The Role of the Cystine Loop in Acetylcholine Receptor Assembly
1997; Elsevier BV; Volume: 272; Issue: 33 Linguagem: Inglês
10.1074/jbc.272.33.20945
ISSN1083-351X
AutoresWilliam N. Green, Christian Wanamaker,
Tópico(s)Receptor Mechanisms and Signaling
ResumoNicotinic acetylcholine receptors (AChRs) are composed of α, β, γ, and δ subunits, assembled into α2βγδ pentamers. A highly conserved feature of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino acid cystine "loop." We find that an intact cystine loop is necessary for complete AChR assembly. By preventing formation of the loop with 5 mm dithiothreitol, AChR subunits assemble into αβγ trimers, but the subsequent steps in assembly are blocked. When α subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when β subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of αβγδ tetramers and before the addition of the second α subunit. After formation of the cystine loop, the α subunit undergoes a conformational change, which buries the loop. This conformational change is concurrent with the step in assembly blocked by removal of the disulfide bond of the cystine loop, i.e. after assembly of αβγ trimers and before the addition of the δ subunit. The data indicate that the α subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits. Nicotinic acetylcholine receptors (AChRs) are composed of α, β, γ, and δ subunits, assembled into α2βγδ pentamers. A highly conserved feature of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino acid cystine "loop." We find that an intact cystine loop is necessary for complete AChR assembly. By preventing formation of the loop with 5 mm dithiothreitol, AChR subunits assemble into αβγ trimers, but the subsequent steps in assembly are blocked. When α subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when β subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of αβγδ tetramers and before the addition of the second α subunit. After formation of the cystine loop, the α subunit undergoes a conformational change, which buries the loop. This conformational change is concurrent with the step in assembly blocked by removal of the disulfide bond of the cystine loop, i.e. after assembly of αβγ trimers and before the addition of the δ subunit. The data indicate that the α subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits. The molecular events involved in subunit folding and assembly of large oligomeric proteins remain largely uncharacterized. The subunit folding and oligomerization events that take place during the assembly of ion channels are particularly complex, since the finished product requires the correct oligomeric arrangement and subunit stoichiometry for proper function (1Green W.N. Millar N.S. Trends Neurosci. 1995; 18: 280-287Abstract Full Text PDF PubMed Scopus (175) Google Scholar). In terms of their structure, the best characterized ion channels are the muscle-type nicotinic acetylcholine receptors (AChRs), 1The abbreviations used are: AChR, acetylcholine receptor; BuTx, α-bungarotoxin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; DTT, dithiothreitol. which are the neurotransmitter receptors responsible for rapid signaling between motor neurons and skeletal muscle. Muscle-type AChRs are composed of four distinct, homologous subunits, α, β, γ, and δ, which assemble into pentamers with the subunit stoichiometry of α2βγδ. Although AChR assembly is a slow process that takes ∼2 h to complete (2Merlie J.P. Lindstrom J. Cell. 1983; 34: 747-757Abstract Full Text PDF PubMed Scopus (127) Google Scholar), assembly intermediates have been difficult to isolate. A number of laboratories turned to expression of less than the full complement of subunits in different heterologous expression systems to isolate assembly intermediates (3Blount P. Smith M.M. Merlie J.P. J. Cell Biol. 1990; 111: 2601-2611Crossref PubMed Scopus (76) Google Scholar, 4Saedi M.S. Conroy W.G. Lindstrom J. J. Cell Biol. 1991; 112: 1007-1015Crossref PubMed Scopus (51) Google Scholar, 5Gu Y. Forsayeth J.R. Verrall S. Yu