Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 Oct 1;91(1):184–194. doi: 10.1083/jcb.91.1.184

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts

PMCID: PMC2111949  PMID: 7028761

Abstract

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte- mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Ames A., 3rd, Parks J. M., Nesbett F. B. Protein turnover in retina. J Neurochem. 1980 Jul;35(1):131–142. doi: 10.1111/j.1471-4159.1980.tb12498.x. [DOI] [PubMed] [Google Scholar]
  2. Arias I. M., Doyle D., Schimke R. T. Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J Biol Chem. 1969 Jun 25;244(12):3303–3315. [PubMed] [Google Scholar]
  3. Ballard F. J., Knowles S. E., Wong S. S., Bodner J. B., Wood C. M., Gunn J. M. Inhibition of protein breakdown in cultured cells is a consistent response to growth factors. FEBS Lett. 1980 Jun 2;114(2):209–212. doi: 10.1016/0014-5793(80)81116-1. [DOI] [PubMed] [Google Scholar]
  4. Bienkowski R. S., Baum B. J., Crystal R. G. Fibroblasts degrade newly synthesised collagen within the cell before secretion. Nature. 1978 Nov 23;276(5686):413–416. doi: 10.1038/276413a0. [DOI] [PubMed] [Google Scholar]
  5. Bradley M. O., Dice J. F., Hayflick L., Schimke R. T. Protein alterations in aging WI38 cells as determined by proteolytic susceptibility. Exp Cell Res. 1975 Nov;96(1):103–112. doi: 10.1016/s0014-4827(75)80042-5. [DOI] [PubMed] [Google Scholar]
  6. Bradley M. O., Hayflick L., Schimke R. T. Protein degradation in human fibroblasts (WI-38). Effects of aging, viral transformation, and amino acid analogs. J Biol Chem. 1976 Jun 25;251(12):3521–3529. [PubMed] [Google Scholar]
  7. Bradley M. O. Regulation of protein degradation in normal and transformed human cells. Effects of growth state, medium composition, and viral transformation. J Biol Chem. 1977 Aug 10;252(15):5310–5315. [PubMed] [Google Scholar]
  8. Cooke R. J., Davies D. D. General characteristics of normal and stress-enhanced protein degradation in Lemna minor (duckweed). Biochem J. 1980 Nov 15;192(2):499–506. doi: 10.1042/bj1920499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davidson S. J., Hughes W. L., Barnwell A. Renal protein absorption into sub-cellular particles. I. Studies with intact kidneys and fractionated homogenates. Exp Cell Res. 1971 Jul;67(1):171–187. doi: 10.1016/0014-4827(71)90633-1. [DOI] [PubMed] [Google Scholar]
  10. Dehlinger P. J., Schimke R. T. Effect of size on the relative rate of degradation of rat liver soluble proteins. Biochem Biophys Res Commun. 1970 Sep 30;40(6):1473–1480. doi: 10.1016/0006-291x(70)90034-3. [DOI] [PubMed] [Google Scholar]
  11. Dice J. F., Dehlinger P. J., Schimke R. T. Studies on the correlation between size and relative degradation rate of soluble proteins. J Biol Chem. 1973 Jun 25;248(12):4220–4228. [PubMed] [Google Scholar]
  12. Dice J. F., Goldberg A. L. A statistical analysis of the relationship between degradative rates and molecular weights of proteins. Arch Biochem Biophys. 1975 Sep;170(1):213–219. doi: 10.1016/0003-9861(75)90112-5. [DOI] [PubMed] [Google Scholar]
  13. Dice J. F., Goldberg A. L. Relationship between in vivo degradative rates and isoelectric points of proteins. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3893–3897. doi: 10.1073/pnas.72.10.3893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dice J. F., Hess E. J., Goldberg A. L. Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points. Biochem J. 1979 Feb 15;178(2):305–312. doi: 10.1042/bj1780305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dice J. F., Walker C. D., Byrne B., Cardiel A. General characteristics of protein degradation in diabetes and starvation. Proc Natl Acad Sci U S A. 1978 May;75(5):2093–2097. doi: 10.1073/pnas.75.5.2093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Epstein D., Elias-Bishko S., Hershko A. Requirement for protein synthesis in the regulation of protein breakdown in cultured hepatoma cells. Biochemistry. 1975 Nov 18;14(23):5199–5204. doi: 10.1021/bi00694a028. [DOI] [PubMed] [Google Scholar]
  17. Fang V. S., Cho H. W., Meltzer H. Y. Labeling of creatine phosphokinase without loss of enzyme activity. Biochem Biophys Res Commun. 1975 Jul 8;65(1):413–419. doi: 10.1016/s0006-291x(75)80109-4. [DOI] [PubMed] [Google Scholar]
