Identification of homo-oligomers as potential intermediates in acetylcholine receptor subunit assembly.

Abstract

We have examined the sedimentation behavior, on sucrose density gradients, of acetylcholine receptor (AcChoR) subunits synthesized in vitro and integrated into heterologous rough microsomal membranes. In media containing nondenaturing detergents such as Triton X-100 or deoxycholate, the subunits appear to self-associate although, as previously reported, no heterologous interactions were detected. The sedimentation profiles assume a broad distribution in the region of 7-13 S. However, the peak fractions occupy the same region of the gradient as does native AcChoR, run in parallel. Such large homo-oligomers were not observed for another membrane protein, opsin, studied in the same way. This indicated that the associations are indeed between the AcChoR subunits and not simply between all newly synthesized membrane proteins. The homologous associations are interpreted to suggest a mechanism for maintaining the ionophore surfaces of the subunits in an energetically preferred, but metastable, configuration during the lengthy period of post-translational assembly.The oligomeric assembly of the acetylcholine receptor (AcChoR) subunits poses a unique problem in membrane protein biogenesis-that is, how four distinct transmembrane glycoproteins, translated from separate mRNAs (1), are assembled into a quaternary complex that forms a hydrophilic ion channel through the lipid bilayer. The problem is further complicated by the observation that AcChoR subunit assembly does not occur immediately upon insertion of the subunits into the rough endoplasmic reticulum (RER) membrane (1) but rather after a delay of at least 30 min, or perhaps even longer (2). This fact implies that (i) newly synthesized AcChoR subunits must be transported to a common subcellular site, perhaps the Golgi apparatus (2, 3), for assembly to occur, and (ii) the individual subunits must assume a stable configuration in the lipid bilayer of intracellular organelles, for a considerable time prior to formation of the ionophore complex.The AcChoR subunits are comprised by four polypeptidesa, ,3, y, and 6, of apparent molecular masses of 40, 50, 60, and 65 kilodaltons (kDa), respectively (4). Their stoichiometry in the mature complex is a2Py8 (5-7). These subunits are synthesized with signal sequences (8-10) and are cotranslationally integrated into the RER membrane (1) by a mechanism that utilizes the recently purified (11, 12) signal recognition particle (8). Although these subunits are not associated with one another at this stage in their biosynthesis (1), they exhibit a transmembrane topology that is similar, at least in its gross aspects, to that observed for their counterparts in the mature hetero-oligomer (13,14).In this study we have examined the sedimentation behavior of these early biosynthetic forms of the AcChoR subunits on sucrose density gradients. The subunits each appear to self-associate, forming homo-oligomers in the range of 7-13 S. Such homologous associations were not observed for another membrane protein, opsin, studied in an ident...

Analysis of early events in acetylcholine receptor assembly.

“…1 D), a 6S sedimentation profile was still obtained. Our 6S profile differs from those obtained by Anderson and Blobel (1983) who found that after synthesis in a cell-free translation system, Torpedo subunits sedimented broadly from ~7 to 13S with a peak at ~9.5S. Using conditions similar to those of the cell-free study (same detergent, buffers, and gradients), we typically observed a 6S sedimentation peak for Torpedo subunits and only obtained peaks >7S if the detergent to protein ratio was reduced (not shown); presumably this is due to insufficient solubilization.…”

“…The 5S sedimentation of unassembled a suggests that newly synthesized subunits do not first associate as large homooligomers, although 5S is larger than expected for a monomer. In a series of cell-free translation studies, Anderson and Blobel (1983) found that each of the four Torpedo subunits formed large, apparently homooligomeric, complexes sedimenting broadly at '~,7-13S. Their data led them to propose that newly synthesized AChR subunits form homooligomers before associating with heterologous subunits.…”

Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein [published errratum appears in Mol Cell Biol 1987 May;7(5):2035]

“…We cannot, therefore, rule out that the orientation achieved in the wheat germ cell-free system is different from that achieved in the brain. However, previous studies of membrane protein biogenesis in cell-free systems have demonstrated fidelity with the early events in living cells (1,21,23), and other studies of the scrapie agent are suggestive of an integral membrane protein (31,32,44 …”

Molecular weight and structural nonequivalence of the mature alpha subunits of Torpedo californica acetylcholine receptor.

“…In this respect, the recent observation (38) that all four subunits of Torpedo AcChoR can form homopolyers during biosynthesis and transport may ultimately lead to understanding how the selective glycosylation we discuss here is achieved.…”