From the Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom and ¶ Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218-2694
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ABSTRACT |
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The serotonin transporter (SERT) is an
N-glycosylated integral membrane protein that is predicted
to contain 12 transmembrane regions. SERT is the major binding site in
the brain for antidepressant drugs, and it also binds amphetamines and
cocaine. The ability of various molecular chaperones to interact with a
tagged version of SERT (Myc-SERT) was investigated using the
baculovirus expression system. Overexpression of Myc-SERT using the
baculovirus system led to substantial quantities of inactive
transporter, together with small amounts of fully active and,
therefore, correctly folded molecules. The high levels of inactive
Myc-SERT probably arose because folding was rate-limiting due, perhaps,
to insufficient molecular chaperones. Therefore, Myc-SERT was
co-expressed with the endoplasmic reticulum (ER) molecular chaperones
calnexin, calreticulin and immunoglobulin heavy chain binding protein
(BiP), and the foldase, ERp57. The expression of functional Myc-SERT, as determined by an inhibitor binding assay, was enhanced nearly 3-fold
by co-expressing calnexin, and to a lesser degree on co-expression of
calreticulin and BiP. Co-expression of ERp57 did not increase the
functional expression of Myc-SERT. A physical interaction between
Myc-SERT-calnexin and Myc-SERT-calreticulin was demonstrated by
co-immunoprecipitation. These associations were inhibited in vivo by deoxynojirimycin, an inhibitor of N-glycan
precusor trimming that is known to prevent the
calnexin/calreticulin-N-glycan interaction. Functional
expression of the unglycosylated SERT mutant, SERT-QQ, was also
increased on co-expression of calnexin, suggesting that the interaction
between calnexin and SERT is not entirely dictated by the
N-glycan. SERT is the first member of the neurotransmitter transporter family whose folding has been shown to be assisted by the
molecular chaperones calnexin, calreticulin, and BiP.
The serotonin transporter
(SERT)1 is a member of a
small family of integral membrane proteins that transport
neurotransmitters and osmolytes into cells by coupling uptake to the
influx of Na+ and Cl In addition to N-glycosylation, the large extracellular loop
between transmembrane domains 3 and 4 may also contain a disulfide bond
between two Cys residues 8 amino acids apart. Alteration of either Cys
residue severely reduced cell surface expression of functional SERT or
the dopamine transporter, but replacement of both Cys residues led to
only a slight reduction in activity (10, 11). The two Cys residues
proposed to form the disulfide bond are absolutely conserved throughout
this neurotransmitter transporter family, suggesting that the disulfide
bond may be another essential structural feature for efficient, stable,
cell surface expression.
Molecular chaperones are a diverse collection of proteins that interact
transiently with unfolded and misfolded proteins, thus preventing
improper protein-protein interactions until the protein folds correctly
(reviewed in Refs. 12 and 13). When the protein reaches its final
correct three-dimensional structure, the molecular chaperone is
released. If the protein never folds correctly, then the interaction
between the molecular chaperone and the misfolded protein may be more
stable (12, 13). A few membrane proteins, including some ion channels
(14, 15), transporters (16-19), and G protein-coupled receptors
(20-22) have been shown to interact in vivo with various
molecular chaperones. Most molecular chaperones, including
immunoglobulin heavy chain binding protein (BiP), interact directly
with the polypeptide chain of the unfolded protein (23), but two
molecular chaperones (calnexin and calreticulin) have been shown to
interact primarily with a precursor N-glycan attached to the
proteins (24-27). Calnexin contains a single transmembrane domain that
anchors it to the ER, where it is thought to play a crucial role in
retention of misfolded proteins (24, 28). Despite being related at the
sequence level, calreticulin and calnexin often show different binding
characteristics to an unfolded protein (27, 29-31). The protein
disulfide isomerase ERp57 also acts only on N-glycosylated
proteins (32, 33), but this is because of its recruitment by either
calnexin or calreticulin (34). The requirement of the neurotransmitter
transporters for N-glycosylation for efficient functional
expression suggests that N-glycan-specific molecular
chaperones and foldases may be involved in their synthesis. In this
paper we test this hypothesis by the co-expression of calnexin,
calreticulin, and ERp57 with SERT in insect cells using the baculovirus
expression system.
The baculovirus expression system is currently used to overexpress many
glycosylated integral membrane proteins in sufficient quantities for
purification and structural studies (35, 36). Expression of SERT in
insect cells using recombinant baculoviruses normally results in about
500,000 copies/cell of functional SERT (determined by a binding assay
using the cocaine analogue 125I-RTI55) and substantially
greater amounts of inactive transporter (6). The amount of inactive,
unglycosylated, transporter increases dramatically after day 2 post-infection when there is no further increase in the amount of
functional SERT expressed. One explanation for this is that the folding
of SERT, presumably co-translational (37, 38), has become uncoupled
from membrane insertion and protein translation. If folding has become
the rate-limiting step during the overexpression of SERT, then
increasing the amounts of specific molecular chaperones or foldases
that help SERT to fold could increase the amount of functional SERT
expressed. Consequently, a selection of different folding assistance
factors, including the N-glycan binding molecular chaperones
(calnexin, calreticulin), an Hsp70 family member (BiP), and the foldase
ERp57 were co-expressed with SERT in insect cells to examine their
roles in the generation of active SERT. The specific folding factors
required by SERT in vivo are not yet known, so any molecular
chaperones or foldases that improve the overexpression of functional
SERT in insect cells can be regarded as potential candidates for
assisting the folding of SERT in native tissues.
