Red Cell-Mediated Microinjection Studies on Protein Degradation

  • M. Rechsteiner
Part of the NATO Advanced Study Institutes Series book series (NSSA, volume 31)


It has long been known that proteins are continually synthesized and degraded within animals (1). While there was once resistance to the idea that this represented protein turnover within cells (2), today there is considerable evidence that this is so (for reviews see 3–7). The physiological significance of protein turnover is not as firmly established. The early studies of Schimke on arginase (8) and Schimke et al. on tryptophan-pyrolase (9) clearly demonstrated that control of degradation could influence enzyme levels. Further support for the idea that selective protein degradation plays a regulatory role has been obtained by comparison of turnover rates for various proteins which has revealed that key metabolic enzymes usually turn over faster than structural proteins (6). Thus there is increasing evidence that this seemingly wasteful process plays a role in metabolic regulation.


HeLa Cell Protein Degradation Protein Turnover Plasma Membrane Protein Diphtheria Toxin 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1).
    Schoenheimer, R. and Rittenberg, D. (1940). The study of intermediary metabolism of animals with the aid of isotopes. Physiol. Revs. Acta, 20, 218.Google Scholar
  2. 2).
    Hogness, D.S., Cohn, M. and Monod, J. (1955). Studies on the induced synthesis of β-galactosidase in Escherichia coli: The kinetics and mechanism of sulfur incorporation. Biochim. Biophys. Acta, 16, 99.PubMedCrossRefGoogle Scholar
  3. 3).
    Schimke, R.T. and Doyle, D. (1970). Control of enzyme levels in animal tissues. Ann. Rev. Biochem., 39, 929.PubMedCrossRefGoogle Scholar
  4. 4).
    Goldberg, A.L. and Dice, J.F. (1974). Intracellular protein degradation in mammalian and bacterial cells. Annu. Rev. Biochem., 43, 835.PubMedCrossRefGoogle Scholar
  5. 5).
    Schimke, R.T. (1975). Turnover of membrane proteins in animal cells. Methods in Membrane Biol., 5, 201.CrossRefGoogle Scholar
  6. 6).
    Goldberg, A.L. and St. John, A.C. (1976). Intracellular protein degradation in mammalian and bacterial cells. Annu. Rev. Biochem., 45, 747.PubMedCrossRefGoogle Scholar
  7. 7).
    Ballard, F.J. (1977). Intracellular protein degradation. Essays in Biochem., 13, 1.Google Scholar
  8. 8).
    Schimke, R.T. (1964). The importance of both synthesis and degradation in the control of arginase levels in rat liver. J. Biol. Chem., 239, 3808.PubMedGoogle Scholar
  9. 9).
    Schimke, R.T., Sweeney, E. and Berlin, M. (1965). The roles of synthesis and degradation in the control of rat liver tryptophan pyrrolase. J. Biol. Chem., 240, 322.PubMedGoogle Scholar
  10. 10).
    Glass, R.D. and Doyle, D. (1972). On the measurement of protein turnover in animal cells. J. Biol. Chem., 247, 5234.PubMedGoogle Scholar
  11. 11).
    Dice, J.F., Dehlinger, P.J. and Schimke, R.T. (1973). Studies on the correlation between size and relative rate of degradation of soluble proteins. J. Biol. Chem., 248, 4220.PubMedGoogle Scholar
  12. 12).
    Dice, J.F. and Goldberg, A.L. (1975). A statistical analysis of the relationship between degradation rates and molecular weights of proteins. Arch. Biochem. Biophys., 170, 213.PubMedCrossRefGoogle Scholar
  13. 13).
    Dice, J.F. and Goldberg, A.L. (1975). Relationship between in vivo degradative rates and isoelectric points of proteins. Proc. Nat. Acad. Sci., 72, 3893.PubMedCrossRefGoogle Scholar
  14. 14).
    Dice, J.F. and Goldberg, A.L. (1976). Structural properties of rat serum proteins which correlate with their degradative rates in vivo. Nature, 262, 514.CrossRefGoogle Scholar
  15. 15).
    Momany, F., Aguanno, J. and Larrabee, A.R. (1976). Correlation of degradative rates of proteins with a parameter calculated from amino acid composition and subunit size. Proc. Nat. Acad. Sci, 23, 3093.CrossRefGoogle Scholar
  16. 16).
