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INTRODUCTION |
Calnexin, an integral membrane protein, and its soluble homolog,
calreticulin, are endoplasmic reticulum
(ER)1 chaperones that bind
transiently to newly synthesized glycoproteins, thereby promoting their
folding and oligomerization (1-4). These chaperones also display a
prolonged interaction with misfolded and aggregated proteins, leading
to their retention in the ER (5-7). Nascent polypeptides are targeted
to the ER membrane by a signal recognition
particle-dependent mechanism. The subsequent translocation
of the polypeptide into the ER lumen and the integration of
transmembrane segments into the ER membrane are mediated by a protein
channel that forms the translocon (8). Calnexin is part of a multimeric
protein complex, including calreticulin, BiP, GRP94, and Erp57, that
"proofreads" the protein folding process in the ER (7, 9-11).
N-Glycosylation is a co-translational event involving the
en bloc transfer of a
Glc3Man9GlcNAc2 oligosaccharide
from a dolichol donor to a lumenal acceptor site
(Asn-X-Ser/Thr) in the nascent chain by the ER
oligosaccharyl transferase (12-14). Co-translational removal of the
terminal glucose residue occurs rapidly by the action of
-glucosidase I, followed by the successive removal of the second and
third glucose residues by
-glucosidase II (15-19). The
monoglucosylated oligosaccharide can be regenerated by a
glucosyltransferase that acts posttranslationally on unfolded proteins
(20). Calnexin and calreticulin are lectins that interact with the
Glc1Man9-5GlcNAc2 oligosaccharide
(21-26), and calnexin-bound monoglucosyl glycoproteins are protected
from
-glucosidase II removal of the final glucose (25). Inhibition
of
-glucosidase I and II with castanospermine (CST) prevents
calnexin binding (27, 28), and calnexin associations are absent in
glucosidase-deficient cell lines (29). The presence of a single
oligosaccaride moiety on a protein is sufficient for the interaction
with calnexin; however, the interaction is enhanced when multiple
N-glycosylated sites are present (30, 31).
It has been proposed that the interaction of calnexin with
glycoproteins may proceed in two stages; the first involves lectin binding to the oligosaccharide, and the second involves a
protein-protein interaction (23, 32, 34). Calnexin can be
co-immunoprecipitated with nonglycosylated proteins (6, 35-39). This
oligosaccharide-independent interaction may, however, be the result of
calnexin binding to aggregated protein (38).
Most studies of calnexin interactions have focused on secreted proteins
or membrane proteins that contain a single transmembrane (TM) segment.
Calnexin and calreticulin can interact with both soluble lumenal
glycoproteins and intrinsic membrane glycoproteins, although some
glycoproteins show a preferential interaction (10, 22, 39-41).
Calnexin is a type I membrane protein with a lumenal lectin domain (42)
that can readily interact with membrane proteins that contain
N-glycosylated sites including those positioned close to the
membrane surface (31). Calnexin is known to bind to newly synthesized,
single-span membrane glycoproteins, such as MHC class I and II (23,
37), hemagglutinin (43), and vesicular stomatitis virus glycoprotein
(38), facilitating their folding and cell-surface expression (2, 34).
Calnexin is also involved in the folding and maturation of polytopic
membrane glycoproteins, such as P-glycoprotein (35), cystic fibrosis
conductance transmembrane regulator (44), the T cell receptor (39), and
the acetylcholine receptor (45, 46). Because most polytopic membrane
proteins are N-glycosylated on a single EC loop, greater
than 30 residues in size (47), these proteins are likely to interact
with calnexin. However, the position and number of
N-glycosylated sites in polytopic membrane proteins vary. We
therefore determined whether calnexin was able to interact with
oligosaccharide chains located at different positions in a polytopic
membrane protein and whether this interaction occurred while the
protein was held within the translocation machinery.
In this study, the interaction of calnexin with
N-glycosylated mutants of the human erythrocyte anion
exchanger, AE1 (band 3) was studied using an in vitro
translation system and in transfected cells. AE1 contains two
structural domains: a large amino-terminal domain located in the
cytoplasm and a carboxyl-terminal, membrane-spanning domain responsible
for the anion exchange function (Fig. 1) (48). Our recent topology
model of AE1 predicts 12 TM segments, with a single site of
N-linked glycosylation at Asn-642, located in the fourth EC
loop (Fig. 1) (49). We show that calnexin interacts with AE1 in a
glycosylation-dependent fashion and that this interaction is not dependent on the position of the oligosaccharide within this
polytopic membrane protein. Truncated constructs of AE1 that retain
tRNA on their nascent chain were tested for co-translational interaction with calnexin. The calnexin interaction was detected by
co-immunoprecipitation when the translationally arrested polypeptide was released by puromycin. This shows that the predominant calnexin-AE1 interaction occurs posttranslationally.
