From the
The nicotinic acetylcholine receptor (AChR) is a pentameric
complex assembled from four different gene products by mechanisms that
are inadequately understood. In this study we investigated the role of
the endoplasmic reticulum (ER)-resident molecular chaperone calnexin in
AChR subunit folding and assembly. We have shown that calnexin
interacts with nascent AChR
Many of the cell surface proteins that participate in
transmembrane signaling, including the ligand-gated ion channel family,
are structural oligomers formed by intracellular assembly of
polypeptide subunits. This assembly process typically takes place in
the endoplasmic reticulum (ER)
Studies utilizing cultured muscle cells expressing AChR
have shown in pulse-chase experiments that an interval of between 15
and 45 min separates the synthesis of AChR subunits from their
subsequent assembly into AChR pentamers(9, 10) . During
this interval AChR subunits have been shown to undergo several
post-translational modifications such as N-linked core
glycosylation, disulfide bond formation, fatty acid acylation, and
phosphorylation (for reviews see Refs. 8 and 11). The use of
transfected cell lines has made it possible to address various aspects
of subunit maturation and assembly, including the order in which the
subunits are assembled (12, 13, 14, 15) and the location of
putative recognition sites for subunit-subunit
interactions(16) . Nevertheless, the mechanisms responsible for
the assembly of AChR in a precise stoichiometry and order of subunits
are poorly understood.
The role of the ER lumen in maintaining a
unique microenvironment that can mediate correct folding of newly
synthesized membrane and secretory proteins and bring about the
assembly of multimeric proteins is now well documented. A related
feature is the capacity to prevent the export of misfolded protein
products and unassembled subunits by their selective retention in the
ER(1) . Recent studies have characterized in increasing detail
the contributions to these functions of a number of ER resident
molecular chaperones, proteins that interact with newly synthesized
polypeptide chains to prevent aggregation and misfolding and possibly
to mediate specific oligomerization events (for reviews see Refs.
17-20). Calnexin, an ER chaperone that is itself a transmembrane
protein(21) , was recently shown to form transient complexes
with individual subunits of several heterooligomeric membrane proteins;
major histocompatibility (MHC) class I (22, 23, 24) and class II molecules(25) ,
B cell and T cell receptors(23, 26, 27) ,
influenza virus hemagglutinin, vesicular stomatitis G
protein(28) , and integrin (29). Together these studies suggest
a causal relationship between association with calnexin and
oligomerization of multisubunit proteins.
In the present study we
show that nascent AChR
The clarified supernatants were
incubated at 4 °C with the specified antiserum for 3 h. Protein
A-Sepharose beads were then added, and incubation at 4 °C was
continued for a further 1 h. When mAb 61 was used as the first
antibody, rabbit anti-rat antisera preabsorbed to protein A-Sepharose
were used in the second incubation. The precipitates were washed five
times with the specified detergent-containing buffer and suspended in
50 µl of SDS sample buffer(36) . After incubation for 5 min
in a boiling water bath, the beads were centrifuged, and the
supernatants were fractionated on 10% SDS-polyacrylamide gels.
Radioactive bands were visualized by radiofluorography and quantitated
by densitometry or PhosphorImaging. In Fig. 6, each data point is
an average of measurements from three separate experiments ±
S.D.
In the
experiment shown in Fig. 1, cultured myotubes were pulse labeled
with Tran
To confirm the specificity of the
association of calnexin with
The
finding that
The
same experimental approach was used to measure the time course of
The
results shown thus far indicate that in the ER of cultured muscle cells
nascent
To investigate further the relationship between
In this study we found that the ER-resident molecular
chaperone calnexin forms complexes with newly synthesized AChR-
In this respect the interaction of calnexin with AChR
appears to be different from the interaction of calnexin with several
other oligomeric membrane proteins where the kinetics support the
possibility that calnexin physically mediates oligomerization. Degen
and Williams (22) reported that calnexin remains complexed with the
heavy chain of MHC class I during the assembly of this oligomeric
membrane protein and dissociates only after other components have been
assembled into the complex. Similarly, calnexin was found to remain
associated with MHC class II complexes throughout the assembly
process(25) . Furthermore, dissociation of calnexin was observed
to be concomitant with the assembly of oligomers in the cases of T cell
receptors (26) and integrins(29) . Finally, it has been
documented that in both MHC class I complexes (22) and T cell
receptors (26) the absence of one of the subunits prevented
assembly of the oligomers and dissociation of calnexin.
