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(Received for publication, August 16, 1995; and in revised form, February 19, 1996) From the
In this study we have used cultured muscle cells to investigate
the role of disulfide bond formation in the sequence of molecular
events leading to nicotinic acetylcholine receptor (AChR) assembly and
surface expression. We have observed that disulfide bond formation in
newly synthesized AChR The extracellular domains of many transmembrane proteins contain
intrachain disulfide bonds, which are thought to contribute to the
formation and stabilization of their mature conformation (for reviews
see (1, 2, 3) ). In some cases these bonds
are established co-translationally, as soon as the participating
cysteine residues emerge into the lumen of the endoplasmic reticulum
(ER), ( The nicotinic acetylcholine receptor (AChR) is a hetero-oligomeric
complex of four different transmembrane glycoproteins assembled in the
stoichiometry The
reducing agent dithiothreitol (DTT) has played a major role in the
molecular characterization of AChR. Before sequence information became
available, the presence of disulfide bonds in AChR was first inferred
from studies demonstrating that DTT treatment altered functional
properties of AChR on the surface of intact electrogenic cells from
electric eel(27) . This effect was demonstrated to be due to
the in situ reduction of a disulfide bond near the
acetylcholine binding site(28) . After AChR Recently, interest in
DTT has been renewed by the demonstration that it can permeate across
cellular membranes and prevent formation of disulfide bonds on nascent
proteins in the ER of living cells(31, 32) . This
approach has been applied to study the contribution of the ER redox
environment to the posttranslational processing and intracellular
transport of individual disulfide-containing secretory and membrane
proteins. Under these conditions DTT blocked disulfide bond formation
in newly synthesized proteins without adverse effects on most cellular
functions, including ATP synthesis and transport of proteins through
the secretory pathway(32, 33) . We have now used this
approach to investigate the relationship between disulfide bond
formation and AChR subunit folding and assembly in cultured myotubes.
Figure 1:
Effect of DTT
treatment on AChR surface appearance. Cultured muscle cells 3 days
after plating were pulse-labeled with
[
Figure 2:
Effect of DTT treatment on AChR assembly.
Cultured muscle cells 3 days after plating were pulse-labeled with
[
Figure 3:
Kinetics of AChR assembly under control
conditions, in the presence of DTT, and after removal of DTT.
Quantitation of AChR assembly time course data was obtained using
scanning densitometry and plotted as a function of chase time. The
curves show the kinetics of AChR assembly in the absence of DTT (squares), in the presence of 5 mM DTT (circles), or upon removal of DTT after 30 min of chase (triangles). Each point represents the average of three
measurements (n = 3, ±S.D.). The timing of DTT
removal is shown by the arrow.
Figure 4:
Effect of DTT treatment on AChR
Figure 5:
Time course of disulfide bond formation on
AChR
In
untreated cultures the shift in migration of
Figure 6:
Effect of DTT treatment on the kinetics of
calnexin-
Finally, we tested whether or not the initial binding of calnexin to
In the present study we have measured the time course of
disulfide bond formation in AChR Our
results show that DTT treatment blocked the appearance of newly made
AChR at the plasma membrane, but only when the reducing agent was
present in the culture medium within the first few minutes after pulse
labeling. Under these conditions DTT blocked disulfide bond formation
on The contribution of disulfide bonds to AChR biogenesis has been
addressed in recent studies using recombinant subunits expressed in Xenopus oocytes (42) or in transfected
fibroblasts(43) . In these experiments mutant We found
that the described effects of DTT on AChR biogenesis are fully
reversible. Removal of the reducing agent leads to the resumption of
disulfide bond formation on the arrested subunits, with subsequent
folding and assembly into pentameric AChR, followed by transport to the
cell surface. Moreover, upon reversal of the DTT effect, the time
course of these events is indistinguishable from that of subunit
folding, assembly, and cell surface appearance in untreated cultures.