Construction of Recombinant Baculoviruses--
A cDNA
encoding Myc-SERT was constructed from SERT-TAG (6) by ligating an
oligonucleotide encoding a calmodulin binding domain in the unique
NcoI site around the initiator Met codon, and an
oligonucleotide encoding a His10 tag was ligated in the NotI/XbaI sites before the termination codon. All
molecular biology techniques were as described in Ausubel et
al. (39). The predicted amino acid sequence of Myc-SERT at the N
and C termini is as follows (SERT amino acid residues are underlined,
and the cMyc tag is in bold).
MGKRRWKKNFIAVSAANRFKKISSSGALMEQKLISEEDLNMETT. . . . .
NAVAAAHHHHHHHHHH. All oligonucleotides ligated into the SERT-TAG
cDNA were checked by DNA sequencing. The Myc-SERT cDNA was
ligated into plasmid pVL1392 (Pharmingen), and a recombinant
baculovirus was constructed using Baculogold (Pharmingen) exactly as
described by the manufacturer. Recombinant viruses were isolated after
one round of plaque purification and amplified using standard
techniques (40, 41). A recombinant baculovirus expressing ERp57 was
constructed from the cDNA ligated in pBlueBacIII (42) (a kind gift
from M. Bourdi) using the Bac-N-Blue transfection kit (Invitrogen). A
baculovirus-expressing calreticulin was constructed from the human
calreticulin cDNA (43) (a kind gift from D. H. Llewellyn) by
ligating it into pVL1392 and using the BacVector-3000 transfection kit
(Novagen) to construct the recombinant baculovirus. Construction of the
murine chaperone BiP baculovirus has been described previously (44).
The virus containing the canine calnexin gene (45) was constructed
using the BacPAK baculovirus system and techniques
(CLONTECH).
Expression of SERT and Molecular Chaperones in Insect
Cells--
Methods for culturing and infecting insect cells (40, 41)
were exactly as described in (6, 46). Two different conditions were
used for expression experiments. For comparing the effects of molecular
chaperones on the expression of SERT, 100-ml cultures containing 1 × 106 cells/ml were infected with recombinant
baculoviruses at an multiplicity of infection of 5. Cultures were grown
at 27 °C in stirred flasks (Techne, Cambridge, UK) that were stirred
at 60 rpm. Aliquots were removed at specific time points, and membranes
were prepared (see below). For experiments involving 1-deoxynojirimycin
(Sigma), 2 × 106 cells were grown in
25-cm2 flasks containing 4 ml of medium; all the cells were
harvested after incubation at 27 °C for a specified time. The medium
used in all these experiments was TNM-FH insect medium (Sigma)
supplemented with 10% bovine calf serum (PAA Laboratories, Austria)
and 0.1% Pluronic F68 (Sigma).
Membrane Preparation and Western Blotting--
Membranes (1 ml)
were prepared from 1 × 107 cells as described by Tate
(46) by shearing the cells through a 26-gauge needle in 0.1×
phosphate-buffered saline containing 1 mM
phenylmethylsulfonyl fluoride and 2.5 µg/ml leupeptin. Western
blotting and SDS-PAGE were performed using standard techniques (39).
Western blots were prepared using ECL-nitrocellulose (Amersham
Pharmacia Biotech) and developed using the enhanced chemiluminescent
system (ECL, Amersham Pharmacia Biotech). Antibodies against mammalian
calnexin (C terminus), BiP (Grp78), and ERp57 were purchased from
StressGen (Victoria, Canada). A culture supernatant of anti-cMyc
antibody was prepared from the hybridoma cell line 9E10 (47) and was used without further purification. An anti-SERT antibody (CT2A) that
recognizes the C terminus of SERT (48) was a kind gift from R. Blakely.
All the antibodies used in Western blots were incubated for 1 h at
room temperature at a dilution of 1:500 in 5% nonfat milk in TBST (20 mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween 20).
Inhibitor Binding Assays for SERT--
Binding assays were
performed as described (6, 46). Briefly, crude membranes were incubated
with 2 nM 125I-RTI55 in the presence or absence
of 200 µM serotonin to determine specific binding.
