on a THF motif located in the intracellular carboxyl end of the protein (44). However, intracellular ends of GLYT2 have been previously shown not to be important in its apical distribution (39).
Some apically directed membrane proteins are associated with
specialized membrane domains that are rich in glycosphingolipids and
are detergent-insoluble (46). These structures, termed "rafts," can
be recovered from low density fractions after centrifugation to
equilibrium in sucrose density gradients. To investigate whether GLYT2
used this sorting mechanism, we centrifuged Triton X-100 extracts from
MDCK cells stably transfected with the wild type GLYT2 to equilibrium
(30). Under these experimental conditions, GLYT2 was completely
solubilized by a brief treatment of the cells with Triton X-100 at
4 °C and appeared in higher density fractions (fractions 1-4) (Fig.
6A). As a control of the
fractionation procedure, we analyzed in parallel the distribution of
the proteolipid MAL (Fig. 6B), a protein associated with
detergent-insoluble membranes that was found in the buoyant,
membrane-insoluble fractions (fractions 7-8). These experiments
indicated that GLYT2 is not associated with membrane rafts and must be
polarized by an alternative mechanism. Proteins that utilize the
glycolipid pathway are heterogeneous in their apical sorting signal
that can be found in N-linked carbohydrates, in the
glycosylphosphatidylinositol anchor, or even in transmembrane domains.
Among them, there are a number of N-glycosylated intestinal hydrolases (47), the alkaline phosphatase (48), or the influenza virus
hemagglutinin (42). Also this pathway is used by other integral
membrane proteins like the MAL proteolipid (31) or caveolin (49).
However, other membrane proteins including both glycosylated
(enteropeptidase) (50) and nonglycosylated proteins (CD3-) (43) are
apically sorted by a proteolipid-independent pathway.
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The nature of the molecular machinery involved in the recruitment of glycoproteins to the apical membrane is largely unknown. A first model propose that N-glycans constitute the apical signal by themselves. Indeed the insertion of ectopic glycosylation sites in secretory or membrane proteins converted nonpolarized secretory proteins or intracellularly retained membrane proteins into apical ones (41, 45). In this model N-glycans would interact with lectins sorters in the trans-Golgi network that would mediate incorporation of glycosylated proteins into apical transport vesicles. Although some candidate lectins have been identified (51, 52), their role in this process is still unclear. Our observation that the triple GLYT2 mutant is distributed in a nonpolarized manner despite the presence of a glycosylation site would not support this model for GLYT2 except that the massive reduction in the glycosylation observed in this mutant would also reduce the affinity for lectins and, consequently, the efficiency of apical recruitment. Indeed, a similar observation has been performed for erythropoietin, a secretory protein where not all its glycosylation sites are equally effective in promoting apical sorting (53). An alternative model where N-glycans would not constitute a sufficient sorting signal per se but would play an indirect role by allowing the correct folding and structural stabilization of proteinaceous sorting signals has been proposed (40). Our observation on the triple GLYT2 mutant would also be compatible with this alternative model.
In summary, in this report we describe the importance of the sugar
moiety of GLYT2 for the transport of the solute and for the arrival to
the membrane in a polarized manner. This could be relevant for the
physiology of the glycinergic neuron, as GLYT2 has to be asymmetrically
distributed to be finally settled in the axonal domain where it
develops its biological function.
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FOOTNOTES |
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* This work was supported by the Spanish Dirección General de Enseñanza Superior e Investigación Científica Grant PM98-0013, the TMR Program Grant FMRX-CT-98-0228 of the European Union, and an institutional grant from the Fundación Ramón Areces.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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain. Tel.: 34 91 3974855; Fax: 34 91 3974799; E-mail: caragon@cbm.uam.es.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M006774200
2 R. Martínez-Maza, B. López-Corcuera, and C. Aragón, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
GLYT1 and GLYT2, glycine transporter 1 and 2;
GABA, -aminobutyric acid;
PBS, phosphate-buffered saline;
MDCK, Madin-Darby canine kidney cells;
PNGaseF, N-glycosidase F.
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REFERENCES |
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