These results indicate that DTT treatment at least for the durations
used in this experiment does not cause irreversible misfolding or
aggregation of the subunits: instead, subunit conformational maturation
is suspended until the oxidizing environment in the ER is restored. It is possible that the prevention of irreversible misfolding of
proteins reduced by DTT is mediated by one or more ER-resident
molecular chaperones, proteins that are thought to prevent misfolding
and facilitate correct folding of nascent polypeptides. The ER
chaperone calnexin forms complexes with newly made AChR Recent studies describe divergent effects of DTT
on calnexin interaction with substrate
proteins(36, 45, 46) . In two instances, the
major secretory glycoprotein in Madin-Darby canine kidney cells (36) and thyroglobulin in thyroid epithelial
cells(45) , DTT has been reported to lock substrates onto
calnexin by blocking the dissociation of the complexes. In contrast, in
the case of HA, the addition of DTT to a cell-free translation system
has been reported to cause detachment of calnexin from unfolded HA,
apparently due to reduction of the disulfide bond(s) in calnexin
itself(46) . Since this susceptibility of calnexin to DTT is
not restricted to cell-free preparations(46) , it is possible
that the ER in different cell types varies with respect to the ability
of DTT to alter its redox potential. In addition, since the structural
requirements for complexing with calnexin apparently differ among its
various substrate proteins(45, 47, 48) ,
calnexin-substrate interactions may also vary with respect to their
sensitivity to disulfide bond reduction. Our present findings
summarized schematically in Fig. 7indicate that formation of
disulfide bonds is an essential aspect of the quality control mechanism
that ascertains that only correctly folded and assembled AChR pentamers
exit the ER and are transported to the cell surface. The ability to
interrupt AChR folding and assembly by preventing disulfide bond
formation in intact cells offers a means for isolation of folding
intermediates as well as the potential for identification of additional
ER-resident proteins that participate in the quality control process.
Figure 7:
Schematic representation of the sequence
of events in AChR biogenesis in the absence and presence of DTT. a, AChR
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10709-10714
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunits occurs 5-20 min after
translation and that this modification can be blocked by dithiothreitol
(DTT), a membrane-permeant thiol-reducing agent. DTT treatment was
found to arrest AChR -subunit conformational maturation, assembly,
and appearance on the cell surface, showing that these events are
dependent on prior formation of disulfide bonds. Subunits prevented
from maturation by the reducing agent do not irreversibly misfold or
aggregate, since upon removal of DTT, AChR -subunits undergo
formation of disulfide bonds and resume folding, oligomerization, and
surface expression. We have previously found that nascent
-subunits form transient complexes with the molecular chaperone
calnexin immediately after subunit synthesis (Gelman, M. S., Chang, W.,
Thomas, D. Y., Bergeron, J. J. M., and Prives, J. M.(1995) J. Biol.
Chem. 270, 15085-15092) and have now observed that both the
formation and the subsequent dissociation of these complexes are
unaffected by DTT treatment. Thus, -subunits appear to dissociate
from calnexin independently of their undergoing disulfide bond
formation and achieving conformational maturation. This finding
together with the absence of irreversible misfolding of DTT-arrested
-subunits suggests that calnexin may act to prevent misfolding by
aiding in the initial folding events and is not an essential
participant in the late stages of -subunit maturation.
)while in other cases the formation of disulfide
bonds does not occur until an appreciable interval after
translation(4, 5, 6, 7) , or even
after assembly of monomers(6, 7) . The oxidizing
microenvironment necessary for this modification is provided by the
lumen of the ER. This organelle also contains the enzymes that
facilitate formation of disulfide bonds and assist in protein folding,
including ``foldases,'' such as protein disulfide isomerase
and peptidylprolyl isomerase, and molecular chaperones such as calnexin
and BiP(8, 9, 10, 11) . In addition,
the ER is the site of assembly of most oligomeric membrane and
secretory proteins, which exit to the Golgi complex and are transported
to the cell surface only after the completion of assembly(12) . 
(for reviews see (13, 14, 15, 16) ). The assembly of
subunits into pentamers in a precise stoichiometry and order of
subunits does not occur immediately upon synthesis of the subunits, but
only after a lag period of 
30 min(17, 18) .
