INTRODUCTION

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The nicotinic acetylcholine receptor is a prototype molecule of a family of ligand-gated ion channels, which include GABA_A, glycine, and 5HT₃ receptors (1, 2). Following binding by agonists to these receptors, a conformational change increases cation permeability through the central pore of the receptor, eliciting depolarization of the cell membrane (1, 2). Peptide backbones of each of the four subunits of the acetylcholine receptor transverse the membrane four times, and possess at least one Asn-X-Ser/Thr glycosylation signal (2, 3). The subunits are thought to undergo a maturation pathway which includes oligosaccharide attachment (4), formation of disulfide bonds (5-7), proline isomerization (8), and intersubunit contacts at specific interfaces (9-11). Members of this family of receptors are composed of a multisubunit complex of glycoproteins which are retained and assembled in the endoplasmic reticulum prior to transport to the cell surface (9, 12). Acetylcholine receptor subunits at the neuromuscular junction assemble into a circular orientation of subunits of ----, to enclose the central ion channel (2, 13; but see Ref. 14).

The endoplasmic reticulum localized protein, calnexin, is associated with unassembled subunits of the acetylcholine receptor (15-17), but calnexin appears to be absent with combinations of assembled subunits (16). Connolly et al. (18) demonstrated that calnexin is associated with subunits of the GABA_A receptor, indicating that calnexin association might be involved in the biogenesis of other multisubunit ion channels. Numerous investigations have established that calnexin associates primarily with monoglucosylated oligosaccharides, which are intermediates in the processing of nascent oligosaccharides or products of reglucosylation by the enzyme UDP-glucose:glycoprotein glucosyltransferase, and that treatment with the glucosidase inhibitor castanospermine can disrupt the interaction (reviewed in Refs. 19 and 20).

Using transient expression of acetylcholine receptor subunits in HEK cells, we find a role for calnexin association in reducing degradation of unassembled subunits by the proteasome, since treatment with castanospermine reduces the subunit-calnexin association and increases the polyubiquitination of the -subunit. Additionally, co-treatment with proteasome inhibitors blocks the degradation. Although unassembled -subunits are degraded at a rapid rate (5), treatment with glucosidase inhibitors substantially promotes degradation (4). Our data also indicate that castanospermine treatment does not cause the -subunit to misfold, as detected by a conformationally sensitive antibody. Therefore, increased degradation by the proteasome appears to be independent of the nascent peptide undergoing misfolding in this system. In contrast, a recent study indicated that treatment of chick myotubes with castanospermine disrupts -subunit folding and assembly (17). Our data also indicate that calreticulin and ERp57, the two other proteins known to have glycoprotein-associating properties similar to calnexin (19, 21), do not appear to be bound to the -subunit.

Assembly with the -subunit reduces the degradation of the -subunit when - and -subunits are co-expressed in the presence of castanospermine, indicating that glucose trimming, calnexin association, assembly and entrance into the proteasome are linked in the expression of the receptor. Connections among these phenomena are also likely to be critical to the fidelity of expression of other multisubunit glycoproteins.

	MATERIALS AND METHODS

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Castanospermine Treatment and Transfections-- Castanospermine (Calbiochem, San Diego, CA) was solubilized in Dulbecco's modified Eagle's medium at a concentration of 100 µg/ml, and immediately added to 10-cm plates of cells. Cells were treated with castanospermine 2 h prior to transfection, transfected with receptor subunit cDNAs, allowed to grow for 16 h, or replenished with fresh castanospermine in Dulbecco's modified Eagle's medium and raised for another 24 h.

Detergent Solubilization, Immunoprecipitation, Electrophoresis, and Western Blots-- In experiments involving immunoprecipitations with antibodies to calnexin, calreticulin, ERp57, and the receptor subunits, the solubilization buffer consisted of 0.5% CHAPS,¹ 150 mM NaCl, 1 mM CaCl₂, 20 mM HEPES, pH 8.0, and the protease inhibitors: benzamidine, aprotinin, leupeptin, and pepstatin A. The characteristics of the antibodies used for these immunoprecipitations, mAb 35 or mAb 61 to precipitate the -subunit, mAb 111 to precipitate the -subunit and mAb 137 to precipitate the -subunit, were described previously (22). Anticalnexin, antipolyubiquitin, and anti-calreticulin were purchased from Stressgen (British Columbia, Canada). Solubilization of cells and immunoprecipitations using anti-polyubiquitin were in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.4% deoxycholate, 1% Nonidet P-40, 0.1% SDS, phenylmethanesulfonyl fluoride, N-ethylmaleimide, and the other protease inhibitors listed above. The ratio of solubilization buffer volume relative to the number of cell plates were the same for each sample within an experiment. Following solubilization, samples were centrifuged at 10,000 × g for 5 min. Dilution ratios for antibody in all immunoprecipitations were approximately 1:100.

