From the
To elucidate the role of N-glycosylation in the
function of the high affinity glycine transporter GLYT1, we have
investigated the effect of the glycosylation inhibitor tunicamycin as
well as the effect of the disruption of the putative glycosylation
sites by site-directed mutagenesis. SDS-polyacrylamide gel
electrophoresis of proteins from GLYT1-transfected COS cells reveals a
major band of 80-100 kDa and a minor one of 57 kDa. Treatment
with tunicamycin produces a 40% inhibition in transport activity and a
decrease in the intensity of the 80-100-kDa band, whereas the
57-kDa band decreases in size to yield a 47-kDa protein corresponding
to the unglycosylated form of the transporter. Simultaneous mutation of
Asn-169, Asn-172, Asn-182, and Asn-188 to Gln also produces the 47-kDa
form of the protein, indicating that there are no additional sites for
N-glycosylation. Progressive mutation of the potential
glycosylation sites produces a progressive decrease in transport
activity and in size of the protein, indicating that the four putative
glycosylation sites are actually glycosylated. N-Glycosylation
of the GLYT1 is not indispensable for the transport activity itself, as
demonstrated by enzymatic deglycosylation of the transporter. Analysis
of surface proteins by biotinylation and by immunofluorescence
demonstrates that a significant portion of the unglycosylated GLYT1
mutant remains in the intracellular compartment. This suggests that the
carbohydrate moiety of glycine transporter GLYT1 is necessary for the
proper trafficking of the protein to the plasma membrane.
The re-uptake of neurotransmitter amino acids into presynaptic
nerve terminals or the neighboring fine glial processes provides one
way of clearing the extracellular space of the neuroactive substances
and so constitutes an efficient mechanism by which the postsynaptic
action can be terminated
(1, 2, 3, 4) .
This process is carried out by integral membrane proteins that use a
chemical or electrochemical gradient as the driving force for the
uphill movements of the transmitter across the plasma membrane. Some of
the Na
Glycine is an important inhibitory
neurotransmitter in the central nervous system of vertebrates, mainly
in the spinal cord and the brain stem. In addition, glycine could
potentiate the action of glutamate, the main excitatory
neurotransmitter in the brain, on postsynaptic
N-methyl-
D-aspartate receptors. Specific high
affinity transport systems for glycine have been identified in nerve
terminals and glial cells
(30, 31, 32, 33, 34, 35, 36, 37) .
To date, two different glycine transporters have been cloned. The first
one, GLYT1
(20, 21, 22, 25) , presents
three isoforms produced by alternative splicing and/or alternative
promoter usage
(23, 25) (termed GLYT1a, GLYT1b, and
GLYT1c, according to Liu et al. (24) and Kim et
al. (25) ). More recently, the existence of a second
glycine transporter (GLYT2)
(24) has been reported, which is
present specifically in the brain stem and spinal cord
(24, 38) , brain areas where strychnine-sensitive
glycine receptors are more represented. The hydropathic profile of
these proteins suggests the presence of 12 transmembrane segments. On
the basis of cDNA sequence of GLYT1, there are seven potential
glycosylation sites for N-linked glycosylation, but only four
are supposed to be extracellular, located in the loop between putative
transmembrane segments three and four
(22) . The presence of
N-glycosylation sites in all the transporters cloned so far
suggests that glycosylation must play an important role in the function
of these proteins. Moreover, it has been recently demonstrated that the
removal of oligosaccharides by enzymatic methods from a purified
glycine transporter affects the transport activity
(39) .
In
the present study, we have used site-directed mutagenesis to
investigate which of the potential glycosylation sites of GLYT1 are
actually utilized and the consequences of the disruption of these sites
on the targeting and transport activity of the protein.
Materials [2-
To determine whether the carbohydrate moiety of GLYT1 plays a
role in the function of the protein, we carried out initial experiments
by using the general inhibitor of glycosylation tunicamycin. When COS
cells transfected with the wild type GLYT1 expression vector were
treated with 10 µg/ml tunicamycin for 24 h, a reduction in the
transport activity from 33.8 pmol/10 min/mg of protein to 20.1 pmol/10
min/mg of protein was observed. A similar decrease in transport
activity has been previously described for norepinephrine and
To
address the role of glycosylation on the GLYT1 function in a more
selective and specific way, we performed site-directed mutagenesis of
the potential glycosylation sites of GLYT1 and studied the effect of
such modification on the properties of the protein in transfected COS
cells. We disrupted 2, 3, or all 4 predicted extracellular
N-glycosylation sites by substituting glutamine for asparagine
in the consensus sites. The resulting mutants were called N2, N3, or
N4. This stands for substitution of two (Asn-182 and Asn-188), three
(Asn-172, Asn-182, and Asn-188), or four (Asn-169, Asn-172, Asn-182,
and Asn-188) putative glycosylation sites, respectively. Besides the
inability of glutamine to function as acceptor of
N-glycosylation, this is a highly conservative amino acid
substitution that should have minimal structural effects on the
protein.