During this interval AChR subunits undergo post-translational
modifications leading to conformational maturation and acquisition of
the capacity to assemble(18, 19, 20) .
Conformational maturation has been studied in the -subunit, which
besides its prominence from a stoichiometric standpoint, has binding
properties distinct from those of the other subunits. The extracellular
domain of each -subunit contains a region essential for
acetylcholine binding, as well as a binding site for the elapid venom
neurotoxin -bungarotoxin (-Bgt) and a characteristic sequence
termed the main immunogenic region, the epitope for most antibodies
made against native AChR(21) . All of these sites are initially
absent in newly translated -subunit polypeptides in intact
cells(22, 23) , nor are they present in -subunit
polypeptides expressed in a cell-free system(24) . In intact
cells the toxin binding site and main immunogenic region epitope are
acquired by the -subunit monomers in the interval between
biosynthesis and subunit assembly, while the agonist binding sites are
not acquired until the assembly of - with - or
-subunits(23) . On this basis the binding of mAb 35, a
monoclonal antibody that selectively recognizes the main immunogenic
region(21) , as well as acquisition of the ability to bind
-Bgt (25, 26) can be used to monitor the course
of -subunit folding toward conformational maturation.-subunit was
cloned and sequenced (29) the precise location of this
disulfide bond was determined by the covalent binding of a radioactive
affinity alkylating agent to a pair of cysteine residues
(Cys
-Cys
) after DTT treatment(30) .
This disulfide bond is unique to -subunit: an additional disulfide
bond between Cys
and Cys
, forming a
15-residue loop in the N-terminal extracellular domain, is conserved
among all four AChR subunits (30) .
Reagents
[
S]methionine/[S]cysteine
protein labeling mix (specific activity 1050-1200 Ci/mmol) and 
I--bungarotoxin (specific activity 13-15
mCi/µg) were purchased 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 chick embryos and plated on
collagen-coated culture dishes (initial density = 6 
10
cells/100-mm culture dish) as described
previously(18, 34) . 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. Under these conditions the
myoblasts fused to form multinucleated myotubes on the second day after
plating, initiated rapid synthesis of AChR on the third day, and were
used 3-4 days postplating.Antibodies
Anti-chick AChR -subunit antibody
and anti-chick AChR -subunit antibody were raised in rabbits
against the individual subunits purified on SDS-polyacrylamide gel
electrophoresis from denervated chick leg muscle and were shown to be
non-cross-reactive(18) . Anti--bungarotoxin antibody was
raised in rabbits and affinity-purified on
-bungarotoxin-Sepharose(18) . The monoclonal antibody mAb
35, which recognizes the main immunogenic region of AChR -subunit (21) was isolated from the supernatant of hybridoma TIB 175
(American Type Culture Collection). Anti-calnexin antibody was raised
against a synthetic peptide corresponding to a highly conserved stretch
near the C terminus (amino acids 487-505) of
calnexin(35, 36) .AChR Surface Labeling
For surface labeling of AChR
on intact muscle cells, 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(18, 37) . At the end of this period,
cultures were washed five times with 3-ml volumes of Dulbecco's
phosphate-buffered saline to remove unbound toxin. Surface labeling was
quantitated by -counting of the cell extracts.Metabolic Labeling and Immunoprecipitation
For
metabolic labeling, cultures were methionine/cysteine-depleted by
incubation with methionine- and cysteine-free DMEM (Life Technologies,
Inc.) for 1 h and then labeled at 37 °C with a mixture of
[S]methionine and
[S]cysteine 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. Where specified, freshly prepared 5 mM DTT was added to the chase medium or the labeling medium. Cells
were harvested as follows. Cultures were washed twice with ice-cold
phosphate-buffered saline, scraped, and extracted for 30 min at 4
°C in STE buffer (150 mM NaCl, 10 mM Tris-HCl, pH
7.4, 2 mM EGTA, 2 mM EDTA) containing 1 mg/ml
aprotinin, 10 mMN-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride and supplemented with 1% Triton
X-100. Clarification of the extracts was achieved by centrifugation for
15 min in the microcentrifuge at 4 °C. The clarified supernatants
were incubated at 4 °C with the specified antiserum for 3 h. Then
protein A-Sepharose beads were added, and incubation at 4 °C was
continued for a further 1 h. Where indicated, sequential
immunoprecipitations were carried out essentially as described
before(20, 38) . Briefly, cells were extracted in HBS
buffer (50 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM CaCl, 10% glycerol, 1% aprotinin, 10 mMN-ethylmaleimide, 1 mM phenylmethylsulfonyl
fluoride) supplemented with 2% sodium cholate. After
immunoprecipitation of cell extracts with anti-calnexin or
anti--subunit antibody, proteins were eluted from the protein
A-Sepharose beads under denaturing conditions by incubating the beads
in 50 µl of HBS buffer containing 1% SDS at 75 °C for 10 min.
