(Received for publication, August 1, 1996, and in revised form, October 5, 1996)
From the Centro de Biología Molecular "Severo Ochoa," Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain
A theoretical 12-transmembrane segment model based on the hydrophobic moment has been proposed for the transmembrane topology of the glycine transporter GLYT1 and all other members of the sodium- and chloride-dependent transporter family. We tested this model by introducing N-glycosylation sites along the GLYT1 sequence as reporter for an extracellular localization and by an in vitro transcription/translation assay that allows the analysis of the topogenic properties of different segments of the protein. The data reported herein are compatible with the existence of 12 transmembrane segments, but support a rearrangement of the first third of the protein. Contrary to prediction, hydrophobic domain 1 seems not to span the membrane, and the loop connecting hydrophobic domains 2 and 3, formerly believed to be intracellular, appears to be extracellularly located. In agreement with the theoretical model, we provide evidence for the extracellular localization of loops between hydrophobic segments 5 and 6, 7 and 8, 9 and 10, and 11 and 12.
Glycine is a major inhibitory neurotransmitter in the spinal cord
and the brain stem of vertebrates. In addition, glycine can potentiate
the action of glutamate, the main excitatory neurotransmitter in the
brain, on postsynaptic N-methyl-D-aspartate
receptors. The re-uptake of glycine into presynaptic nerve terminals or
the neighboring fine glial processes provides one way of clearing the
extracellular space of this neuroactive substance and so constitutes an
efficient mechanism by which the postsynaptic action can be terminated
(1, 2, 3). This process is carried out by two different glycine
transporters, named GLYT1 and GLYT2, which belong to the
Na+- and Cl-dependent
neurotransmitter transporter family (4, 5, 6, 7, 8, 9). GLYT1 and GLYT2 present a
differential expression pattern among central nervous system cells (7,
10, 11, 12, 13, 14).
The hydropathic profiles of the Na+- and
Cl-dependent neurotransmitter transporters
reveal the presence of 12 hydrophobic segments that have been suggested
to form transmembrane
-helices (15). With the aid of
sequence-specific antibodies, immunofluorescence, and electron
microscopy, it has been shown that both the amino- and
carboxyl-terminal ends of GLYT1 are intracellularly located (11, 16).
Additional topological data have been obtained from the study of the
glycosylation pattern of GLYT1. Site-directed mutagenesis has shown
that GLYT1 is heavily glycosylated at four asparagine residues
(Asn169, Asn172, Asn182, and
Asn188) (17). This fact involves an extracellular
localization of the hydrophilic loop placed between hydrophobic
segments 3 and 4.
In this report, we present an extensive experimental evaluation of a neurotransmitter carrier protein transmembrane topology. The data included are compatible with a 12-membrane spanning segment model, with evidence for the extracellular localization of loops connecting hydrophobic domains 5 and 6, 7 and 8, 9 and 10, and 11 and 12. However, data herein favor an extracellular localization of the loop connecting HD2 1 and HD3, intracellularly located in the theoretical model. These observations, together with the previous demonstration of intracellular N and C termini and the extracellular localization of the loop placed between HD3 and HD4, provide an extensive experimental description of the GLYT1 transmembrane topology.
The production and characterization of antibodies against a fusion protein containing the 76 C-terminal amino acids of GLYT1 have been described (12).
Site-directed MutagenesisThe polymerase chain reaction
(PCR)-based site-directed mutagenesis strategy followed to insert new
glycosylation sites in GLYT1 was a modification of the method of
Higuchi (18) as described (17). A deglycosylated form of the rB20a
clone that had been previously subcloned in the
XhoI-XbaI sites of pBluescript (construct N4) was
used as template. Oligonucleotides (Isogen Bioscience) were designed to
introduce the mutations listed in Fig. 2. The different mutated PCR
fragments were introduced into construct N4 with the adequate flanking
restriction sites. Mutant clones were identified either by sequencing
or by restriction analysis: the glycosylation cluster sequence NNST
introduced a ScaI restriction site, and the cluster NNTS
introduced a SpeI site. Mutant clones were characterized by
sequencing. Finally, the full-length clones were transferred from
pBluescript to the XhoI-XbaI sites of the pSVL
expression vector.
