©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of the Endoplasmic Reticulum Chaperone Calnexin in Subunit Folding and Assembly of Nicotinic Acetylcholine Receptors (*)

Marina S. Gelman (1)(§), Weise Chang (1), David Y. Thomas (2) (3), John J. M. Bergeron (2), Joav M. Prives (1)(¶)

From the (1)Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794, the (2)Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H4P 2R2, Canada, and the (3)Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, H4P 2R2 Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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 .


INTRODUCTION

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)()(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).

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 -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.


MATERIALS AND METHODS

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 (10M) 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 (TranS-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.

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.


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.


RESULTS

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).

In the experiment shown in Fig. 1, cultured myotubes were pulse labeled with TranS-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 TranS-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 TranS-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- and Calnexin: Evidence for Association of Calnexin with the -Subunit

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 TranS-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 TranS-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 TranS-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.

To confirm the specificity of the association of calnexin with 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 TranS-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 TranS-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 TranS-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 TranS-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.

The finding that -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.

The same experimental approach was used to measure the time course of -subunit assembly with -subunit. Muscle cultures were pulse labeled with TranS-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).

The results shown thus far indicate that in the ER of cultured muscle cells nascent -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.

To investigate further the relationship between -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 TranS-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.




DISCUSSION

In this study we found that the ER-resident molecular chaperone calnexin forms complexes with newly synthesized AChR- 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.

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- 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.

Several considerations support the possibility that in chick muscle cells calnexin interacts with 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.

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 -subunit before AChR assembly point to the participation of other yet unidentified ER components in assembly and export of AChR from the ER.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant NS25945 (to J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by National Institutes of Health Training Grant in Pharmacological Sciences GMO7518.

To whom correspondence should be addressed: Dept. of Pharmacological Sciences, Health Sciences Center, SUNY Stony Brook, Stony Brook, NY 11794. Tel.: 516-444-3139; Fax: 516-444-3218.

The abbreviations used are: ER, endoplasmic reticulum; AChR, nicotinic acetylcholine receptor; MHC, major histocompatibility complex; Bgt, bungarotoxin; DMEM, Dulbecco's modified Eagle's medium; mAb, monoclonal antibody; BFA, brefeldin A; BiP, immunoglobulin heavy chain-binding protein.

W. Chang and J. M. Prives, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Jon Lindstrom for generously supplying mAb 61 and Dr. Jim Boulter for the gift of AChR- 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.


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