Membranes were pelleted by centrifugation (13,000 × g,
5 min), the supernatant was removed, and the radioactivity in the
membranes was determined using a gamma counter. Assays were performed
in triplicate. Total binding represented 10% or less of the total
available 125I-RTI55. Typically 1 µl of membranes
containing Myc-SERT (as prepared above) was used per binding assay, but
10 µl of membranes containing SERT-QQ were used, because it is
expressed at about 20-fold lower levels than glycosylated SERT.
Immunoprecipitations and Their Analysis--
Membrane samples
were treated with 1% digitonin (Sigma-Aldrich) buffer containing 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) to
solubilize membrane proteins. Crude membrane samples were clarified at
16,000 × g for 15 min to separate aggregates from
soluble fractions. Protein A-Sepharose beads (50 µl/sample)were
washed twice with cold phosphate-buffered saline and incubated with 5 µl of anti-cMyc antibody (9E10) or 1% anti-SERT antibody in 50 µl
of phosphate-buffered saline for 1 h at 4 °C on a rotator.
After incubation, 100 µl of clarified sample was added to each tube
and immunoprecipitated overnight on a rotator at 4 °C. Samples were
then centrifuged and washed four times with cold phosphate-buffered
saline. Immunoprecipitates were analyzed by SDS-PAGE under reducing and
denaturing conditions. The gel was then transferred onto a
nitrocellulose membrane (Life Technologies, Inc.) and incubated
overnight in 10% skim milk (ICN) in TBST on an orbital shaker.
Subsequent to blocking, nitrocellulose membranes were washed three
times for 15 min in TBST and then incubated for 1 h at room
temperature in primary antibody solution (2% skim milk (ICN), 20%
fetal bovine serum (Life Technologies, Inc.), 1 mM
phenylmethylsulfonyl fluoride (Sigma-Aldrich), 0.02% Thimerosol
(Sigma-Aldrich) containing primary antibody (either anti-calnexin C
terminus (StressGen) at 1:6000 or anti-calreticulin (StressGen) at
1:8000). After 3 15-min washes in TBST, the blots were incubated for
1 h in 1:15,000 dilution of peroxidase-conjugated goat anti-rabbit
IgG (Pierce) in 5% skim milk in TBST. After 3 more washes in TBST for
15 min each, blots were developed with the Super Signal
chemiluminescence system (Pierce).
Co-expression of Molecular Chaperones Affects the Amount of
Functional SERT Expressed--
The serotonin transporter and the
molecular chaperones calnexin, calreticulin and BiP, and the foldase
ERp57 were incorporated into separate baculovirus vectors for
expression in insect cells. The serotonin transporter used in this
study was altered to contain a cMyc tag at the N terminus (Myc-SERT) to
allow easy identification of SERT using an anti-cMyc antibody (9E10)
(47). Myc-SERT also contained a calmodulin binding domain at the N
terminus and a His10 tag at the C terminus; both tags are
used for the purification of
Myc-SERT.2 Placing tags at
the N and C termini of SERT does not affect either the
Kd for RTI55 binding or the Km
for serotonin uptake into insect cells (6, 46). The amino acid
sequences of the molecular chaperones were unaltered. All the molecular chaperones and Myc-SERT were expressed under the control of the polyhedrin promoter. Sf9 insect cells were infected with these recombinant baculoviruses at a multiplicity of infection of 5. Total
cellular membranes were prepared from aliquots of cells up to 4 days
post-infection (p.i.) and assayed for the amount of functional Myc-SERT
using the inhibitor 125I-RTI55. It was assumed that if
125I-RTI55 bound to Myc-SERT in a serotonin-protectable
manner, then Myc-SERT was functional i.e. if it was present
at the plasma membrane, then it could transport serotonin. It was not
possible to quantitate Myc-SERT levels by measuring
[3H]serotonin uptake, because the cells become leaky on
Myc-SERT expression, preventing any serotonin transport (6).
To examine the role of molecular chaperones and foldases in the folding
of the serotonin transporter, Myc-SERT was initially expressed in the
presence of either calnexin, calreticulin, ERp57, BiP or the control
virus, bv-pVL. The control virus (bv-pVL (6)) does not express any
protein from the polyhedrin promoter and was used to maintain a
constant multiplicity of infection per experiment, because increasing
the amount of virus infecting the cells can decrease levels of Myc-SERT
expression. Western blots of insect cells expressing mammalian
molecular chaperones were probed with antibodies to each molecular
chaperone and confirmed that they were expressed (results not shown).
The amount of functional Myc-SERT expressed, as determined by inhibitor
binding, increased over time, with a maximum functional expression 2-3
days p.i. depending on which molecular chaperone was present (Fig.
1a). Calnexin consistently
increased the amount of functional Myc-SERT expressed nearly 3-fold
(Fig. 1b), with calreticulin and BiP giving only a slight
increase in functional SERT expression (1.3-fold and 1.4-fold,
respectively). Co-expression of ERp57 did not enhance the amount of
functional SERT expressed.