    Simon, L.D., Tomczak, K. and St. John, A.C. (1978). Bacteriophages inhibit degradation of abnormal proteins in E. coli. Nature, 275, 424.PubMedCrossRefGoogle Scholar
  17. 17).
    Capecchi, M., Capecchi, N.E., Hughes, S. and Wahl, G.M. (1974). Selective degradation of abnormal proteins in mammalian tissue. Proc. Nat. Acad. Sci., 71, 4732.PubMedCrossRefGoogle Scholar
  18. 18).
    Knowles, S.E., Gunn, J.M., Hanson, R.W. and Ballard, F.J. (1975). Increased degradation rates of protein synthesized in hepatoma cells in the presence of amino acid analogues. Biochem. J., 146, 595.PubMedGoogle Scholar
  19. 19).
    Prouty, W.F., Karnovsky, J.M. and Goldberg, A.L. (1975). Degradation of abnormal proteins in Escherichia coli. J. Biol. Chem., 250, 1112.PubMedGoogle Scholar
  20. 20).
    Hendil, K.B. (1975). Degradation of abnormal proteins in HeLa cells. J. Cell. Physiol., 87, 289.CrossRefGoogle Scholar
  21. 21).
    Knowles, S.E. and Ballard, F.J. (1976). Selective control of the degradation of normal and aberrant proteins in Reuber H35 hepatoma cells. Biochem. J., 156, 609.PubMedGoogle Scholar
  22. 22).
    Ballard, F.J., Hopgood, M., Reshef, L. and Hanson, R. (1974). Degradation of phosphoenol pyruvate carboxykinase (guanosine triphosphate) in vivo and in vitro. Biochem. J., 144, 531.Google Scholar
  23. 23).
    Hopgood, M. and Ballard, F.J. (1974). The relative stability of liver cytosol enzymes incubated in vitro. Biochem. J., 144, 371.PubMedGoogle Scholar
  24. 24).
    Segal, H.L., Winkler, J.R. and Miyagi, M. (1974). Relationship between degradation rates of proteins in vivo and their susceptibility to lysosomal proteases. J. Biol. Chem., 249, 6364.PubMedGoogle Scholar
  25. 25).
    Segal, H.L., Rothstein, D. and Winkler, J. (1976). A correlation between turnover rates and lipophilic affinities of soluble rat liver proteins. Biochem. Biophys. Res. Commun., 73, 79.PubMedCrossRefGoogle Scholar
  26. 26).
    Poole, B. and Wibo, M. (1973). Protein degradation in cultured cells. J. Biol. Chem., 248, 6221.PubMedGoogle Scholar
  27. 27).
    Warburton, M.J. and Poole, B. (1977). Effect of medium composition on protein degradation and DNA synthesis in rat embryo fibroblasts. Proc. Nat. Acad. Sci., 74, 2427.PubMedCrossRefGoogle Scholar
  28. 28).
    Lee, G. T.-Y. and Engelhardt, D.L. (1977). Protein metabolism during growth of Vero cells. J. Cellul. Physiol., 92, 293.PubMedCrossRefGoogle Scholar
  29. 29).
    Hendil, K.B. (1977). Intracellular protein degradation in growing, in density inhibited, and in serum-restricted fibroblast cultures. J. Cellul. Physiol., 92, 353.PubMedCrossRefGoogle Scholar
  30. 30).
    Tanaka, K. and Ichihara, A. (1977). Effect of the growth state on protein turnover in two lines of cultured BHK cells. J. Cellul. Physiol., 93, 407.PubMedCrossRefGoogle Scholar
  31. 31).
    Bradley, M.O. (1977). Regulation of protein degradation in normal and transformed human cells. J. Biol. Chem., 252, 5310.PubMedGoogle Scholar
  32. 32).
    Bradley, M.O., Dice, J.F., Hayflick, L. and Schimke, R.T. (1975). Protein alterations in aging W138 cells as determined by proteolytic susceptibility. Exp. Cell Res., 96, 103.PubMedCrossRefGoogle Scholar
  33. 33).
    Kaftory, A., Hershko, A. and Fry, M. (1978). Protein turnover in senescent cultured chick embryo fibroblasts. J. Cellul. Physiol., 94, 147.PubMedCrossRefGoogle Scholar
  34. 34).