Time courses of [2-
Fig. 4 shows a kinetic analysis of the N4 mutant using a wide
range of substrate concentrations (0.2 µ
M to 2
m
M). The Eadie-Hofstee plot of the data (Fig. 4 B)
revealed that the transport characteristics of the
glycosylation-defective N4 mutant were significantly different from
those of the wild type. Whereas the apparent K
When the expression of GLYT1 was
analyzed by Western blot, two bands were observed in the wild type and
in the mutants N2 and N3, and a single band was observed in N4. The
intensity of the upper band of these mutants was comparable with the
wild type (except for N4 mutant). In addition, the mobility of the
bands became progressively increased by increasing the number of
mutated asparagines (Fig. 5, left blot). This
experiment demonstrated that the lower band was indeed an intermediate
in the synthesis of the transporter that was already glycosylated,
corresponding probably to the endoplasmic reticulum form of the protein
after accepting the core of 14 sugars from the dolychol-linked
oligosaccharide
(52) . The scale of sizes of the lower band,
ranging from 57 kDa in the wild type, to 52 kDa in N2, to 49 kDa in N3,
and to 47 kDa in N4 mutants, was compatible with this possibility (the
molecular mass of each oligosaccharide core is approximately 2500 Da)
and indicated that the four putative glycosylation sites are actually
glycosylated and extracellularly located. The N4 mutant was expressed
as a single band whose size coincides with that of the lower band
observed in the presence of tunicamycin (Fig. 2), indicating that
there are no additional sites for N-glycosylation. The two
asparagines placed in the carboxyl-terminal part of the protein
(Asn-578 and Asn-628), and that located close to the amino terminus
(Asn-29) are therefore not used as glycosylation targets, as
corresponds to the intracellular location of both amino and carboxyl
termini of the protein
(43) .
The effect of
the deglycosylation on the transport activity itself was studied in an
experiment of solubilization, reconstitution, and deglycosylation of
the transporter. GLYT1-transfected COS cells underwent detergent
extraction, and the solubilized proteins reconstituted into liposomes.
The reconstituted proteins were treated with N-glycosidase F,
an enzyme which hydrolyzes most types of N-linked carbohydrate
groups from glycoproteins. Fig. 7 shows the effect of
N-glycosidase F both on the specific glycine transport
activity (Fig. 7 A) and on the electrophoretic mobility
of the transporter (Fig. 7 B). Results show that
N-glycosidase F treatment does not affect transport of
glycine. When deglycosylated proteins were analyzed by Western blot, a
single band of 47 kDa appeared. The size of the band agrees with that
observed in the expressed N4 mutant. These results indicate that after
3 or 5 h of incubation, the deglycosidase was able to remove almost
completely the N-linked oligosaccharide chains of the
reconstituted GLYT1 (Fig. 7 B). From these data, we can
conclude that glycosylation is not involved in the GLYT1 transport
activity itself. Modifications of the oligosaccharide structure of
glycoproteins related to changes in the biological activity of
transporter proteins have been described. The
Na
Cells were transfected with an expression
vector containing the wild type GLYT1 (clon rB20a) or the indicated
mutants. After 48 h of incubation at 37 °C in the CO
We thank Drs. K. E. Smith and R. L. Weinshank from
Synaptic Pharmaceutical Corp. for sending the rB20a clone. We also
thank E. Nez for expert technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- and Cl
-dependent
neurotransmitter transporters have been purified from mammalian brain
(5, 6, 7) , and more recently, cDNA clones
encoding transporters for
-aminobutyric acid
(8, 9, 10, 11, 12) ,
catecholamines
(13, 14, 15, 16, 17) , serotonin
(18, 19) , glycine
(20, 21, 22, 23, 24, 25) ,
glutamate
(26, 27, 28) , and proline
(29) have been isolated.