X.M. Hall Z.W. J. Cell Biol. 1991; 114: 799-807Crossref PubMed Scopus (91) Google Scholar, 6Kreienkamp H.J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Based on their findings, the "heterodimer" model was proposed, where the α subunit must first fold or "mature," as assayed by the formation of the α-bungarotoxin (BuTx) binding site and antigenic epitopes, before assembling with other subunits. The mature α subunit assembles with γ or δ subunits in parallel to form αγ and αδ heterodimers, and the heterodimers associate together and with β subunits to form α2βγδ pentamers. We have developed techniques that have allowed isolation of assembly intermediates in cells stably expressing all four AChR subunits (7Claudio T. Green W.N. Hartman D.S. Hayden D. Paulson H.L. Sigworth F.J. Sine S.M. Swedlund A. Science. 1987; 238: 1688-1694Crossref PubMed Scopus (68) Google Scholar, 8Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar) and have obtained results at odds with the heterodimer model. Instead of heterodimers, two partially assembled complexes, αβγ trimers and αβγδ tetramers, were isolated. αβγ trimers, which assemble extremely rapidly, were assembled first into αβγδ tetramers and then into α2βγδ pentamers. Our data demonstrated that assembly occurs sequentially, each step being the addition of an uncomplexed subunit. We also demonstrated that the α subunit maturation steps, which were thought to precede its assembly, occurred after assembly into αβγ trimers but prior to the addition of the δ subunit. These folding events require a specific combination of subunits and correlate in time with the δ subunit addition. The data led us to suggest that the α subunit maturation steps are folding events forming the δ subunit recognition site,i.e. the site where the δ subunit associates with the αβγ trimer. Our goal has been to identify posttranslational processing sites and regions on the AChR subunits involved in AChR subunit folding and assembly. A good candidate is the highly conserved region defined by a pair of cysteine residues separated by a stretch of 13 amino acids, which is found on the neurotransmitter-binding, extracellular domain of the subunits (Fig. 1, A and B). The two cysteines form a disulfide bridge on all four Torpedo AChR subunits (9Kao P.N. Karlin A. J. Biol. Chem. 1986; 261: 8085-8088Abstract Full Text PDF PubMed Google Scholar, 10Kellaris K.V. Ware D.K. Biochemistry. 1989; 28: 3469-3482Crossref PubMed Scopus (27) Google Scholar). Analogous cystine "loops" appear to form on other neurotransmitter-gated ion channel subunits, which include all muscle and neuronal AChR subunits and all GABAA, glycine, and 5HT3 receptor subunits. Other residues in the loop are identically conserved across species from Caenorhabditis elegans to mammals and are even conserved on some of the glutamate receptor subunits (11Hollmann M. O'Shea G.A. Rogers S.W. Heinemann S. Nature. 1989; 342: 643-648Crossref PubMed Scopus (778) Google Scholar). Site-directed mutations of the cysteines in the α subunit prevented BuTx binding site formation and reduced AChR expression (12Mishina M. Tobimatsu T. Tanaka K. Fujita Y. Fukuda K. Kurasake M. Takahashi H. Morimoto Y. Hirose T. Inayama S. Takahasi T. Kuno M. Numa S. Nature. 1985; 313: 364-369Crossref PubMed Scopus (274) Google Scholar), which suggested that the cystine loop is critical for subunit conformational stability or assembly. A recent study where the conserved proline in the cystine loop (see Fig.1, A and B) was mutated also suggested that the cystine loop may play a role in subunit assembly (13Fu D.