Formation of the alpha-bungarotoxin binding site and assembly of the nicotinic acetylcholine receptor subunits occur in the endoplasmic reticulum

During the process by which newly synthesized subunits of the nicotinic acetylcholine receptor (stoichiometry = alpha 2 beta gamma delta) mature and acquire the properties of the fully functional cell surface receptor, they undergo numerous covalent and noncovalent modifications. Using ligand-mediated and subunit-specific immunoprecipitation, four forms in the maturation of the alpha subunit can be detected: the primary translation product; alpha subunit that can bind alpha-bungarotoxin; alpha subunit assembled with the other subunits; and surface receptor. The alpha subunit acquires the ability to bind alpha-bungarotoxin with a t1/2 of approximately 40 min after translation and becomes assembled with a t1/2 of 80 min after translation. Using metabolic labeling and sucrose gradient fractionation, we have determined the subcellular location of alpha subunit when it acquires the ability to bind alpha-bungarotoxin and when it is assembled. Golgi membranes were identified across the gradient by the enzymatic activities UDP-galactose:N-acetylglucosamine galactosyltransferase and alpha-mannosidase. Endoplasmic reticulum membranes were identified by the enzymatic activity glucose-6-phosphatase and by the presence of newly synthesized alpha and beta subunits. Pulse-labeled alpha subunit that bound alpha-bungarotoxin was first detected co-migrating in the gradient with the glucose-6-phosphatase activity. Therefore, the capacity to bind alpha-bungarotoxin was acquired while the alpha subunit was in the endoplasmic reticulum. Assembled alpha subunit was detected by immunoprecipitating with an anti-beta subunit-specific monoclonal antibody. By this method, assembled receptor was first detected 15 min after translation in both the endoplasmic and Golgi portions of the gradient. To validate this method of detecting assembled receptor, we determined the sedimentation coefficient of the receptor subunits in the endoplasmic reticulum. Both unassembled subunits with sedimentation coefficients of 5 S and assembled receptor with a sedimentation coefficient of 9 S were recovered from the endoplasmic reticulum portion of the gradient. Thus, our data concerning the subcellular site of assembly are consistent with assembly occurring in the endoplasmic reticulum followed by rapid transport to the Golgi.     

Architectural editing: determining the fate of newly synthesized membrane proteins

Many integral membrane proteins exist on the plasma membrane as part of multicomponent complexes. In addition to correctly transporting newly synthesized proteins from their site of synthesis in the endoplasmic reticulum to the plasma membrane, the cell must possess mechanisms to ensure that the complexes expressed on the cell surface are accurately assembled. The cell appears to accomplish this feat by superimposing a set of constraints on the newly synthesized membrane proteins whereby the structure and state of assembly of the protein determine its intracellular fate. These processes impose a dramatic level of post-translational regulation on the expression of surface membrane protein complexes. By and large, the cell uses these mechanisms to dispose of, or "edit out," newly synthesized proteins that are not correctly assembled or folded. This review will describe current views of the processes of architectural editing, with an emphasis on the regulation of cell surface expression of the multicomponent T-cell antigen receptor complex.     

A C2 muscle cell variant defective in transport of the acetylcholine receptor to the cell surface

We have previously reported the isolation of variants of the C2 mouse muscle cell line that express reduced amounts of acetylcholine receptors (AChRs) on their surface (Black, R. A., and Hall, Z. W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 124-128). One of the variants, T-, makes an approximately normal amount of the AChR but accumulates most of it in an intracellular pool. This pool is stable and does not serve as precursor for surface AChR. Surface levels of insulin receptor and transferrin receptor are normal in T- cells, and a normal proportion of total hemagglutinin is expressed on the surface after infection of the T- variant with influenza virus. Pulse-chase experiments and kinetic analysis show: 1) that T- cells synthesize a normal amount of the alpha subunit but degrade it much more slowly than do wild-type cells; and 2) that newly synthesized alpha subunit is assembled into the AChR at a normal rate. A small fraction of the assembled AChR in T- cells is transported to the surface with normal kinetics, but most of it remains in an internal pool. This variant may provide an important tool for investigation of the factors that regulate AChR assembly and transport to the surface membrane.     