The supernatants were then diluted with 1 ml of HBS containing 1%
Triton X-100 and precipitated with second antibody.SDS-Polyacrylamide Gel Electrophoresis
The
precipitates were washed five times with STE, 1% Triton and suspended
in 50 µl of 2 sample buffer(39) . For fractionation
of proteins under nonreducing conditions -mercaptoethanol was
omitted from the sample buffer. After incubation for 5 min in a boiling
water bath, the protein A-Sepharose beads were centrifuged, and the
supernatants were fractionated on 10% SDS-polyacrylamide gels.
Radioactive bands were visualized by radiofluorography and quantitated
by scanning densitometry or phosphor imaging.
DTT Treatment Blocks AChR Surface Appearance
To
determine the effect of DTT on the cell surface expression of newly
made AChR, cultured myotubes were pulse-labeled with
[S]methionine/[S]cysteine
for 15 min and then incubated for the specified intervals in chase
medium in the absence or presence of DTT (5 mM). To monitor
the appearance of labeled receptors on the external surface of
myotubes, the pulse-labeled cells were incubated with the AChR ligand
-Bgt and subsequently immunoprecipitated with anti--Bgt
antibody. As can be seen in Fig. 1, in the absence of DTT,
labeled AChR -subunit was detected at the cell surface after chase
intervals of 3 h and longer following its synthesis (lanes 2 and 3). No labeled AChR was detected at the cell surface
after chase intervals of 2 h or less (not shown), consistent with a
duration of between 2 and 3 h between AChR biosynthesis and its surface
appearance(40, 41) . When DTT was present in the chase
medium (lanes 4 and 5), significant amounts of
labeled receptors could not be immunoprecipitated from the cell
surface, showing that DTT treatment blocks the appearance of newly made
AChR at the plasma membrane. The faint bands in lanes 4 and 5 that comigrate with -subunit probably correspond to the
small population of pulse-labeled subunits that underwent oxidation and
assembly during the 15-min pulse period before application of DTT. It
should be noted that DTT was removed immediately prior to labeling of
DTT-treated cells with -Bgt to prevent reduction of the disulfide
bonds in -Bgt and its consequent inactivation. The block of AChR
surface appearance by DTT was reversible: upon removal of the reducing
agent from the chase medium and incubation of myotubes in DTT-free
medium for an additional 3 or 4 h, labeled AChR was detected at the
cell surface (lanes 6 and 7). To test whether the
block in AChR surface expression upon exposure to DTT occurred before
or after AChR assembly, DTT was added to the culture medium 90 min
after the end of the pulse, at a time after completion of AChR assembly
but before the receptor had reached the cell surface. As seen in lanes 8 and 9, application of DTT after assembly does
not block surface appearance of AChR (or impair the binding of
-Bgt to these receptors). Therefore, in order to block AChR
surface expression DTT must be present before the assembly of AChR
takes place.