  18. Goldberg A. L., Dice J. F. Intracellular protein degradation in mammalian and bacterial cells. Annu Rev Biochem. 1974;43(0):835–869. doi: 10.1146/annurev.bi.43.070174.004155. [DOI] [PubMed] [Google Scholar]
  19. Goldberg A. L., St John A. C. Intracellular protein degradation in mammalian and bacterial cells: Part 2. Annu Rev Biochem. 1976;45:747–803. doi: 10.1146/annurev.bi.45.070176.003531. [DOI] [PubMed] [Google Scholar]
  20. Hendil K. B. Intracellular degradation of hemoglobin transferred into fibroblasts by fusion with red blood cells. J Cell Physiol. 1980 Dec;105(3):449–460. doi: 10.1002/jcp.1041050309. [DOI] [PubMed] [Google Scholar]
  21. Hendil K. B. Intracellular protein degradation in growing, in density-inhibited, and in serum-restricted fibroblast cultures. J Cell Physiol. 1977 Sep;92(3):353–364. doi: 10.1002/jcp.1040920304. [DOI] [PubMed] [Google Scholar]
  22. Heward C. B., Yang Y. C., Sawyer T. K., Bregman M. D., Fuller B. B., Hruby V. J., Hadley M. E. Iodination associated inactivation of beta-melanocyte stimulating hormone. Biochem Biophys Res Commun. 1979 May 14;88(1):266–273. doi: 10.1016/0006-291x(79)91725-x. [DOI] [PubMed] [Google Scholar]
  23. Hod Y., Hershko A. Relationship of the pool of intracellular valine to protein synthesis and degradation in cultured cells. J Biol Chem. 1976 Jul 25;251(14):4458–4457. [PubMed] [Google Scholar]
  24. Hubbard A. L., Cohn Z. A. The enzymatic iodination of the red cell membrane. J Cell Biol. 1972 Nov;55(2):390–405. doi: 10.1083/jcb.55.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. KALNITSKY G., HUMMEL J. P., DIERKS C. [Some factors which affect the enzymatic digestion of ribonucleic acid]. J Biol Chem. 1959 Jun;234(6):1512–1516. [PubMed] [Google Scholar]
  26. Kaltoft K., Celis J. E. Ghost-mediated transfer of human hypoxanthine-guanine phosphoribosyl transferase into deficient Chinese hamster ovary cells by means of polyethylene glycol-induced fusion. Exp Cell Res. 1978 Sep;115(2):423–428. doi: 10.1016/0014-4827(78)90299-9. [DOI] [PubMed] [Google Scholar]
  27. Knowles S. E., Ballard F. J. Selective control of the degradation of normal and aberrant proteins in Reuber H35 hepatoma cells. Biochem J. 1976 Jun 15;156(3):609–617. doi: 10.1042/bj1560609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  29. Morrison M., Schonbaum G. R. Peroxidase-catalyzed halogenation. Annu Rev Biochem. 1976;45:861–888. doi: 10.1146/annurev.bi.45.070176.004241. [DOI] [PubMed] [Google Scholar]
  30. Naya J., Vigne J. L., De Castro F. T. The dynamic state of Tetrahymena pyriformis cytosol proteins during culture development. FEBS Lett. 1977 Apr 15;76(2):269–273. doi: 10.1016/0014-5793(77)80166-x. [DOI] [PubMed] [Google Scholar]
  31. Neff N. T., DeMartino G. N., Goldberg A. L. The effect of protease inhibitors and decreased temperature on the degradation of different classes of proteins in cultured hepatocytes. J Cell Physiol. 1979 Dec;101(3):439–457. doi: 10.1002/jcp.1041010311. [DOI] [PubMed] [Google Scholar]
  32. Neff N. T., Ross P. A., Bartholomew J. C., Bissell M. J. Leucine in cultured cells: its metabolism and use as a marker for protein turnover. Exp Cell Res. 1977 Apr;106(1):175–183. doi: 10.1016/0014-4827(77)90254-3. [DOI] [PubMed] [Google Scholar]
  33. Nichols W. W., Murphy D. G., Cristofalo V. J., Toji L. H., Greene A. E., Dwight S. A. Characterization of a new human diploid cell strain, IMR-90. Science. 1977 Apr 1;196(4285):60–63. doi: 10.1126/science.841339. [DOI] [PubMed] [Google Scholar]
  34. Olden K., Pratt R. M., Yamada K. M. Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts. Cell. 1978 Mar;13(3):461–473. doi: 10.1016/0092-8674(78)90320-3. [DOI] [PubMed] [Google Scholar]
  35. Opresko L., Wiley H. S., Wallace R. A. Proteins iodinated by the chloramine-T method appear to be degraded at an abnormally rapid rate after endocytosis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1556–1560. doi: 10.1073/pnas.77.3.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pittman R. C., Green S. R., Attie A. D., Steinberg D. Radiolabeled sucrose covalently linked to protein. A device for quantifying degradation of plasma proteins catabolized by lysosomal mechanisms. J Biol Chem. 1979 Aug 10;254(15):6876–6879. [PubMed] [Google Scholar]