Regulation of acetylcholinesterase synthesis and assembly by muscle activity. Effects of tetrodotoxin

The abundance and distribution of acetylcholinesterase (AChE) oligomeric forms expressed in skeletal muscle is strongly dependent upon the activity state of the cells. In this study, we examined several stages of AChE biogenesis to determine which ones were regulated by muscle activity. Inhibiting spontaneous contraction of tissue-cultured quail myotubes with tetrodotoxin (TTX) reduces AChE activity by approximately 30% of the levels found in actively contracting cells. This decrease is due primarily to the loss of 20 S asymmetric (collagen-tailed) AChE from TTX-treated cultures and is reflected in reduced pool sizes for both cell surface and intracellular AChE molecules. Using monoclonal anti-AChE antibodies to immunoprecipitate and quantify isotopically labeled enzyme molecules, we show that AChE down-regulation by TTX is not mediated through changes in the rates of synthesis or degradation of AChE polypeptide chains. Newly synthesized AChE polypeptides acquire enzymatic activity at the same rate in TTX-treated cultures as in actively contracting cells, however, a larger percentage of catalytically active dimers and tetramers are secreted from TTX-treated cultures compared with controls. These results suggest that TTX-induced down-regulation of asymmetric AChE occurs at the level of assembly of globular AChE molecules with collagen-like tail subunits in the Golgi apparatus, rather than through changes in the availability of catalytic subunits. Thus, post-translational mechanisms appear to play an important role in regulating the abundance and distribution of this important synaptic component in skeletal muscle.     

The effects of inhibiting oligosaccharide trimming by 1-deoxynojirimycin on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor has a subunit stoichiometry of alpha 2 beta gamma delta; all 5 subunits contain N-linked oligosaccharides. We investigated what role trimming of the oligosaccharides played in the post-translational processing of the subunits and assembly of the receptor by examining the receptor synthesized in the presence of an inhibitor of oligosaccharide trimming, 1-deoxynojirimycin. BC3H-1 cells express one-third fewer receptors when grown in the presence of 1-deoxynojirimycin. The receptor subunits that are expressed have decreased mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating an inhibition of oligosaccharide trimming. In control cells, 40% of the translated alpha subunit acquires the capacity to bind alpha-bungarotoxin with a half-time of 40 min before assembly with the other subunits; the rest is rapidly degraded. In 1-deoxynojirimycin-treated cells approximately the same amount of alpha subunit is translated as in control cells, but that alpha subunit is degraded more rapidly, and only 25% acquires the capacity to bind alpha-bungarotoxin. From these results, we conclude that oligosaccharide processing either may aid in protecting the alpha subunit primary translation product from degradation or may be required for the conformational change or other post-translational modification(s) necessary for formation of the alpha-bungarotoxin binding form of the alpha subunit, which is then protected from proteolytic degradation. The cell surface receptor that is expressed in the presence of 1-deoxynojirimycin, however, is not altered in its affinity for cholinergic ligands. Thus, we conclude that differential N-linked oligosaccharide trimming of the 2 alpha subunits does not appear to play a part in the differences in affinities of the 2 alpha subunits for cholinergic ligands.     

Transforming growth factor beta differentially regulates expression of integrin subunits in guinea pig airway epithelial cells.

The integrin family is composed of a large number of heterodimers, each one mediating distinct interactions with extracellular matrix and/or cell surface ligands. The expression of integrins appears to be tightly regulated in vivo, but the mechanisms by which cells control the formation and surface expression of specific pairs of subunits have not been well characterized. Two integrin subunits, the alpha subunit alpha v, and the beta subunit beta 1, could pose special problems in regulation because of their capacity to associate with multiple partners. In the present study, we have examined the effects of the cytokine transforming growth factor beta 1 (TGF-beta 1) on the expression of alpha v- and beta 1-containing integrins in primary cultures of guinea pig airway epithelial cells, e.g. cells that we have previously found to express multiple potential partners for both alpha v and beta 1. TGF-beta 1 increased the surface expression of both alpha v- and beta 1-containing heterodimers after periods of stimulation from 24 to 72 h. These increases in surface expression were associated with significant increases in the concentrations of mRNA encoding each of the partners of alpha v and beta 1, but with only minimal increases in mRNA encoding alpha v and beta 1 themselves. Airway epithelial cells metabolically labeled with [35S]methionine during stimulation with TGF-beta 1 demonstrated only a minimal increase in the synthesis of new alpha v protein at a time when synthesis of alpha v's beta subunit partners and surface expression of alpha v-containing heterodimers were dramatically increased. These data suggest that, at least in some cells, promiscuous integrin subunits (both alpha and beta) may normally be synthesized in excess. Thus, the surface expression of specific integrin heterodimers can be regulated primarily through regulation of the synthesis of the specific partners of these subunits.     