S]methionine/[S]cysteine
(200 µCi/ml, 15 min) and then incubated for the times shown in
chase medium in the absence (lanes 2 and 3) or
presence (lanes 4 and 5) of 5 mM DTT. Where
specified, DTT was only present during the initial 60 min of chase (lanes 6 and 7) or added after 90 min of chase (lanes 8 and 9). During the final 1 h of chase, cells
were surface-labeled with -Bgt (10 nM). DTT was removed
immediately prior to labeling of DTT-treated cells with -Bgt to
prevent reduction of the disulfide bonds in -Bgt and its
consequent inactivation. Cells were then extracted with STE buffer
supplemented with 1% Triton X-100 and immunoprecipitated with
anti--Bgt antibody as described under ``Materials and
Methods.'' Lane 1 shows a nonimmune control in which
cells were immunoprecipitated with anti-Bgt antibody in the absence of
prior labeling with -Bgt. Immunoprecipitates were resolved on 10%
SDS-polyacrylamide gels. The band corresponding to the -subunit is
denoted by an arrow. Higher molecular weight bands represent
nonspecifically immunoprecipitated proteins. Molecular weight standards
are shown at the left.
DTT Treatment Arrests AChR Assembly
To test
directly the effect of DTT treatment on AChR subunit assembly, myotubes
were pulse-labeled and then chased in the absence or presence of DTT,
and AChR assembly was measured by coimmunoprecipitation of
-subunit with a non-cross-reactive antibody directed against the
-subunit. Fig. 2(left panel) shows the time
course of AChR assembly under control conditions, as monitored by the
accumulation of -subunit immunoprecipitated with
anti--subunit antibody with increasing chase times. The
-subunit itself is not visible in
[S]methionine-labeled preparations due to its
diffuse migration as a heterogeneous band, high susceptibility to
proteolysis, and nonspecific backgrounds in this region of the gel, as
noted previously by ourselves (18, 20) and
others(43) . However, the -subunit is phosphorylated and
is clearly visible in immunoprecipitates from cultures labeled with 
P
(18) . As can be seen, appreciable
AChR assembly was not detected immediately after the pulse but was
clearly evident by 40 min and continued to increase with the further
chase intervals. In contrast, when DTT was present in the chase medium (right panel) no labeled -subunit was precipitated by
anti--subunit antibody at any time point. The ability of
anti--subunit antibody to recognize -subunit was not impaired
by this DTT treatment, as determined by immunoprecipitation of this
subunit from cultures P-labeled in the
presence of DTT (data not shown). Therefore, we conclude that the
absence of -subunit in anti--subunit immunoprecipitates
reflects a block in AChR assembly in the ER in the presence of the
reducing agent. The effect of DTT treatment on the rate of AChR
assembly is shown in Fig. 3. As can be seen, upon removal of DTT
from the culture medium, the assembly of the labeled AChR subunits
resumed at the same rate as in untreated cultures (Fig. 3). This
finding indicates that DTT treatment does not induce the subunits to
misfold permanently, but rather arrests them in a partially folded
conformation that remains conducive to assembly upon withdrawal of the
reducing agent. When DTT treatment was followed by brief exposure to
the cell-permeant alkylating agent N-ethylmaleimide (20
mM), the assembly of the AChR subunits was not restored even
after prolonged incubation in the absence of both drugs (not shown),
likely due to the alkylation of the -SH groups and indicating that the
recovery of assembly requires the formation of disulfide bonds.
S]methionine/[S]cysteine
(200 µCi/ml, 15 min) and then chased for the specified intervals in
the absence (lanes 1-5) or presence (lanes
6-10) of 5 mM DTT. For measurement of AChR
assembly, cells were extracted with STE, 1% Triton X-100 and
immunoprecipitated with anti--subunit antibody. The band
corresponding to the -subunit is denoted by an arrow.
Higher molecular weight bands represent nonspecifically
immunoprecipitated proteins.
DTT Treatment Prevents AChR
To determine if the arrest of AChR assembly by DTT
treatment is due to impaired subunit folding, pulse-labeled cultures
were chased in the absence or presence of 5 mM DTT as
described above, and cellular extracts were then immunoprecipitated
with mAb 35, an antibody specific for conformationally mature AChR
-Subunit
Folding-subunit(21) . As shown in Fig. 4, -subunits
were not recognized by mAb 35 to an appreciable extent immediately
after the pulse (lane 1), but became immunoprecipitable with
this antibody by 60 min of chase in untreated cells (lane 2).