Expression in COS Cells
Transient expression in COS cells was carried out using DEAE-dextran (Pharmacia Biotech Inc.) with dimethyl sulfoxide according to the method of Kaufman (19) with minor modifications (16).
Electrophoresis and BlottingSDS-polyacrylamide gel electrophoresis (PAGE) and blotting were performed as described previously (16). Bands were visualized with the ECL detection method (Amersham Corp.) and quantified by densitometry (Molecular Dynamics ImageQuant Version 3.0).
H+,K+-ATPase CloningA cDNA coding for the 177 C-terminal amino acids of the rat H+,K+-ATPase was cloned by PCR from a rat gastric cDNA, obtained by reverse transcription from total RNA. cDNA was amplified with Taq polymerase, using as primers the following oligonucleotides: GCGGATCCAGCTTCTTAGCGGGCTACACCCCAGCA (ATP1) and GCGAATTCTTACTTCTGTATTGTGAGCTTGAACT (ATP2). The 561-base pair fragment was digested with BamHI and EcoRI and ligated into a Bluescript vector digested with BamHI and EcoRI (vector ATPase-Bluescript).
Vector ConstructionTo analyze the topogenic properties of different GLYT1 fragments, a series of vectors was constructed using the ATPase-Bluescript vector as the starting point. To generate diverse GLYT1-ATPase fusion proteins, various GLYT1 fragments were produced by PCR and ligated in the correct reading frame upstream of the ATPase, under the control of the T7 promoter. All the GLYT1 fragments started with the 37 N-terminal amino acids, which would anchor the N terminus of the different fusion proteins to the cytoplasmic side of the membrane. Between the amino-terminal segment and the ATPase, we introduced different internal segments of GLYT1. Two possibilities existed. If the truncated GLYT1 segment was the natural continuation of the 37 N-terminal amino acids, a single GLYT1 PCR fragment including both the N-terminal segment and the desired amino acids was produced for each construct. These PCR fragments were flanked by restriction sites for XbaI and BamHI and were ligated in the XbaI-BamHI sites of the ATPase-Bluescript vector. Alternatively, if the variable GLYT1 segment was not contiguous to the N-terminal fragment, vectors were constructed in two steps. First, a fragment corresponding to the 37 N-terminal amino acids was introduced in the SacI-XbaI sites of the ATPase-Bluescript vector, and then the desired GLYT1 fragments were ligated into the XbaI-BamHI sites of the latter construct. Oligonucleotides were flanked with the above-mentioned restriction sites, and after digestion and ligation, the reading frame of the fusion protein was always correct. Constructs were confirmed by DNA sequencing with the fmolTM kit (Promega).
In Vitro Transcription/TranslationFollowing linearization of the plasmids by EcoRI, RNA was synthesized using T7 RNA polymerase with the RNA synthesis and capping kit from Stratagene. Protein was synthesized from 0.5 µg of synthetic RNA using a reticulocyte lysate system (Promega) in the presence of [35S]methionine (Amersham Corp.) for 1 h at 30 °C according to the manufacturer's instructions. Translation products were separated by 10% SDS-PAGE (minigels), fixed in methanol/acetic acid, dried, and subjected to autoradiography. Translation reactions containing canine microsomes (Promega) were centrifuged for 15 min at 15,0000 × g through 100 mM KCl, 0.5 M sucrose, and 50 mM HEPES, pH 7.5. The pellets were resuspended in electrophoresis loading buffer.
Enzymatic DeglycosylationTo confirm the variation in the
electrophoretic mobility of the different COS cell-expressed
glycosylation tagged mutants or in vitro translated fusion
proteins, transfected COS cells or microsomal pellets were resuspended
in denaturing buffer (0.5% SDS, 1% -mercaptoethanol) and boiled
for 10 min. Then, sodium phosphate and Nonidet P-40 were added (50 mM and 1% final concentrations, respectively), followed by
5 units of peptide N-glycosidase F (New England Biolabs
Inc.). Samples were incubated for 2 h at 30 °C prior to
SDS-PAGE.