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EXPERIMENTAL PROCEDURES |
Materials--
The following is a list of products and their
sources: ModulisTM in vitro transcription system
and canine pancreatic microsomes (MBI Fermentas), Flexi rabbit
reticulocyte lysate and RNasin (Promega), [35S]Met and
[
-35S]ATP (NEN Life Science Products),
TransformerTM mutagenesis kit
(CLONTECH), T7 sequencing kit (Amersham Pharmacia Biotech), digitonin and Triton X-100 (Roche Molecular Biochemicals), Brij 96 (Sigma), dropping bottle with 20-30 µm filter unit
(Wheaton). Polyclonal rabbit anti-calnexin antibodies directed against
a carboxyl-terminal peptide (antibody C) corresponding to the last 14 residues of canine calnexin (5) or against ER lumenal region of
calnexin (antibody N) expressed and purified from Sf9 cells (residues 1-461) as described in Ref. 26 were kind gifts from Dr.
David Williams (University of Toronto). Polyclonal rabbit anti-AE1
antibody directed against a carboxyl-terminal peptide corresponding to
the last 12 residues of human AE1 (50) was employed to detect AE1.
Oligonucleotide-directed Mutagenesis--
The cDNA of human
Band 3 in Bluescript vector BSSK+ was a kind gift from Drs.
A. M. Garcia and H. Lodish (Whitehead Institute). The endogenous
N-glycosylation site at Asn-642 was mutated to Asp (N642D)
in the membrane domain construct of AE1-AE1md (residues 386-911), as
described (51). The Full-length AE1 and the AE1md were subcloned into
pcDNA3 vector as described (50). A series of unique
N-glycosylation acceptor sites were created in the AE1md or
in the N642D construct (Fig. 1) using oligonucleotide-directed mutagenesis and the double-stranded mutagenesis system from
CLONTECH. A series of mutants were made containing
unique restriction enzyme sites that cut at amino acid positions 696 (NcoI), 714 (NotI), 754 (XbaI), 785 (XbaI), and 820 (XbaI) in the AE1md construct.
Insertion of EC Loop 4 into EC Loops 1, 2, and 7--
Short EC
loops in TM were expanded by insertion of a 35-amino acid sequence
corresponding to residues 626-659 of EC loop 4, containing the
endogenous or mutated (N642D) N-glycosylation acceptor site,
as described (49, 52). The insertion mutants are illustrated in Fig. 1.
In insertion mutants ins.EC1 and ins.EC7, the sequence of EC loop 4 containing the endogenous acceptor site, Asn-642, was inserted into EC
loop 1 and EC loop 7, respectively, whereas the endogenous site was
mutated to N642D. Insertion construct ins.EC2a contained an EC loop 4 insertion in EC loop 2 with both N-glycosylation acceptor
sites mutated. Insertion construct ins.EC2b contained one
N-glycosylation acceptor site in the inserted sequence only,
whereas ins.EC2c had the endogenous acceptor site only. The ins.EC2d
construct contained two N-glycosylation acceptor sites, one
endogenous and one in the insert. All mutations were confirmed by DNA
sequencing (53).
In Vitro Transcription and
Translation--
ModulisTM in vitro
transcription system and plasmid DNA (0.5 µg) in a final volume of 10 µl were used to synthesize mRNA. The transcription mixture (3 µl) was used for in vitro translation using Flexi rabbit
reticulocyte lysate as described by the supplier; the lysate was
supplemented with 2 units of canine pancreatic microsomes in a final
volume of 30 µl. Membrane integration of the translation products was
determined by extracting samples (10 µl) with 100 µl of ice-cold
0.1 M Na2CO3, pH 11.5, followed by
recovery of the stripped microsomes by centrifugation (16,000 × g for 20 min). Alternatively, 35 µM
Ac-NYT-NH2 (to block the N-glycosylation of AE1)
or CST (to inhibit
-glucosidase I and II) was included in the
translation mixture when indicated. For co-translational interaction,
the cDNA corresponding to the AE1md was linearized within the
coding region at amino acid positions 696 (NcoI), 714 (NotI), 754 (XbaI), 785 (XbaI), 820 (XbaI), and 854 (NaeI, the endogenous unique
restriction site). The insertion mutant ins.EC1 (Fig. 1) was linearized
at position 854 (NaeI). The translation reaction was
performed at 27 °C for 30 min. Following the translation reaction,
two separate additions were made: (i) 1.5 mM puromycin,
followed by an additional 10-min incubation at 27 °C, or (ii) 35 µM Ac-NYT-NH2, 1 mM CST, followed
by incubation on ice for 5 min. The stability of attached ribosomes was
time- and temperature-dependent. Lower temperatures
(25 °C) and shorter translation times (30 min) favored the stability
of the complex but significantly reduced protein biosynthesis (54).