-X. Sine S.M. J. Biol. Chem. 1996; 271: 31479-31484Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). However, mutated α or β subunits lacking the cystine loop disulfide bond assembled with other wild type subunits (14Blount P. Merlie J.P. J. Cell Biol. 1990; 111: 2613-2622Crossref PubMed Scopus (93) Google Scholar, 15Sumikawa K. Gehle V.M. J. Biol. Chem. 1992; 267: 6286-6290Abstract Full Text PDF PubMed Google Scholar) and produced functional receptors (15Sumikawa K. Gehle V.M. J. Biol. Chem. 1992; 267: 6286-6290Abstract Full Text PDF PubMed Google Scholar). Based on these data, it was suggested that formation of the cystine loop is not required for subunit assembly but instead plays an important role in the rate that subunits are degraded and in the efficiency of transport of AChRs to the cell surface. Contrary to this viewpoint, we demonstrate in this paper that an intact cystine loop is essential for proper AChR subunit assembly. Elimination of the cystine loop separately on the α and β subunits blocks different steps during assembly. For the α subunit, the cystine loop undergoes a conformational change, which appears to be an event required for assembly to continue.Figure 5Elimination of the cystine loop disulfide on the β subunit. A, AChR subunit assembly in the αβ128γδ cell line. αβ128γδ cells were pulse-labeled with [35S]methionine and chased for the indicated times. Labeled subunits were immunoprecipitated with a mixture (cocktail) of α, β, γ, and δ subunit-specific antibodies (lane 1), the β subunit-specific mAb 148 (lanes 2–6), or mAb 14 (lane 7). As in Fig. 2, A and B, the band labeled as α′ coprecipitates with the other subunits. The band (∼43 kDa) that migrates between the α and β subunit bands is believed to be actin. B, the rate of β128 subunit degradation. Displayed are the scanned values for the β128 subunit bands and the α subunits that coprecipitated with the β128 subunits in Fig.5 A. The data are plotted on a semilog scale. The half-life was estimated to be 15.4 h for β128 subunits based on a least squares fit of an exponential function to the data, which is within the range of values found for the wild type subunits under the same conditions. C, sedimentation of αβ128γδ subunit complexes. αβ128γδ and αβγδ cells were bound with125I-BuTx as in Fig. 3 B and size-fractionated on a 5–20% linear sucrose gradient. Shown are 125I counts from the 125I-BuTx-bound intracellular complexes in fractions 4–14, which were immunoprecipitated with the β subunit-specific mAb 148. αβγδ cell surface complexes were first bound with cold BuTx to block 125I-BuTx binding to the surface AChRs. Also displayed on the figure are the standards, alkaline phosphatase (5.4 S), catalase (11 S), and surface α2βγδ complexes (9 S; dashed line). The αβ128γδ complexes peak at 8 S as estimated from a least squares linear regression fit to the S values of the standards. The shape of the intracellular αβγδ profile can be duplicated by the sum of the intracellular αβ128γδ profile reduced by 60% plus the 9 S peak cell-surface α2βγδ complexes. This indicates that intracellular αβ128γδ complexes are similar in size and composition to intracellular αβγδ complexes with the exception of the α2βγδ complexes in the 9 S peak. The broad profile observed for the intracellular AChR complexes relative to the cell surface α2βγδ 9 S peak has been seen in other studies both for the Torpedosubunits at reduced temperature (4Saedi M.S. Conroy W.G. Lindstrom J. J. Cell Biol. 1991; 112: 1007-1015Crossref PubMed Scopus (51) Google Scholar, 8Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 40Ross A.F. Green W.N. Hartman D.S. Claudio T. J. Cell Biol. 1991; 113: 623-636Crossref PubMed Scopus (56) Google Scholar) and the mouse subunits at 37 °C (3Blount P. Smith M.M. Merlie J.P. J. Cell Biol. 1990; 111: 2601-2611Crossref PubMed Scopus (76) Google Scholar, 5Gu Y. Forsayeth J.R. Verrall S. Yu X.M. Hall Z.W. J. Cell Biol. 1991; 114: 799-807Crossref PubMed Scopus (91) Google Scholar, 6Kreienkamp H.J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 8Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar). D, the effects of the α128/142 and β128 subunits on subunit assembly are consistent with the model shown. The α128/142 subunit and DTT block assembly after the formation of trimers but before the formation of the BuTx binding site and mAb 14 epitope. The β128 subunit blocks assembly after the addition of the δ subunit but before the addition of the second α subunit.