Biogenesis of acetylcholinesterase molecular forms in muscle. Evidence for a rapidly turning over, catalytically inactive precursor pool

Tissue-cultured chicken embryo muscle cells synthesize several molecular forms of acetylcholinesterase (AChE) which differ in oligomeric structure and fate as membrane-bound or secreted molecules. Using irreversible inhibitors to inactivate AChE molecules we show that muscle cells rapidly synthesize and assemble catalytically active oligomers which transit an obligatory pathway through the Golgi apparatus. These oligomers acquire complex oligosaccharides and are ultimately localized on the cell surface or secreted into the medium. Immunoprecipitation of isotopically labeled AChE shows that the oligomers are assembled shortly after synthesis from two allelic polypeptide chains. About two-thirds of the newly synthesized molecules are assembled into dimers and tetramers, and once assembled these forms do not interconvert. Comparison of newly synthesized catalytically active AChE molecules with isotopically labeled ones indicates that a large fraction of the immature molecules are catalytically inactive. Pulse-chase studies measuring both catalytic activity and isotopic labeling indicate that only the catalytically active oligomers are further processed by the cell, whereas inactive molecules are rapidly degraded intracellularly by an as yet unknown mechanism. Approximately 70-80% of the newly synthesized AChE molecules are degraded in this manner and do not transit the Golgi apparatus. These studies indicate that muscle cells synthesize an excess of this important synaptic component over that which is necessary for maintaining normal levels of this protein. In addition, these studies indicate the existence of an intracellular route of protein degradation which may function as a post-translational regulatory step in the control of exportable proteins.     

Biogenesis of the avian erythroid membrane skeleton: receptor-mediated assembly and stabilization of ankyrin (goblin) and spectrin

Ankyrin is an extrinsic membrane protein in human erythrocytes that links the alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin ( goblin ) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton. In contrast, continuous labeling of erythroid cells with [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions. The soluble pools of ankyrin and beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.     

Biosynthesis of titin in cultured skeletal muscle cells

Although significant progress has been made regarding the structure and function of titin, little data exist on the biosynthesis of this large protein in developing muscle. Using pulse-labeling with [35S]methionine and immunoprecipitation with an anti-titin mAb, we have examined the biosynthesis of titin in synchronized cultures of skeletal muscle cells derived from day 12 chicken embryos. We find that: (a) titin synthesis increases greater than 4-fold during the first week in culture and during this same time period, synthesis of muscle-specific myosin heavy chain increases greater than 12-fold; (b) newly synthesized titin has a t1/2 of approximately 70 h; (c) titin is resistant to extraction with Triton X-100 both during and immediately after its synthesis. These observations suggest that newly synthesized titin molecules are stable proteins that rapidly associate with the cytoskeleton of developing myotubes.     

The transmembrane topology of the amino terminus of the alpha subunit of the nicotinic acetylcholine receptor

We have investigated the transmembrane topology of the amino-terminal domain of the alpha subunit of the mouse muscle nicotinic acetylcholine receptor synthesized in vitro and in vivo. Using oligonucleotide-directed mutagenesis we introduced new glycosylation consensus sequences at alpha 154 and at alpha 200. For each novel site, additional constructs were made in which the original site at alpha N141 was eliminated. Glycosylation at the new sites, as exhibited in a rabbit reticulocyte cell-free translation system supplemented with canine pancreatic microsomes and in a transient transfection system with COS cells, was taken as evidence of the transmembrane translocation of the new site. Each of the new sites was glycosylated in both systems. In separate experiments we found that an alpha subunit fragment terminating at alpha M207 could be extracted from microsomal membranes with sodium carbonate after in vitro translation, indicating that this fragment is not an integral membrane protein. Our results, taken together with previous experiments, indicate that the amino terminus of the alpha subunit up to at least residue alpha 207 is translocated across the membrane of the endoplasmic reticulum. This topology probably represents the orientation of the amino terminus of the alpha subunit in the assembled receptor.     