Immunoprecipitation--
For co-immunoprecipitation, 20 µl of
the translation reaction was solubilized in 600 µl of PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM
KH2PO4, pH 7.4) containing 1% (w/v) digitonin,
1 mM phenylmethylsulfonyl fluoride, 1 µM
aprotinin, 1 µM leupeptin, 1 µM pepstatin
A. Alternatively, 1% Triton X-100, Brij 96, or CHAPS was used as
indicated. After 3-4 h of incubation at 4 °C with anti-calnexin
antibody (5 µl), 20 µl of protein G-Sepharose beads in PBS buffer
(1:1) was added, followed by a 2 h incubation at 4 °C. Beads
were washed with PBS buffer containing 0.2% digitonin and bound
proteins were solubilized in 20 µl of 2× SDS sample buffer (4%
SDS), at room temperature.
Expression of AE1 in Transfected Cells--
HEK293 cells were
grown in Dulbecco's modified Eagle's medium and transfected with 5 µg of DNA/10-cm dish, using the DEAE-dextran method as described in
Ref. 50. 20-24 h following transfection, cells were labeled with
[35S]Met (0.1 mCi/ml) for 20-25 min. Labeled cells were
washed once with PBS buffer, and cells were solubilized in PBS
containing 1% (w/v) digitonin and protease inhibitors (as described
above). Samples were filtered through a large pore filter unit, divided into three equal aliquots (700 µl), and subjected to
immunoprecipitation using anti-calnexin antibody (3 µl) or anti-AE1
antibody (3 µl). In the case of double immunoprecipitation, lysates
were first immunoprecipitated using anti-calnexin antibody, followed by
incubation with protein G-Sepharose beads. Beads were washed, followed
by incubation with 1% SDS, 5 mM EDTA at 30 °C for 30 min, and then diluted with 10 volumes of PBS buffer containing 1%
Triton X-100. Beads were pelleted, and the supernatant was used for
anti-AE1 immunoprecipitation.
The radiolabeled proteins were examined by SDS-gel electrophoresis
followed by autoradiography. The efficiency of
N-glycosylation was determined by scanning autoradiographs
of SDS gels using a ScanMaker IIHR (Microtec) scanner and the Adobe
PhotoshopTM program, version 3.0. The pixel density was determined
using NIH Image software. The percentage of N-glycosylation
was calculated from the areas under the two peaks corresponding to the
N-glycosylated and nonglycosylated products. The fraction of
protein that co-immunoprecipitated with calnexin was estimated by
determining the intensity of the band interacting with calnexin in the
immunoprecipitation fraction divided by the total protein integrated
into the microsomes.
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RESULTS |
AE1 Associates with Calnexin in a
Glycosylation-dependent Manner--
The interaction of
calnexin with human AE1 was studied in a cell-free translation system,
using rabbit reticulocyte lysate supplemented with canine pancreatic
microsomal membranes. This in vitro system has been used
extensively to study the biosynthesis of polytopic membrane proteins,
including AE1 (49, 51, 52, 55). Due to its lower protein mass, the
AE1md construct (51) was used to more readily distinguish the
N-glycosylated and nonglycosylated forms of the protein on
SDS gels. The AE1md construct contains the entire transmembrane domain
of AE1, encompassing residues 386-911 (Fig.
1). AE1md is sufficient to carry out the
anion exchange function (56, 57), and it has been functionally
expressed in Xenopus oocytes (58).

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Fig. 1.
Top panel, folding model of the AE1md of
human AE1 (residues 386-911) showing the 12 transmembrane segments
(49). Numbered black circles indicate the positions of the
acceptor Asn in the introduced N-glycosylation sites;
triangles indicate the positions of EC loop 4 insertions;
solid squares indicate limits of truncations. Lower
panel, Y symbol indicates the position of the
oligosaccharide chain in the linear form of the indicated AE1 mutant.
Box represents the inserted sequence of the EC loop 4. Carboxyl-terminally truncated forms (t) were generated at
the indicated amino acid positions, and the chain length following the
glycosylation site is indicated in parentheses.
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The AE1md can be synthesized and glycosylated at Asn-642 in an in
vitro translation system in the presence of microsomal membranes (Fig. 2, lane 1). The position
of the full-length AE1md protein (48 kDa) is indicated by the
open circle in Fig. 2, and the glycosylated, higher
molecular mass band (50 kDa) is indicated by the closed circle. The efficiency of N-glycosylation in this
particular experiment was 60%. The glycosylated form was previously
shown to be sensitive to endoglycosidase H, and
N-glycosylation can be inhibited by the presence of the
Ac-NYT-NH2 tripeptide (49, 51). Both the N-glycosylated and nonglycosylated bands were resistant to
alkaline extraction (Fig. 2, lane 1) and therefore had been
integrated into the microsomal membrane. The N642D mutant, with the
mutated endogenous N-glycosylation acceptor site, was not
glycosylated but was also integrated into the membrane (Fig. 2,
lane 4).

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Fig. 2.