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Elimination of the cystine loop disulfide on the α subunit. A, AChR subunit assembly in the α128/142βγδ cell line. α128/142βγδ cells were pulse-labeled with [35S]methionine and chased for the indicated times.Lane 1, labeled subunits were immunoprecipitated with a mixture (cocktail) of α, β, γ, and δ subunit-specific antibodies, which consisted of α and β subunit-specific polyclonal antiserum (39Claudio T. Raftery M. Arch. Biochem. Biophys. 1977; 181: 484-489Crossref PubMed Scopus (55) Google Scholar) and the γ and δ subunit-specific mAb 88b (American Tissue Culture Collection).Lanes 2–6, labeled subunits were immunoprecipitated with the α subunit-specific polyclonal antiserum, eluted from protein G-Sepharose with a 0.1 m glycine buffer (pH 2.5), and precipitated a second time with the α subunit-specific polyclonal antiserum. Only significant amounts of the β and γ subunits coprecipitate with the α128/142 subunit, and the efficiency of these interactions was reduced 2–3-fold relative to wild type subunit assembly (see Fig. 2 A, lane 2, and Fig. 6 C, lane 1). B, formation of the mAb 35 epitope during subunit assembly in the α128/142βγδ cell line. α128/142βγδ cells were pulse-labeled and chased for the same times as in Fig. 4 A (lanes 2–6) except that labeled subunits were immunoprecipitated with mAb 35.C, the rate of α128/142subunit degradation. Displayed are the scanned values for the α128/142 subunit bands (Fig. 4 A). The data are plotted on a semilog scale. The half-life was estimated to be 10.9 h for α128/142 based on a least squares fit of an exponential function to the data, which is within the range of values found for the wild type subunits under the same conditions (8Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Reduction of disulfide bonds blocks AChR subunit assembly. A and B, AChR subunit assembly in the absence or presence of 5 mm DTT. A mouse L cell line, stably expressing all four Torpedo AChR subunits (the αβγδ cell line; Ref. 7Claudio T. Green W.N. Hartman D.S. Hayden D. Paulson H.L. Sigworth F.J. Sine S.M. Swedlund A. Science. 1987; 238: 1688-1694Crossref PubMed Scopus (68) Google Scholar), was pulse-labeled with [35S]methionine for 30 min at 37 °C and chased at 20 °C in the absence or presence of 5 mm DTT for the indicated times. Labeled subunits were immunoprecipitated with either the γ subunit-specific mAb 168 or the α subunit, conformation-dependent mAb 14 (24Tzartos S.J. Lindstrom J.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 755-759Crossref PubMed Scopus (334) Google Scholar). The band labeled as α′ has previously been shown to be different from the δ subunit (8Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar) (see also Fig. 5 B), although it migrates just above the δ subunit. Further evidence that this band is not the δ subunit is displayed in lane 1 of Fig. 2 A. Cells stably expressing only the Torpedo α, β, and γ subunits were subjected to the same pulse-chase protocol as the αβγδ cells and immunoprecipitated with mAb 168. The α′ band coprecipitates with the other subunits in the absence of any δ subunit expression.C, time course of mAb 14 epitope formation. Displayed is the quantification of the mAb 14-precipitated α, β, γ, and δ subunit bands analyzed by SDS-polyacrylamide gel electrophoresis and quantified from resultant fluorographs by scanning densitometry. Also displayed for comparison are the scanned values of the δ subunits coprecipitating with the precipitated γ subunits. The percentage assembled values are the scanned values divided by the value for the β subunit at the 48-h chase time (100%) and are shown to emphasize the time course of the α subunit doubling. D, the rate of γ subunit degradation in the absence or presence of DTT. Displayed are the scanned values for the γ subunit bands chased in the absence or presence of DTT and precipitated by the γ subunit-specific mAb (Fig. 2, A and B). Also displayed are values for the β subunits that coprecipitate with the γ subunits in the absence of DTT (Fig. 2 A). The data are plotted on a semilog scale. In the absence of DTT, the decay of the γ subunit signal is biphasic. The slowly decaying component corresponds to assembled γ subunits as shown by the similar rate of decay of the assembled β subunits. To estimate the rate of decay of unassembled γ subunits, the 48-h value was subtracted from the other values and plotted as the γ adjusted symbols. E, the presence of DTT blocks BuTx site formation. 