Calnexin and calreticulin can recruit ERp57 to promote the folding of
proteins containing disulfide bonds (34), so co-expression experiments
were conducted using combinations of Myc-SERT, calnexin, calreticulin,
and ERp57. No further increase in functional Myc-SERT expression above
that observed in the presence of calnexin was observed (Fig.
1c). Similar experiments co-expressing Myc-SERT, BiP, and
calnexin showed that the individual effects of BiP and calnexin on
improving functional Myc-SERT expression were not additive and actually
resulted in a decrease in functional Myc-SERT expression (Fig.
1c). However, this result may be linked to the simultaneous
expression of three different recombinant proteins from the polyhedrin
promoter, leading to an overload of the protein synthesis capacity of
the insect cells and a reduction in total SERT produced.
All the experiments above were performed on Myc-SERT, which differs
from the native SERT by having tags at the N and C termini (see
"Materials and Methods"). The effect of co-expression of molecular
chaperones with untagged SERT was also studied (Fig. 1d).
Calnexin clearly increases the level of functional SERT, but the
co-expression of BiP and CRT was not helpful. In addition, calnexin did
not improve SERT activity as much as was observed for Myc-SERT,
suggesting that the effects of BiP and CRT on Myc-SERT activity would
likely be too small to be detected on untagged SERT.
The amount of total Myc-SERT expressed in the presence of the molecular
chaperones was assessed by Western blotting using an anti-cMyc antibody
(Fig. 2). Quantitation was performed
using the program Geltrak (49, 50) by simple integration of the area
under the peaks. Both glycosylated and unglycosylated Myc-SERT were
always expressed regardless of the type or presence of other co-expressed proteins. However, the amount of glycosylated Myc-SERT expressed on day 2 p.i. decreased slightly when BiP and calnexin were co-expressed (62 and 91% of the control, respectively). This contrasts with the increase in functional Myc-SERT expressed (the binding assays for these samples are shown in Fig. 1a)
i.e. 110 and 180%, respectively. This phenomenon is most
apparent in the experiment co-expressing Myc-SERT, calnexin, and BiP on
day 1 p.i. compared with the control (Fig. 2b). On day
1 p.i. there were identical amounts of functional SERT (Fig.
2b), yet the Western blotting signal for glycosylated
Myc-SERT co-expressed with calnexin and BiP was considerably weaker
than the control. Quantitation of these bands by scanning densitometry
showed that glycosylated and unglycosylated Myc-SERT represented 8 and
3%, respectively, of the levels observed in the absence of
co-expressed chaperones, despite equal amounts of functional SERT
present in both cases. In this instance, co-expression of calnexin and
BiP with Myc-SERT resulted in a 18-fold increase in the ratio of active
to inactive transporter, assuming that all the Myc-SERT expressed in
the presence of calnexin and BiP is active.
Another effect of co-expressing Myc-SERT with calnexin is observed when
SERT is solubilized from membranes with the detergent digitonin. The
total and soluble Myc-SERT levels with and without co-infection are
shown in Fig. 3a. As noted
previously, co-infection with multiple baculoviruses reduces the total
SERT expression level. However, the soluble Myc-SERT levels in Fig.
3a actually increase following co-infection with pVL,
although not to the level observed with CXN co-expression. As has been
observed previously with insect cells and Escherichia coli,
reducing expression levels can often increase the solubility of
heterologous proteins (51, 52). Comparing the two infections with pVL
and CXN indicated comparable total SERT levels according to
densitometric analysis, but a 2-fold enhancement in the soluble SERT
levels was detected following CXN co-expression. This increase is
comparable with the 2-fold increase observed on day 2 p.i. in the
activity measurements following CXN coexpression (Fig.
1a).
Direct Interaction of Myc-SERT with Calnexin and Calreticulin Shown
by Co-immunoprecipitation--
To examine the possible association of
the molecular chaperones calnexin and calreticulin with SERT,
co-immunoprecipitation experiments were performed. Insect cell
membranes from day 2 p.i. were solubilized in digitonin, and the
insoluble fraction was removed by centrifugation. The digitonin-soluble
fraction was then used in the co-immunoprecipitations. Digitonin
preferentially solubilizes the glycosylated form of SERT and does not
solubilize any of the unglycosylated SERT (Fig. 3a). It is
likely that all the unglycosylated SERT is inactive (6) and forms a
digitonin-resistant aggregate in the ER, which can only be solubilized
using harsher detergents.2 The soluble fraction also
contained the molecular chaperones e.g. calnexin (Fig.
3b).
Digitonin extracts of membranes containing Myc-SERT and calnexin were
immunoprecipitated with the anti-cMyc antibody, and the
immunoprecipitate was separated by SDS-PAGE and Western blotted using
an anti-calnexin antibody (Fig. 3c). Calnexin was
immunoprecipitated with anti-cMyc antibody when Myc-SERT and calnexin
were co-expressed, but calnexin was not precipitated when it was
expressed alone (Fig. 3c). These data indicate that there is
a specific interaction between Myc-SERT and calnexin. Similarly,
co-immunoprecipitation of calreticulin and Myc-SERT with the anti-cMyc
antibody showed that calreticulin also interacts with Myc-SERT (Fig.