Autoradiograph of the cell-free translation
products of the AE1md (N642) and N642D constructs, in the presence or
absence of 1 µM or 1 mM
CST, as indicated. The asterisk indicates that CST was
added posttranslationally. Total, after the cell-free
translation, translation mixture was subjected to alkaline extraction
of microsomes (lanes 1-6). IP fraction,
immunoprecipitation with anti-carboxyl-terminal calnexin antibody
(lanes 7-14). Also shown are the nonglycosylated AE1md
( ), glycosylated AE1md ( ), AE1md7-12 nonglycosylated
form ( ), and AE1md7-12 glycosylated form ( ). The
positions of molecular mass markers are indicated to the
left of the figure in kDa.
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The association of AE1md with calnexin was determined by solubilization
of microsomes with digitonin and immunoprecipitation with the
anti-carboxyl-terminal calnexin antibody. The presence of both
N-glycosylated and nonglycosylated products in the
translation mixture allowed us to determine the glycosylation
dependence of the interaction with calnexin in the same sample. The
results of co-immunoprecipitation experiments are shown in Fig. 2,
lanes 7-14. The N-glycosylated form of the AE1md
(Fig. 2, lane 1 versus lane 7, closed circle) was readily
co-immunoprecipitated with the anti-calnexin antibody. In contrast, the
lower, nonglycosylated form (Fig. 2, lane 7, open circle)
that was present in the same translation mixture was poorly
immunoprecipitated. In a second immunoprecipitation of the same sample,
only minor amounts of either form were present, indicating a
quantitative recovery of calnexin in the initial immunoprecipitation.
Very little of the nonglycosylated construct, N642D, was
co-immunoprecipitated with the anti-calnexin antibody (Fig. 2,
lane 4 versus lane 11).
The interaction of N-glycosylated AE1md with calnexin could
be inhibited if translation was carried out in the presence of 1 mM CST, an inhibitor of
-glucosidases I and II (Fig. 2,
lane 9), whereas posttranslational addition of CST did not
prevent the interaction (Fig. 2, lane 10). It has been shown
that low concentrations of deoxynojirimycin stabilizes the
calnexin-protein complex by slowing down the trimming of the glucose
residues (59). We were not able to detect any increase in the number of
calnexin/AE1md complexes in the presence of 1 µM CST
(Fig. 2, lane 8). Dose dependence experiments showed that 50 µM CST was sufficient to inhibit the interaction of
calnexin with AE1 (data not shown). The weak calnexin interaction with
the nonglycosylated form of the protein was unaffected by the presence
of CST (Fig. 2, lane 9 versus lane 13).
In addition to the full-length AE1md, lower molecular weight products
were observed in cell-free translations of the AE1md (Fig. 2,
diamonds). We have shown that these products are the result
of nonspecific translation initiation at a cluster of Met residues
positioned at the end of putative TM segment 6 (51). In the resulting
protein, TM7 acts as a signal sequence and the rest of the protein is
stably integrated into microsomal membranes (Fig. 1). This truncated
AE1md7-12 was glycosylated at Asn-642 (Fig. 2, lane
1, filled diamond) and was efficiently co-immunoprecipitated with
the anti-calnexin antibody (Fig. 2, lane 7). CST prevented
this interaction (Fig. 2, lane 9). The truncated
nonglycosylated mutant was not co-immunoprecipitated (Fig. 2,
lane 11, open diamond). The results indicate that calnexin binds to AE1md or truncated AE1md7-12 in a
N-glycosylation-dependent manner and that the
interaction requires a glucose trimmed carbohydrate structure.
Quantification of the percentages of co-immunoprecipitation of AE1md
from at least three separate translations is summarized in Table
I. On average, 46% of the glycosylated
form of the AE1md (Asn-642) was co-immunoprecipitated with
anti-calnexin antibody, whereas 20% of the nonglycosylated form was
co-immunoprecipitated. Only 12% of the total translated protein of
N642D construct was co-immunoprecipitated with calnexin. Similar
results were obtained when detergents 1% CHAPS or 1% BRIJ 96 were
used instead of digitonin. Triton X-100 disrupted the interaction of
AE1md with calnexin, as solubilization with 1% Triton X-100 resulted
in reduced efficiency of co-immunoprecipitation of AE1 (12 versus 46%).
Calnexin Has Access to Various EC Regions of AE1md--
The single
site of N-linked glycosylation (Asn-642) in the AE1md is
located in the fourth EC loop (Fig. 1). To test whether calnexin can
bind to oligosaccharide chains located on other EC loops in AE1md, a
series of glycosylation mutants that possess a single
N-glycosylation site in other loops were made using the N642D mutant. Novel N-glycosylation sites in EC loop 3 at
position 556 and in the loop structure at position 743 were introduced by point mutations (Fig. 1). N-Glycosylation sites were
introduced into EC loops 1, 2, and 7 by insertion of 35 amino acids of
EC loop 4 (ins.EC1, ins.EC2b, and ins.EC7, respectively) (49) (Fig. 1).