H]Glycine (1757.5 GBq/mmol) was supplied
by DuPont NEN. Ligase and restriction enzymes were from Boehringer
Mannheim. Taq polymerase was from Perkin-Elmer Corp.
DEAE-dextran, pGEX-2, pSVL, and glutathione S-Sepharose 4B
were from Pharmacia Biotech Inc. (Upsala, Sweden).
Sulfo- N-succinimidyl-6-biotinamido hexanoate was from Pierce.
Recombinant N-glycosidase F was from New England Biolabs.
Citifluor, FITC
(
)
-coupled goat anti-rabbit IgG,
peroxidase-linked anti-rabbit IgG, and ECL reagent were from Amersham
Corp. (Bucks, United Kingdom). Immobilized streptavidin and cholic acid
were obtained from Sigma. Cholic acid was recrystallized and
neutralized as described
(7) . All other reagents were obtained
in the purest form available. Methods
Expression in COS Cells
Transient
expression of COS cells was carried out using DEAE-dextran with
dimethyl sulfoxide according to the method of Kaufman
(40) with
minor modifications. COS cells were grown in 24-well plates at 37
°C and 5% COin 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. Cells
were used 2 days after transfection.
Glycine Transport Assay
Glycine transport
into reconstituted liposomes was measured using an inwardly directed
NaCl gradient in the presence of a negative membrane potential, as
described
(7, 41) . Transport assays in transfected COS
cells were performed at 37 °C in HEPES-buffered saline (150 m
M NaCl, 10 m
M HEPES, 1 m
M CaCl, 10
m
M glucose, 5 m
M KCl, 1 m
M MgCl
,
pH 7.4) as described
(42) . All incubations were carried out in
triplicate. Each experiment was repeated at least three times with
different cell cultures. For estimating statistical differences, the
data were compared using the Student's t test;
differences at the 0.05 level were considered statistically
significant.
Solubilization and Reconstitution
Procedure
For each reconstitution experiment, transfected
cells from one dish (10-cm diameter) were used. Cells were scraped and
collected by centrifugation. Solubilization of cells was performed in
the presence of 25% saturated ammonium sulfate and sodium cholate at a
1:1 detergent/protein ratio. After 10 min on ice, the mixture was
centrifuged for 5 min. The supernatant was reconstituted with
asolectin/brain lipids, and transport was measured as described
(41) .
Preparation of Glutathione S-Transferase-Glycine
Transporter Fusion Proteins
Antibodies against a fusion
protein of the glutathione S-transferase and the carboxyl
terminus of GLYT1 were produced as described
(43) . In brief, a
PCR fragment corresponding to the 76 amino acids of the carboxyl
terminus of GLYT1 was cloned in the BamHI- EcoRI
restriction sites of pGEX-2 plasmid, keeping the correct reading frame.
Transformant Escherichia coli XL-1 were selected, and the
expression of the fusion protein was induced by
isopropylthiogalactoside. The protein was purified in a
glutathione-Sepharose 4B column following the instructions of the
supplier.
Rabbit Immunization
Two rabbits were
immunized with GST-GLYT1 fusion protein. Each rabbit received
intradermic injection of the fusion protein in complete Freund's
adjuvant (100 µg at day 0). Subsequent immunizations were done
subcutaneously with the same amount of protein in incomplete
Freund's adjuvant at days 14, 28, 42, and 56. The antibodies have
been characterized elsewhere.(
)
Preparation of SDS
Extracts
SDS extracts from whole rat brains or specific
regions were prepared by homogenizing and solubilizing the tissue in
PBS medium (137 m
M NaCl, 0.9 m
M CaCl,
2.68 m
M KCl, 1.47 m
M KH
PO
,
0.49 m
M MgCl
, 7.37 m
M Na
HPO
, pH 7.4) with SDS (10 mg/ml) and 1
m
M phenylmethanesulfonyl fluoride and removing unsolubilized
material by centrifugation (50,000
g, 10 min, 10
°C).