Characterization of the mRNA for mouse muscle acetylcholine receptor alpha subunit by quantitative translation in vitro

The nicotinic acetylcholine receptor of mammalian skeletal muscle is a multisubunit membrane glycoprotein whose synthesis is regulated by developmental and physiological cues. We report here the identification and characterization of the primary translation product of alpha subunit mRNA. The alpha subunit synthesized in rabbit reticulocyte lysate is approximately 2000 larger in apparent molecular weight than the native alpha subunit polypeptide found in acetylcholine receptor. Evidence from peptide maps and the effect of co-translational incubation with dog pancreas microsomes suggests that the in vitro product differs in two ways from native alpha subunit: 1) it is synthesized with an NH2-terminal signal peptide which is removed in vivo, and 2) the in vitro product is not glycosylated. We have characterized the alpha subunit mRNA activity by using a quantitative the membrane-bound polysome fraction. It is poly(A+) and approximately 2000 nucleotides long. Finally, we have shown that in BC3H-1 cells, alpha subunit mRNA is regulated developmentally. We detected a 10-fold increase in the relative abundance of alpha subunit mRNA in cells which had undergone the transition from log phase growth to differentiated myoblast.     

16S acetylcholinesterase of the extracellular matrix is assembled within mouse muscle cells in culture.

The present paper examines where the extracellular-matrix (ECM) 16S acetylcholinesterase (AChE, EC 3.1.1.7) is assembled in muscle cells in culture. The existence of an internal pool of 16S AChE was detected by using AChE inhibitors of differing membrane permeability. After irreversible inhibition of all cellular esterase, the newly synthesized 16S form appears in an intracellular compartment and is only later detected on the cell surface. Results show that the ECM 16S AChE is assembled within muscle cells.     

Assembly in vivo of mouse muscle acetylcholine receptor: identification of an alpha subunit species that may be an assembly intermediate

We have studied assembly of acetylcholine receptor in vivo using subunit-specific monoclonal antibodies and immunoprecipitation with alpha-bungarotoxin and antitoxin. We have identified three distinct forms of the alpha subunit. The newly synthesized alpha subunit species has a sedimentation coefficient of 5S and is recognized only by antibody specific for SDS-denatured alpha subunit. We have called this species alpha 61. The 5S alpha Tx species is not associated with beta subunits and is probably monomeric. alpha Tx is formed from alpha 61 with a half-time of 15 min and an efficiency of approximately equal to 30%. Formation of alpha Tx involves a conformational change, and we suggest that this conformation is dependent upon or stabilized by disulfide bond formation. The assembly of alpha Tx with beta subunits (and probably gamma and delta) into a 9S complex appears to be an efficient but slow process requiring more than 90 min. Unassembled alpha 61 subunits are degraded rapidly. However, subunit degradation is a result of failure to assemble, rather than its cause.     

Assembly intermediates of the mouse muscle nicotinic acetylcholine receptor in stably transfected fibroblasts

We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor alpha and gamma, and alpha and delta subunits to measure the kinetics of subunit assembly, and to study the properties of the partially assembled products that are formed. We demonstrate by coimmunoprecipitation that assembly intermediates in fibroblasts coexpressing alpha and delta subunits are formed in a time-dependent manner. The alpha and gamma- and the alpha and delta-producing transfected cells form complexes that, when labeled with 125I-alpha- bungarotoxin, migrate in sucrose gradients at 6.3S, a value consistent with a hetero-dimer structure. An additional peak at 8.5S is formed from the alpha and gamma subunits expressed in fibroblasts suggesting that gamma may have more than one binding site for alpha subunit. The stability and specificity of formation of these partially assembled complexes suggests that they are normal intermediates in the assembly of acetylcholine receptor. Comparison of the binding of 125I-alpha- bungarotoxin to intact and detergent-extracted fibroblasts indicate that essentially all of the binding sites are retained in an intracellular pool. The fibroblast delta subunit has the electrophoretic mobility in SDS-PAGE of a precursor that does not contain complex carbohydrates. In addition, alpha gamma and alpha delta complexes had lectin binding properties expected of subunits lacking complex oligosaccharides. Therefore, fibroblasts coexpressing alpha and gamma or alpha and delta subunits produce discrete assembly intermediates that are retained in an intracellular compartment and are not processed by Golgi enzymes.     