    Rannels, D.E., Kao, R. and Morgan, H.E. (1975). Effect of insulin on protein turnover in heart muscle. J. Biol. Chem., 250, 1694.PubMedGoogle Scholar
  35. 35).
    Demartino, G.N. and Goldberg, A.L. (1978). Thyroid hormones control lysosomal enzyme activities in liver and skeletal muscle. Proc. Nat. Acad. Sci., 75, 1369.PubMedCrossRefGoogle Scholar
  36. 36).
    Hubbard, A. and Cohn, Z. (1975). Externally disposed plasma membrane protein. J. Cell. Biol., 64, 461.PubMedCrossRefGoogle Scholar
  37. 37).
    Doyle, D., Baumann, H., England, B., Friedman, E., Hau, E. and Tweto, J. (1978). Biogenesis of plasma membrane glycoproteins in hepatoma tissue culture cells. J. Biol. Chem., 253, 965.PubMedGoogle Scholar
  38. 38).
    Baumann, H. and Doyle, D. (1978). Turnover of plasma membrane glycoproteins and glycolipids of hepatoma tissue culture cells. J. Biol. Chem., 253, 4408.PubMedGoogle Scholar
  39. 39).
    Fambrough, D.M. and Devreotes, P.N. (1976). In: Biogenesis and Turnover of Membrane Macromolecules (ed., J.S. Cook) p. 121. Development of chemical excitability in skeletal muscle. Raven Press, New York.Google Scholar
  40. 40).
    Gorden, P., Carpenter, J.L., Cohen, S. and Orci, L. (1978). Epidermal growth factor: morphological demonstration of binding, internalization and lysosomal association in human fibroblasts. Proc. Nat. Acad. Sci., 25, 5025.CrossRefGoogle Scholar
  41. 41).
    Etlinger, J.D. and Goldberg, A.L. (1977). A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Proc. Nat. Acad. Sci., 74, 54.PubMedCrossRefGoogle Scholar
  42. 42).
    Ballard, F.J. and Hopgood, M. (1976). Inactivation of phosphoenolpyruvate carboxykinase (GTP) by liver extracts. Biochem. J., 154, 717.PubMedGoogle Scholar
  43. 43).
    Varandani, P.T. (1973). Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane. Biochim. Biophys. Acta, 304, 642.PubMedCrossRefGoogle Scholar
  44. 44).
    Ansorge, S., Bokley, P., Kirschke, H., Langner, J., Wiedewanders, B. and Hanson, H. (1973). Metabolism of insulin and glycagon. 32, 27.Google Scholar
  45. 45).
    Ashford, T.P. and Porter, K.R. (1962). Cytoplasmic components in hepatic cell lysosomes. J. Cell. Biol., 12, 198.PubMedCrossRefGoogle Scholar
  46. 46).
    Novikoff, A.B. and Shin, W.-Y. (1978). Endoplasmic reticulum and autophagy in rat hepatocytes. Proc. Nat. Acad. Sci., 75, 5039.PubMedCrossRefGoogle Scholar
  47. 47).
    Holtzman, E. (1976). Lysosomes: A survey. Springer Verlag, New York.CrossRefGoogle Scholar
  48. 48).
    Pfeifer, U. (1978). Inhibition by insulin of the formation of autophagic vacuoles in rat liver. J. Cell. Biol., 78, 152.PubMedCrossRefGoogle Scholar
  49. 49).
    Dice, J.F., Walker, C.D., Byrne, B. and Cardill, A. (1978). General characteristics of protein degradation in diabetes and starvation. Proc. Nat. Acad. Sci., 75, 2093.PubMedCrossRefGoogle Scholar
  50. 50).
    Stacey, D.W. and Allfrey, V.G. (1977). Evidence for the autophagy of microinjected proteins in HeLa cells. J. Cell. Biol., 75, 807.PubMedCrossRefGoogle Scholar
  51. 51).
    Banno, Y., Shiotani, T., Towatari, T., Yoskikawa, D., Katsunuma, T., Afting, E.-G. and Katunuma, N. (1975). Studies of new intracellular proteases in various organs of rat. Eur. J. Biochem., 52, 59.PubMedCrossRefGoogle Scholar
  52. 52).
    Katunuma, N., Kominami, E., Banno, Y., Kito, K., Aoki, Y. and Urata, G. (1976). Concept on mechanism and regulation of intra-cellular eznyme degradation in mammalian tissues. Adv. Reviews Enzyme Regul., 14, 325.CrossRefGoogle Scholar
  53. 53).
