Journal of Muscle Research & Cell Motility

, Volume 12, Issue 2, pp 161–170 | Cite as

Cell-free incorporation of newly synthesized myosin subunits into thick myofilaments

  • Steven M. Goldfine
  • Steven Einheber
  • Donald A. Fischman


Although a substantial literature exists on thein vitro polymerization of purified myosin, little is known about native thick filament assembly, remodeling or turnover. We have recently described a cell-free system (Boucheet al., 1988) to examine the interactions between thick filaments and soluble, newly synthesized myofibrillar proteins. In the present manuscript we describe our studies on myosin heavy (MHC) and light chain (LC) incorporation into myofibrils or native and synthetic thick filaments.35S-labeled myofibrillar proteins or myosin subunits were synthesized in a reticulocyte lysate translation system after which myofibrils or myofilaments were added and incubated with these proteins in the lysate. The added filaments were then sedimented and analyzed by SDS-PAGE and fluorography to establish which of the labeled protein subunits were co-pelleted. Operationally, this co-sedimentation of labeled proteins with myofilaments has been termed ‘protein incorporation’. We observed that newly synthesized MHC, LCs 1, 2 and 3 all incorporated into the thick filaments. However, the quantity and specificity of LC incorporation depended upon the structure or composition of the filaments. LCs 1 and 3 were preferentially incorporated into myofibrils and native thick filaments, whereas LC2 was selectively taken up by synthetic filaments prepared from purified myosin. These results suggest that soluble MHCs and LCs interact independently with myofilaments. This hypothesis is supported by the observation that selective removal of soluble MHCs, or of a single LC, did not alter the incorporation of the remaining myosin subunits. Similarly, MHCs synthesized in the absence of LCs also incorporated into myofilaments or myofibrils. We propose that myosin subunits are capable of independent incorporation into and exchange from myofilaments.