125I-BuTx binding to the cell lysate of αβγδ cells was measured for cells grown in the absence or presence of 5 mm DTT for different lengths of time. The 0 time point is the time at which the cultures were shifted from 37 to 20 °C to start subunit assembly. A single 10-cm culture was used for each time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1The cystine loop region of AChR subunits. A, comparison of the cystine loop region in different neurotransmitter-gated ion channel subunits. Shown are sequences from the four Torpedo nAChR subunits (30Popot J.-L. Changeux J.-P. Physiol. Rev. 1984; 64: 1162-1239Crossref PubMed Scopus (246) Google Scholar), the rat brain nAChR α2, β2 (31Deneris E.S. Boulter J. Swanson L.W. Patrick J. Heinemann S. J. Biol. Chem. 1989; 264: 6268-6272Abstract Full Text PDF PubMed Google Scholar) and α7 (32Seguela P. Wadiche J. Dineley M.K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar) subunits, the C. elegans nAChR subunit deg-3 (33Treinin M. Chalfie M. Neuron. 1995; 14: 871-877Abstract Full Text PDF PubMed Scopus (159) Google Scholar), the calf brain GABAA receptor α1 and β1 subunits (34Schofield P.R. Darlison M.G. Fujita N. Burt D.R. Stephenson F.A. Rodriguez H. Rhee L.M. Ramachandran J. Reale V. Glencorse T.A. Seeburg P.H. Barnard E.A. Nature. 1987; 328: 221-227Crossref PubMed Scopus (1269) Google Scholar, 35Levitan E.S. Schofield P.R. Burt D.R. Rhee L.M. Wisden W. Kohler M. Fujita N. Rodriguez H.F. Stephenson A. Darlison M.G. Barnard E.A. Seeburg P.H. Nature. 1988; 335: 76-79Crossref PubMed Scopus (490) Google Scholar), the rat brain glycine receptor α1 subunit (36Grenningloh G. Rienitz A. Schmitt B. Methfessel C. Zensen M. Beyreuther K. Gundelfinger E.D. Betz H. Nature. 1987; 328: 215-220Crossref PubMed Scopus (535) Google Scholar), the mouse 5HT3 receptor subunit (37Maricq A.V. Peterson A.S. Brake A.J. Myers R.M. Julius D. Science. 1991; 254: 432-437Crossref PubMed Scopus (884) Google Scholar), and the rat brain glutamate receptor subunit Glu R1 (11Hollmann M. O'Shea G.A. Rogers S.W. Heinemann S. Nature. 1989; 342: 643-648Crossref PubMed Scopus (778) Google Scholar). B, the cystine loop is located within the subunit's N-terminal, extracellular region. The putative secondary structure of AChR subunits is displayed, showing the four membrane-spanning regions, M1–M4 (for a recent review, see Ref. 38Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (564) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The cystine loop mutations, α128, α142, and β128 (15Sumikawa K. Gehle V.M. J. Biol. Chem. 1992; 267: 6286-6290Abstract Full Text PDF PubMed Google Scholar), where the subscript indicates which cystine residues were replaced by serine residues, were a generous gift from Dr. Sumikawa. The cystine loop mutation, α128/142, was created by inserting the α142 BclI fragment back into α128. α128/142 and β128 were subcloned into theEcoRI site of pSVDF4 (16Takeyasu K. Tamkun M.M. Siegel N.R. Fambrough D.M. J. Biol. Chem. 1987; 262: 10733-10740Abstract Full Text PDF PubMed Google Scholar). The α128/142βγδ and αβ128γδ cell lines were established by stably transfecting each of the two cystine loop mutations along with the appropriate wild type subunit constructs plus the thymidine kinase gene (tk) into mouse fibroblast L cells deficient in tk as described previously (7Claudio T. Green W.N. Hartman D.S. Hayden D. Paulson H.L. Sigworth F.J. Sine S.M. Swedlund A. Science. 