To confirm insertion of translated proteins, products obtained by translation in the presence of microsomes were diluted to 50 µl with HEPES, pH 11.5. After 10 min on ice, the samples are loaded onto a 100-µl cushion of 0.2 M sucrose, 60 mM HEPES, pH 11, 150 mM potassium acetate, 2.5 mM magnesium acetate, and 1 mM dithiothreitol in a nitrocellulose tube for the TL100.1 rotor and centrifuged for 10 min at 4 °C in a Beckman TL100 centrifuge at 50,000 rpm. The pellet fractions were taken up in SDS gel sample buffer, and samples were electrophoresed as described above.
Glycine Transport AssayTransport assays in transfected COS cells were performed as described previously (20).
Protein DeterminationProtein concentration was measured by the method of Bradford (21) using bovine serum albumin as a standard.
We
designed a series of mutants of GLYT1 by introducing
N-glycosylation consensus sequences (NX(S/T))
(22) at different sites along the entire length of the protein and
analyzed the mutant transporter for glycosylation at the newly
engineered sites in a COS cell expression system. Glycosylation of
membrane and secretory glycoproteins occurs only on the luminal side of
the endoplasmic reticulum and Golgi apparatus membranes (23), which coincides with the extracellular face of the protein. Crude protein fractions obtained from transiently transfected COS cells were separated by SDS-PAGE, blotted onto nitrocellulose filters, and probed
with an antibody directed against the carboxyl-terminal end of the
protein (11, 12). In this experimental system, native GLYT1 is
expressed as two bands (Fig. 1). Previous studies from
our laboratory have shown that the upper band, ranging from 70 to 100 kDa, corresponds to the fully processed protein, whereas the lower band
of 57 kDa corresponds to a partially glycosylated form of GLYT1 (17).
Owing to the heterogeneous pattern of glycosylation of mature GLYT1,
which could complicate interpretation of results, we did not use native
GLYT1 as a starting point, but instead used a form of the protein in
which the four native glycosylation sites (N169Q, N172Q, N182Q, and
N188Q) had been eliminated by mutation. This form of the protein
(mutant N4) (Fig. 1), with a molecular mass of 47 kDa, has previously
been shown not to be properly delivered to the cell surface, but is
fully functional in transport assays after solubilization and
reconstitution into liposomes, indicating that the oligosaccharide
moiety is not required for proper folding of the protein (17). Since
some of the newly introduced glycosylation sites were often not used,
usually we had to introduce two to five clustered sites within a narrow
region of the protein (Fig. 2) in order to maximize the
probability that at least one of the sites would become glycosylated.
When the mobility of the various GLYT1 mutants was compared with that
of mutant N4, a variation was observed in constructs IL1b, EL3, EL4,
and EL5 (named according to the theoretical model) (Fig.
3). However, the size of GLYT1 protein from mutants IL3,
IL4, IL5, EL1, and EL6 was similar to that of N4 protein (Fig. 3). The
increase in the size of the band observed in constructs IL1, EL3, EL4,
and EL5 was due to glycosylation, as treatment of the crude protein
fraction with peptide N-glycosidase F prior to SDS-PAGE
yielded the fully deglycosylated 47-kDa form of the protein (Fig.
4). Negative results obtained with the rest of the
constructs were not further investigated, as the lack of glycosylation
can be due either to intracellular localization of the epitope or to
inability of the glycosylation machinery to recognize the new
sites.
To assay the function of constructs IL1b, EL3, EL4, and EL5, the natural glycosylation sites (N169Q, N172Q, N182Q, and N188Q) were reintroduced in the constructs, and the transport activity was determined in transfected COS cells. Biotinylation of cell-surface proteins with the impermeant reagent N-succinimidyl 6-biotinamidohexanoate indicated that proteins IL1b, EL3, EL4, and EL5 were delivered to the membrane with an efficiency similar to that observed with native GLYT1 (data not shown). However, the rates of [3H]glycine transport suffered a dramatic reduction as compared with the wild type (Table I).
