Analysis of early events in acetylcholine receptor assembly.
Mammalian cell lines expressing nicotinic acetylcholine receptor (AChR) subunit cDNAs from Torpedo californica were used to study early events in AChR assembly. To test the hypothesis that individual subunits form homooligomeric intermediates before assembling into alpha 2 beta gamma delta pentamers, we analyzed the sedimentation on sucrose density gradients of each subunit expressed separately in cell lines. We have shown previously that the acute temperature sensitivity of Torpedo AChR subunit assembly is due, in part, to misfolding of the polypeptide chains (Paulson, H.L., and T. Claudio. 1990. J. Cell Biol. 110:1705-1717). We use this phenomenon to further analyze putative assembly-competent intermediates. In nonionic detergent at an assembly-permissive temperature, the majority of alpha, beta, gamma, and delta subunits sediment neither as 3-4S monomers nor as 9S complexes, but rather as 6S species whether synthesized in fibroblasts, myoblasts, or differentiated myosyncytia. Several results indicate that the 6S species are complexes comprised predominantly of incorrectly folded subunit polypeptides. The complexes represent homoaggregates which form rapidly within the cell, are stable to mild SDS treatment and, in the case of alpha, contain some disulfide-linked subunits. The coprecipitation of alpha subunit with BiP or GRP78, a resident protein of the ER, further indicates that at least some of these internally sequestered subunits also associated with an endogenous protein implicated in protein folding. The majority of subunits expressed in these cell lines appear to be aggregates of subunits which are not assembly intermediates and are not assembly-competent. The portion which migrates as monomer, in contrast, appears to be the fraction which is assembly competent. This fraction increases at temperatures more permissive for assembly, further indicating the importance of the monomer as the precursor to assembly of alpha 2 beta gamma delta pentamers.
Paulson
H L
HL
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Ross
A F
AF
Green
W N
WN
Claudio
T
T
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015151:Immunoblotting; D008970:Molecular Weight; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D011233:Precipitin Tests; D011950:Receptors, Cholinergic / Q000096:biosynthesis*; D014101:Torpedo
D004220:Disulfides; D011950:Receptors, Cholinergic