Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Apr 1;104(4):939–946. doi: 10.1083/jcb.104.4.939

300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses

PMCID: PMC2114440  PMID: 3558487

Abstract

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post- synaptic membrane.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson M. J., Cohen M. W. Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):757–773. doi: 10.1113/jphysiol.1977.sp011880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BIRKS R., HUXLEY H. E., KATZ B. The fine structure of the neuromuscular junction of the frog. J Physiol. 1960 Jan;150:134–144. doi: 10.1113/jphysiol.1960.sp006378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrantes F. J. Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the Mr 43,000 nonreceptor peptide. J Cell Biol. 1982 Jan;92(1):60–68. doi: 10.1083/jcb.92.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bloch R. J., Hall Z. W. Cytoskeletal components of the vertebrate neuromuscular junction: vinculin, alpha-actinin, and filamin. J Cell Biol. 1983 Jul;97(1):217–223. doi: 10.1083/jcb.97.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Branton D., Cohen C. M., Tyler J. Interaction of cytoskeletal proteins on the human erythrocyte membrane. Cell. 1981 Apr;24(1):24–32. doi: 10.1016/0092-8674(81)90497-9. [DOI] [PubMed] [Google Scholar]
  6. Burden S. J., DePalma R. L., Gottesman G. S. Crosslinking of proteins in acetylcholine receptor-rich membranes: association between the beta-subunit and the 43 kd subsynaptic protein. Cell. 1983 Dec;35(3 Pt 2):687–692. doi: 10.1016/0092-8674(83)90101-0. [DOI] [PubMed] [Google Scholar]
  7. Burden S. J., Sargent P. B., McMahan U. J. Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve. J Cell Biol. 1979 Aug;82(2):412–425. doi: 10.1083/jcb.82.2.412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burden S. J. The subsynaptic 43-kDa protein is concentrated at developing nerve-muscle synapses in vitro. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8270–8273. doi: 10.1073/pnas.82.23.8270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burden S. Identification of an intracellular postsynaptic antigen at the frog neuromuscular junction. J Cell Biol. 1982 Sep;94(3):521–530. doi: 10.1083/jcb.94.3.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. COUTEAUX R. THE DIFFERENTIATION OF SYNAPTIC AREAS. Proc R Soc Lond B Biol Sci. 1963 Nov 19;158:457–480. doi: 10.1098/rspb.1963.0058. [DOI] [PubMed] [Google Scholar]
  11. Couteaux R., Pécot-Dechavassine Particularités structurales du sarcoplasme sous-neural. C R Acad Sci Hebd Seances Acad Sci D. 1968 Jan 3;266(1):8–10. [PubMed] [Google Scholar]
  12. Couteaux R. Structure of the subsynaptic sarcoplasm in the interfolds of the frog neuromuscular junction. J Neurocytol. 1981 Dec;10(6):947–962. doi: 10.1007/BF01258523. [DOI] [PubMed] [Google Scholar]
  13. Davis J., Bennett V. Brain spectrin. Isolation of subunits and formation of hybrids with erythrocyte spectrin subunits. J Biol Chem. 1983 Jun 25;258(12):7757–7766. [PubMed] [Google Scholar]
  14. Dennis M. J. Development of the neuromuscular junction: inductive interactions between cells. Annu Rev Neurosci. 1981;4:43–68. doi: 10.1146/annurev.ne.04.030181.000355. [DOI] [PubMed] [Google Scholar]
  15. Elliott J., Blanchard S. G., Wu W., Miller J., Strader C. D., Hartig P., Moore H. P., Racs J., Raftery M. A. Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins. Biochem J. 1980 Mar 1;185(3):667–677. doi: 10.1042/bj1850667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fertuck H. C., Salpeter M. M. Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. J Cell Biol. 1976 Apr;69(1):144–158. doi: 10.1083/jcb.69.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Froehner S. C. Peripheral proteins of postsynaptic membranes from Torpedo electric organ identified with monoclonal antibodies. J Cell Biol. 1984 Jul;99(1 Pt 1):88–96. doi: 10.1083/jcb.99.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Godfrey E. W., Nitkin R. M., Wallace B. G., Rubin L. L., McMahan U. J. Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells. J Cell Biol. 1984 Aug;99(2):615–627. doi: 10.1083/jcb.99.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  20. Hall Z. W., Lubit B. W., Schwartz J. H. Cytoplasmic actin in postsynaptic structures at the neuromuscular junction. J Cell Biol. 1981 Sep;90(3):789–792. doi: 10.1083/jcb.90.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hamilton S. L., McLaughlin M., Karlin A. Formation of disulfide-linked oligomers of acetylcholine receptor in membrane from torpedo electric tissue. Biochemistry. 1979 Jan 9;18(1):155–163. doi: 10.1021/bi00568a024. [DOI] [PubMed] [Google Scholar]
  22. Hartig P. R., Raftery M. A. Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes. Biochemistry. 1979 Apr 3;18(7):1146–1150. doi: 10.1021/bi00574a004. [DOI] [PubMed] [Google Scholar]
  23. Hartwig J. H., Stossel T. P. Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages. J Biol Chem. 1975 Jul 25;250(14):5696–5705. [PubMed] [Google Scholar]