4). The protein observed at lower
molecular weights in all the lanes of the immunoprecipitation blot in
Fig. 4 is the immunoglobulin heavy chain from the anti-cMyc antibody.
The Interaction of Calnexin with SERT Is Predominantly via the
N-Glycan--
Previous studies have indicated that the interaction
between calnexin and N-glycosylated proteins is mainly
dependent on the presence of a monoglucosylated N-glycan
precursor (GlcNac2Man9Glc). If glucose trimming
by glucosidases I and II is inhibited by the presence of
1-deoxynojirimycin (dNJM), calnexin binding to the N-glycan
can be prevented (24). Insect cells infected with baculoviruses expressing Myc-SERT and calnexin were, therefore, grown in the presence
or absence of dNJM. Membranes from the cells were assayed for
functional expression of Myc-SERT and the association between calnexin
and Myc-SERT. In the absence of calnexin, dNJM has little effect on
functional Myc-SERT expression (Fig.
5a). When Myc-SERT is
co-expressed with calnexin, an increase in functional Myc-SERT expressed per cell is observed; in the presence of dNJM, functional Myc-SERT expressed is decreased to the level seen in the absence of
calnexin (Fig. 5a). In these experiments, calnexin did not increase the amount of functional SERT as much as in the experiments depicted in Fig. 1. This is probably because the experiments using dNJM
were conducted on cells adhered to tissue culture flasks containing 4 ml of medium, and the experiments described in Fig. 1 were grown in
well aerated spinner cultures. An immunoprecipitation of Myc-SERT
co-expressed in the presence of calnexin and dNJM showed minimal levels
of calnexin associated with Myc-SERT compared with Myc-SERT and
calnexin expressed in the absence of dNJM (Fig. 5b). The
background levels of observed calnexin may be because of the inability
to block all glucosidase-mediated N-glycan trimming in
insect cells under the conditions employed, or perhaps there is a weak
association between calnexin and Myc-SERT when the N-glycans are in the glucosylated form. Similar experiments showed no association between calreticulin and Myc-SERT in the presence of dNJM (Fig. 5c).
A second approach we used to look at the interaction between SERT and
calnexin was to use an unglycosylated SERT mutant (SERT-QQ) that had
both Asn residues (N-208 and N-217) normally glycosylated changed to
Gln (Q) residues. SERT-QQ was expressed at 20-fold lower levels than
glycosylated SERT but was still fully functional according to binding
assays (6). Surprisingly, co-expression of SERT-QQ with calnexin led to
a small but reproducible increase in the amount of SERT-QQ expressed
(Fig. 6a), which was
inhibitable by dNJM (Fig. 6b). Immunoprecipitations failed
to show any stable interaction between SERT-QQ and calnexin (Fig.
6b).
The serotonin transporter is a complex integral membrane protein
composed of 12 transmembrane regions that requires
N-glycosylation for efficient folding and stability (6) and
the presence of a putative disulfide bond for activity (11). We have
shown here that the production of Myc-SERT is enhanced by at least
three molecular chaperones. The interactions between Myc-SERT and the molecular chaperones was investigated in insect cells using the baculovirus expression system. The presence of large quantities of
inactive Myc-SERT expressed in insect cells suggested that folding
might be rate-limiting for the production of functional Myc-SERT using
the baculovirus expression system. In this study, the role of molecular
chaperones in generating active Myc-SERT was investigated, and we found
that co-expression of three molecular chaperones increased the amount
of functional SERT expressed. The role of these chaperones may be to
retain Myc-SERT in a folding-competent conformation and prevent
nonspecific aggregation. Calnexin was the most successful of the
co-expressed molecular chaperones, improving functional Myc-SERT
expression by nearly 3-fold. Smaller increases in functional Myc-SERT
expression were also seen on co-expression of calreticulin and BiP. In
addition, specific interactions between Myc-SERT and calnexin, and
Myc-SERT and calreticulin, were confirmed in co-immunoprecipitation
experiments. It was not possible to demonstrate an interaction between
BiP and Myc-SERT in the same way because of nonspecific interactions in
the immunoprecipitation. The N-glycan-specific protein
disulfide isomerase, ERp57, did not increase functional Myc-SERT
expression either in the presence or absence of other molecular chaperones.