Proteasome Inhibitors-- Proteasome inhibitors were solubilized in Me₂SO and added 3 h after transfection. Equivalent concentrations of Me₂SO were added to untreated cells. The final concentration of Me₂SO in a plate of cells was at most 0.3%. Cells were grown for an additional 16 h and then solubilized. The proteasome inhibitor benzyloxycarbonyl-Leu-Leu-phenylalaninal (Z-LLF-CHO) was obtained from Dr. F. Mercurio (Signal Pharmaceuticals, San Diego, CA) and used at a final concentration of 20 µM. MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) and lactacystin were purchased from Calbiochem (San Diego, CA) and used at 50 and 10 µM, respectively. Calpain inhibitor I (N-Ac-Leu-Leu-norleucinal; Calbiochem, San Diego, CA) was used at final concentration of 100 µM.

¹²⁵I--Bungarotoxin Binding and Density Scans of Western Blots-- The snake toxin -bungarotoxin (-Bgt) binds to both unassembled and assembled -subunits, whereas carbamoylcholine recognizes only -subunits assembled with -, -, or -subunits (10). Transfected and untransfected cells were permeabilized in a 0.1% saponin buffer (11), exposed to an excess of ¹²⁵I--bungarotoxin (¹²⁵I--Bgt) for 3 h, washed, and -radiation was counted. The binding of ¹²⁵I--Bgt to untransfected cells was subtracted from its binding to transfected cells. Data were standardized to the average of ¹²⁵I--Bgt bound to cells untreated with castanospermine. Bands on Western blots were scanned with Deskscan (Hewlett Packard) and integrated with the program Collage (Fotodyne, New Berlin, WI).

	RESULTS

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Castanospermine Diminishes Accumulation of -, -, and -Subunits in the Cell, without Generally Influencing the Accumulation of Other Cellular Proteins-- Cells were treated with the glucosidase inhibitor castanospermine, to identify roles for glucose trimming in the biogenesis of acetylcholine receptors expressed in HEK cells. Treatment with castanospermine, prior to and during the transfection period, inhibits the glucosidase I and glucosidase II enzymes, leaving oligosaccharides capped with three glucose residues. Castanospermine treatment, therefore, maintains larger sized oligosaccharides which are attached to glycoproteins confined to the endoplasmic reticulum. Since unassembled receptor subunits and - dimers are retained intracellularly (23, 24), treatment with castanospermine should maintain larger oligosaccharides attached to the receptor subunits. A side reaction, which may result from a residual fraction of glucosidase enzymes remaining active, is that, nascent oligosaccharide chains whose glucosyl residues have been trimmed, in turn, become reglucosylated by UDP-glucose:glycoprotein-dependent glucosyltransferase. Reglucosylated receptor subunits may accumulate transiently because most glucosidase enzymes are inhibited (20).

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Castanospermine treatment reduces the intracellular accumulation of -, -, and -subunits but not a random sample of cellular proteins. A, cells were treated or untreated with castanospermine and transfected (TFT) separately with plasmid DNAs encoding -subunits (lanes 1 and 2), -subunits (lanes 3 and 4), or -subunits (lanes 5 and 6). Detergent extracts were immunoprecipitated with antibodies specific for  (mAb 61),  (mAb 111), or  (mAb 137), and receptor subunits were detected using mAb 210 for -subunits, mAb 111 for -subunits, and mAb 137 for -subunits. Western blots were developed with chemiluminescent techniques. Equivalent volumes were loaded in each lane, consisting of approximately 5% of the immunoprecipitation mixture. Note in this and all subsequent blots that castanospermine treatment decreases slightly the mobilities of the receptor subunit bands, consistent with inhibition of glucose trimming. B, HEK cells were untreated (lanes 1 and 3) or treated with 100 µg/ml CST (lanes 2 and 4), grown for 18 h, cells were washed and detergent extracts were resolved in gels. Gels were either developed with Coomassie Blue stain (lanes 1 and 2) or protein was transferred to nitrocellulose and exposed to a high concentration of antimouse IgG-peroxidase to detect a random sample of cellular proteins (lanes 3 and 4). Equivalent volumes of detergent extract were loaded in each lane.