The apparent
``early'' dissociation of calnexin from AChR-
Several
considerations support the possibility that in chick muscle cells
calnexin interacts with
Calnexin may constitute
a part of the ER quality control mechanism by forming complexes with
nascent subunits and selectively releasing correctly folded subunits
while irreversibly misfolded subunits are complexed with BiP and
eventually get degraded. In the case of the AChR there is evidence that
ER quality control mechanisms may operate not only at the level of
subunit folding but also at subsequent levels of assembly and
postassembly. For instance, a C2 muscle cell subline has been described
in which a fully assembled variant form of AChR does not exit the
ER(41, 50) . Our kinetic data suggesting that calnexin
dissociates from the
We thank Dr. Jon Lindstrom for generously supplying
mAb 61 and Dr. Jim Boulter for the gift of AChR-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunits (AChR-
) in muscle cell
cultures and in COS cells transfected with mouse AChR-
. In chick
muscle cells maximal association of labeled
-subunits with
calnexin was observed immediately after a 15-min pulse with
[
S]methionine/cysteine and subsequently declined
with a t of approximately 20 min. The decrease in association
with calnexin was concomitant with the folding of the
-subunit to
achieve conformational maturation shortly before assembly. Brefeldin A
did not inhibit AChR subunit assembly or the dissociation of calnexin
from the assembling subunits, confirming that the ER is the site of
AChR assembly and that calnexin dissociation is not affected under
conditions in which the exit of assembled AChR from the ER is blocked.
These results indicate that calnexin participates directly in the
molecular events that lead to AChR assembly .
(
)(for review see
Ref. 1) or in some cases in the Golgi apparatus (2, 3) and involves highly specific folding,
recognition, and association of polypeptides producing hetero- or
homooligomers that are then transported to the cell surface. The muscle
nicotinic acetylcholine receptor (AChR) is a ligand-gated membrane ion
channel that mediates neuromuscular synaptic transmission upon binding
the neurotransmitter acetylcholine released by the motor neuron. This
binding alters the conformation of the receptor causing increased
cation permeability and consequent membrane
depolarization(4, 5) . The AChRs are 250-kDa pentameric
complexes of four discrete AChR subunits in the stoichiometry
, with each subunit encoded by a
different gene and translated from a separate mRNA (for reviews see
Refs. 6 and 7). These subunits assemble intracellularly, and the
resulting pentamers are exported through the Golgi complex to the cell
surface (8).
-subunit (AChR-
) forms complexes with
calnexin in the ER of cultured muscle cells and COS cells transfected
with mouse AChR-
. In muscle cells this association is transient
with conformationally mature subunits undergoing dissociation from
calnexin. Our results indicate that transient interaction with calnexin
is an early post-translational event in AChR biogenesis, contributing
to the folding and assembly of AChR subunits in the ER.
Reagents
TranS-label
(specific activity 1,050-1,200 Ci/mmol) was purchased from ICN
Radiochemicals.
I-
-Bungarotoxin (
I-
-Bgt) (specific activity 13-15
µCi/µg) was from DuPont NEN. Polyacrylamide gel electrophoresis
reagents were from Bio-Rad. All other reagents were from Sigma.
Cell Culture
Muscle primary cultures were
prepared from breast muscle of 12-day-old chick embryos as described
previously(10, 30) . The cells were plated on
collagen-coated culture dishes at initial densities of 6
10
cells/100-mm culture dish. Cultures were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
10% horse serum and 2% chick embryo extract at 37 °C in an
atmosphere of 92% air, 8% CO
. COS cells (African green
monkey kidney cells, CRL 1650, American Type Culture Collection) were
cultured in DMEM supplemented with 10% fetal calf serum.
Antibodies
Anti-chick AChR- antibody
and anti-chick AChR
-subunit (AChR-
) antibody were raised in
rabbits against the respective subunits purified on SDS-polyacrylamide
gel electrophoresis from denervated chick leg muscle and were shown to
be noncross-reactive(10) . Anti-
-Bgt antibody was raised in
rabbits and affinity purified on
-Bgt-Sepharose(10) . The
monoclonal antibody mAb 35, which recognizes the main immunogenic
region of AChR-
(31) , was isolated from the supernatant of
hybridoma TIB 175 (American Type Culture Collection). The monoclonal
antibody mAb 61, which recognizes mouse AChR-
, was provided by Dr.
Jon Lindstrom. Anti-calnexin antibody was raised against a synthetic
peptide corresponding to a highly conserved stretch near the COOH
terminus (amino acids 487-505) of
calnexin(32, 33) .
Transfections
Full-length cDNA coding for
the mouse AChR- (provided by Dr. Jim Boulter) was subcloned into
the pRc/CMV expression vector (Invitrogen), which contains a simian
virus 40 origin and is driven by the cytomegalovirus promoter.