Electrophoresis and Blotting
SDS-PAGE was
done in the presence of 2-mercaptoethanol. The gels were run slowly
(overnight) at constant current starting at 30 V. After
electrophoresis, samples were transferred by electroblotting onto a
nitrocellulose membrane in a semidry electroblotting system (Life
Technologies, Inc.) at 1.2 mA/cmfor 2 h. The transfer
buffer consisted of 192 m
M glycine and 25 m
M Tris-HCl, pH 8.3. Nonspecific protein binding to the blot was
blocked by the incubation of the filter with 3% non-fat milk protein in
10 m
M Tris-HCl, pH 7.5, 150 m
M NaCl for 4 h at 25
°C. The blot was then probed with the indicated dilutions of crude
antisera overnight at 4 °C. After washing, blots were then probed
with a peroxidase-linked anti-rabbit IgG, and bands were visualized
with the ECL detection method (Amersham Corp.) and quantified by
densitometry (Molecular Dynamics Image Quant v. 3.0).
Site-directed Mutagenesis
A PCR-based
site-directed mutagenesis strategy was followed using as template the
rB20a clone that had been previously subcloned in the
XhoI- XbaI sites of pBluescript. Glycosylation sites
were eliminated sequentially, starting with mutations N182Q and N188Q
(mutant N2). Mutant N2 was used as template for mutation N172Q (mutant
N3), and mutant N3 was used as template for mutation N169Q (mutant N4).
This mutagenesis strategy is a modification of the method of Higuchi
(44) and is summarized in Fig. 1. The ``left'' PCR
product for the N2, N3, and N4 mutants was generated by using
oligonucleotides T3 (ATTAACCCTCACTAAAG) (sequence from the vector) and
B1 (CACTTTCCCTGAAGACTTGACTCCTCG) (sequence complementary to bases
715-741 of rB20a). The ``right'' PCR product for the N2
mutant was generated by using the oligonucleotides A2
(CTGTCTGGCCAACTGTCTCACCTGTTCCAATACACCTTG) (bases 535-573 of
rB20a) and KV1 (GGATGCCATGGTGATGAGG) (complementary to bases
930-948 of rB20a). For mutant N3, the ``right'' PCR was
produced with oligonucleotides A3 (TTCCAATCTCACCCAAGGCTCCCGGCCC) (bases
501-528) and KV1. For mutant N4, the ``right'' PCR was
produced with oligonucleotides A4 (GGATGCTTCCCAACTCACCCAAGGCTCCC)
(bases 495-523) and KV1. Underlined bases correspond to mutated
codons. For every mutant, the corresponding ``left'' and
``right'' PCR products were purified, denatured (94 °C,
10 min), annealed each other (50 °C, 5 h), and elongated with
Taq polymerase (72 °C, 10 min) in a volume of 50 µl.
Then, DNA contained in 5 µl of the elongation reaction was
reamplified by PCR using the external oligonucleotides T3 and KV1. The
amplified products were digested with XhoI and NcoI,
and the XhoI -NcoI fragment of the wild type rB20a
clone was exchanged for the mutated one. The XhoI site was
located between the T3 oligonucleotide and the starting ATG of rB20a,
and the NcoI restriction site was located in the KV1
oligonucleotide. This procedure yields 50% mutants and 50% of wild
type. Mutant clones were identified by sequencing, and then the
full-length clone was subcloned in the XhoI- XbaI
sites of the pSVL expression vector.
Immunofluorescence of Transfected
Cells
48 h after transfection, COS cells were washed three
times in PBS, fixed for 20 min at room temperature in 2%
paraformaldehyde in PBS, and rewashed three times in PBS. The fixed
cells were then incubated for 1 h at room temperature in PBS containing
1% bovine serum albumin and 0.02% digitonin. After that, fixed cells
were incubated for 2 h at room temperature in the same medium
containing rabbit anti-GLYT1 antibody diluted 1:1000. The cells were
washed, and bound primary antibodies were detected with FITC-coupled
goat anti-rabbit IgG for 60 min at room temperature. Cells were
thoroughly washed, and the cover glasses were mounted in 90% glycerol
plus Citifluor. Samples were visualized on a microscope Zeiss Axioskop.
Cell Surface Labeling
Cell surface
proteins of transfected COS cells were labeled with the cell membrane
impermeable reagent sulfo- N-succinimidyl-6-biotinamido
hexanoate as described
(45) . In brief, cells were washed with
PBS and incubated for 30 min in the presence of 0.5 mg/ml
sulfo- N-succinimidyl-6-biotinamido hexanoate in ice-cold PBS.
