(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 -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.
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), ()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) .
The nicotinic acetylcholine receptor (AChR) is a hetero-oligomeric
complex of four different transmembrane glycoproteins assembled in the
stoichiometry (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.
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 -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) .
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
[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.
Figure 2:
Effect of DTT treatment on AChR assembly.
Cultured muscle cells 3 days after plating were pulse-labeled with
[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.
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
-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.
Figure 5:
Time course of disulfide bond formation on
AChR -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).
In
untreated cultures the shift in migration of -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.
Figure 6:
Effect of DTT treatment on the kinetics of
calnexin--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).
Finally, we tested whether or not the initial binding of calnexin to
-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.
In the present study we have measured the time course of
disulfide bond formation in AChR -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.
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 -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.
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 -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.
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 -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) .
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 -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.