Polyubiquitinated -Subunits Accumulate when Cells Are Treated with Proteasome Inhibitors-- To identify mechanisms which regulate receptor subunit degradation, experiments were designed to ascertain whether treatment with castanospermine increases conjugation of polyubiquitin to -subunits. Cells were treated or untreated with castanospermine, transfected with cDNA encoding the -subunit, and maintained in the presence of proteasome inhibitors: calpain inhibitor I and benzyloxycarbonyl-Leu-Leu-phenylalaninal (Z-LLF-CHO; 26). Calpain inhibitor I and Z-LLF-CHO were used in this experiment, because other investigations have demonstrated their efficacy for detecting the conjugation of polyubiquitin chains to proteins (27, 28). Detergent extracts were immunoprecipitated with an antibody to polyubiquitin, and Western blots were developed with mAb 210 to detect -subunits (Fig. 2). The appearance of a characteristic ladder pattern near the top of the gel indicates that the proteins are polyubiquitinated (27-30). A high molecular weight ladder pattern is observed when -subunits are expressed in the presence of castanospermine (Fig. 2A, lane 2), indicating that the -subunits become polyubiquitinated. In comparison, -subunits expressed in the absence of castanospermine are also polyubiquitinated, but the high molecular weight ladder pattern is fainter (Fig. 2A, compare lanes 1 and 2). The high molecular weight pattern is absent when Z-LLF-CHO is omitted in the experiment (Fig. 2B); this finding indicates that treatment with Z-LLF-CHO inhibits the degradation of polyubiquitinated -subunits in the proteasome, thereby allowing the conjugated intermediates to accumulate. Treatment with calpain inhibitor I alone does not lead to the detection of polyubiquitinated -subunits.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   A, castanospermine treatment enhances polyubiquitination of the -subunit. Cells were incubated with or without castanospermine, and transfected with plasmid DNA encoding the -subunit. All cells were treated with the proteasome inhibitors: calpain inhibitor I (CI) and Z-LLF-CHO. Extracts were immunoprecipitated with an antibody to polyubiquitin and the Western blot was developed with mAb 210, which detects the -subunit. B, similar experiment to that described in A, but Z-LLF-CHO was omitted.