Transfection of COS cells for transient expression of AChR-
was
carried out by DNA-calcium phosphate precipitation(34) .
Briefly, 60-mm dishes of cells at 30-50% confluence were
incubated for 6-16 h at 37 °C with 5 µg of the
-subunit expression vector in a mixture containing CaCl
and HEPES-buffered saline solution. Cultures were then washed
twice with phosphate-buffered saline and incubated in fresh DMEM
containing 10% fetal calf serum for 1 more day before cells were
harvested.
AChR Surface Labeling
AChR on intact
muscle cells was monitored by the binding of I-
-Bgt
as described previously(10, 35) . Cultures were washed
once with DMEM and incubated with
I-
-Bgt
(10
M) in DMEM containing bovine serum
albumin (1 mg/ml) for 1 h at 37 °C. At the end of this period,
cultures were washed five times with 3-ml volumes of DMEM to remove
unbound toxin. Cells were solubilized in 1 N NaOH containing
1% Triton X-100, and labeling was quantitated by
-counting.
Metabolic Labeling and
Immunoprecipitation
Cultures were methionine-depleted by
incubation with methionine-free DMEM for 1 h and then labeled at 37
°C with a mixture of [S]methionine and
[
S]cysteine (Tran
S-label; for
specific activities see figure legends) in methionine-free DMEM for the
specified time. In the pulse-chase experiments, chase was performed by
washing cells once with DMEM followed by incubation in DMEM
supplemented with 5 mML-methionine. Cells were
harvested as follows. Cultures were washed twice with ice-cold
Dulbecco's phosphate-buffered saline, scraped, and extracted for
30 min at 4 °C in HBS buffer (50 mM HEPES, pH 7.5, 200
mM NaCl, 1 mM CaCl
, 10% glycerol, 1
mM phenylmethylsulfonyl fluoride, 1% aprotinin, 10 µg/ml
leupeptin, 10 mMN-ethylmaleimide) supplemented with
2% sodium cholate. Alternatively, cells were harvested exactly as
above, except that instead of HBS cholate, the lysis buffer was STE
(150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 2 mM EGTA, 2 mM EDTA) supplemented with 1% Triton X-100.
Clarification of the extracts was achieved by centrifugation for 30 min
in the microcentrifuge at 4 °C.
Figure 6:
Temporal relationship between calnexin
dissociation and folding and assembly of AChR- in chick myotubes.
Values obtained by scanning densitometry of the time course data of
calnexin dissociation (circles), folding (squares),
and assembly (triangles) are plotted against the chase time.
Each point represents the mean of triplicate determinations
± S.D.
Sequential immunoprecipitations were carried out as described
by Ou et al.(37) . After immunoprecipitation with
anti-calnexin or anti- antibody, proteins were eluted from the
protein A-Sepharose beads under denaturing conditions by suspending in
50 µl of HBS containing 1% SDS and heating at 75 °C for 10 min.
The supernatants were then diluted with 1 ml of HBS containing 1%
Triton X-100 and precipitated with the second antibody. Where
specified, elution was carried out under nondenaturing conditions by
incubating the precipitates twice sequentially with 0.5 ml of HBS
containing 1% Triton X-100 for 15 min at room temperature.
Assembly of AChR Is Insensitive to Brefeldin A
(BFA), whereas Cell Surface Expression of Assembled AChR Is Blocked by
BFA
We have shown earlier that the assembly into oligomeric
AChR of [S]methionine-labeled
-subunit can
be monitored accurately in pulse-chase experiments by its
coprecipitation with antiserum specific for the
-subunit and
noncross-reactive with
-subunit(10) . These measurements
revealed that AChR assembly in chick muscle cells is initiated
15-30 min after subunit biosynthesis and is completed within the
next 60 min(10) . As the object of our present study was to
determine the contribution of the ER resident protein calnexin to AChR
assembly, we first ascertained that assembly takes place in the ER of
cultured chick myotubes. For this purpose, we measured the effects on
AChR biogenesis of BFA, an antiviral antibiotic that blocks the
transport of newly synthesized proteins from the ER to the Golgi
apparatus and cell surface while causing vesiculation of Golgi
cisternae and Golgi-ER fusion (for review see Ref. 38).