The mutants with novel N-glycosylation sites were translated in the presence of microsomes, and all were N-glycosylated
(Fig. 3A). Mutant 556 was
glycosylated with efficiency of 36 ± 5%, whereas the
N-glycosylation efficiency at position 743 was 38 ± 8%, as previously reported for sites located at positions 735-750
(49). The ins.EC1, ins.EC2b, and ins.EC7 mutants were glycosylated with efficiencies of 61 ± 5, 52 ± 6, and 54 ± 3%,
respectively. The insertion mutant lacking N-glycosylation
acceptor sites (ins.EC2a) was not glycosylated (Fig. 3A, lane
5).

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Fig. 3.
A, autoradiograph of the cell-free
translation products of constructs with a single site for
N-linked glycosylation. B, constructs containing
none, one, or two sites of N-glycosylation. The presence of
a novel site introduced by point mutation or by insertion is indicated
with a closed circle or a closed triangle,
respectively. The open symbols indicate the absence of an
N-glycosylation site. Cell-free translation was carried out
in the presence of microsomes, followed by alkaline extraction of
microsomes (upper panel, total), or anti-carboxyl-terminal
calnexin immunoprecipitation (lower panel, IP fraction).
Upper and lower panels represent equivalent
amounts of the original translation mixture, and images are from the
same film exposure. Quantification of mutants is presented in Table I.
The mutants are as described in the legend to Fig. 1.
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Immunoprecipitation with the calnexin antibody indicated that calnexin
interacted with all the different N-glycosylated mutants of
AE1md (Fig. 3A, lanes 11-17). The percentage of
N-glycosylated AE1 mutants that could be immunoprecipitated
with calnexin was in the range of 40-55% (Table I). The mutant with a
single acceptor site at 639 (Fig. 3B) co-immunoprecipitated
with calnexin with high efficiency (77%) (Table I). The interactions
were N-glycosylation-dependent because the
nonglycosylated bands, in the same translation reaction, were
precipitated with lower efficiencies (10-20%) (Table I). The ins.EC2a
mutant, which was not N-glycosylated, co-immunoprecipitated with efficiency of 16% (Table I).
Multiple N-Glycosylation Enhances Calnexin
Binding--
Introduction of two N-glycosylation acceptor
sites into AE1 allows double glycosylation to occur (52). We tested the
effect of the additional glycosylation site on calnexin interaction
with AE1md. Two types of doubly glycosylated mutants were made in the AE1md construct: (i) a mutant with two oligosaccharides in the same
loop EC4 (639 + 642) and (ii) mutants with oligosaccharide chains in
different EC loops, with combinations EC2 + EC4 (ins.EC2d) and EC3 + EC4 (556 + 642) (Fig. 1). Translation of mutants with two
N-glycosylation sites resulted in a doubly glycosylated form of the protein (Fig. 3B, lanes 4, 6, and 9). The
efficiency of double N-glycosylation matched the lower
efficiency of the two individual sites. This pattern was observed
whether the two N-glycosylation sites were positioned in the
same or in different EC loops.
Calnexin co-immunoprecipitation of doubly N-glycosylated
mutant 556 + 642 (Fig. 3B, lane 15) was more efficient
(63%) compared with the individual efficiencies of
co-immunoprecipitation of the 556 mutant (Fig. 3B, lane 14)
(47%) or the wild-type Asn-642 (Fig. 3B, lane 11) (46%)
(Table I). The co-immunoprecipitation efficiency was also higher for
the doubly N-glycosylated ins.EC2d (Fig. 3B, lane
18) (Table I). In the case of the 639+642 mutant (Fig. 3B,
lane 13 versus lane 12), the co-immunoprecipitation efficiency did
not increase (Table I), probably because it already reached the maximum
level. In secreted proteins, it has been shown that increasing the
number of oligosaccharide chains from one to two increases the
efficiency of co-immunoprecipitation with calnexin severalfold (30,
38). Here, we demonstrate that calnexin could interact with
oligosaccharides located on different EC loops in a polytopic membrane
protein and that increasing the number of oligosaccharide chains from
one to two led to a modest increase in the efficiency of
co-immunoprecipitation.
Calnexin Interaction with Truncated AE1md Constructs--
To test
whether the interaction of calnexin with AE1md occurs
co-translationally, restriction enzymes were used to linearize the
AE1md cDNA to produce truncated mRNAs that lacked a stop codon. During translation, this results in the production of
translocation-arrested intermediates at defined positions in the AE1md
sequence that retained the final tRNA (60). These intermediates can be
N-glycosylated if the distance from the P-site in the
ribosome to the active site on the oligosaccharyl transferase is
greater then 67 residues (61). This number of residues increased to 71 if an intervening transmembrane segment was present (61). The
intermediates can be released from their ribosomes upon the addition of
puromycin, which allows the polypeptide chain to move from the
translocon into the lipid bilayer (62, 63) and allows
N-glycosylation of short fragments to occur (61). The
truncated mRNAs at amino acid positions 696, 716, 754, 820, and 854 were constructed in order to vary the number of amino acid residues
following the endogenous N-glycosylation acceptor site at
Asn-642 (Fig. 1). The resulting polypeptide chain lengths, following
Asn-642, were 54, 74, 112, 178, and 212 residues. Another construct was
made by truncating the insertion mutant EC1 at position 854, 444 amino acid residues after the acceptor site in the expanded EC1. The constructs were translated in a cell-free system in the presence or
absence of Ac-NYT-NH2.