    Haas, R., Heinrich, P.C., Tesch, R. and Witt, I. (1978). Cleavage specificity of the serine proteinase from the rat liver mitochondria. Biochem., Biophys. Res. Commun., 85, 1039.CrossRefGoogle Scholar
  54. 54).
    Dean, R.T. (1975). Concerning a possible mechanism for selective capture of cytoplasmic proteins by lysoscmes. Biochem. Biophys. Res. Commun., 67, 604.PubMedCrossRefGoogle Scholar
  55. 55).
    Dean, R.T. (1975). Lysosomal enzymes as agents of turnover of soluble cytoplasmic proteins. Eur. J. Biochem., 58, 9.PubMedCrossRefGoogle Scholar
  56. 56).
    Lloyd, J.B. (1978). The role of lysosomes in turnover of cytoplasmic exogenous proteins. J. Biochem. Soc. Trans., 6, 500.Google Scholar
  57. 57).
    Neely, A.N. and Mortimore, G.E. (1974). Localization of products of endogenous proteolysis in lysosomes of perfused rat liver. Biochem. Biophys. Res. Commun., 59, 680.PubMedCrossRefGoogle Scholar
  58. 58).
    Haider, M. and Segal, H.L. (1972). Some characteristics of the alanine aminotransferase and arginase inactivating system of lysosomes. Arch. of Biochemistry and Biophys., 148, 228.CrossRefGoogle Scholar
  59. 59).
    Dean, R.T. (1975). Direct evidence of lysosomes in degradation of intracellular proteins. Nature, 257, 414.PubMedCrossRefGoogle Scholar
  60. 60).
    Rudek, D., Dien, P. and Schneider, D.L. (1978). Identification of tryptophan pyrrolase in liver lysosomes after treatment of rats with hydrocortisone and chloroquine. Biochem. Biophys. Res. Commun., 82, 342.PubMedCrossRefGoogle Scholar
  61. 61).
    Furusawa, M., Nishimura, T., Yamaizumi, M. and Okada, Y. (1974). Injection of foreign substances into single cells by cell fusion. Nature, 249, 449.PubMedCrossRefGoogle Scholar
  62. 62).
    Schlegel, R. and Rechsteiner, M. (1975). Microinjection of thymidine kinase and bovine serum albumin into mammalian cells by fusion with red blood cells. Cell, 5, 371.PubMedCrossRefGoogle Scholar
  63. 63).
    Loyter, A., Zakai, N. and Kulka, R. (1975). “Ultramicroinjection” of macromolecules or small particles into animal cells. J. Cell. Biol., 66, 292.PubMedCrossRefGoogle Scholar
  64. 64).
    Ryser, H. J.-P. (1963). Comparison of the incorporation of tyrosine and its iodinated analogs into the proteins of Ehrlich ascites tumor cells and rat liver slices. Biochem. Biophys. Acta, 78, 759.PubMedCrossRefGoogle Scholar
  65. 65).
    Schlegel, R.A., Iverson, P. and Rechsteiner, M. (1978). The turnover of tRNAs microinjected into animal cells. Nucl. Acid. Res., 5, 3715.CrossRefGoogle Scholar
  66. 66).
    Rechsteiner, M. (1978). Red cell-mediated microinjection. Nat. Cancer Inst. Monogr., 48, 57.PubMedGoogle Scholar
  67. 67).
    Zavortink, M., Thacher, T. and Rechsteiner, M. (1979). Degradation of proteins microinjected into cultured mammalian cells. J. Cellul. Physiol., 100, 175.PubMedCrossRefGoogle Scholar
  68. 68).
    Wasserman, M., Kulka, R.G. and Loyter, A. (1977). Degradation and localization of IgG injected into friend erythroleukemia cells by fusion with erythrocyte ghosts. FEBS Lett., 83, 48.PubMedCrossRefGoogle Scholar
  69. 69).
    Fritz, P.J., Vesell, E.S., White, E.L. and Pruitt, K.M. (1969). The roles of synthesis and degradation in determining tissue concentrations of lactate dehydrogenase 5. Proc. Nat. Acad. Sci., 62, 558.PubMedCrossRefGoogle Scholar
  70. 70).