Treatment with the Proteasome Inhibitors MG-132 or Lactacystin Inhibits the Degradation of -Subunits-- To further characterize the mechanisms of degradation, cells were treated with the proteasome inhibitors lactacystin or MG-132, to examine whether degradation of -subunits is reduced. Lactacystin is a irreversible inhibitor which binds specifically to the proteasome, as demonstrated by affinity labeling and peptide sequencing (31). MG-132 is a congener of Z-LLF-CHO, also a peptide-aldehyde that blocks the proteolytic activities of the proteasome (32). Cells were treated or untreated with castanospermine, transfected with the same plasmid DNA transfection mixture in all plates, and then treated or untreated with a proteasome inhibitor. Following an incubation period, cells were solubilized, and -subunits were immunoprecipitated and detected on Western blots. As displayed in Fig. 3, lanes 3 and 4, and in previous experiments, treatment with castanospermine results in a loss of -subunit protein; however, inclusion of lactacystin diminishes the loss of -subunit protein (Fig. 3, lanes 1 and 2). Similar results were obtained with MG-132 (Fig. 4C, lanes 3 and 4). These data indicate that -subunits, which may have been polyubiquitinated and subjected to isopeptidase activity (33), are degraded in the proteasome in cells treated with castanospermine.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Lactacystin, a selective inhibitor of the proteasome, inhibits the degradation of -subunits expressed in the presence of castanospermine. Cells were treated (+) or untreated () with CST, transfected with plasmid DNA encoding the -subunit, treated (+) or untreated () with 10 µM lactacystin (LAC); an equivalent concentration of Me2SO was added to untreated cells, since LAC was solubilized in Me2SO. Samples were immunoprecipitated with mAb 61 and the Western blot developed with mAb 210. Equivalent numbers of cells were solubilized and subjected to immunoprecipitation and equivalent volumes were loaded in lanes, which consisted of approximately 5% of the total sample.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Characteristics of calnexin recognition of -subunits. A, cells were treated or untreated with CST and transfected (TFT) with 15 µg of plasmid DNA encoding the -subunit or also co-transfected with 15 µg of plasmid DNA encoding the -subunit. Cells were not treated with a proteasome inhibitor. Sequential immunoprecipitation (IP to:) was first to calnexin (c), followed by immunoprecipitation of the unbound material with anti- (mAb 61). Lanes 1 and 3, 2 and 4, 5 and 7, and, 6 and 8 are pairs of sequentially immunoprecipitated samples. The Western blot was developed with mAb 210 to detect -subunits. The density ratios were calculated by scanning and integrating the density of -subunit bands on the Western blot and dividing the CST-treated sample by the CST-untreated sample for -subunits precipitated by one of the antibodies. B, similar to A, except cells were transfected with 3.0 µg of plasmid DNA encoding the -subunit to reduce expression levels: cells were also treated with lactacystin. C, similar to A, except cells were treated with 50 µM MG-132. Blot was exposed to film 10 times longer than in A and B.

Treatment with Castanospermine Disrupts an Interaction between Calnexin and the -Subunit-- To ascertain whether glucose trimming influences interactions between calnexin and -subunits, cells were treated with castanospermine, and then transfected with various amounts of plasmid DNA encoding the -subunit (Fig. 4). In some experiments, cells were also treated with proteasome inhibitors to reduce degradation and to facilitate the distinction between altered recognition by calnexin from increased degradation. Sequential immunoprecipitations were conducted with an antibody to calnexin (c), followed by immunoprecipitation of the unbound material with an anti-- subunit antibody (mAb 61), to assess the extent of calnexin--subunit recognition. In Fig. 4, sequentially precipitated -subunits are displayed, with lane 1 in sections A-C displaying -subunits bound to calnexin, and lane 3 exhibiting the -subunits not cleared with calnexin; likewise, lane 2 in sections A-C displays -subunits bound to calnexin and lane 4 exhibits -subunits not cleared with calnexin. Densities of -subunit bands were quantified, and density ratios for -subunits expressed in the presence relative to the absence of castanospermine were calculated. Each histogram bar was calculated separately for -subunits precipitated with anti-calnexin or anti--subunit antibodies; for example, in Fig. 4, A-C, the histogram bars designated TFT:  and IP to: c, were calculated for -subunits co-immunoprecipitated with calnexin by anti-calnexin antibody, by dividing the band densities for -subunits expressed in the presence (shown in lane 2) and absence of castanospermine (shown in lane 1). Similarly, the histogram bar designated as TFT:  and IP to: , was calculated for -subunits immunoprecipitated with anti--subunit antibody, by dividing the band densities for -subunits expressed in the presence and absence of castanospermine (displayed in lane 3). A comparison in the density ratios for -subunits co-immunoprecipitated with calnexin relative to those immunoprecipitated with anti--antibody provides an estimate of the extent of calnexin--subunit recognition.

Assembly with -Subunits Reduces the Loss of -Subunits Expressed in the Presence of Castanospermine-- To examine whether -subunit assembly with the -subunit ameliorates the loss of -subunits expressed in the presence of castanospermine, the mass of cDNA encoding the -subunit was kept constant at 15 µg of plasmid DNA/plate and the plasmid DNA encoding the -subunit was transfected at two different ratios to -subunit. Accumulation of -subunits was measured using ¹²⁵I--Bgt binding to permeabilized cells (Fig. 5): ¹²⁵I--Bgt binding was also quantified in a similar manner with untransfected cells. Standardized values for ¹²⁵I--Bgt binding are displayed in Fig. 5, where -toxin binding to untransfected cells is subtracted from the values in transfected cells.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Increasing the mass of plasmid DNA encoding the -subunit in the transfection increases the accumulation of -subunits when subunits are coexpressed in the presence of castanospermine. Accumulation of folded -subunits in the absence () or presence (+) of CST is measured by 125I--Bgt binding to permeabilized cells. : ratios refer to the masses of plasmid DNAs in the transfection mixture, where the amount of plasmid DNA encoding the -subunit is kept constant at 15 µg of DNA/plate and plasmid DNA encoding the -subunit is varied. Radioactivity (cpm) is standardized to the binding of 125I--Bgt to untransfected cells, and each histogram bar displays an average from two or three samples. The maximum counts/min are calculated as the average among samples untreated with castanospermine, determined separately for the two transfection ratios.