  37. Rae P. A., Schimmer B. P. Iodinated derivatives of adrenocorticotropic hormone. Preparation of iodinated alpha 1-24-ACTH with full hormonal activity. J Biol Chem. 1974 Sep 10;249(17):5649–5653. [PubMed] [Google Scholar]
  38. Rechsteiner M. C. Uptake of proteins by red blood cells. Exp Cell Res. 1975 Jul;93(2):487–492. doi: 10.1016/0014-4827(75)90478-4. [DOI] [PubMed] [Google Scholar]
  39. Rechsteiner M., Kuehl L. Microinjection of the nonhistone chromosomal protein HMG1 into bovine fibroblasts and HeLa cells. Cell. 1979 Apr;16(4):901–908. doi: 10.1016/0092-8674(79)90105-3. [DOI] [PubMed] [Google Scholar]
  40. Rice R. H., Means G. E. Radioactive labeling of proteins in vitro. J Biol Chem. 1971 Feb 10;246(3):831–832. [PubMed] [Google Scholar]
  41. SHUGAR D. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochim Biophys Acta. 1952 Mar;8(3):302–309. doi: 10.1016/0006-3002(52)90045-0. [DOI] [PubMed] [Google Scholar]
  42. Schlegel R. A., Rechsteiner M. C. Red cell-mediated microinjection of macromolecules into mammalian cells. Methods Cell Biol. 1978;20:341–354. doi: 10.1016/s0091-679x(08)62026-9. [DOI] [PubMed] [Google Scholar]
  43. Stacey D. W., Allfrey V. G. Evidence for the autophagy of microinjected proteins in HeLA cells. J Cell Biol. 1977 Dec;75(3):807–817. doi: 10.1083/jcb.75.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tack B. F., Dean J., Eilat D., Lorenz P. E., Schechter A. N. Tritium labeling of proteins to high specific radioactivity by reduction methylation. J Biol Chem. 1980 Sep 25;255(18):8842–8847. [PubMed] [Google Scholar]
  45. Taylor J. M., Dehlinger P. J., Dice J. F., Schimke R. T. The synthesis and degradation of membrane proteins. Drug Metab Dispos. 1973 Jan-Feb;1(1):84–91. [PubMed] [Google Scholar]
  46. Tweto J., Doyle D. Turnover of the plasma membrane proteins of hepatoma tissue culture cells. J Biol Chem. 1976 Feb 10;251(3):872–882. [PubMed] [Google Scholar]
  47. Udenfriend S., Stein S., Böhlen P., Dairman W., Leimgruber W., Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972 Nov 24;178(4063):871–872. doi: 10.1126/science.178.4063.871. [DOI] [PubMed] [Google Scholar]
  48. Van Zile J., Henderson L. A., Baynes J. W., Thorpe S. R. [3H]Raffinose, a novel radioactive label for determining organ sites of catabolism of proteins in the circulation. J Biol Chem. 1979 May 10;254(9):3547–3553. [PubMed] [Google Scholar]
  49. Warburton M. J., Poole B. Effect of medium composition on protein degradation and DNA synthesis in rat embryo fibroblasts. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2427–2431. doi: 10.1073/pnas.74.6.2427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wasserman M., Kulka R. G., Loyter A. Degradation and localization of IgG injected into Friend erythroleukemic cells by fusion with erythrocyte ghosts. FEBS Lett. 1977 Nov 1;83(1):48–52. doi: 10.1016/0014-5793(77)80639-x. [DOI] [PubMed] [Google Scholar]
  51. Wilkinson K. D., Urban M. K., Haas A. L. Ubiquitin is the ATP-dependent proteolysis factor I of rabbit reticulocytes. J Biol Chem. 1980 Aug 25;255(16):7529–7532. [PubMed] [Google Scholar]
  52. Williams K. E., Lloyd J. B., Davies M., Beck F. Digestion of an exogenous protein by rat yolk-sac cultured in vitro. Biochem J. 1971 Nov;125(1):303–308. doi: 10.1042/bj1250303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Woodhead J. S., O'Riordan J. L., Keutmann H. T., Stoltz M. L., Dawson B. F., Niall H. D., Robinson C. J., Potts J. T., Jr Isolation and chemical properties of porcine parathyroid hormone. Biochemistry. 1971 Jul 6;10(14):2787–2792. doi: 10.1021/bi00790a021. [DOI] [PubMed] [Google Scholar]
  54. Yamaizumi M., Uchida T., Mekada E., Okada Y. Antibodies introduced into living cells by red cell ghosts are functionally stable in the cytoplasm of the cells. Cell. 1979 Dec;18(4):1009–1014. doi: 10.1016/0092-8674(79)90213-7. [DOI] [PubMed] [Google Scholar]
  55. Yamaizumi M., Uchida T., Okada Y., Furusawa M., Mitsui H. Rapid transfer of non-histone chromosomal proteins to the nucleus of living cells. Nature. 1978 Jun 29;273(5665):782–784. doi: 10.1038/273782a0. [DOI] [PubMed] [Google Scholar]
  56. Zavortink M., Thacher T., Rechsteiner M. Degradation of proteins microinjected into cultured mammalian cells. J Cell Physiol. 1979 Jul;100(1):175–185. doi: 10.1002/jcp.1041000118. [DOI] [PubMed] [Google Scholar]

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

RESOURCES