1987; 238: 1688-1694Crossref PubMed Scopus (68) Google Scholar). To establish the α128 cell line, mouse NIH3T3 cells were infected with the retroviral recombinant, pDOJ, which contained the α128 subunit cDNA in the EcoRI site as well as the neomycin resistance gene. The αβγδ and αβγ cell lines, which stably express the indicated Torpedo AChR subunits, were previously described (7Claudio T. Green W.N. Hartman D.S. Hayden D. Paulson H.L. Sigworth F.J. Sine S.M. Swedlund A. Science. 1987; 238: 1688-1694Crossref PubMed Scopus (68) Google Scholar, 8Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar). The assembly of the Torpedo subunits is temperature-dependent (7Claudio T. Green W.N. Hartman D.S. Hayden D. Paulson H.L. Sigworth F.J. Sine S.M. Swedlund A. Science. 1987; 238: 1688-1694Crossref PubMed Scopus (68) Google Scholar, 17Paulson H.L. Claudio T. J. Cell Biol. 1990; 110: 1705-1717Crossref PubMed Scopus (32) Google Scholar). To allow assembly to occur, the temperature was dropped from 37 to 20 °C. The transfectedTorpedo subunit cDNAs in the αβγδ cell line are under the control of SV40 promoters (7Claudio T. Green W.N. Hartman D.S. Hayden D. Paulson H.L. Sigworth F.J. Sine S.M. Swedlund A. Science. 1987; 238: 1688-1694Crossref PubMed Scopus (68) Google Scholar). To enhance expression ofTorpedo AChR subunits, the medium (Dulbecco's modified Eagle's medium (DMEM; JRH Scientific) plus 10% calf serum (Hyclone) and HAT (15 μg/ml hypoxanthine, 1 μg/ml aminopterin, and 5 μg/ml thymidine), was replaced with fresh DMEM supplemented with 20 mm sodium butyrate (NB medium; Baker) 36–48 h prior to the experiment. To pulse-label the subunits, 10-cm cultures were first washed with PBS and starved of methionine for 15 min in methionine-free DMEM. Cultures were incubated at 5% CO2 and labeled in 2 ml of methionine-free DMEM, supplemented with 20 mm sodium butyrate and 333 μCi of [35S]methionine for 30 min at 37 °C. The labeling was stopped with the addition of DMEM containing 5 mmmethionine and, if not "chased," two washes of ice-cold PBS. To follow the subsequent changes in the labeled subunits, the cells were chased. Specifically, these cells were washed two more times with DMEM containing 5 mm methionine and incubated for various times in NB medium at 20 °C in the absence or presence of 5 mmdithiothreitol (DTT). After a [35S]methionine pulse chase, cultures were rinsed with ice-cold PBS, scraped, and pelleted by centrifugation at 5,000 × g for 3 min, and the pellets were resuspended in lysis buffer: 150 mm NaCl, 5 mm EDTA, 50 mm Tris, pH 7.4, 0.02% NaN3, 2 mm phenylmethylsulfonyl fluoride, 2 mm N-ethylmaleimide plus 1% solubilizing agent. In some of the experiments, 1 mm MgATP replaced the 5 mm EDTA to reduce the amount of actin that nonspecifically precipitated with the subunits. To solubilize the labeled subunits, a Lubrol-phosphatidylcholine mixture composed of 1.83 mg/ml phosphatidylcholine and 1% Lubrol was used. Antibody-subunit complexes were precipitated with Protein G-Sepharose and electrophoresed on 7.5% SDS-polyacrylamide gels, fixed, enhanced for 30 min, dried on a gel dryer, and exposed to film at −70 °C with an intensifying screen. Autoradiographs were quantified by scanning densitometry using a flatbed scanner and analyzed with the Intelligent Quantifier software from BioImage. To measure cell surface125I-BuTx binding, cultures were grown at 37 °C for 36 h in NB medium and then shifted to 20 °C for 48 h. Cultures were washed with PBS and incubated at room temperature in PBS containing 4 nm125I-BuTx (140–170 cpm/fmol) for 2 h, which results in the saturation of binding. Cultures were washed again and solubilized, and the cell surface counts were determined by γ-counting, or counts were immunoprecipitated with appropriate antibodies and counted. Total cell 125I-BuTx binding was measured after solubilization. In this case, cell lysates were incubated in 10 nm125I-BuTx at 4 °C overnight to saturate binding at this lower temperature. The125I-BuTx in the lysates was then immunoprecipitated with the indicated antibodies. To distinguish subunit complexes on the basis of their size, solubilized subunits (labeled with [35S]methionine and/or bound by 