In vitro synthesis, glycosylation, and membrane insertion of the four subunits of Torpedo acetylcholine receptor.

Abstract

We have characterized the early biosynthetic forms of the Torpedo electroplax acetylcholine receptor by using a cell-free protein synthesizing system. We obtained primary translation products of =38, 50, 49, and 60 kilodaltons for the a, (3; y, and 8 polypeptides, respectively, by using immunoprecipitation with subunit-specific antisera. These chains could each be labeled by the formylated initiator [35S]Met-tRNA. On cotranslational incubation with pancreatic rough microsomes, glycosylated forms of each subunit were obtained that had molecular weights close to those of their mature authentic counterparts. Extensive trypsinization reduced the glycosylated forms of the receptor subunits to glycosylated membrane-protected fragments of =35 (a), 37 ( (3), 45 (y), and 44 (8) kilodaltons. In this system, then, each receptor chain spans the membrane at least once. This in vitrosynthesized material apparently exhibited neither oligomeric assembly nor a-bungarotoxin binding.

Formation of a Ligand-binding Site for the Acetylcholine Receptor in Vitro

“…1A, lane 4). These results are consistent with those previously reported in conventional in vitro translation systems (8,43). Although all forms of the ␣ subunit were immunoprecipitated with the monoclonal antibody specific for the AChR ␣ subunit (mAb 61) (38), the antibody bound with highest affinity to the fully mature (44-kDa) form (Fig.…”

“…Subunit folding and assembly have been previously observed only in intact cells (5,10,11), in which it is difficult to manipulate conditions. Although subunit synthesis and processing have been reported in an in vitro translation system, correct folding of the ␣ subunit and heterooligomerization have not been previously observed (8,9,35). Using an in vitro translation system, we have established conditions under which the ␣ subunit of the AChR folds to acquire BTX binding activity through a process that shows sensitivity to the redox potential and requires core glycosylation but does not require oligosaccharide trimming.…”

“…Each of the subunits of the muscle AChR is synthesized as a separate polypeptide chain (8) that is translocated into the ER, where the signal sequence is cleaved (8), the core glycosyl residues are added (8,9), and the N-terminal domain is folded (10,11). The folded, glycosylated polypeptide chains then associate with each other to form the pentameric AChR (11,12).…”

Arrest of Subunit Folding and Assembly of Nicotinic Acetylcholine Receptors in Cultured Muscle Cells by Dithiothreitol

“…The extracellular domain of each ␣-subunit contains a region essential for acetylcholine binding, as well as a binding site for the elapid venom neurotoxin ␣-bungarotoxin (␣-Bgt) and a characteristic sequence termed the main immunogenic region, the epitope for most antibodies made against native AChR (21). All of these sites are initially absent in newly translated ␣-subunit polypeptides in intact cells (22,23), nor are they present in ␣-subunit polypeptides expressed in a cell-free system (24). In intact cells the toxin binding site and main immunogenic region epitope are acquired by the ␣-subunit monomers in the interval between biosynthesis and subunit assembly, while the agonist binding sites are not acquired until the assembly of ␣-with ␥-or ␦-subunits (23).…”

Structural models of the nicotinic acetylcholine receptor and its toxin-binding sites

“…1980: Schindler and Quast, 1980. Studies with cell-free synthesis of AChR subunits suggest that each of the subunits spans the membrane and that the N-terminal ends are on the extracellular side of the membrane (Anderson and Blobel, 1980).…”