The cell-free translation results are shown in Fig.
4A, including the full-length
AE1md (Asn-642), which contained the original stop codon (Fig.
4A, lanes 1-3). Inclusion of Ac-NYT-NH2 in the translation reaction produced the nonglycosylated protein (Fig. 4A, lane 2, open circle). The posttranslational addition of
puromycin did not change the level of N-glycosylation of the
full-length AE1md (Fig. 4A, lane 1 versus lane 3, closed
circles).

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Fig. 4.
A, autoradiograph of cell-free
translation products of the AE1md (N642) and translocation arrested
constructs lacking the stop codon (numbers 54, 74, 112, 178, 212, and
444 indicate number of residues after N-glycosylation site).
Cell-free translation was carried out in the presence of microsomes,
and Ac-NYT-NH2 (35 µM) was included in the
translation reaction as indicated. Addition of puromycin (1.5 mM) was 30 min after the start of the translation reaction.
Microsomes were alkali-extracted and resolved on SDS-polyacrylamide
gels. Closed circles indicate the position of the
glycosylated band, and open circles indicate the
nonglycosylated band. The positions of molecular mass markers are
indicated to the left of the figure in kDa. B,
percentage of N-glycosylation for translocation arrested
constructs lacking the stop codon and AE1md (N642) as indicated,
without (black bars) or with (hatched bars) added
puromycin. C, autoradiograph of cell-free translation
products of the AE1md (N642) and translocation arrested constructs
lacking the stop codon (numbers 54, 74, 212, and 444 indicate number of
residues after N-glycosylation site). Microsomes were
alkali-extracted (lanes 1-3, 7-9, 13-15, 19-21, and
25-27) or subjected to anti-calnexin immunoprecipitation,
as indicated. Antibody directed against the carboxyl terminus of
calnexin is indicated as C; antibody directed against the
soluble, ER lumenal region of calnexin is indicated as N.
Closed circles indicate the position of the glycosylated
band, and open circles indicate the nonglycosylated band.
The positions of molecular mass markers are indicated to the
left of the figure in kDa.
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When truncation 54 was translated in the presence of microsomes less
than 10% of the protein was N-glycosylated (Fig. 4A, lane 4, closed circle). The presence of a small amount of a
glycosylated band indicated that under the experimental conditions 10%
of the ribosomes were not firmly attached to the translocon complex, resulting in the release and N-glycosylation of nascent
chains. The addition of puromycin after 30 min resulted in the release of the nascent chain and increased N-glycosylation of
truncation 54 (Fig. 4A, lane 6). The chain length of
truncation 54 was too short to be N-glycosylated
co-translationally. Translation of truncation mutant 74 also resulted
in some co-translational N-glycosylation (Fig. 4A,
lane 7), but the level of glycosylation increased after the
addition of puromycin (Fig. 4A, lane 9). Truncations 112, 178, 212, and 444 resulted in co-translational glycosylation (Fig. 4A, lanes 10, 13, 16, and 19). In these
truncations, the level of glycosylation did not increase upon the
addition of puromycin (Fig. 4A, lanes 12, 15, 18, and
21). This indicates that the nascent chains of these
truncations were of sufficient length for the acceptor site to reach
the oligosaccharyltransferase in the ER lumen. Fig. 4B shows
that 112 residues were sufficient for efficient co-translational
N-glycosylation of AE1. Short constructs (truncations 54 and
74) were efficiently N-glycosylated after their release from
the ribosome.
Anti-calnexin immunoprecipitation with the antibody directed against
the carboxyl terminus of calnexin showed that calnexin interaction with
the nascent chains of the truncated constructs could not be detected
(Fig. 4C, lanes 10, 16, 22, and 28). This was as
expected for the short constructs (truncations 54 and 74), which were
poorly N-glycosylated. The longer constructs were
efficiently N-glycosylated, yet they were not
co-immunoprecipitated with the anti-calnexin antibody. Because this
antibody is directed against the cytoplasmic tail of calnexin, the
ribosome may sterically block the epitope. We therefore also performed
co-immunoprecipitation with a polyclonal anti-calnexin antibody
directed against the soluble, lumenal region of calnexin. Although this
antibody efficiently co-immunoprecipitated the full-length AE1md
similar to the anti-carboxyl terminus calnexin antibody (Fig. 4C,
lane 5), it could not co-immunoprecipitate any of the truncated
constructs (Fig. 4C, lanes 11, 17, 23, and 29).
The interaction of N-glycosylated truncated chains with
calnexin could be detected using the amino-terminal antibody after the
nascent chains had been from released from their ribosomes with
puromycin (Fig. 4C, lanes 12, 18, 24, and 30).