Then, cells were washed, and the excess of reagent was quenched with 10
m
M lysine in PBS for 10 min. Cells were lysed in
immunoprecipitation buffer (150 m
M NaCl, 50 m
M HEPES-Tris, 5 m
M EDTA, 1% Triton X-100, 0.25%
deoxycholate, 0.1% SDS, pH 7.4), and biotinylated proteins were
precipitated with agarose-streptavidin. Precipitated proteins were
fractionated by SDS-PAGE and analyzed by Western blot for
immunoreactivity with anti-GLYT1 antibodies, as described above.
Protein Determination
Protein
concentration was measured by the method of Bradford
(46) or
Peterson
(47) using bovine serum albumin as a standard.
-aminobutyric acid transporters in different cell types after
treatment with tunicamycin
(48, 49) . Fig. 2 shows
immunoblotting data with a specific antibody raised against the
carboxyl terminus of GLYT1. When proteins from GLYT1-transfected COS
cells were probed with this antibody, two bands of 57 and 80-100
kDa appeared in controls. Molecular mass of the GLYT1 transporter
expressed in COS cells slightly differs from that of native GLYT1 from
rat spinal cord membranes,
indicating differences in host
cell-specific posttranslational processing, a phenomenon that has been
also shown for the dopamine transporter
(50) . After treatment
with tunicamycin, a new band of 47 kDa was observed, while the
80-100-kDa band decreased in intensity, and the 57-kDa band
almost disappeared. The broad shape of the upper band is characteristic
of glycoproteins, reflecting in some cases heterogeneity in the
glycosylation pattern
(51) . This experiment suggests that the
57-kDa band of the wild type corresponds to a partially glycosylated
intermediate that disappears in the absence of further glycosylation to
become the 47-kDa fully deglycosylated protein. This intermediate
lasted much shorter than the upper form, whose amount was only reduced
by 39% after a 24-h treatment. A 24-h half-life has been reported for
the fully glycosylated form of the closely related norepinephrine
transporter
(49) . However, the interpretation of the effects of
tunicamycin is complicated by the fact that tunicamycin affects
glycosylation of many other proteins and reduces protein synthesis.
Thus, the observed effects on the transport activity could be
indirectly mediated by changing the activity of ion channels or the
(Na
-K
)-ATPase function and hence
altering ion concentrations both inside and outside of the cell.
H]glycine
transport in COS cells transfected with the wild type or the mutated
GLYT1 expression vector are presented in Fig. 3. The results show that
transport activity could be detected in all cases, confirming that each
of these forms of the transporter was expressed in the cells. However,
progressive mutation of the potential glycosylation sites produced a
progressive decrease in transport activity. When the four potential
glycosylation sites were mutated, only approximately 30% of the
activity was retained (average percent of initial rate steady state of
the wild type activity). All the active or partially active transporter
forms retained the characteristic sodium and chloride dependence (Table
I).
of the transport process did not change significantly (195
µ
M in the wild type versus 167 µ
M in
the N4 mutant), the V
decreased dramatically
(19.2 nmol/4 min/mg of protein in wild type versus 3.5 nmol/4
min/mg of protein in N4 mutant). According to enzyme kinetics,
V
is an indicator of total enzyme activity.
Thus, in the case of uptake kinetics, a decrease in V
of the transport system could be explained by defects in the
synthesis and/or turnover of the protein, by a decrease in the number
of available transport systems at the plasma membrane, or by a reduced
functioning of the transporter.
Figure 2:
Effect of tunicamycin on GLYT1 expression.
COS cells were transfected with GLYT1 (clone rB20a). After 48 h of
incubation at 37 °C in the COincubator, cells were
treated for 24 h with 10 µg/ml tunicamycin ( TN). Solvent
(ethanol) was added to control cells ( CT). MT,
mock-transfected cells. Cell protein was subjected to SDS-PAGE,
electroblotted onto nitrocellulose, and incubated with anti-GLYT1
antibody (1:250). Bands were visualized with the ECL detection
method.
Next, we investigated the
reasons for a decrease in the transport activity with deglycosylation.
In the experiment shown in Fig. 5( right blot),
we measured the amount of protein reaching the plasma membrane. For
that purpose, proteins from the cell surface were biotinylated with the
impermeant reagent sulfo- N-succinimidyl-6-biotinamido
hexanoate, and the biotinylated proteins were precipitated with
immobilized streptavidin, followed by quantitation in Western blot with
the anti-GLYT1 antibody. The results show that the protein arrived to
the plasma membrane with progressively more difficulties as the degree
of glycosylation decreased. This was especially clear in the mutant N3,
where the amount of total cellular GLYT1 protein was similar to that in
the wild type, whereas membrane GLYT1 was only 55% of the wild type
(Fig. 5). In the case of N4 mutant, there was also an important
decrease in the protein reaching the membrane. Considering as 100% the
fraction of total protein that reached the membrane in the wild type
(compare lane 2 in Fig. 5 versus lane
6), only 42% of total N4 protein arrived to the cell surface
( lane 5 in Fig. 5 versus lane 9).