125I-BuTx) were sedimented on sucrose gradients. For this procedure, lysates were layered on a 5-ml 5–20% linear sucrose gradient prepared in the appropriate lysis buffer. Gradients were centrifuged in a Beckman SW 50.1 rotor at 40,000 rpm to ω2 t = 9.0 × 1011. ∼17 fractions (300 μl) were collected from the top of the gradient, and the appropriate antibodies were then added to the fractions to be assayed. When added extracellularly to cultured cells, DTT reaches the endoplasmic reticulum and prevents the formation of protein disulfide bonds without altering most other cellular functions (18Braakman I. Helenius J. Helenius A. EMBO J. 1992; 11: 1717-1722Crossref PubMed Scopus (331) Google Scholar). Cells stably expressing the four Torpedo AChR subunits were subjected to an [35S]methionine pulse-chase protocol in the absence or presence of 5 mm DTT (Fig. 2,A and B). Labeled subunits were immunoprecipitated with either a γ subunit-specific monoclonal antibody (mAb 168) or α subunit, conformation-dependent mAb 14, to assay for subunit assembly and formation of the mAb 14 epitope. During a 30-min pulse of [35S]methionine, αβγ trimers formed as shown by the coprecipitation of predominantly α and β subunits with the γ subunits (Fig.2 A). During the chase in the absence of DTT, progressively more δ subunits are added to the trimers, followed by the addition of the second α subunit as shown by the doubling in the amount of α subunit relative to the other coprecipitated subunits. These two subunit additions are better resolved by the mAb 14 immunoprecipitations (Fig. 2, A and C). Since the mAb 14 epitope forms on αβγ trimers just prior to the addition of the δ subunit (8Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar), these immunoprecipitations show the complete time course of both the addition of the δ and second α subunits to the αβγ trimers. In the presence of DTT, the subunits clearly retain the ability to assemble into αβγ trimers and with approximately the same efficiency as occurs in the absence of DTT (Fig. 2 B). This result is at odds with a recent study (19Gelman M.S. Prives J.M. J. Biol. Chem. 1996; 271: 10709-10714Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), which suggested that 5 mm DTT completely blocks AChR subunit assembly. There are several differences between this and the other study that explain the conflicting results. Probably the most important difference in terms of the ability to observe αβγ trimers was that their only assay for measuring the assembly of α subunits with other subunits was an immunoprecipitation with δ subunit-specific antibodies. Obviously, αβγ trimers could never be observed with this assay. Another difference was the protocol used to solubilize the AChR subunit complexes. We have shown previously that solubilization in 1% Triton X-100 causes the assembly intermediates to dissociate and that the subunit associations are stable when solubilized with a combination of Lubrol PX and phosphatidylcholine (8Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar). After αβγ trimers assemble in the presence of 5 mmDTT, the ensuing subunit associations, the addition of the δ and second α subunits, fail to occur, and the αβγ trimers that had assembled were degraded 48 h after the [35S]methionine pulse (Fig. 2 B). γ subunits in the presence of DTT degrade at about the same rate as unassembled γ subunits in the absence of DTT (Fig. 2 D). Subunit complexes degrade more rapidly in the presence of DTT because the complexes are no longer stabilized by events subsequent to trimer formation. Although there are almost as many αβγ trimers present with DTT at the times when δ subunits normally assemble with αβγ trimers (Fig. 2, A and B; 3- and 6-h chase), the formation of αβγδ tetramers and α2βγδ pentamers is inhibited in the presence of DTT. Furthermore, the BuTx binding site (Figs. 2 E and3 A) and the mAb 14 epitope (Fig. 2 B) do not form on the αβγ trimers in the presence of DTT. We conclude that the additio
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