Similar results were obtained using the carboxyl-terminal antibody
(data not shown). The immunoprecipitates were enriched in the
N-glycosylated forms over nonglycosylated, consistent with a
glycosylation-dependent interaction. Although truncations
112, 178, 212, and 444 were efficiently glycosylated
co-translationally, the interaction with calnexin was not detected
until their release from the ribosome. This suggested that even the
extension of the nascent chain by 444 amino acid residues after the
utilized N-glycosylation site was not sufficient to allow
co-translational interaction with calnexin. The interaction was
detected after incubation with puromycin, showing that the most
prominent interaction of AE1md with calnexin was posttranslational.
Calnexin Interacts with AE1 in Transfected Cells--
To test
whether the AE1 interaction with calnexin also occurs in
vivo, we transiently transfected HEK293 cells with full-length AE1, the nonglycosylated N642D mutant, and the Asn-555 mutant with a
single novel N-glycosylation site at position 555. HEK293 cells endogenously express calnexin and were previously used in studies
with other membrane glycoproteins (35, 45, 46). Mock transfected cells
were used as a control (Fig. 5, lanes C). Cells labeled with
[35S]Met for 20 min were lysed in digitonin and subjected
to immunoprecipitation. Immunoprecipitation with anti-AE1 antibody is
shown in Fig. 5 (lanes 1-4).
Transfected cells expressed comparable amounts of the AE1 protein that
corresponds to the expected size of full-length AE1 (95 kDa) (50).
Immunoprecipitation with anti-calnexin antibody detected a number of
labeled proteins (Fig. 5, lanes 5-8), as expected for
calnexin interaction with newly synthetized glycoproteins. The major
band was at 95 kDa in AE1-transfected (lane 6) and
Asn-555-transfected (lane 8) cells, but not in control
(lane 5) or N642D-transfected (lane 7) cells. The
amount of co-immunoprecipitated AE1 indicates that AE1 is the major
newly synthetized glycoprotein in these transfected cells (lane
6). When anti-calnexin immunoprecipitation was followed by a
second immunoprecipitation using anti-AE1 antibody (Fig. 5, lanes
9-12) a faint band was detected in the control sample (lane
9). This is likely residual calnexin, which has a molecular mass
(90 kDa) similar to that of deglycosylated AE1. Double
immunoprecipitation detected the two N-glycosylated forms, AE1 and Asn-555. The nonglycosylated mutant of AE1, N642D, interacted poorly with calnexin (lane 11). Some of the protein band in
this double immunoprecipitation may be contributed by calnexin as in the control sample (lane 9). This N642D-calnexin interaction
was consistently weaker then the AE1-calnexin interaction in four independent transfections. Therefore, we conclude that AE1-calnexin interaction in vivo is glycosylation-dependent.
Pulse-chase experiments show that AE1-calnexin interaction is transient
(data not shown). The interaction of AE1 and Asn-555 mutant with
calnexin was diminished after a 1-h chase and completely lost after a
5-h chase, similar to findings with other membrane glycoproteins (5,
44, 64).

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Fig. 5.
Transfected HEK293 cells were labeled with
[35S]Met for 20 min, lysed in 1% digitonin PBS buffer
and subjected to immunoprecipitation, using anti-AE1 antibody
( -AE1) or anti-calnexin antibody directed against
the ER lumenal region of calnexin ( -calnexin), or
to double immunoprecipitation, in which anti-calnexin immunoprecipitate
was reprecipitated using anti-AE1 antibody
( -calnexin/ -AE1). Samples were run on 8%
SDS-polyacrylamide gels and exposed to autoradiography. Closed
circle indicates the position of the 95-kDa
N-glycosylated AE1, and the open circle indicates
the position of the nonglycosylated N642D mutant.
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DISCUSSION |
N-Glycosylation-dependent Interaction of Calnexin with
AE1--
Although calnexin acts primarily as a lectin,
glycosylation-independent interactions of calnexin with a number of
membrane proteins (6, 35, 37, 65) and some secretory proteins (36) have
been reported. The significance of these interactions is controversial.
The glycosylation-independent interaction may be an additional mode of
interaction (23) or alternatively an interaction with accumulated
aggregates of the protein (38). In our in vitro and
in vivo experiments, interaction of AE1 with calnexin was
glycosylation-dependent and glycosylation-independent interactions were at least 3-fold lower. Major changes to the AE1
structure introduced by insertion into EC loop 1, 2, or 7 did not
increase the glycosylation-independent interactions.
In previously described interactions of calnexin with membrane
proteins, the glycosylated region of the membrane protein was restricted to a single EC domain. In a polytopic membrane protein, various EC domains may not be equally accessible to interact with calnexin. Here, we systematically introduced N-glycosylation
sites into EC loops in AE1, a protein with 12-14 TM segments. Our
study shows that there is no spatial restriction for calnexin to
approach any EC loop in AE1. The dimer of the AE1md of AE1 is 110 Å long and 60 Å wide, as viewed by electron microscopy of
two-dimensional crystals (66). Also, it is known that oligosaccharide
positioned very close (12 residues) to the membrane were not restricted
in their interaction with calnexin (31). Together with our results, we
suggest that calnexin can interact with different glycosylated lumenal
regions of polytopic membrane proteins.