S-label for 15 min and then incubated for the
specified intervals in chase medium either in the absence or presence
of BFA (1 µg/ml). AChR assembly was monitored by
coimmunoprecipitation of labeled
-subunit with anti-
antibody. The appearance of AChR on the external surface of muscle
cells was monitored by labeling intact cells with the AChR ligand
-Bgt and subsequent immunoprecipitation with anti-
-Bgt
antibody. As can be seen in Fig. 1, the newly synthesized
-subunit is clearly discernible as a 40-kDa band upon
SDS-polyacrylamide gel electrophoresis of Triton X-100 extracts of
radiolabeled cultures that have been immunoprecipitated with anti-
antibody (lane 1). Under these conditions, in which cultures were
extracted and immunoprecipitated immediately after the 15-min pulse,
only a minimal amount of
-subunit is precipitated with anti-
antibody, reflecting the low level of assembly at this early time point (lane 2, also see Fig. 5B). However, after a
90-min chase period a major proportion of the labeled
-subunit has
undergone assembly and is consequently coprecipitated with anti-
antibody (lane 3). We have previously observed that AChR
assembly in chick muscle cells is highly efficient, with practically
all nascent
-subunit being chased into oligomeric complexes under
these experimental conditions(10) . As noted by ourselves (10) and others(39) , the
-subunit, which migrates
at 55 kDa, is difficult to resolve in
[
S]methionine-labeled preparations because of
its high susceptibility to proteolysis and nonspecific backgrounds in
this region of the gel. Therefore,
-subunit could not be
identified as a distinct band in this figure. At the end of a 3-h chase
period the radiolabeled assembled
-subunits have been transported
to the cell surface, and can now be precipitated from
-Bgt
surface-labeled cultures with anti-
-Bgt antibody (Fig. 1, lane 5). Treatment of cultures with BFA completely abolished
the transport of pulse-labeled AChR to the cell surface (lane
6) but had no effect on the assembly of labeled
-subunit with
-subunit in the same cultures (lanes 3 and 4).
These results demonstrate that AChR assembly in chick myotubes takes
place within a BFA-insensitive compartment, likely the ER. Our
subsequent experiments focused on investigating the interactions within
the ER which contribute to AChR subunit folding and assembly.
Figure 1:
Effects of BFA on
AChR- assembly with
-subunit and on the appearance of AChR on
the cell surface. Cultured muscle cells 3 days after plating were pulse
labeled with Tran
S-label (150 µCi/ml, 15 min) and
chased for 0 h (lanes 1 and 2), 1.5 h (lanes 3 and 4), or 4 h (lanes 5 and 6) in the
absence (lanes 1-3 and 5) or presence (lanes 4 and 6) of BFA (1 µg/ml). Cultures used
for the immunoprecipitations (IP) shown in lanes 1-4 were extracted with STE buffer supplemented with 1% Triton X-100
and precipitated with anti-
(lane 1) or anti-
antibody (lanes 2-4). Cultures used for the
immunoprecipitations shown in lanes 5 and 6 were
surface labeled with
-Bgt (10 nM) during the final 1 h of
chase and then extracted with STE buffer supplemented with 1% Triton
X-100 and precipitated with anti-
-Bgt antibody as described under
``Materials and Methods.''
Figure 5:
Panel A, time course of maturation of
nascent AChR-. Cultured muscle cells were pulse labeled with
Tran
S-label (200 µCi/ml, 15 min) and chased for the
indicated times. Immunoprecipitations were carried out as described
under ``Materials and Methods,'' using nonimmune serum (lane 1) or mAb 35, a conformation-sensitive antibody that
selectively recognizes folded AChR-
(lanes 2-6). Panel B, time course of assembly of nascent AChR-
with
AChR-
. Cultured muscle cells were pulse labeled and chased exactly
as described in panel A. Immunoprecipitations were carried out
using nonimmune serum (lane 1) or anti-chick AChR-
antibody (lanes 2-6). Panel C, measurement of
the relative amount of labeled nascent AChR-
after increasing
chase intervals. Cells were pulse labeled and chased as in panels A and B, and cell extracts were immunoprecipitated exactly
as above, with the exception that anti-chick AChR-
antibody was
used.