  24. Heuser J. E., Salpeter S. R. Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell Biol. 1979 Jul;82(1):150–173. doi: 10.1083/jcb.82.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hooper J. E., Carlson S. S., Kelly R. B. Antibodies to synaptic vesicles purified from Narcine electric organ bind a subclass of mammalian nerve terminals. J Cell Biol. 1980 Oct;87(1):104–113. doi: 10.1083/jcb.87.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kordeli E., Cartaud J., Nghiêm H. O., Pradel L. A., Dubreuil C., Paulin D., Changeux J. P. Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes. J Cell Biol. 1986 Mar;102(3):748–761. doi: 10.1083/jcb.102.3.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  28. Lindstrom J., Walter B., Einarson B. Immunochemical similarities between subunits of acetylcholine receptors from Torpedo, Electrophorus, and mammalian muscle. Biochemistry. 1979 Oct 16;18(21):4470–4480. doi: 10.1021/bi00588a004. [DOI] [PubMed] [Google Scholar]
  29. Marchesi V. T. Stabilizing infrastructure of cell membranes. Annu Rev Cell Biol. 1985;1:531–561. doi: 10.1146/annurev.cb.01.110185.002531. [DOI] [PubMed] [Google Scholar]
  30. Neubig R. R., Krodel E. K., Boyd N. D., Cohen J. B. Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides. Proc Natl Acad Sci U S A. 1979 Feb;76(2):690–694. doi: 10.1073/pnas.76.2.690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nitkin R. M., Wallace B. G., Spira M. E., Godfrey E. W., McMahan U. J. Molecular components of the synaptic basal lamina that direct differentiation of regenerating neuromuscular junctions. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):653–665. doi: 10.1101/sqb.1983.048.01.069. [DOI] [PubMed] [Google Scholar]
  32. Pruss R. M., Mirsky R., Raff M. C., Thorpe R., Dowding A. J., Anderton B. H. All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell. 1981 Dec;27(3 Pt 2):419–428. doi: 10.1016/0092-8674(81)90383-4. [DOI] [PubMed] [Google Scholar]
  33. Rosenbluth J. Synaptic membrane structure in Torpedo electric organ. J Neurocytol. 1975 Dec;4(6):697–712. doi: 10.1007/BF01181631. [DOI] [PubMed] [Google Scholar]
  34. Sanes J. R., Carlson S. S., Von Wedel R. J., Kelly R. B. Antiserum specific for motor nerve terminals in skeletal muscle. Nature. 1979 Aug 2;280(5721):403–404. doi: 10.1038/280403a0. [DOI] [PubMed] [Google Scholar]
  35. Sealock R. Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting. J Cell Biol. 1982 Feb;92(2):514–522. doi: 10.1083/jcb.92.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sealock R., Kavookjian A. Postsynaptic distribution of acetylcholine receptors in electroplax of the torpedine ray, Narcine brasiliensis. Brain Res. 1980 May 19;190(1):81–93. doi: 10.1016/0006-8993(80)91161-0. [DOI] [PubMed] [Google Scholar]
  37. Sealock R. Visualization at the mouse neuromuscular junction of a submembrane structure in common with Torpedo postsynaptic membranes. J Neurosci. 1982 Jul;2(7):918–923. doi: 10.1523/JNEUROSCI.02-07-00918.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sealock R., Wray B. E., Froehner S. C. Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies. J Cell Biol. 1984 Jun;98(6):2239–2244. doi: 10.1083/jcb.98.6.2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shulman M., Wilde C. D., Köhler G. A better cell line for making hybridomas secreting specific antibodies. Nature. 1978 Nov 16;276(5685):269–270. doi: 10.1038/276269a0. [DOI] [PubMed] [Google Scholar]
  40. Sobel A., Heidmann T., Hofler J., Changeux J. P. Distinct protein components from Torpedo marmorata membranes carry the acetylcholine receptor site and the binding site for local anesthetics and histrionicotoxin. Proc Natl Acad Sci U S A. 1978 Jan;75(1):510–514. doi: 10.1073/pnas.75.1.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sobel A., Weber M., Changeux J. P. Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ. Eur J Biochem. 1977 Oct 17;80(1):215–224. doi: 10.1111/j.1432-1033.1977.tb11874.x. [DOI] [PubMed] [Google Scholar]
  42. Spiegel J. E., Beardsley D. S., Southwick F. S., Lux S. E. An analogue of the erythroid membrane skeletal protein 4.1 in nonerythroid cells. J Cell Biol. 1984 Sep;99(3):886–893. doi: 10.1083/jcb.99.3.886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. St John P. A., Froehner S. C., Goodenough D. A., Cohen J. B. Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides. J Cell Biol. 1982 Feb;92(2):333–342. doi: 10.1083/jcb.92.2.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tyler J. M., Reinhardt B. N., Branton D. Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin. J Biol Chem. 1980 Jul 25;255(14):7034–7039. [PubMed] [Google Scholar]
  46. Walker J. H., Boustead C. M., Witzemann V. The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein. EMBO J. 1984 Oct;3(10):2287–2290. doi: 10.1002/j.1460-2075.1984.tb02127.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wennogle L. P., Changeux J. P. Transmembrane orientation of proteins present in acetylcholine receptor-rich membranes from Torpedo marmorata studied by selective proteolysis. Eur J Biochem. 1980 May;106(2):381–393. doi: 10.1111/j.1432-1033.1980.tb04584.x. [DOI] [PubMed] [Google Scholar]
  48. Ziskind-Conhaim L., Geffen I., Hall Z. W. Redistribution of acetylcholine receptors on developing rat myotubes. J Neurosci. 1984 Sep;4(9):2346–2349. doi: 10.1523/JNEUROSCI.04-09-02346.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES