Crosslinking of proteins in acetylcholine receptor-rich membranes: Association between the β-subunit and the 43 kd subsynaptic protein

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four subunits (α, β, γ, δ) of the acetylcholine receptor (AChR) and for polypeptides at 43 kd and 270 kd. Reacton of these membranes with ³H-N-ethylmaleimide (³H-NEM) demonstrates that most of the available free sulfhydryls reside on the 43 kd protein. Cross-linking reagents that contain NEM as one reactive group, and N-hydroxysuccinimide as the other, were used to study the topography of the 43 kd protein in AChR-rich membranes. Proteins from cross-linked membranes were resolved by SDS-PAGE and the composition of crosslinked products was determined by Western blots and monoclonal antibodies. A crosslinked product at 110 kd was labeled by a monoclonal antibody to the β-subunit and by a monoclonal antibody to the 43 kd protein, but not by monoclonal antibodies to the α, γ, or δ subunits. The 110 kd crosslink was not produced in the presence of 10 mM lithium diiodosalicylate, which dissociates the 43 kd protein from the membrane. Thus the 43 kd protein is intimately associated with the AChR and in close proximity to the β-subunit.