Accumulation of - Dimers Expressed in the Presence of Castanospermine Is Dependent on the Transfected Ratios of - and -Subunit cDNAs-- To ascertain whether treatment with castanospermine and the stoichiometric ratios of cDNAs encoding - and -subunits in the transfection influence the accumulation of - dimers, cells were treated with castanospermine and co-transfected with plasmid DNAs encoding the - and -subunits at ratios of 8:1 and 3:1. The mass of plasmid DNA encoding the -subunit was kept constant at 15 µg of plasmid DNA/plate and the mass of plasmid DNA encoding the -subunit was varied. The -subunit was immunoprecipitated with anti--antibody (mAb 61), and the Western blot was developed with antibodies to the - and -subunits (mAb 210 and 137, respectively; Fig. 6A). The appearance of co-immunoprecipitated - and -subunit bands appear to display correspondingly similar changes in density when coexpressed in the absence or presence of castanospermine, indicating that subunit assembly is not disrupted (Fig. 6A). The density of the -subunit band on the blot provides an indication of the accumulation of - dimers. When cells are co-transfected with a large stoichiometric imbalance of subunits, using plasmid DNAs encoding - and -subunits at a 8:1 ratio, there are significantly diminished - and -subunit bands in the sample expressed in the presence of castanospermine, indicating - dimers are less prevalent (Fig. 6A, compare lanes 1 to 2). However, treatment with castanospermine has a smaller impact on altering the density of - and -subunit bands when plasmid DNAs are co-transfected at a 3:1 ratio, a closer stoichiometric balance of subunits (Fig. 6A, compare lanes 3 to 4). Similar results with respect to changes in the accumulation of assembled -subunits are displayed in Fig. 6B, in an experiment where plasmid DNAs encoding  and  are co-transfected at ratios of 8:1 and 1:1. These data indicate that when -subunits have a higher probability of contacting -subunits, glucose trimming has a smaller impact on the accumulation of - dimers. These data also indicate that the ratios of nascent unassembled subunits and glucose trimming have a combined influence on the accumulation of assembled receptor subunits, which should ultimately dictate the expression levels of fully assembled receptors at the cell surface.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   The influence of castanospermine on the accumulation of - subunit dimers depends on the transfection ratios of plasmid DNAs encoding the - and -subunits. The transfected :-subunit ratios refer to the masses of plasmid DNAs present in the transfection mixture, where the amount of plasmid DNA encoding the -subunit is kept constant at 15 µg of DNA/plate and plasmid DNA encoding for the -subunit is varied. A, lanes 1 and 2 display samples transfected at a 8:1 ratio and lanes 3 and 4 display samples transfected at a 3:1 ratio of plasmid DNAs encoding the - and -subunits. The numbers of cells, antibody dilutions for immunoprecipitations, and volumes loaded in each lane were the same among samples, and consisted of approximately 5% of the total sample. The Western blot was developed first with mAb 210 to detect the -subunit and then reprobed with mAb 137 to detect the -subunit, and the exposures on films were overlaid to show - and -subunits together. The -subunits displayed in the Western blot are assembled with -subunits, since the immunoprecipitating antibody recognized the -subunit (mAb 61). B, Western blot revealing assembled -subunits from a similar experiment as displayed in A, developed with mAb 137.