Immunoprecipitation of AChR-
Calnexin is a newly characterized ER resident
transmembrane protein that functions as a molecular chaperone to
nascent secretory and membrane proteins and has been implicated in the
oligomerization of several surface proteins (22-29). To
investigate the possibility that calnexin participates in AChR subunit
folding and assembly, we first determined if recently synthesized AChR
subunits are bound to calnexin. Cultured chick muscle cells were
labeled with Tran and Calnexin:
Evidence for Association of Calnexin with the
-Subunit
S-label for 1 h, and cell lysates were
immunoprecipitated with anti-calnexin antisera under conditions shown
to preserve the interaction between calnexin and associated
proteins(37) , resulting in the presence in the precipitate of a
large number of labeled proteins (not shown). The anti-calnexin
immunoprecipitate was then solubilized by incubating the protein
A-Sepharose beads in SDS-containing buffer and reimmunoprecipitated
with anti-calnexin antibody. Radiolabeled calnexin is prominent,
migrating as a doublet at
90 kDa (Fig. 2A, from left, first lane), consistent with the migration
pattern observed by others(40) . When the solubilized
anti-calnexin immunoprecipitate was reimmunoprecipitated with
anti-
-subunit antibody, labeled
-subunit was clearly resolved (Fig. 2A, fourth lane). For comparison, the fifth lane of Fig. 2A shows the result of the
immunoprecipitation of these cells using the anti-
-subunit
antibody twice in sequence. Together, these immunoprecipitations
indicate that a measurable amount of the AChR-
synthesized by
chick muscle cells during the labeling period becomes physically
associated with calnexin.
Figure 2:
Association of calnexin with AChR- in
chick muscle cells and COS cells transfected with mouse
-subunit. Panel A, cultured muscle cells were labeled with
Tran
S-label (200 µCi/ml) for 1 h. The labeled cultures
were extracted and immunoprecipitated (IP) in HBS buffer
containing 2% sodium cholate with anti-calnexin followed by
anti-calnexin (cal) (from left, first lane);
anti-calnexin antibody followed by nonimmune (NI) serum (second lane); nonimmune serum followed by anti-
(third lane); anti-calnexin followed by anti-
(fourth
lane); anti-
followed by anti-
(fifth lane). Arrows on the left indicate the positions of
calnexin. Panel B, COS cells were transfected with mouse
-subunit cDNA as described under ``Materials and
Methods'' and 2 days later were labeled with
Tran
S-label (200 µCi/ml) for 4 h, extracted in HBS
buffer supplemented with 2% sodium cholate, and immunoprecipitated with
anti-calnexin followed by anti-calnexin (left lane);
anti-calnexin followed by anti-
(mAb 61) (center lane);
anti-
(mAb 61) followed by anti-
(mAb 61) (right
lane). Molecular mass markers are shown at the left of
the panels.
We repeated these measurements with COS
cells transfected with mouse AChR-. In contrast to muscle cell
cultures, which express all four AChR subunits and transport assembled
AChR to the cell surface, the COS cells expressed only the one
transfected subunit, which does not assemble into pentameric AChR and
is restricted to the ER (41).
(
)As shown in Fig. 2B, in these cells a portion of the AChR-
is
coimmunoprecipitated by antibody directed against calnexin, comparable
to the results obtained with the myotubes. These results show that
calnexin and
-subunit association is not a phenomenon specific to
muscle cells, nor is it restricted to
-subunit that is undergoing
assembly into pentameric AChR.
subunit, this interaction was also
investigated using an alternative order of sequential
immunoprecipitations, and the results are shown in Fig. 3A. To visualize calnexin associated with
-subunit, cells were metabolically labeled for 15 h and then
extracted and immunoprecipitated first with anti-
antibody and
then with anti-calnexin antibody (lane 3). The long labeling
period was utilized since calnexin is relatively stable with a
metabolic t > 24 h(20) . Under these conditions
calnexin coimmunoprecipitated by anti-
antibody was clearly
visible (though constituting only a small fraction of total cellular
calnexin; compare lanes 2 and 3). When the monoclonal
antibody mAb 35, which selectively recognizes conformationally mature
-subunit(31) , was used instead of anti-
antibody in
the first immunoprecipitation, the amount of calnexin precipitated was
not significantly above background (lane 4). These data showed
that anti-
antibody, which recognizes unfolded as well as folded
AChR-
, was more effective than mAb 35 in precipitating
-subunit-calnexin complexes, suggesting that calnexin does not
bind appreciably to folded
.
Figure 3:
Panel A, association of AChR- with
calnexin (cal) in chick muscle cells. Cultured muscle cells
were labeled with Tran
S-label (200 µCi/ml) for 15 h in
order to label calnexin. The labeled cultures were extracted and
immunoprecipitated (IP) in HBS cholate buffer with nonimmune (NI) serum followed by anti-calnexin (lane 1);
anti-calnexin antibody followed by anti-calnexin (lane 2);
anti-
antibody followed by anti-calnexin (lane 3); mAb 35
followed by anti-calnexin (lane 4). Panel B,
relationship between calnexin association and conformational maturation
of
-subunit. Cultured muscle cells were labeled with
Tran
S-label (200 µCi/ml) for 1 h. The labeled cultures
were extracted and immunoprecipitated in HBS buffer containing 2%
sodium cholate with nonimmune serum followed by anti-
antibody
(from left, first lane); nonimmune serum followed by
mAb35 (second lane); anti-calnexin antibody followed by
anti-
(third lane); anti-calnexin antibody followed by
mAb 35 (fourth lane); anti-
followed by anti-
(fifth lane). In the first four lanes, the elution of
protein complexes resulting from the first immunoprecipitation was
carried out under nondenaturing conditions as described under
``Materials and Methods.''