In secreted proteins, multiple oligosaccharide chains increase the
amount of protein co-immunoprecipitated with calnexin (30, 38). The
presence of two oligosaccharide chains in AE1 also increased the
proportion of protein complexed with calnexin, regardless of the
positions of the oligosaccharide chains. There are reports that
calnexin exists as a homotetramer/pentamer (11); therefore, multiple
oligosaccharides on a glycoprotein could increase the avidity of
binding. Alternatively, calnexin may be monomeric (7). In this case,
multiple calnexin molecules could bind simultaneously to several
oligosaccharides. It is also possible that multiple lectin domains
within calnexin could interact simultaneously with several
oligosaccharides on one glycoprotein. Our data suggest that several
calnexin molecules are required to interact with multiple
oligosaccharide chains placed in AE1 because the interaction is
enhanced regardless of the relative locations of the two
N-glycosylated sites in the protein.
Posttranslational Interaction of Calnexin with AE1--
Our
results indicate that calnexin-AE1 interactions occur after release of
the nascent chain from the ribosome. Most calnexin interactions with
newly synthesized proteins were characterized using full-length
proteins, i.e. posttranslationally, where even multiple
rounds of interaction with substrate proteins occur (9, 21, 67, 68).
There are few reports in which calnexin was shown to interact with
nascent chains co-translationally (43, 69, 70). Two papers (69, 70)
describe the calnexin interaction with a truncated construct of glucose
transporter Glut 1 (GT155). However, the yield of the
calnexin-nascent chain complex immunoprecipitated with the
anti-calnexin antibody was low compared with the total amount of
nascent chain (70). In the other experiment, in which calnexin
interacted with a population of incomplete type I membrane protein
chains of influenza hemagglutinin, the level of calnexin interaction
was estimated to be about 20% (43). An interaction with nascent chains
was first detected ~250 residues after the first glycosylation site.
This length of soluble hemagglutinin fragment could be sufficient to
span the distance between the P-site in the ribosome and the binding
site on calnexin. In our experiments on a polytopic membrane protein,
AE1, a length of 444 residues was not sufficient to detect the
calnexin-nascent chain interaction. Our results show that although
calnexin can interact with any EC region of AE1 posttranslationally, it
may not be positioned to interact with the translocating chains of this
polytopic membrane protein.
Sec61p,
subunit of translocon-associated protein, and
oligosaccharyl transferase complexes are directly associated with ribosomes (71). A high molecular weight form of calnexin has been
reported to associate with ribosomes and Sec61p (72), the core
component of the translocon apparatus. Calnexin has also been
co-purified with the translocon-associated protein
(42). This
suggests that a fraction of calnexin may be associated with the protein
translocation complex in the ER. Calnexin in the vicinity of the
translocon may be available to interact with N-glycosylated proteins upon their release from the translocon.
The journey of a nascent chain from the translocon to be a fully
integrated membrane protein can be viewed through the fate of a
oligosaccharide chain. After its co-translational attachment to nascent
chains, the Glc3Man9GlcNAc2
oligosaccharide meets two
-glucosidases. The removal of glucose
residues has been shown to occur co-translationally on a nascent
vesicular stomatitis virus G protein (16). Removal of the outer
two glucose residues primes the glycoprotein for the subsequent
rendezvous with calnexin. It has been shown that the re-glucosylation
reaction by UDP-glucose glycoprotein glucosyltransferase occurs
posttranslationally (20, 73); therefore, calnexin must interact with
re-glucosylated unfolded glycoproteins posttranslationally. Our results
suggest that calnexin interacts with newly synthesized membrane
glycoproteins posttranslationally, after their release from the
ribosome and movement into the lipid bilayer. Once in the bilayer,
polytopic membrane proteins such as AE1 are free to interact with
calnexin. We have shown that calnexin can bind to oligosaccharides on
different loops in AE1, consistent with the interaction occurring
within the bilayer. This is in agreement with findings in which
multiple TM segments can be accommodated within the translocon before
being released into the lipid bilayer (54). Also, in the experiments with single TM constructs, the TM segment was not released to the lipid
bilayer until the stop codon was reached, regardless of the length of
the carboxyl-terminal tail (62, 63). These results suggest a sequential
and regulated movement of newly synthesized membrane proteins from
Sec61p and translocating-chain-associating membrane proteins to
chaperones such as calnexin. Functional expression of complementary
fragments of polytopic membrane proteins, including AE1 (55, 74-79),
suggests that the final folding likely occurs in the lipid bilayer and
supports the "two-stage model" of membrane proteins assembly (33).
In this process, the transient interaction with chaperones may be a
required step to prevent aggregation and nonspecific interactions
between the incompletely folded membrane proteins that are released
from the translocon.