Calreticulin and ERp57 Association with -Subunits Is not Detectable under Conditions where Calnexin Association Is Evident-- Similar to the binding properties of calnexin, calreticulin and ERp57 are thought to preferentially associate with newly synthesized glycoproteins which possess oligosaccharides capped with a single glucose residue (19, 21). Immunoprecipitation and Western blotting experiments were performed to ascertain whether calreticulin and ERp57 are associated with -subunits. Calreticulin is expressed in HEK cells, as detected in a Western blot of a detergent extract (Fig. 7A, lane 1) and upon immunoprecipitation with an antibody to calreticulin (Fig. 7A, lane 4). However, co-immunoprecipitation of -subunits with calreticulin was not appreciably detected (Fig. 7B, lane 2), although calreticulin was immunoprecipitated in this sample (Fig. 7A, lane 4), -subunits were present in the extraction mixture (Fig. 7B, lane 4), and these conditions reveal co-immunoprecipitation of -subunits with calnexin (Fig. 7B, lane 1). Association of -subunits with calreticulin was also not detected when the detergent extract was immunoprecipitated with anti--antibody (mAb 61) and the Western blot developed with an antibody to calreticulin (data not shown). Experiments with the antibodies available to us indicate calnexin is more prevalently associated with -subunits than is calreticulin.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   The -subunit is not observed to associate appreciably with calreticulin (cr) and ERp57 under conditions which detect calnexin (c), yet all three proteins are present in HEK cells. Each sample represents equivalent numbers of cells, and equivalent volumes were loaded in gel lanes. A, Western blot developed with an antibody to calreticulin. Lane 1, Western blot of a detergent extract demonstrating calreticulin is present in HEK cells; lanes 2 and 3, immunoprecipitation using an antibody to calnexin without (lane 2) and with (lane 3) treatment of castanospermine; lane 4, immunoprecipitation using an antibody to calreticulin (TFT; transfected). B, cells were transfected with plasmid DNA encoding the -subunit and the Western blot developed with an antibody to the -subunit (mAb 210). Lane 1, detergent extract immunoprecipitated with an antibody to calnexin; lane 2, detergent extract immunoprecipitated with an antibody to calreticulin. This sample is also shown in A, lane 4, demonstrating immunoprecipitation of calreticulin; lane 3, immunoprecipitation of the unbound material using an antibody to the -subunit (mAb 61; designated as c/61). This step followed the first immunoprecipitation to calnexin in lane 1; lane 4, immunoprecipitation of the unbound material using mAb 61, which followed the first immunoprecipitation to calreticulin in lane 2 (designated as cr/61). C, cells were transfected separately with plasmid DNAs encoding the -subunit (lane 1) or the -subunit (lane 2), extracts were immunoprecipitated with antibodies to the receptor subunits (mAb 61 for  and mAb 137 for ) and the Western blot was developed with an antibody to ERp57.

Castanospermine Treatment Does Not Appear to Alter the Folding of -Subunits, as Detected with a Conformationally Sensitive Antibody, whereas Assembly with -Subunits Increases the Fraction of Folded -Subunits in the Cell-- We used mAb 35 reactivity to identify whether treatment with castanospermine influences the folding of unassembled -subunits expressed in HEK cells. Cells were treated or untreated with castanospermine, and transfected with 15 µg of plasmid DNA/plate encoding the -subunit. Sequential immunoprecipitations using mAb 35, which binds primarily to folded -subunits (5), followed by an immunoprecipitation of the unbound material with mAb 61, which binds independently of conformation (5), were used to assess folding (Fig. 8A). Only a fraction of the -subunit pool is recognized by mAb 35, indicating the -subunit pool is predominantly unfolded when expressed in the absence of other subunits in HEK cells (Fig. 8A, lanes 1-4); this observation is consistent with previous observations in other cells (10, 34). An estimate of the fraction of folded -subunits was obtained by measuring the density of -subunit bands in Fig. 8 and dividing the respective mAb 35 density by the mAb 61 density (Fig. 8C). The fractions of -subunits immunoprecipitated with mAb 35 relative to mAb 61 are similar when expressed in the absence or presence of castanospermine (22 and 29%, respectively; Fig. 8C). Within resolution limits of our assay, and the capacity of mAb 35 to detect folded subunits, glucose trimming does not appear to influence -subunit folding.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   CST does not influence the folding of the -subunit as detected by a conformation sensitive antibody (mAb 35); however, assembly with the -subunit increases the fraction of folded -subunits. A, Western blot developed with an antibody to the -subunit (mAb 210). Cells were transfected with plasmid DNA encoding the -subunit or co-transfected with plasmid DNAs encoding the - and -subunits at a 1:1 ratio of plasmid masses (15 µg of plasmid DNA/plate for each subunit). Sequential immunoprecipitations initially used mAb 35, which has a higher affinity for folded -subunits, and then unbound material was immunoprecipitated with mAb 61. Both antibodies, at a concentration of 5 mg/ml, were diluted 1:100 prior to a 1.5-h immunoprecipitation. The intensity of the -subunit band immunoprecipitated by mAb 35 relative to mAb 61 provides an indication of the fraction of folded -subunits. B, same Western blot as in A, but reprobed with an antibody to  (mAb 137), indicating the relative amounts of assembled -subunits. Lanes 5-8 in B correspond to lanes 5-8 in A. C, quantitation of the fraction of folded -subunits in the Western blot displayed in A, calculated as the ratio of densities for -subunits immunoprecipitated with mAb 35 relative to -subunits immunoprecipitated with mAb 61; - and + refer to cells transfected with plasmid DNA encoding the -subunit, untreated or treated with castanospermine, respectively, and - and + refer to cells co-transfected with plasmid DNAs encoding the - and -subunits, untreated and treated with castanospermine, respectively.