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The absence of information about the
topology of a part of the protein and the necessity of confirmation of
the results obtained by the former technique prompted us to use an
independent methodology that allowed us to analyze the topogenic
properties of presumptive transmembrane segments. Vectors were
constructed to allow insertion of different GLYT1 segments, presumed to
be membrane spanning sequences, between the cytoplasmically anchored
N-terminal sequence of GLYT1 and a reporter sequence containing several
N-linked glycosylation consensus sequences (Fig.
5, A and B). mRNAs from the
different constructs were obtained by in vitro transcription
and then translated in a rabbit reticulocyte lysate system in either
the absence or presence of canine microsomes. The 177-amino acid
carboxyl terminus of the H+,K+-ATPase
-subunit, which contains five glycosylation sites, was chosen as a
reporter because it has been previously used in a similar system and
shown to contain neither anchoring nor stop transfer signals (24).
Glycosylation of the fusion protein by the microsomes was visualized by
a shift in relative molecular mass after SDS-PAGE and autoradiography
of the gels. The increase in molecular mass indicates insertion of the
protein in the microsomal membrane and translocation of the reporter
present in the C-terminal part of the fusion protein. This involves the
presence of at least an anchor signal within the analyzed sequence.
Absence of glycosylation indicated either the absence of membrane
insertion or the presence of an even number of membrane spanning
segments, the last one containing a stop transfer sequence (Fig. 5,
A and B). Table II summarizes the
amino acid sequences of the different constructs used in this
study.
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To study the
transmembrane topology of the segment of the protein placed between the
intracellular N terminus and the extracellular loop containing the
native N-glycosylation sites, we produced a series of
plasmids as shown in Fig. 5C. First, we analyzed the ability
of individual predicted membrane spanning segments to promote
translocation of the ATPase (Fig. 6A). Our
data indicated that, contrary to the prediction of the theoretic model,
hydrophobic domain 1 (amino acids 38-64 in the variable region;
construct 1) was not able to promote translocation of the ATPase to the microsomal lumen. Similar results were obtained with a longer construct
(amino acids 38-69 in the variable region) (data not shown). However,
hydrophobic domain 2 (construct 2) as well as hydrophobic domains 3 (construct 3) and 4 (construct 4), when assayed individually, were able
to traverse the membrane, indicating that these segments on their own
are able to act as signal anchor sequences. Interestingly, construct 5, which contained only the predicted loop EL2, was glycosylated,
indicating the existence of an unpredicted anchor sequence in this
loop. Next, we placed in the variable region of the ATPase vector
segments containing several hydrophobic domains of GLYT1 (Fig.
6B). Translation products from constructs 6 and 7, carrying
HD2 and HD3, were glycosylated independently of the inclusion of the
natural glycosylation sites (construct 7) or not (construct 6). The
inclusion of HD1 in the constructs did not affect the glycosylation
pattern; for instance, fusion proteins containing HD1 and HD2
(construct 9) or HD1, HD2, and HD3 (construct 10) were glycosylated.
The inability of HD1 to traverse the membrane and the presence of an
anchor signal in EL2 strongly support a rearrangement of the topology
for this region of GLYT1, with IL1 being extracellular and the flanking transmembrane segments HD2 and HD3 displaying an orientation reversed from that proposed by the theoretical model. According to the new
model, HD3 should be the second membrane spanning segment, and thus, it
should contain a stop transfer sequence. However, this signal was not
identified in our experiments, as glycosylation of the reporter was not
prevented by HD3 in constructs 6 and 9.
The insertion into the membrane of all the constructs was routinely assessed by alkaline extraction (at pH 11.5) of the microsomes. This method is known to strip the membrane of peripheral proteins and hence is a useful empirical procedure to distinguish between integral membrane proteins and soluble or peripheral proteins. Products from the constructs, depicted in Fig. 6 (A and B), either glycosylated or not, were recovered in the microsomal pellet, indicating that these fusion proteins are integral membrane proteins. The elevation in the relative molecular mass, when observed, was due to glycosylation, as treatment of translated protein in microsomal membranes with peptide N-glycosidase F reduced the molecular mass to that observed in the absence of microsomes. Fig. 6C shows the deglycosylation pattern produced by peptide N-glycosidase F on fusion proteins obtained from constructs 1-3 and 5. Similarly, deglycosylation was observed with constructs 4 and 7-10 (data not shown).