To verify this finding, the
sequential immunoprecipitations were repeated but this time with
anti-calnexin antibody followed by mAb 35. To preserve the mAb 35
epitope, -subunit coprecipitated with calnexin was resolubilized
under nondenaturing conditions using Triton X-100. As can be seen in Fig. 3B, a readily detectable amount of radiolabeled
-subunit was precipitated with anti-calnexin followed by
anti-
antibody (third lane), whereas only a trace of
-subunit was precipitated with anti-calnexin followed by mAb 35 (fourth lane). Since under these conditions both anti-chick
-subunit antibody and mAb 35 were equally efficient in
immunoprecipitating mature
-subunit in assembled AChR (data not
shown), these results support the idea that calnexin preferentially
associates with unfolded
-subunit that has not acquired mAb 35
epitope.
Pulse-Chase Analysis of the Association with
Calnexin, Maturation and Assembly of AChR-
We next
measured the time course of the interaction of -subunit with
calnexin in the context of subunit folding and assembly. For this
purpose cultures were labeled for 15 min with Tran
S-label
and then chased in DMEM supplemented with 5 mML-methionine for 0, 15, 30, and 60 min. Shown in Fig. 4A (lanes 3-6) are the results of
sequential anti-calnexin, anti-
immunoprecipitations after
increasing chase intervals. The maximal levels of radiolabeled
-subunit associated with calnexin were evident immediately after
the 15-min pulse period (Fig. 4A, lane 3) and
could be seen to decrease rapidly in a time-dependent manner during the
chase period (Fig. 4A, lanes 4-6).
Scanning densitometry and PhosphorImager measurements showed that the
-subunit band precipitated by anti-calnexin antibody at the end of
the pulse period consistently contained 10-20% of the
radioactivity found in a similar band precipitated by anti-AChR-
antibody twice sequentially. Titration of radiolabeled calnexin
precipitated with increasing amounts of anti-calnexin antibody (not
shown) revealed that the amount of anti-calnexin antibody used in these
experiments was sufficient for precipitation of approximately 20% of
the total cellular calnexin. Taking this into account, as well as the
weak nature of calnexin binding to substrate proteins which may lead to
some dissociation upon cell lysis, it is likely that a major proportion
of nascent AChR-
undergoes transient association with calnexin in
the ER of chick myotubes.
Figure 4:
Kinetics of association of calnexin with
nascent AChR-. Panel A, cultured muscle cells 3 days
postplating were pulse labeled with Tran
S-label (400
µCi/ml, 15 min) and chased for the indicated times. Extractions and
sequential immunoprecipitations were carried out as described under
``Materials and Methods'' with nonimmune serum followed by
anti-
(lane 1); anti-
followed by anti-
(lane 2); anti-calnexin followed by anti-
(lanes
3-6). Panel B, the amounts of radiolabeled
-subunit in lanes 3-6 of panel A were
quantified by scanning densitometry and plotted versus time on a
semilogarithmic scale.
Fig. 4B shows the rate at
which AChR- dissociates from calnexin in myotubes as measured in
pulse-chase experiments. When plotted semilogarithmically this rate can
be fitted by a straight line, characteristic of first-order kinetics.
As calculated from this curve the initial half-time of dissociation of
calnexin from newly synthesized AChR-
is 20 ± 2 min.
-subunit-calnexin complexes exist transiently led us
to investigate the temporal relationship between AChR-
-calnexin
dissociation and the two major developmental changes that the
-subunit undergoes in the ER of myotubes: conformational
maturation and assembly. The folding of AChR-
was measured by the
ability of this subunit to be recognized by the conformation-specific
antibody mAb 35(31) . As shown in Fig. 5A, the
amount of labeled
-subunit precipitated by mAb 35 increases as a
function of chase time (lanes 2-6), reflecting the fact
that nascent
-subunits in chick myotubes undergo time-dependent
post-translational changes leading to conformational maturation.