	DISCUSSION

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Of the three proteins thought to be involved in the biogenesis of glycoproteins, and to associate primarily with glycoproteins possessing oligosaccharides capped with one glucose residue (19, 21), only calnexin was found to associate with the receptor -subunits. Calreticulin and ERp57 could be associated with -subunits in vivo, but the interaction was undetectable in vitro, by using antibodies available to us and techniques which readily detect calnexin in association with -subunits. Based on data presented in this study, the processing events in receptor biosynthesis which are altered by castanospermine treatment appear to be related to calnexin association.

Recognition of -subunits by calnexin appears to be rather complex, with calnexin displaying association to both oligosaccharide structures and the polypeptide backbone. Calnexin binding to the -subunit oligosaccharide is dependent on oligosaccharide trimming, because castanospermine treatment reduces the interaction. The importance for oligosaccharide trimming in this interaction becomes apparent when expression levels are low, and degradation is inhibited with a proteasome inhibitor (Fig. 4, B and C). These observations may indicate that the calnexin-oligosaccharide interaction displays a higher affinity with unassembled subunits, than the calnexin-polypeptide interaction. In fact, several investigations have suggested that calnexin displays a sufficient affinity to bind directly to oligosaccharide structures irrespective of the polypeptide backbone (19, 35).

The additional interaction between calnexin and the -subunit polypeptide backbone appears to be dependent on subunit assembly, because assembly with the -subunit reduces calnexin association, and this interaction is not significantly altered by castanospermine treatment (Fig. 4A, lanes 5-8). Subunit assembly may inhibit the calnexin-polypeptide interaction, because the newly formed contact interface between subunits occludes the lower affinity association with calnexin. Alternatively, -subunits which fold upon assembly may bury an interface which is required for calnexin attachment.

In agreement with our observations that castanospermine increases receptor degradation, an earlier study with BC3H-1 cells showed that, treatment with the glucosidase inhibitor 1-deoxynojirimycin decreased the stability of unassembled -subunits expressed from the endogenous gene (4). Investigations on other transmembrane spanning glycoproteins have also revealed a decreased stability of glycoproteins expressed under conditions which impair glucose trimming (36-38). In contrast, studies of secretory protein processing indicate that calnexin association correlates with the instability of these proteins (39, 40).

The ubiquitin-proteasome pathway appears to be involved in the degradation of -subunits expressed in the presence of castanospermine, because polyubiquitinated -subunits accumulate in cells treated with proteasome inhibitors. Moreover, the degradation of -subunits is inhibited when cells are treated with the specific proteasome inhibitor lactacystin (31), localizing the proteasome as the site for the degradation. Several investigations have established that proteins embedded in the ER membrane are susceptible to degradation in the proteasome, with examples including the cystic fibrosis transmembrane conductance regulator (29, 41), major histocompatibility complex subunits (42-44), and T-cell receptor sub