Increased expression of functional Myc-SERT was a consequence of
molecular chaperone activity and not because of increases in
transcription or translation. This was apparent from Western blots of
membranes prepared from cells co-expressing Myc-SERT and the molecular
chaperones calnexin and BiP (Fig. 2). The levels of unglycosylated and
glycosylated Myc-SERT did not increase in parallel with the increases
in functional Myc-SERT determined from inhibitor binding, suggesting
that the increases were because of better folding of Myc-SERT in the
presence of molecular chaperones. In fact co-expression of calnexin
and/or BiP actually decreased the amount of inactive Myc-SERT
expressed. This was particularly apparent on co-expression of Myc-SERT,
calnexin, and BiP, where on day 1 p.i., glycosylated and
unglycosylated Myc-SERT represented 8 and 3%, respectively, of the
control levels on a Western blot, despite containing equal amounts of
functional SERT. A number of possible mechanisms could explain this
decrease in nonfunctional Myc-SERT expression, including increased
proteolysis of misfolded Myc-SERT or perhaps decreased rates of
translation because of better coupling between folding and translation.
The simplest explanations for the large increase in functional Myc-SERT
expression on co-expression of calnexin are that there is little or no
calnexin expressed in insect cells or that insect calnexin does not
interact with Myc-SERT. This was suggested from expressing Myc-SERT in
the presence of dNJM. The generation of the monoglucosylated
N-glycan precursor is prevented by dNJM; hence, dNJM is
expected to prevent calnexin from interacting with Myc-SERT in
vivo, as we have observed. However, in the absence of co-expressed
calnexin, dNJM has little effect on functional Myc-SERT expression,
suggesting that the folding of Myc-SERT in insect cells is not
dependent on an insect calnexin. At this stage, it cannot be determined
whether there is no endogenous insect calnexin in the cells or whether
there is calnexin and that it does not interact with Myc-SERT like
mammalian calnexin. Western blots of insect cell membranes using
antibodies to mammalian calnexin showed weakly cross-reacting proteins,
but it is not clear whether they represent calnexin homologues.
Sf9 cells may not be the only insect cells to lack calnexin
activity, because this has also been reported for a
Drosophila cell line (53).
The large increase in levels of functional Myc-SERT caused by
co-expressing calnexin warranted a further investigation of the
interaction between them. Therefore, an unglycosylated SERT mutant,
SERT-QQ, was co-expressed with calnexin. Surprisingly, calnexin was
capable of increasing the functional expression of SERT-QQ.
Co-immunoprecipitations suggested that there was no measurable interaction between calnexin and SERT-QQ. However, this does not mean
that there was never any transient association between SERT-QQ and
calnexin, because any such interaction may well have been disrupted by
digitonin solubilization before immunoprecipitation. This would be
likely if calnexin interacted with SERT via its transmembrane region,
as has been suggested for the association between calnexin and
P-glycoprotein (54). The interaction between SERT-QQ and
calnexin also seemed to be inhibited by dNJM. The most straightforward
conclusion is that dNJM can interact directly with calnexin and prevent
its function as a molecular chaperone; this may not be surprising given
that glucosidase II, which is inhibited by dNJM, recognizes the
identical monoglucosylated N-glycan precursor as calnexin.
An alternative explanation is that a small proportion of SERT-QQ is
N-glycosylated, but this seems unlikely as there are no
other consensus N-glycosylation sites predicted to be in
SERT facing the ER lumen, including the rarely used consensus Asn-X-Cys (see for example Ref. 55). These results could
also be explained by an indirect effect of calnexin on SERT-QQ
i.e. calnexin improved the folding of another unidentified
molecular chaperone in insect cells that then helped SERT-QQ to fold.
The experiments described in Fig. 1, a-c all use a tagged
version of SERT (Myc-SERT), with the tags at the N and C termini predicted to be in the cytoplasm. The molecular chaperones and foldases
BiP, CRT, and ERp57 are all present in the ER lumen and, therefore,
cannot interact directly with tags. The functional domain of calnexin
is in the ER lumen, but it also has a single transmembrane domain and a
short C-terminal domain that is in the cytoplasm. Thus the tags on SERT
could only potentially interact directly with the C terminus of
calnexin. However, although the C terminus of calnexin is essential for
the proper localization of calnexin in the ER, it is not believed to be
required for function of calnexin (34). Given the intracellular
distribution of the molecular chaperones, it was surprising that the
presence of the tags on SERT affected the ability of molecular
chaperones to improve the expression of the transporter (Fig. 1). In
addition, the amount of functional Myc-SERT per cell does not differ
appreciably from the amount of functional untagged SERT per cell when
they are expressed independently. Yet co-expression of calnexin with
Myc-SERT led to a 3-fold improvement in functional activity as compared with a 1.4-fold improvement in expression of untagged SERT. It is
possible that the tags on SERT are having a subtle effect on either the
folding pathway or kinetics of folding, which provide a greater
opportunity for the co-expressed molecular chaperones to bind and have
an effect. Presumably, in the absence of additional tags, SERT can be
folded sufficiently using the molecular chaperones naturally found in
insect cells, and additional molecular chaperones therefore have little effect.