-subunit assembly with
-subunit. Muscle cultures were pulse
labeled with Tran
S-label and then chased for the specified
intervals. Immunoprecipitations were carried out with anti-chick
AChR-
antibody. Fig. 5B shows the time-dependent
increase in the amount of
S-labeled
-subunit
coimmunoprecipitated with anti-
subunit antibody (lanes
2-6). To verify that this rise reflects increased AChR
subunit assembly, we determined that the amount of labeled
-subunit directly immunoprecipitated by anti-
antibody does
not change with increasing chase times (Fig. 5C).
-subunits participate in three separate processes: the
reversible interaction with calnexin, folding leading to conformational
maturation, and assembly with other subunits. The temporal sequence of
these events is shown in Fig. 6. The association of nascent
-subunit with calnexin is the first of these processes, occurring
immediately after biosynthesis. This association is transient and is
followed by the relatively rapid dissociation of the
calnexin-
-subunit complex (Fig. 4). Also, the results
suggest that under these experimental conditions folding precedes
assembly by an interval of 10-20 min. As can be seen in Fig. 6, after a 30-min chase interval approximately 70% of the
-subunit has dissociated from calnexin, 65% has achieved
conformational maturation, but only about 35% has undergone assembly.
These measurements indicate that the conformational maturation of the
-subunit occurs concomitantly with the dissociation of the
calnexin-
-subunit complex, whereas assembly of
- with
-subunits occurs only after the
-subunit has dissociated from
calnexin.
-subunit interaction with calnexin on the one hand and AChR
assembly and exit from the ER on the other, we tested the possibility
that calnexin-
-subunit dissociation is a consequence of the exit
of the assembled AChR from the ER. Muscle cells were pulse labeled for
15 min and then chased for the specified intervals and
immunoprecipitated sequentially with anti-calnexin antibody followed by
anti-chick AChR-
antibody. In this experiment, the chase medium
contained BFA, which does not affect AChR assembly but prevents the
transport of newly assembled receptors out of the ER to the cell
surface (Fig. 1). As can be seen in Fig. 7, BFA treatment
did not alter the rate of calnexin dissociation from the
-subunit.
Thus, the dissociation of the calnexin-
-subunit complex is not
dependent on the transport of the subunit from the ER, similar to
findings reported for MHC class I-calnexin complex
dissociation(22) .
Figure 7:
Effects of BFA treatment on kinetics of
calnexin dissociation from AChR- in cultured chick muscle cells.
The scanning densitometry data of labeled
-subunit precipitated by
anti-calnexin antibody in the presence (inset) or absence
(Fig. 4A) of BFA are plotted against chase time. The solid
line represents the time course of calnexin dissociation in the
presence of BFA. The broken line corresponds to the time
course of calnexin dissociation in untreated cultures. Inset,
cultured muscle cells were pulse labeled with Tran
S-label
(400 µCi/ml, 15 min) and then chased in the absence (lane
1) or presence (lanes 2-7) of BFA (1 µg/ml) for
either 4 h (lanes 1 and 2) or 0 min (lanes 3 and 4), 15 min (lane 5), 30 min (lane
6), or 60 min (lane 7). Cultures used for the
immunoprecipitations shown in lanes 1 and 2 were
surface labeled with
-Bgt (10 nM) during the final 1 h of
chase and then extracted with STE buffer supplemented with 1% Triton
X-100 and precipitated with anti-
-Bgt antibody as described under
``Materials and Methods.'' Cultures used for the
immunoprecipitations shown in lanes 3-7 were extracted
in HBS buffer containing 2% sodium cholate and precipitated with
anti-chick AChR-
antibody followed by anti-chick AChR-
(lane 3), or anti-calnexin antibody followed by anti-chick
AChR-
(lanes 4-7). The extra (unnumbered) lane shows a precipitation with
anti-calnexin antibody followed by nonimmune
serum.
in
cultured chick muscle cells as well as in COS cells transfected with
mouse AChR-
. Using pulse-chase analysis we observed that nascent
-subunits associate with calnexin immediately or soon after their
synthesis, before the folding of the subunits into a mature
conformation takes place. These complexes are transient; shortly after
complex formation the
-subunit dissociates from calnexin with a
half-time of approximately 20 min. A comparison of the kinetics of
-subunit-calnexin dissociation with those of
-subunit folding
and subsequent assembly indicates that
-subunits remain associated
with calnexin during the process of conformational maturation. The
onset of AChR assembly occurs only after a major proportion of calnexin
has dissociated from
-subunits. Thus, calnexin appears to be
directly involved in the folding rather than in the assembly of
AChR-
. However, it is important to note that our measurements of
the time course of AChR assembly detected only high affinity
interactions between
- and
-subunits, and these might be
preceded by weak interactions that take place in the presence of
calnexin.
as
compared with MHC class I complexes and T cell receptors could reflect
the possibility that whereas the assembly of the immunological
receptors takes place in the ER, the assembly of AChR might not be
completed until after the subunits have been transported from the ER to
the Golgi. If the later stages of AChR assembly occur in the Golgi
apparatus, the direct participation of calnexin would not be
anticipated since the chaperone is apparently restricted to the ER by
means of a COOH-terminal ER retention motif(27, 42) .