In contrast, when DTT was present during the chase -subunit was
not recognized by mAb 35 even after 60 min of chase (lane 4),
although equivalent amounts of labeled -subunit were
immunoprecipitable by anti-chick subunit antibody from untreated
and DTT-treated cell extracts (not shown). Replicate cultures were
chased in the presence of DTT for 30 min and then rinsed and shifted to
medium lacking DTT. Upon removal of the reducing agent, the labeled
-subunits acquired the mAb 35 epitope at a rate and to an extent
comparable with that observed under control conditions. These results
show that, as in the cases of AChR subunit assembly and surface
expression, -subunit folding is dependent on disulfide bond
formation and thus susceptible to DTT. Furthermore, in all three cases
the DTT arrest is reversed when the oxidizing environment in the ER is
restored.
-subunit folding. Cultured muscle cells 3 days after plating were
pulse-labeled with
[S]methionine/[S]cysteine
(200 µCi/ml, 15 min) and then either extracted immediately (lanes 1 and 3) or chased for the times specified.
The chases were carried out in the absence of DTT (lane 2), in
the presence of 5 mM DTT (lanes 4 and 5), or
for 30 min in the presence of 5 mM DTT, after which cells were
rinsed and chased for an additional 1 h in the absence of DTT (lane
6). For measurement of AChR folding, cells were extracted with
STE, 1% Triton X-100 and immunoprecipitated with mAb 35. The arrow denotes the -subunit. The higher molecular weight band
represents nonspecifically immunoprecipitated
protein.
Temporal Relationship between
In order
to determine the time course of the formation of disulfide bonds on the
nascent -Subunit Translation,
Disulfide Bond Formation, and Conformational Maturation-subunits, we used nonreducing SDS-polyacrylamide gel
electrophoresis in an attempt to distinguish the oxidized and reduced
forms of -subunit. Cells were pulse-labeled for 5 min to isolate a
relatively synchronous population of newly made subunits, chased for
the specified intervals, and then extracted and immunoprecipitated with
anti--subunit antibody. The immunoprecipitates were fractionated
in SDS-polyacrylamide gels under nonreducing conditions. To detect the
difference in migration between the nascent and disulfide-bonded forms
of -subunit, samples harvested immediately after pulse were run
next to each sample harvested after the specified chase interval (Fig. 5, lanes 1-10). To compare the migration
pattern of the fully reduced and oxidized forms of -subunit,
replicate cultures were chased in the presence or absence of 5 mM DTT for 1 h, an interval sufficient for the completion of
-subunit folding in the untreated cultures. As can be seen in Fig. 5, lanes 11 and 12, a small but clearly
detectable difference in the mobility of reduced versus oxidized -subunit is evident under these conditions.
-subunit. Cultured chick muscle cells were pulse-labeled with
[S]methionine/[S]cysteine
(200 µCi/ml, 5 min) and then incubated in chase medium for the
indicated times. To obtain completely oxidized and reduced forms of
-subunit, pulse-labeled cells were chased for 60 min either in the
absence (lane 12) or presence of 5 mM DTT (lane
11). 20 mMN-ethylmaleimide was added to the
culture medium during the final 1 min to prevent the formation of
disulfide bonds upon extraction. All cultures were extracted with STE,
1% Triton buffer and immunoprecipitated with anti--subunit
antibody, and immunoprecipitates were resolved on 10% nonreducing gels.
To detect the difference in migration between the nascent and
disulfide-bonded forms of -subunit, samples at various chase
intervals (lanes 2, 4, 6, 8, and 10) were run next to the samples harvested immediately after
pulse (lanes 1, 3, 5, 7, and 9).