We have shown that the folding of Myc-SERT in vivo can be
assisted by the molecular chaperones calnexin, calreticulin, and BiP.
In addition, it has recently been shown that the foldase NinaA, a
membrane-bound peptidyl-prolyl cis-trans isomerase from Drosophila, can increase the cell surface expression of the
dopamine transporter (56), a homologue of SERT. Elucidating the role of
molecular chaperones and foldases in the folding of SERT will not only
lead to an understanding of how complex membrane proteins fold, but it
will also help us to overexpress proteins for subsequent purification
and structural analysis. Many membrane proteins are expressed very
poorly in heterologous expression systems (35, 36), and this may be a
direct consequence of the lack of specific molecular chaperones in the
cell lines used. Alternatively, there may be insufficient amounts of
molecular chaperones to cope with the high level of protein translation
induced in most heterologous expression systems. For example,
recombinant IgG secretion from insect cells is enhanced by
co-expressing BiP or protein disulfide isomerase (57, 58).
Unfortunately, co-expressing multiple molecular chaperones
simultaneously has not been successful, suggesting that their relative
levels of expression may require careful modulation. The inclusion of
several chaperones and foldases on one virus, and the use of vectors
including lower strength promoters, such as IE1 or the basic protein
promoter, may help to rectify this limitation.
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MATERIALS AND METHODS
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ions down their
concentration gradients (reviewed in Refs 1-3). SERT is of particular
pharmacological interest, because it is the primary binding site in the
brain for antidepressant drugs, and it also interacts with cocaine and
amphetamines (4, 5). All members of this family share two structural
characteristics, namely 12 putative transmembrane domains and a large
extracellular loop between transmembrane domain 3 and transmembrane
domain 4. This particular loop is glycosylated in all members of the
family, although the number of potential N-glycans varies
between 1 and 4, and the sites of glycosylation are not absolutely
conserved. SERT has two N-glycans in this region, and their
importance for its activity has been tested by altering the consensus
N-glycosylation sequence from Asn-X-Ser/Thr to
Gln-X-Ser/Thr; deletion of both N-glycosylation
sites in SERT neither changed the Km for serotonin
uptake nor the Kd for binding of the inhibitor RTI55
(6). However, the number of functional molecules of SERT/cell decreased
to 5% that of the levels of fully glycosylated SERT, suggesting that
N-glycosylation plays an important role in either the
initial folding of SERT or for its stability in the membrane (6). Other
members of the neurotransmitter transporter family were later shown to
share this characteristic with SERT (7-9).
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RESULTS
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Fig. 1.
Expression levels of functional Myc-SERT
co-expressed with molecular chaperones. Insect cultures (100 ml)
co-infected with baculoviruses expressing Myc-SERT and various
molecular chaperones were grown at 27 °C. Membranes were prepared
from 10-ml aliquots of cells at various time intervals and assayed for
functional Myc-SERT using the inhibitor 125I-RTI55.
a, time course of Myc-SERT expression. Myc-SERT was
co-expressed with baculoviruses expressing molecular chaperones
calnexin (black squares), calreticulin (open
squares), ERp57 (open circles), BiP (open
triangles), control virus bv-pVL (black circles).
b, comparison of functional Myc-SERT expression on day 3 post-infection. Myc-SERT was co-expressed with the molecular chaperones
as indicated, and the control was Myc-SERT co-infected with bv-pVL.
Error bars represent the S.E. with n = 6 for
CXN, n = 4 for BiP, and n = 2 for CRT
and ERp57. c, comparison of functional Myc-SERT expression
on day 3 post-infection when co-expressed with calnexin and additional
molecular chaperones as indicated. Error bars represent the
S.E. (n = 2). d, comparison of functional
SERT expression on day 3 post-infection when co-expressed with the
molecular chaperones as indicated. Error bars represent the
S.E. (n = 3).
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Fig. 2.
Western blots of Myc-SERT co-expressed with
various molecular chaperones. a, membranes from the
experiment shown in Fig. 1a were separated by SDS-PAGE,
transferred to nitrocellulose, and probed with an anti-cMyc antibody.
b, insect cultures (100 ml) co-infected with baculoviruses
expressing Myc-SERT, calnexin, and BiP were grown at 27 °C; the
control contained Myc-SERT and bv-pVL. Membranes were separated by
SDS-PAGE, transferred to nitrocellulose, and probed with an anti-cMyc
antibody. Membranes prepared from the same number of cells were
loaded/lane. Numbers below each lane
refer to the day post-infection the cells were harvested. All the blots
shown in a and b were transferred to
nitrocellulose simultaneously, and they were all developed in parallel.
Glycosylated SERT (G) and unglycosylated SERT
(UG) are indicated by arrows. The lower
panel in b represents the activity of Myc-SERT in the
membrane samples directly above, in the Western blot. Samples were
assayed using 125I-RTI55 as described under "Materials
and Methods."
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Fig. 3.