The results of our experiments utilizing BFA do not support the
possibility that the Golgi apparatus is the site of AChR assembly. BFA
causes disruption of the Golgi apparatus (43) and was shown to
abolish the assembly of connexin, a multisubunit surface membrane
protein that undergoes assembly in the Golgi apparatus(3) . In
contrast, in the present study BFA treatment that totally blocked
transport of AChR between ER and cell surface had no effect on AChR
subunit assembly. Likewise, we found that the kinetics of calnexin
dissociation from AChR-
were not altered by the BFA treatment.
Together these results strongly indicate that the ER is the site of
AChR assembly and that dissociation of calnexin-
subunit complexes
is not due to the transport of AChR out of the ER. This complements
earlier evidence, based on subcellular fractionation (13) and
susceptibility to endoglycosidase digestion (41), which pointed to the
ER as the site of AChR assembly in mouse muscle cell lines.
subunits that are destined to assemble.
According to our current results, calnexin in these cells forms
complexes with the major proportion of newly made
-subunits. It is
unlikely that this large pool of calnexin-bound
-subunits
represents a nonassembling population of
, since AChR
oligomerization in chick myotubes appears to be highly efficient with
practically all nascent
-subunits undergoing assembly(10) .
Moreover, both the transient nature of
-subunit-calnexin complexes
and the timing of their dissociation suggest that the complexes
represent intermediates in AChR subunit folding and assembly. These
conclusions are summarized in the model shown in Fig. 8, which
incorporates calnexin association-dissociation into the sequence of
events leading to AChR assembly. According to the scheme, the existence
of the transient calnexin-subunit complexes occurs during the lag time
between subunit biosynthesis and assembly. During this interval
calnexin may both prevent nonproductive aggregation and mediate correct
folding of the subunits.
Figure 8:
Model
for the participation of calnexin in AChR subunit folding and assembly.
Nascent -subunit is cotranslationally inserted into the ER
membrane and binds to calnexin soon after synthesis. The folding of
-subunit to achieve conformational maturation occurs while it is
complexed to calnexin during the lag time between synthesis and
assembly. Correctly folded
-subunit dissociates from calnexin at
the onset of assembly.
Calnexin is the second ER molecular
chaperone shown to complex with AChR subunits. BiP, a soluble ER
molecular chaperone that forms transient complexes with several nascent
proteins and more stable complexes with misfolded
proteins(44, 45, 46) , was recently reported to
bind to AChR subunits expressed in the muscle-like cell line BC3H1,
transfected fibroblasts(47) , C2 mouse myotubes, and transfected
COS cells(48) . As in the present case of calnexin, BiP
selectively binds to unassembled -subunits and does not bind to
conformationally mature or assembled AChR-
. However, the kinetics
of the high affinity association of
-subunit with BiP are
completely different from those of calnexin-
-interaction. The
formation of BiP-
-subunit complexes has been reported to occur
with a time course that is slower than folding and assembly. Moreover,
the amount of
-subunit stably associated with BiP was observed to
increase during chase times of up to 6 h(48) . These differences
in kinetics suggest that BiP and calnexin play distinct roles in AChR
biogenesis. Whereas BiP forms complexes with subunits that are
misfolded and unable to assemble(48) , our data are consistent
with the possibility that calnexin facilitates the correct folding of
AChR subunits. Although it is possible that BiP also complexes with
AChR subunits en route to assembly by means of reversible, low affinity
binding, such interactions have not been described. Recently it has
been proposed that the unassembled monomer of vesicular stomatitis
virus G protein can bind to both BiP and
calnexin(28, 42, 49) , indicating that the
participation of both of these chaperones in the biogenesis of membrane
oligomers may be a more general phenomenon.
-subunit before AChR assembly point to the
participation of other yet unidentified ER components in assembly and
export of AChR from the ER.
cDNA. We are
grateful to Dr. Ari Helenius for a sample of anti-calnexin antibody and
for helpful discussions. We are indebted to Dr. Wei-Jia Ou for valuable
advice on the immunoprecipitation protocols. We thank Sandeep Mody for
preparation of muscle cell cultures.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.