Characterization and Localization of the Mr = 43,000 Proteins Associated with Acetylcholine Receptor-rich Membranes

A protein of Mr = 43,000 (43K protein) is a major component of highly purified postsynaptic membranes isolated from Torpedo electric organ. It can be removed from these membranes by alkaline treatment or with 10 mM lithium diiodosalicylate, conditions which extract peripheral membrane proteins without solubilizing the acetylcholine receptor. Two-dimensional polyacrylamide gel electrophoresis of the lithium diiodosalicylate extract shows it to contain several major Mr = 43,000 components of isoelectric points between 7.0 and 8.0 and a minor component of approximately pI 5.6. The pI 5.6 polypeptide co-migrates with skeletal muscle actin on a two-dimensional gel and gives a peptide map pattern very similar to that of actin. The three major spots of pI 7.0-8.0 yield peptide maps which are indistinguishable from one another but quite different from that of actin. The membrane-bound forms of both the basic 43K protein and actin can be iodinated in a lactoperoxidase-catalyzed reaction only if the membrane vesicles are first permeabilized with saponin, a treatment which permits entry of macromolecules to the interior of the vesicles. An immunoblot of a two-dimensional gel shows that a rabbit antiserum made against alkaline extract reacts strongly with the basic 43K protein and to lesser extent with an acidic component of Mr = 55,000 but not with actin. Immunofluorescence studies have shown that this antiserum reacts with the innervated face of Torpedo electrocytes and recognizes a component of rat diaphragm muscle which is highly concentrated at the neuromuscular junction (Froehner, S.C., Gulbrandsen, V., Hyman, C., Jeng, A. Y., Neubig, R. R., and Cohen, J. B. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5230-5234). Similar experiments on intact rat muscle indicate that, like the 43K protein of Torpedo postsynaptic membranes, the immunologically related component in mammalian muscle endplates is located on the cytoplasmic side of the postsynaptic membrane.