    Deschatrette, J. and Weiss, M.C. (1975). Extinction of liver-specific functions in hybrids between differentiated and dedifferentiated rat hepatoma cells. Som. Cell. Gen., 1, 279.CrossRefGoogle Scholar
  71. 71).
    Gardner, D.A., Sato, G.H. and Kaplan, N.O. (1972). Pyridine nucleotides in normal and nicotinamide depleted adrenal tumor cell cultures. Dev. Biol., 28, 84.PubMedCrossRefGoogle Scholar
  72. 72).
    Aoyagi, T. and Umezawa, H. (1975). Proteases and biological control (eds, E. Reich, D.B. Rifkin and E. Shaw). Cold Spring Harbor Laboratory Press, p. 429.Google Scholar
  73. 73).
    Schnebli, H.P. (1975). Proteases and biological control (eds, E. Reich, D.B. Rifkin and E. Shaw). Cold Spring Harbor Laboratory Press, p. 785.Google Scholar
  74. 74).
    Overath, P., Schairer, H.U. and Stoffel, W. (1974). Correlation of in vivo and in vitro phase transitions of membrane lipids in Escherichia coli. Proc. Nat. Acad. Sci., 67, 606.CrossRefGoogle Scholar
  75. 75).
    Wilson, G. and Fox, C.F. (1971). Biogenesis of microbial transport systems: evidence for coupled incorporation of newly synthesized lipids and proteins into membrane. J. Mol. Biol., 55, 49.PubMedCrossRefGoogle Scholar
  76. 76).
    Lagunoff, D. and Wan, H. (1974). Temperature dependence of most cell histamine secretion. J. Cell. Biol., 61, 809.PubMedCrossRefGoogle Scholar
  77. 77).
    Petit, V. and Edidin, M. (1974). Lateral phase separation of lipids in plasma membranes: effect of temperature on the mobility of membrane antigens. Science, 184, 1183.PubMedCrossRefGoogle Scholar
  78. 78).
    Plagemann, P.G.W. and Richey, D.P. (1974). Transport of nucleosides, nucleic acid bases, choline and glucose by animal cells in culture. Biochim. Biophys. Acta., 344, 263.PubMedGoogle Scholar
  79. 79).
    Wisnieski, B.J., Parkes, J.G., Huang, Y.O. and Fox, C.F. (1974). Physical and physiological evidence for two phase transitions in cytoplasmic membranes of animal cells. Proc. Nat. Acad. Sci., 71, 4381.PubMedCrossRefGoogle Scholar
  80. 80).
    Steinman, R.M., Silver, J.M. and Cohn, Z.A. (1974). Pinocytosis in fibroblasts. J. Cell. Biol., 63, 949.PubMedCrossRefGoogle Scholar
  81. 81).
    Mahoney, E.M., Hamill, A.L., Scott, W.A. and Cohn, Z.A. (1977). Response of endocytosis to altered fatty acyl composition of macrophage phospholipids. Proc. Nat. Acad. Sci., 74, 4895.PubMedCrossRefGoogle Scholar
  82. 82).
    Sandvig, K. and Olsnes, S. (1979). Effect of temperature on the uptake, excretion and degradation of abrin and ricin by HeLa cells. Exp. Cell. Res., 121, 15.PubMedCrossRefGoogle Scholar
  83. 83).
    Bergmann, M. and Fruton, J.S. (1941). The specificity of proteinases. Adv. in Enzymol., 1, 63.Google Scholar
  84. 84).
    Pittman, R.C. and Steinberg, D. (1978). A new approach for assessing cumulative lysosomal degradation of proteins or other macromolecules. Biochem. Biophys. Res. Commun., 81, 1254.PubMedCrossRefGoogle Scholar
  85. 85).
    Ehrenreich, B.A. and Cohn, Z.A. (1969). The fate of peptides pinocytosed by macrophages in vitro. J. Exp. Med., 129, 227.PubMedCrossRefGoogle Scholar
  86. 86).
    Yamaizumi, M., Uchida, Y., Okada, Y. and Furusawa, M. (1978). Neutralization of diphtheria toxin in living cells by microinjection of antifragment A contained within resealed erythrocyte ghosts. Cell, 13, 227.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1980

Authors and Affiliations

  • M. Rechsteiner
    • 1
  1. 1.Department of BiologyUniversity of UtahSalt Lake CityUSA

Personalised recommendations