-subunit indicative
of disulfide bond formation was only marginally detectable after 5 min
of chase (Fig. 5, lanes 1 and 2), was more
clearly seen after 10 min (lanes 3 and 4), and
appeared to be maximal at 15 min of chase and longer (lanes
5-10). These results indicate that disulfide bonds on
-subunits are established before the acquisition of mAb 35
epitope. Since DTT treatment that prevents formation of these disulfide
bonds results in the block of folding and assembly it is likely that
the formation of intrachain disulfides is essential for conformational
maturation of the individual subunits.Relationship between Disulfide Bond Formation and the
Interaction of AChR
We have recently
found that nascent AChR -Subunit with Calnexin-subunit binds to the molecular chaperone
calnexin immediately after -subunit synthesis(20) . The
time course of -subunit-calnexin dissociation was observed to
coincide with the conformational maturation of -subunit,
consistent with a role for calnexin in mediating -subunit folding.
Since DTT has now been shown to block the folding of -subunit (Fig. 4), it is of interest to determine the fate of
a-subunit-calnexin complexes in the presence of DTT. In the experiment
shown in Fig. 6cells were pulse-labeled with
[S]methionine/[S]cysteine
for 15 min and chased in the absence or presence of 5 mM DTT.
To determine the amount of radiolabeled -subunit associated with
calnexin at each time point, cells were extracted in HBS, 2% sodium
cholate buffer, which preserves interactions of calnexin with substrate
proteins, and immunoprecipitated with anti-calnexin antibody. The
immunoprecipitated calnexin-substrate complexes were eluted from the
pellet and reprecipitated with anti--subunit antibody. The rate of
-subunit dissociation from calnexin in DTT-treated cells, with a
half-time of 18 min, is indistinguishable from that observed in the
absence of DTT(20) . Thus, no difference is seen in the amounts
of subunit bound to calnexin after 15 min of chase in DTT-treated
and untreated cultures (Fig. 6, lanes 7 and 8). Therefore, -subunit-calnexin dissociation does not
appear to be altered by the presence of DTT in the chase medium.
-subunit dissociation. Cultured chick muscle cells were
pulse-labeled with
[S]methionine/[S]cysteine
(400 µCi/ml, 15 min) and then incubated in chase medium for the
indicated times in the absence (lane 7) or presence of 5
mM DTT (lanes 1-6 and 8). Cells were
then extracted in HBS, 2% cholate buffer and immunoprecipitated
sequentially with nonimmune antibody followed by anti--subunit
antibody (lane 1), anti--subunit antibody twice (lane
2), or anti-calnexin antibody followed by anti--subunit
antibody (lanes 3-8).
-subunit is affected by DTT when the reducing agent is present in
the medium during the pulse period. A measurable amount of
-subunit was coprecipitated with anti-calnexin antibody after
pulses in the absence and presence of DTT (not shown), and quantitation
by Phosphor imager established that the proportion of -subunits
complexed to calnexin was unchanged in the presence of DTT. Together
these results indicate that DTT treatment does not alter appreciably
both association and dissociation of nascent AChR -subunit with
the ER-resident chaperone calnexin.
-subunits and have shown that
this posttranslational modification is crucial for further folding,
assembly, and surface expression of AChR. As established previously
under similar experimental conditions, AChR -subunit
conformational maturation is first detectable at 30 min after
translation (interval measured from the beginning of the pulse) and
increases linearly, achieving a maximum by 60-75 min after
translation(20) . Assembly into pentameric complexes takes
place shortly after maturation, beginning 30-45 min after
translation and attaining completion by 90
min(18, 20) . The assembled AChR reaches the cell
surface 2.5-3 h after subunit synthesis ( (40) and (41) ; our present results). We have now determined that
disulfide bond formation on -subunit is completed by 20 min after
subunit translation, preceding subunit maturation as measured with the
conformation-specific antibody. Our observation that the AChR
-subunit needs to undergo additional folding after this disulfide
bond formation to acquire the mAb 35 epitope indicates that the newly
oxidized subunit constitutes a discrete folding intermediate that is a
precursor of the mature, assembly-competent -subunit.-subunit and arrested subsequent subunit folding and assembly.