Analysis of the interaction between Myc-SERT
and calnexin. Membranes were prepared from cells infected with
recombinant baculovirues that expressed combinations of the following
proteins: serotonin transporter (Myc-SERT), calnexin (CXN), control
virus not expressing any protein (pVL). Equal numbers of cell
equivalents were loaded per lane. Membranes were used
directly (Whole membranes) or were first
solubilized with digitonin and centrifuged, and the soluble fraction
was used (soluble fraction). a, Western blot probed with
anti-cMyc recognizing Myc-SERT. Glycosylated (G) and
unglycosylated (UG) SERT are indicated by arrows.
b, Western blot of the identical membrane samples used in
a but probed with an anti-calnexin antibody. c,
co-immunoprecipitation of the identical membrane samples used in
a using an anti-cMyc antibody. The Western blot of the
immunoprecipitates was probed with an anti-calnexin antibody.
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Fig. 4.
Co-immunoprecipitation of SERT and
calreticulin. Membranes were prepared from cells infected with
combinations of baculoviruses expressing the serotonin transporter
(Myc-SERT), CRT, and the control virus (pVL). The immunoprecipitations
were performed on digitonin-solubilized membranes using the anti-cMyc
antibody. The immunoprecipitates (IP) were separated by
SDS-PAGE, Western blotted, and probed with an anti-calreticulin
antibody. The band corresponding to calreticulin is
indicated by an arrow.
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Fig. 5.
The effect of deoxynojirimycin on the
expression of Myc-SERT in the presence and absence of calnexin.
Membranes were prepared from cells infected with Myc-SERT in the
presence (+) or absence ( ) of either CXN and/or dNJM. Cells were
harvested 3 days post-infection. a, functional Myc-SERT
expression measured by 125I-RTI55 binding assays.
Error bars represent the S.E. of 4 independent experiments
assayed in triplicate. Co-immunoprecipitations of Myc-SERT and calnexin
(b) and Myc-SERT and calreticulin (c) after
expression in cells treated with deoxynojirimycin is shown.
Immunoprecipitations (IP) were performed on a digitonin
solubilizate. The immunoprecipitates were separated by SDS-PAGE,
Western blotted, and probed with an anti-calnexin antibody
(b) or with an anti-calreticulin antibody
(c).
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Fig. 6.
Functional expression of SERT-QQ in the
presence of calnexin. a, functional SERT-QQ expression,
measured by 125I-RTI55 binding assays, using membranes
prepared from cells infected with SERT-QQ in the presence (+) or
absence ( ) of either CXN and/or dNJM. Cells were harvested 3 days
post-infection. Error bars represent the S.E. of four
independent experiments assayed in triplicate. Note that the 100%
values for SERT (Fig. 5a) and SERT-QQ are not comparable;
SERT-QQ is expressed at 20-fold lower levels than Myc-SERT.
b, co-immunoprecipitation of SERT and SERT-QQ co-expressed
with calnexin. Membranes were prepared from cells infected with
combinations of recombinant baculoviruses expressing the following
proteins: serotonin transporter with two N-glycosylation
sites (Myc-SERT), unglycosylated SERT (SERT-QQ), CXN, CRT, and the
control virus (pVL). The membranes were solubilized with digitonin, and
the solubilizate was immunoprecipitated with an anti-SERT antibody
(CT2A). The immunoprecipitate was separated by SDS-PAGE, Western
blotted, and probed with an anti-calnexin antibody.
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ABSTRACT
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ACKNOWLEDGEMENTS |
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We are indebted to R. Blakely for providing the anti-SERT antibody (CT2A), D. Williams for the canine calnexin cDNA, M. Bourdi for the ERp57 cDNA clone, and D. Llewellyn for the calreticulin cDNA clone.
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FOOTNOTES |
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* This work was funded by a Medical Research Council Career Development Award and European Union Training and Mobility of Researchers Grant CT98-0228 (to C. G. T.) and by a National Science Foundation Young Investigator Award (to M. J. B.) Grant BES-9258453.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.
These authors contributed equally to this work and are listed in
alphabetical order.
§ To whom correspondence should be addressed: MRC Laboratory of Molecular biology, Hills Rd., Cambridge CB2 2QH, UK. Tel.: 44-1223-402291; Fax: 44-1223-213556; E-mail cgt{at}mrc-lmb.cam.ac.uk.
2 C. G. Tate, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
SERT, serotonin
transporter;
SERT-QQ, unglycosylated serotonin transporter mutant;
dNJM, 1-deoxynojirimycin;
CXN, calnexin;
CRT, calreticulin;
BiP, immunoglobulin heavy chain binding protein;
RTI55 (-CIT), 2
-carbomethoxy-3
-(4-iodophenyl)tropane;
ER, endoplasmic
reticulum;
PAGE, polyacrylamide gel electrophoresis;
TBST, Tris-buffered saline-Tween 20;
p.i., post-infection.
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