Light Chain Protein Subunit Label Protein Myofibrillar Protein Translation System 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Auffray, C. &Rougen, R. (1980) Purification of mouse immunoglobin heavy chain messenger RNAs from total myeloma tumor RNA.Eur. J. Biochem. 107, 303–14.PubMedGoogle Scholar
  2. Bader, D., Masaki, T. &Fischman, D. (1982) Immunocytochemical analysis of myosin heavy chain during avian myogenesisin vivo andin vitro.J. Cell Biol. 95, 763–70.PubMedGoogle Scholar
  3. Bahler, M., Eppenberger, H. &Walliman, T. (1985) Novel thick filament protein of chicken pectoralis muscle: the 86 kD protein. II. Distribution and localization.J. Mol. Biol. 186, 393–401.PubMedGoogle Scholar
  4. Benian, G., Kiff, J., Neckelmann, N., Moerman, D. &Waterston, R. (1989) The sequence of twitchin: An unusually large protein implicated in regulation of myosin activity inCaenorhabditis elegans.Nature 342, 45–50.PubMedGoogle Scholar
  5. Bennett, P., Craig, R., Starr, R. &Offer, G. (1986) The ultrastructural location of C-protein, X-protein, and Hprotein in rabbit muscle.J. Muscle Res. Cell Motil. 7, 550–67.Google Scholar
  6. Bouche, M., Goldfine, S. &Fischman, D. (1988) Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system.J. Cell Biol. 107, 587–96.PubMedGoogle Scholar
  7. Burke, M. &Sivaramakrishnan, M. (1981) Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Formation and properties of thermal hybrids.Biochem. 20, 5908–13.Google Scholar
  8. Cheng, M., Hartl, F.-U., Martin, J., Pollock, R., Kalousek, F., Neupert, W., Hallberg, E., Hallberg, R. &Horwich, A. (1989) Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria.Nature 337, 620–5.PubMedGoogle Scholar
  9. Cooper, J. &Trinick, J. (1984) Binding and location of AMP-deaminase in rabbit psoas muscle myofibrils.J. Mol. Biol. 177, 137–52.PubMedGoogle Scholar
  10. Craig, R. &Offer, G. (1976) The location of C-protein in rabbit skeletal muscle.Proc. R. Soc. Lond. B192, 451–61.Google Scholar
  11. Crisona, N. &Strohman, R. (1983) Inhibition of contraction of cultured muscle fibers results in increased turnover of myofibrillar proteins but not intermediate-filament proteins.J. Cell. Biol. 105, 371–80.Google Scholar
  12. Davis, J. (1981) Pressure jump studies on the length regulation kinetics of the self-assembly of myosin from vertebrate skeletal muscle into thick filaments.Biochem. J. 197, 309–414.PubMedGoogle Scholar
  13. Davis, J. (1988a) Interaction of C-protein with pH 8.0 synthetic thick filaments prepared from the myosin of vertebrate skeletal muscle.J. Muscle Res. Cell. Motil. 9, 174–83.PubMedGoogle Scholar
  14. Davis, J. (1988b) Assembly process in vertebrate skeletal thick filament formation.Ann. Rev. Biophys. Biophys. Chem. 17, 217–39.Google Scholar
  15. Dennis, J., Shimizu, T., Reinach, F. &Fischman, D. (1984) Localization of C-protein isoforms in chicken skeletal muscle: Ultrastructural detection using monoclonal antibodies.J. Cell Biol. 98, 1514–22.PubMedGoogle Scholar
  16. Dhoot, G., Hales, M., Grail, B. &Perry, S. (1985) The isoforms of C-protein and their distribution in mammalian skeletal muscle.J. Muscle Res. Cell Motil. 6, 487–505.PubMedGoogle Scholar
  17. Einheber, S., Raman, G., Nickowitz, R. &Fischman, D. (1988) Isolation and characterization of a chicken ‘perinatal’specific myosin heavy chain cDNA.J. Cell Biochem. (suppl.)12, 366.Google Scholar
  18. Einheber, S. &Fischman, D. (1990) Isolation and characterization of a cDNA clone encoding avian skeletal muscle Cprotein: An intracellular member of the immunoglobulin superfamily.Proc. Natl. Acad. Sci. (USA) 87, 2157–61Google Scholar
  19. Gagnon, J., Kurowski, T. &Zak, R. (1989) Synthesis and assembly of native myosin on muscle polyribosomes.FEBS Letters 250, 549–55.PubMedGoogle Scholar
  20. Garcia, A., Coudrier, E., Carboni, J., Anderson, J., Vandekerkhove, J., Mooseker, M., Lounard, D. &Arpin, M. (1989) Partial deduced sequence of the 110-kdcalmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family.J. Cell Biol. 109, 2895–903.PubMedGoogle Scholar
  21. Goldfine, S. &Fischman, D. (1987) Myosin light chain exchange differs in native and synthetic thick filaments.Biophys. J. 51, 319a.Google Scholar
  22. Goldfine, S., Einheber, S. &Fischman, D. (1987) Independent association of myosin heavy and light chains with thick filaments.J. Cell Biol. 105, 117a.Google Scholar