Alpha and beta subunits of the nicotinic acetylcholine receptor contain covalently bound lipid

Labeling of the BC3H1 muscle-like cell line with [3H] palmitate, followed by immunoprecipitation of the acetylcholine receptor, indicated that the alpha and beta subunits of the receptor contain covalently bound fatty acid. After acid hydrolysis, fatty acid methyl esters could be recovered from the isolated [3H]palmitate-labeled alpha subunit. Treatment of differentiated BC3H1 cells with cerulenin, an inhibitor of fatty acid and sterol synthesis and fatty acid acylation of proteins, resulted in a 50% inhibition in expression of the acetylcholine receptor on the cell surface under conditions where there was minimal inhibition of protein synthesis. We conclude that this previously undetected post-translational modification may play a role in assembly and/or surface expression of the acetylcholine receptor.     

Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells

The glycoprotein hormones lutropin (LH) and chorionic gonadotropin (CG) share a common structure consisting of an identical alpha subunit noncovalently linked to a hormone-specific beta subunit. While LH is produced in the anterior pituitary, CG is synthesized in placenta. To compare the assembly, processing, and secretion of human LH and CG in the same cell type, we have expressed their subunits, individually and together, in mouse C-127 mammary tumor cells. Analysis of transfected clones revealed an unexpected difference in the secretion of individually expressed subunits. Whereas alpha and CG beta subunits were rapidly and quantitatively secreted, only 10% of newly synthesized LH beta subunit reached the medium. The remaining subunit was found in an intracellular, endoglycosidase H (endo H)-sensitive pool that had a turnover rate of approximately 8 h. Coexpression with alpha subunit resulted in "rescue" of LH beta subunit by formation of LH dimer, which was efficiently secreted. However, combination of LH beta with alpha was slow, with an overall efficiency of only 50% despite the presence of excess alpha. In contrast, CG beta was rapidly assembled with the alpha subunit after synthesis. The two beta subunits also differed in their influence on the N-linked oligosaccharide processing of combined alpha. The oligosaccharides of LH dimer were endo H resistant, while those of CG dimer remained partially endo H sensitive. Thus, despite a high degree of homology between LH beta and CG beta, the two subunits differ in their secretion as free subunits, their rate of assembly with alpha subunit, and in their effect on the N-linked oligosaccharide processing of combined alpha.     

The roles of the alpha and gamma subunits in proton conduction through the Fo sector of the proton-translocating ATPase of Escherichia coli

Previous genetic and biochemical studies have shown that the Fo sector of the Escherichia coli H+-ATPase is synthesized and assembled in a nonleaky form from plasmid-borne genes. The proton channel then appears to be opened by an interaction of F1 subunits, especially the alpha subunit, with the nonleaky Fo (Brusilow, W. S. A. (1987) J. Bacteriol. 169, 4984-4990; Solomon, K. A., and Brusilow, W. S. A. (1988) J. Biol. Chem. 263, 5402-5407). To study the role of the alpha and gamma subunits in proton conduction, we constructed an inducible alpha plasmid. In an alpha-gamma- background, the induction of alpha synthesis caused lethal proton leakiness, as assayed by the loss of respiration-dependent acridine orange fluorescence quenching of E. coli membranes. The presence of a gamma subunit counteracted the lethal effects as if gamma were blocking the opened channel.     

Increases in mRNA concentrations of the alpha and beta subunits of prolyl 4-hydroxylase accompany increased gene expression of type IV collagen during differentiation of mouse F9 cells.

Mouse F9 teratocarcinoma stem cells differentiate in monolayer cultures in the presence of retinoic acid, dibutyryl cAMP, and isobutyl methylxanthine. This differentiation is associated with a marked increase in the synthesis rates and mRNA concentrations of basement membrane proteins such as type IV collagen. We report here that the differentiation also involves an increase of up to 50-fold in the concentrations of the mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase, the enzyme required for the cotranslational and post-translational hydroxylation of proline residues in collagens. The time courses and magnitudes of increases in these two mRNA concentrations were similar to those observed in the same experiments for the mRNA of the alpha chain of type IV collagen. In the differentiated F9 cells the concentration of the alpha subunit mRNA was about 30% of the beta subunit mRNA concentration. Northern blot analyses indicated that the sizes of the alpha and beta subunit mRNAs in the differentiated mouse F9 cells are similar to those in human skin fibroblasts. The F9 cell differentiation system appears to provide a useful model for studies on the regulation of prolyl 4-hydroxylase synthesis.