Structure of the Central and Carboxyl-terminal Parts of GLYT1The topogenic properties of sequences representing HD5
through HD12 were analyzed first by inserting each individual
hydrophobic sequence into the ATPase vector. The results are summarized
in Fig. 7. Fusion proteins containing any of the
individual segments from HD5 to HD12, except HD6, were inserted into
the membrane and glycosylated. The results indicate that all these
hydrophobic sequences, except HD6, individually considered were able to
act as signal anchor sequences. According to the theoretical model, the
even-numbered hydrophobic domains should act as stop transfer signals
for the previous domain. We analyzed the glycosylation pattern of
fusion proteins produced by translation of constructs carrying pairs of
hydrophobic domains: constructs 19 (HD5 and HD6), 20 (HD7 and HD8), 21 (HD9 and HD10), and 22 (HD11 and HD12). None of these fusion proteins
resulted glycosylated (Fig. 7) in the presence of microsomes, but they
were all inserted into the membrane as they were resistant to alkaline
extraction. These results indicate that, as expected, HD6, HD8, HD10,
and HD12 acted as stop transfer sequences when placed after HD5, HD7,
HD9, and HD11, respectively. These data support results obtained by the glycosylation scanning technique, confirming the extracellular localization of EL3, EL4, and EL5 and supporting as well the
extracellular localization of EL6.
We have made a systematic investigation of the membrane topology of GLYT1 by inserting new glycosylation consensus sequences along the entire length of this protein, followed by analysis of the mutant protein expressed in COS cells. The analysis has been complemented by an independent biochemical method consisting of the insertion of a glycosylation reporter after proposed transmembrane domains and analysis of the glycosylation pattern in an in vitro translation system. On the basis of hydropathy plots, it has been proposed that GLYT1, as all other members of its gene family, contains 12 transmembrane segments, a number also shared by carrier proteins without sequence homology to the sodium- and chloride-dependent transporter family (25, 26, 27). Our results are consistent with a 12-transmembrane model, but support a rearrangement in the first third of the protein.
As with most of the available techniques to study the transmembrane
topology of proteins, the two techniques used in this report present
some limitations. For instance, the introduction of new glycosylation
sites throughout the protein has the potential to alter the topology.
In addition, the transmembrane topology could also be altered by
truncation and addition of the large reporter protein. Thus, the most
compelling conclusions would be those supported by convergent results
of independent methods. In this report, data from both methodologies
are coincident with respect to the structure of the second third of the
protein, and they support the theoretical model. The glycosylation
tagging technique shows that loops EL3, EL4, and EL5 are glycosylated after the introduction of a new glycosylation consensus sequence, suggesting an extracellular location. In vitro transcription
experiments support this structure, with HD6, HD8, and HD10 acting as
stop transfer signals for the respective preceding hydrophobic domains HD5, HD7, and HD9, which would act as membrane anchor signals. Consistent with these observations, loop EL4 of the norepinephrine transporter has also been shown to be extracellular by using
anti-peptide antibodies (28). With the glycosylation tagging technique,
we did not observe glycosylation of loop EL6. However, absence of glycosylation does not disprove an extracellular localization, as it
may simply be a consequence of steric hindrance (22, 29, 30). In fact,
data from in vitro transcription support an extracellular localization for this loop, as both HD11 and HD12 are able to traverse
the membrane, with acting HD12 as a stop transfer signal when placed
after HD11. Moreover, in the accompanying paper, Bennett and Kanner
(31) have obtained evidence for glycosylation of EL6 of the
-aminobutyric acid transporter GAT-1 by using the glycosylation
tagging technique. These results are consistent with the intracellular
location of the C terminus of GLYT1 (17).