Figure 5:
Subcellular distribution of GLYT1
immunoreactivity in transfected COS cells. COS cells were transfected
with the wild type GLYT1 (clone rB20a) ( WT), N2, N3, or N4
mutants or mock transfected ( C). After 48 h of incubation at
37 °C in the COincubator, total cell protein (total
protein) was solubilized, or cell surface proteins were biotinylated
and isolated with streptavidin-agarose beads (membrane protein).
Proteins were subjected to SDS-PAGE, electroblotted onto
nitrocellulose, and incubated with anti-GLYT1 antibody (1:250). Bands
were visualized with the ECL detection
method.
Further evidence of this difficulty of deglycosylated protein to be
transported to the plasma membrane was obtained by immunofluorescence
(Fig. 6). Whereas the plasma membrane was clearly labeled in wild type
cells, fluorescence remained mainly in the endoplasmic reticulum in the
N4 mutant. Considering together all these data, it can be concluded
that glycosylation is necessary for proper targeting of GLYT1 to the
plasma membrane, although a certain leakage exists, and a fraction of
the deglycosylated protein can reach the cell surface.
-H
antiporter of renal brush-border
membrane
(53) , the GLUT1 glucose transporter
(54, 55) , the organic cation transporter of renal
brush-border membrane
(56) , and a purified glycine transporter
probably corresponding to GLYT2
(39) are examples of such a
correlation. Moreover, N-glycosylation plays an important role
in the targeting to the membrane for the GLUT1 glucose transporter
(57) , the renal Na
-P
cotransporter
(58) , or the serotonin transporter
(59) . However,
glycosylation is not required for the transport activity of the
serotonin transporter
(59) or the intestinal
Na
-glucose transporter
(60, 61) .
Figure 7:
Effect
of glycosidase treatment on transport activity and electrophoretic
mobility of reconstituted native GLYT1. Panel A, COS cells
transfected with wild type GLYT1 were solubilized, reconstituted into
liposomes, and treated with N-glycosidase F (500 units/µg
of protein). 1, mock-transfected COS cells; 2,
control non-treated GLYT1-transfected COS cells; 3, control
GLYT1-transfected COS cells after 3 h at 37 °C; 4,
GLYT1-transfected COS cells after 3 h of treatment with
N-glycosidase F; 5, control GLYT1-transfected COS
cells after 5 h at 37 °C; 6, GLYT1-transfected COS cells
after 5 h of treatment with N-glycosidase F. The results are
means ± S.E. of two triplicate determinations. Values were
compared with control values. Result in bar 4 was not
significant versus its control in bar 3. Result in
bar 6 was not significant versus its control in
bar 5. Uptake value for nontreated GLYT1-transfected cells is
20.02 pmol/15 min/mg of protein (100%). Panel B, 25 µg of
protein of the same fractions used in A were electrophoresed,
electroblotted onto a nitrocellulose membrane, and incubated with
anti-GLYT1 antibody (1:250). Bands were visualized with the ECL
detection method.
In
summary, our data demonstrate that the mature form of the glycine
transporter GLYT1 contains four Asn-linked glycosylation sites located
extracellularly in the hydrophilic domain between the predicted third
and fourth membrane-spanning segments. Although impairment of the
transporter glycosylation either by tunicamycin treatment or by
site-directed mutagenesis leads to a decrease in glycine transport,
N-glycosylation of the GLYT1 glycine transporter is not
absolutely essential for the transport activity itself. Instead,
glycosylation appears to play an important role in the proper
trafficking of the transporter to the plasma membrane.
Table: Ionic dependence of glycine transport
mutants in COS cells
incubator, uptake was assayed at 37 °C in the presence of 0.2
µ
M [
H]glycine for 10 min in
HEPES-buffered saline containing 150 m
M NaCl (HBS) or a
modified HBS where NaCl had been isotonically substituted by LiCl
(-Na
) or sodium gluconate
(-Cl
). The results are the means ± S.E.
of two triplicate determinations. Values were compared with control
values by using Student's t test.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.