  23. Goldfine, S., Peng, I., Bouche, M. &Fischman, D. (1989) Incorporation of newly synthesized protein by myofibrils and myofilaments in a cell-free system.U.C.L.A. Symp. Mol. Biol. 93N.S., 271–84.Google Scholar
  24. Harrington, W. &Rodgers, M. (1984) Myosin.Ann. Rev. Biochem. 53, 35–71.PubMedGoogle Scholar
  25. Holt, J. &Lowey, S. (1975) An immunological approach to the role of the low molecular weight subunits in myosin. I. Physical-chemical and immunological characterization of the light chains.Biochem. 14, 4600–9.Google Scholar
  26. Horvath, G. &Gaetjens, E. (1972) Immunohistochemical studies on the light chains from skeletal muscle myosin.Biochim. Biophys. Acta 263, 779–93.PubMedGoogle Scholar
  27. Huxley, H. (1963) Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle.J. Molec. Biol. 7, 281–308.Google Scholar
  28. Johnson, C., Mckenna, N. &Wang, Y.-L. (1988) Association of microinjected myosin and its subfragments with myofibrils in living muscle cells.J. Cell Biol. 107, 2213–22.PubMedGoogle Scholar
  29. Josephs, R. &Harrington, W. (1966) Studies on the formation and physical chemical properties of synthetic myosin filaments.Biochem. 7, 2834–47.Google Scholar
  30. Katayama, E., Wakabayashi, T., Reinach, F., Masaki, T. &Fischman, D. (1984) Proximity of reactive lysyl residue to the antigenic site in rabbit skeletal myosin against the monoclonal antibody (MF-18) generated to chicken skeletal myosin.J. Biochem. (Tokyo) 95, 721–27.Google Scholar
  31. Kerwin, B. &Bandman, E. (1988) Association of nascent myosin heavy and light chain subunits in a RNA-dependent reticulocyte lysate system.J. Cell. Biol. 107, 37a.Google Scholar
  32. Kessler, S. (1975) Rapid isolation of antigens from cells with a Staphylococcal protein A-antibody adsorbent: Parameters of the interaction of antibody-antigen complexes with protein A.J. Immunol. 115, 1617–24.PubMedGoogle Scholar
  33. Knight, P. &Trinick, J. (1987) The myosin molecule. InFibrous Protein Structure (edited byJ. M. Squire andP. J. Vibert, pp. 247–82. London: Academic Press.Google Scholar
  34. Koretz, J. (1979) Effects of C-protein on synthetic myosin filament structure.Biophys. J. 27, 433–46.PubMedGoogle Scholar
  35. Labeit, S., Barlow, D., Gautel, M., Gibson, T., Holt, J., Hsieh, C.- L., Francke, U., Leonard, K., Wardale, J., Whiting, A. &Trinick, J. (1990) A regular pattern of two types of 100-residue motif in the sequence of titin. Nature345, 273–6.PubMedGoogle Scholar
  36. Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophageT.Nature 227, 680–85.PubMedGoogle Scholar
  37. Maniatis, T., Fritsch, E. &Sambrook, J. (1982)Molecular Cloning. A Laboratory Manual, pp. 196–8. Cold Spring Harbor, NY: Cold Spring Harbor Laboratories.Google Scholar
  38. Margossian, S. &Lowey, S. (1982) Preparation of myosin and its subfragments from rabbit skeletal muscle.Meth. Enzymol. 85, 55–71.PubMedGoogle Scholar
  39. Masaki, T. &Takaiti, O. (1974) M-protein.J. Biochem. (Tokyo)75, 367–80.Google Scholar
  40. Mikawa, T., Takeda, S., Shimizu, T. &Kitaura, T. (1981) Gene expression of myofibrillar proteins in single muscle fibers of adult chicken: Micro two-dimensional gel electrophoresis analysis.J. Biochem. (Tokyo) 89, 1951–62.Google Scholar
  41. Miller, J., Paterson, B., Ricciardi, R., Cohen, L. &Robers, B. (1983) Methods in cell-free protein-synthesizing system for the identification of recombinant DNA molecules.Meth. Enzymol. 101, 650–74.PubMedGoogle Scholar
  42. Mittal, B., Sanger, J. &Sanger, J. (1987) Visualization of myosin in living cells.J. Cell Biol. 105, 1753–60.PubMedGoogle Scholar
  43. Moss, P., Micou-Eastwood, J. &Strohman, R. (1986) Altered synthesis of myosin light chains is associated with contractility in cultures of differentiating chick embryo breast muscle.Dev. Biol. 114, 311–14.PubMedGoogle Scholar
  44. Noda, H. &Ebashi, S. (1960) Aggregation of myosin A.Biochim. Biophys. Acta 41, 386–92.PubMedGoogle Scholar
  45. Obinata, T. (1985) Changes in myofibrillar protein isoform expression during chicken skeletal muscle development.Zool. Sci. 2, 833–47.Google Scholar
  46. Offer, G. (1987) Myosin filaments. InFibrous Protein Structure (edited byJ. M. Squire andP. J. Vibert), pp. 307–56. London: Academic Press.Google Scholar
