Arrest of subunit folding and assembly of nicotinic acetylcholine receptors in cultured muscle cells by dithiothreitol.
In this study we have used cultured muscle cells to investigate the role of disulfide bond formation in the sequence of molecular events leading to nicotinic acetylcholine receptor (AChR) assembly and surface expression. We have observed that disulfide bond formation in newly synthesized AChR alpha-subunits occurs 5-20 min after translation and that this modification can be blocked by dithiothreitol (DTT), a membrane-permeant thiol-reducing agent. DTT treatment was found to arrest AChR alpha-subunit conformational maturation, assembly, and appearance on the cell surface, showing that these events are dependent on prior formation of disulfide bonds. Subunits prevented from maturation by the reducing agent do not irreversibly misfold or aggregate, since upon removal of DTT, AChR alpha-subunits undergo formation of disulfide bonds and resume folding, oligomerization, and surface expression. We have previously found that nascent alpha-subunits form transient complexes with the molecular chaperone calnexin immediately after subunit synthesis (Gelman, M.S., Chang, W., Thomas, D. Y., Bergeron, J. J. M., and Prives, J. M. (1995) J. Biol. Chem. 270, 15085-15092) and have now observed that both the formation and the subsequent dissociation of these complexes are unaffected by DTT treatment. Thus, alpha-subunits appear to dissociate from calnexin independently of their undergoing disulfide bond formation and achieving conformational maturation. This finding together with the absence of irreversible misfolding of DTT-arrested alpha-subunits suggests that calnexin may act to prevent misfolding by aiding in the initial folding events and is not an essential participant in the late stages of alpha-subunit maturation.
Gelman
M S
MS
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794, USA.
Prives
J M
JM
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin; D002478:Cells, Cultured; D002642:Chick Embryo; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D009132:Muscles / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism
D002135:Calcium-Binding Proteins; D004220:Disulfides; D011978:Receptors, Nicotinic; D037281:Calnexin; D004229:Dithiothreitol