As far as the first third of the protein is concerned, previous studies
on GLYT1, GLYT2, dopamine, and serotonin transporter topologies have
shown that the N terminus of this family of proteins is intracellular
(11, 16, 27, 32). In addition, the four native glycosylation sites of
GLYT1 and those of the serotonin transporter, in the loop connecting
HD3 and HD4, are extracellularly located (17, 33). These observations
imply an odd number of transmembrane domains preceding the first
natural glycosylation site, Asn169. The glycosylation
tagging technique supports an extracellular localization of the loop
connecting HD2 and HD3, a structure that is contrary to that predicted
by the theoretical model (Fig. 8, A and
B). Similar observations have been made in the accompanying paper by Bennett and Kanner (31) for the -aminobutyric acid transporter GAT-1. Consistent with this model, in vitro
translation indicates that HD1 on its own is not able to traverse the
membrane. Moreover, we have been able to identify three segments with
ability to span the membrane in this region of the protein: HD2, HD3, and a stretch of amino acids in the glycosylated loop EL2.
Nevertheless, still some caution should be observed in the
interpretation of these results, as the model in Fig. 8A,
which fulfills the above-mentioned observations, predicts HD3
containing a stop transfer signal. However, constructs carrying HD2 and
HD3 (constructs 6 and 9) were glycosylated in the presence of
microsomes, indicating the absence of stop transfer signals in the
assayed sequence. Nevertheless, one has to keep in mind the limitation
of the in vitro transcription technique, and perhaps the
behavior of these sequences in the context of the native protein would
be as predicted by this model. Model in Fig. 8A proposes HD1
as a reentrant loop, similar to the one described for the glutamate
receptors (34, 35) or the potassium channel (36), as opposed to a
completely intracellular HD1. Although the data in this report are
insufficient to support this arrangement of HD1, the fusion protein
obtained from translation of construct 1 was not separated from the
membrane by the alkaline wash of the microsomes, indicating a close
association of this domain with the membrane. Moreover, some data
obtained for other members of the gene family support the idea that HD1
is associated with the membrane. This domain is highly conserved among
the members of the sodium- and chloride-dependent
transporter family. Arginine 69 of the
-aminobutyric acid
transporter GAT-1, which is located in this domain and also present in
GLYT1, seems to be important for the control of the permeation process
(37). Moreover, aspartate 59, present in the equivalent domain of the
dopamine transporter, has been suggested to interact with the amine
group of dopamine during the translocation process (38). These data
suggest that this domain must be close to the permeation pore and thus
probably is associated with the membrane.
In spite of the above-mentioned methodological limitations, the
transmembrane structure of various eukaryotic proteins has been
analyzed with these techniques. The glycosylation tagging method has
been used to study the structure of the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type of
glutamate receptor (34), the cystic fibrosis transmembrane conductance
regulator (39), and the Na+/glucose cotransporter SGLT1
(40). The gene fusion system has been used extensively for mapping the
transmembrane topology not only of bacterial proteins, but also of
eukaryotic proteins. For instance, a similar gene fusion technique
using the ATPase
-subunit as reporter has been used to determine the
transmembrane structure of the H+,K+-ATPase
-subunit (24). The same technique has also been employed with other
reporters, notably the large prolactin epitope, used to determine the
topology of the
- and
-subunits of the nicotinic receptor (41),
or the structure of the glutamate receptor subunit GluR3 (35). It is
clear that all these techniques provide indirect evidence regarding
membrane topology; however, in the absence of more direct information
provided by x-ray crystallography or averaging of two-dimensional high
resolution electron micrographs, the transmembrane topology of
polytopic proteins must be deduced from the sidedness of identified
protein regions.
Knowledge of the correct transmembrane topology of GLYT1 is critical for understanding important aspects of the structure-function relationships such as the determination of substrate-binding sites, the membrane spanning domains that participate in forming the pore, or post-translational modifications. Moreover, much of the available topological data suggest that other members of the sodium- and chloride-dependent neurotransmitter transporter family may show a similar profile.
We thank Drs. K. E. Smith and R. L. Weinshank (Synaptic Pharmaceutical Corp.) for providing the rB20a clone. We also thank E. Nuñez for excellent technical assistance.