  47. Offer, G., Moos, C. &Starr, R. (1973) A new protein of the thick filaments of vertebrate skeletal myofibrils. Extraction, purification and characterization.J. Mol. Biol. 74, 653–76.PubMedGoogle Scholar
  48. O'Farrell, P. (1975) High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 250, 4007–21.PubMedGoogle Scholar
  49. O'Farrell, P., Goodman, J. &O'Farrell, P. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins.Cell 12, 1133–41.PubMedGoogle Scholar
  50. Pepe, F., Drucker, B. &Chowrashi, P. (1986) The myosin filament: XI. Filament assembly.Prep. Biochem. 16, 99–132.PubMedGoogle Scholar
  51. Price, M. (1987) Skelemins: Cytoskeletal proteins located at the periphery of M-discs in mammalian skeletal muscle.J. Cell Biol. 104, 1325–36.PubMedGoogle Scholar
  52. Reinach, F., Masaki, T. &Fischman, D. (1983) Characterization of the C-protein from posterior latissimus dorsi muscle of the adult chicken: Heterogeneity within a single sarcomere.J. Cell Biol. 96, 297–300.PubMedGoogle Scholar
  53. Saad, A., Pardee, J. &Fischman, D. (1986a) Dynamic exchange of myosin molecules between thick filaments.Proc. Natl. Acad. Sci. USA.,83, 9483–87.PubMedGoogle Scholar
  54. Saad, A., Pardee, J. &Fischman, D. (1986b) Fluorescence energy transfer studies of myosin thick filament assembly.Biophys. J. 49, 140–2.Google Scholar
  55. Samarel, A., Fergusen, A., Vander-Heide, R., Davison, R. &Ganote, C. (1986) Release of unassembled rat cardiac myosin light chain 1 following the calcium paradox.Circ. Res. 58, 166–71.PubMedGoogle Scholar
  56. Schimizu, T., Dennis, J., Masaki, T. &Fischman, D. (1985) Axial arrangement of the myosin rod in vertebrate thick filaments: Immunoelectron microscopy with a monoclonal antibody to light meromyosin.J. Cell Biol. 101, 1115–23.PubMedGoogle Scholar
  57. Silver, G. &Etlinger, J. (1985) Regulation of myofibrillar accumulation in chick muscle cultures: Evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile protein.J. Cell Biol. 101, 2383–91.PubMedGoogle Scholar
  58. Starr, R. &Offer, G. (1983) H-protein and X-protein: Two new components of the thick filaments of vertebrate skeletal muscle.J. Mol. Biol. 170, 675–98.PubMedGoogle Scholar
  59. Tokano-Ohmuro, H., Goldfine, S., Kojima, T., Obinata, T. &Fischman, D. A. (1989) Size and charge heterogeneity of C-protein isoforms in avian skeletal muscle. Expression of six different isoforms in chicken muscle.J. Muscle Res. Cell Motil. 10, 369–78.PubMedGoogle Scholar
  60. Taylor, L. &Bandman, E. (1989) Distribution of fast myosin heavy chain isoforms in thick filaments of developing chicken pectoralis muscle.J. Cell Biol. 108, 533–42.PubMedGoogle Scholar
  61. Trybus, K. &Lowey, S. (1988) Subunit exchange between smooth muscle myosin filaments.J. Cell Biol. 105, 3021–30.Google Scholar
  62. Wagner, P. (1982) Preparation and fractionation of myosin light chains and exchange of the essential light chains.Meth. Enzymol. 85, 72–80.PubMedGoogle Scholar
  63. Wang, K., McClure, J. &Tu, A. (1979) Titin: Major myofibrillar components of striated muscle.Proc. Natl. Acad. Sci. USA76, 3698–702.PubMedGoogle Scholar
  64. Wang, K. (1985) Sarcomere-associated cytoskeletal lattices in striated muscle. InCell and Muscle Motility (edited byJ. Shay), pp. 315–69. New York: Plenum Press.Google Scholar
  65. Waller, G. &Lowey, S. (1985) Myosin subunit interaction. Localization of the alkali light chains.J. Biol. Chem. 260, 14368–73.PubMedGoogle Scholar
  66. Weeds, A. &Lowey, S. (1971) Substructure of the myosin molecule. II. The light chains of myosin.J. Mol. Biol. 61, 701–25.PubMedGoogle Scholar
  67. Wenderoth, M. &Eisenberg, B. (1987) Incorporation of nascent myosin heavy chains into thick filaments of cardiac myocytes in thyroid-treated rabbits.J. Cell Biol. 105, 2771–80.PubMedGoogle Scholar
  68. Zak, R., Martin, A. &Blough, R. (1979) Assessment of protein turnover by use of radioisotopic tracers.Physiol. Rev. 59, 407–47.PubMedGoogle Scholar

Copyright information

© Chapman and Hall Ltd 1991

Authors and Affiliations

  • Steven M. Goldfine
    • 1
  • Steven Einheber
    • 1
  • Donald A. Fischman
    • 1
  1. 1.Department of Cell Biology and AnatomyCornell University Medical CollegeNew YorkUSA

Personalised recommendations