The addition of DTT after the disulfide bonds have formed (at 30 min
after the pulse; data not shown) no longer had any effect on the extent
and timing of assembly. Similarly, adding DTT after the completion of
assembly (90 min) did not affect the transport of pulse-labeled AChR to
the cell surface. Although the block of disulfide bond formation on
nascent secretory and membrane proteins in the ER is a general
consequence of DTT treatment, the time course data strongly suggest
that DTT exerts its effects on AChR directly, by preventing the
oxidation of AChR subunits. Together these results indicate that
formation of disulfide bonds on AChR -subunit is a fundamental
step required for subunit maturation and assembly into pentameric AChR. -subunits
lacking the conserved disulfide bond between Cys and
Cys failed to form -Bgt binding sites, which like
the mAb 35 epitope, are dependent on the acquisition of the correctly
folded conformation(25, 26) . However, both studies
reported association of these mutant subunits with normal
subunits, suggesting that formation of this disulfide bond is not an
absolute prerequisite for AChR assembly. In contrast, under the present
experimental conditions DTT treatment abolished detectable assembly of
- and -subunits. How can these apparent differences be
reconciled? First, elimination of disulfide bond formation either by
DTT or by mutagenesis of the -subunit may result in a significant
decrease in the affinity between subunits. Consequently, in DTT-treated
cultured muscle cells the assembly of endogenously expressed AChR
subunits is diminished to undetectable levels, while in transfected
cells association between mutant - and -subunits, although
markedly less efficient, could remain detectable due to overexpression
of the recombinant subunits. In addition, since DTT is anticipated to
prevent formation of the conserved disulfide bond in all four AChR
subunits, it is possible that the impaired folding of other subunits as
well as the -subunit contributes to the block in assembly. As
-subunits directly assemble with both - and
-subunits(23) , it would be of interest to determine if
- binding is also vulnerable to DTT treatment.-subunit,
and these complexes subsequently dissociate with a t
of approximately 20 min, concomitantly with -subunit
folding(20) . In the present study we have observed that
formation of -subunit-calnexin complexes was not impaired by DTT.
Moreover, the kinetics of calnexin dissociation were apparently
unchanged in the presence of DTT, although the folding and assembly of
the -subunit remained arrested for as long as DTT was present,
indicating that calnexin dissociation from -subunit is independent
of the completion of -subunit folding. This finding supports the
possibility that calnexin performs its function mainly during the
translational and early posttranslational stages of AChR subunit
folding, when nascent subunits may be most susceptible to misfolding
and aggregation. In a similar manner, calnexin has been reported to
mediate early folding of influenza virus hemagglutinin (HA),
dissociating prior to conformational maturation of HA
monomer(44) . In an earlier study a mechanism has been proposed
in which the ER-resident molecular chaperone BiP forms transient
complexes with nascent HA during the folding process and prevents its
misfolding by blocking formation of inappropriate disulfide
bonds(7) .
-subunit is translated on ER-bound polysomes and
is cotranslationally inserted into the ER membrane. b,
immediately or soon after synthesis -subunit binds to the
ER-resident chaperone calnexin. c-f, disulfide bonds are
established 5-20 min after -subunit translation.
Disulfide-bonded -subunit dissociates from calnexin and continues
to fold, achieving conformational maturation by 60-75 min after
translation. Correctly folded subunits assemble into pentameric AChR
and exit the ER. g-j, in the presence of DTT, formation
of disulfide bonds on -subunit is blocked, but calnexin
dissociation is not impaired. Subunit folding and assembly are
suspended for as long as DTT is present in the medium. Aggregation or
permanent misfolding of subunits do not occur during this interval.
Upon removal of DTT, disulfide bond formation is restored, and the
subunits undergo conformational maturation and
oligomerization.
)-Bgt, -bungarotoxin; DTT, dithiothreitol; mAb,
monoclonal antibody.
We are grateful to Dr. John J. M. Bergeron for helpful
discussions and a generous gift of anti-calnexin antibody. We thank
Sandeep Mody for preparation of muscle cell cultures.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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