Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Apr;86(8):2597–2601. doi: 10.1073/pnas.86.8.2597

Involvement of the proteasome in various degradative processes in mammalian cells.

W Matthews 1, J Driscoll 1, k Tanaka 1, A Ichihara 1, A L Goldberg 1
PMCID: PMC286964  PMID: 2539595

Abstract

Eukaryotic cells contain a 700-kDa proteolytic complex (the "proteasome" or multicatalytic endopeptidase complex), whose role in intracellular protein breakdown is unclear. It has been suggested that the proteasome functions in the rapid degradation of oxidant-damaged proteins and in the ATP-dependent proteolytic pathway. To test these possibilities, oxidant-damaged hemoglobin and albumin were produced by treating hemoglobin and albumin with phenylhydrazine, with hydroxyl radicals, or with both hydroxyl and superoxide radicals. After oxidant damage, these proteins were degraded more rapidly in erythrocyte extracts and also by the purified proteasome. However, complete removal of proteasomes from these extracts by immunoprecipitation (or inhibitors of its proteolytic activity) did not reduce the breakdown of oxidant-damaged hemoglobin and decreased degradation of hydroxyl- and superoxide-treated proteins by only 30-40%. Thus, erythrocytes must contain another proteolytic system for degradation of oxidant-damaged proteins. In contrast, immunoprecipitation of proteasomes with polyclonal or monoclonal antibodies prevented the ATP/ubiquitin-dependent degradation of lysozyme and also blocked the ATP-stimulated degradation of ubiquitin-conjugated lysozyme in reticulocyte and skeletal muscle extracts. These data indicate a critical role of the proteasome in the degradation of ubiquitin-conjugated proteins and suggest that the proteasome is associated with or is a component of the larger ubiquitin-conjugate-degrading enzyme complex.

Full text

PDF
2597

Images in this article

2599Image
on p.2599
2599Image
on p.2599
2600Image
on p.2600
2600Image
on p.2600
2600Image
on p.2600
2600Image
on p.2600

Selected References

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

  1. Arrigo A. P., Darlix J. L., Khandjian E. W., Simon M., Spahr P. F. Characterization of the prosome from Drosophila and its similarity to the cytoplasmic structures formed by the low molecular weight heat-shock proteins. EMBO J. 1985 Feb;4(2):399–406. doi: 10.1002/j.1460-2075.1985.tb03642.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arrigo A. P., Simon M., Darlix J. L., Spahr P. F. A 20S particle ubiquitous from yeast to human. J Mol Evol. 1987;25(2):141–150. doi: 10.1007/BF02101756. [DOI] [PubMed] [Google Scholar]
  3. Arrigo A. P., Tanaka K., Goldberg A. L., Welch W. J. Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome). Nature. 1988 Jan 14;331(6152):192–194. doi: 10.1038/331192a0. [DOI] [PubMed] [Google Scholar]
  4. Boches F. S., Goldberg A. L. Role for the adenosine triphosphate-dependent proteolytic pathway in reticulocyte maturation. Science. 1982 Feb 19;215(4535):978–980. doi: 10.1126/science.7156977. [DOI] [PubMed] [Google Scholar]
  5. Bolton A. E., Hunter W. M. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. doi: 10.1042/bj1330529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  8. Castaño J. G., Ornberg R., Koster J. G., Tobian J. A., Zasloff M. Eukaryotic pre-tRNA 5' processing nuclease: copurification with a complex cylindrical particle. Cell. 1986 Aug 1;46(3):377–385. doi: 10.1016/0092-8674(86)90658-6. [DOI] [PubMed] [Google Scholar]
  9. Chin D. T., Carlson N., Kuehl L., Rechsteiner M. The degradation of guanidinated lysozyme in reticulocyte lysate. J Biol Chem. 1986 Mar 15;261(8):3883–3890. [PubMed] [Google Scholar]
  10. Dahlmann B., Kuehn L., Reinauer H. Identification of three high molecular mass cysteine proteinases from rat skeletal muscle. FEBS Lett. 1983 Aug 22;160(1-2):243–247. doi: 10.1016/0014-5793(83)80975-2. [DOI] [PubMed] [Google Scholar]
  11. Dahlmann B., Kuehn L., Rutschmann M., Reinauer H. Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle. Biochem J. 1985 May 15;228(1):161–170. doi: 10.1042/bj2280161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davies K. J., Goldberg A. L. Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes. J Biol Chem. 1987 Jun 15;262(17):8220–8226. [PubMed] [Google Scholar]
  13. Davies K. J., Goldberg A. L. Proteins damaged by oxygen radicals are rapidly degraded in extracts of red blood cells. J Biol Chem. 1987 Jun 15;262(17):8227–8234. [PubMed] [Google Scholar]
  14. Davies K. J., Lin S. W., Pacifici R. E. Protein damage and degradation by oxygen radicals. IV. Degradation of denatured protein. J Biol Chem. 1987 Jul 15;262(20):9914–9920. [PubMed] [Google Scholar]
  15. Davies K. J. Protein damage and degradation by oxygen radicals. I. general aspects. J Biol Chem. 1987 Jul 15;262(20):9895–9901. [PubMed] [Google Scholar]
  16. DeMartino G. N., Goldberg A. L. Identification and partial purification of an ATP-stimulated alkaline protease in rat liver. J Biol Chem. 1979 May 25;254(10):3712–3715. [PubMed] [Google Scholar]
  17. Dean R. T. A mechanism for accelerated degradation of intracellular proteins after limited damage by free radicals. FEBS Lett. 1987 Aug 17;220(2):278–282. doi: 10.1016/0014-5793(87)80829-3. [DOI] [PubMed] [Google Scholar]
  18. Driscoll J., Goldberg A. L. Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin. Proc Natl Acad Sci U S A. 1989 Feb;86(3):787–791. doi: 10.1073/pnas.86.3.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Edmunds T., Pennington R. J. A high molecular weight peptide hydrolase in erythrocytes. Int J Biochem. 1982;14(8):701–703. doi: 10.1016/0020-711x(82)90005-2. [DOI] [PubMed] [Google Scholar]
  20. Etlinger J. D., Goldberg A. L. A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Proc Natl Acad Sci U S A. 1977 Jan;74(1):54–58. doi: 10.1073/pnas.74.1.54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fagan J. M., Waxman L., Goldberg A. L. Red blood cells contain a pathway for the degradation of oxidant-damaged hemoglobin that does not require ATP or ubiquitin. J Biol Chem. 1986 May 5;261(13):5705–5713. [PubMed] [Google Scholar]
  22. Fagan J. M., Waxman L., Goldberg A. L. Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system. Biochem J. 1987 Apr 15;243(2):335–343. doi: 10.1042/bj2430335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Falkenburg P. E., Haass C., Kloetzel P. M., Niedel B., Kopp F., Kuehn L., Dahlmann B. Drosophila small cytoplasmic 19S ribonucleoprotein is homologous to the rat multicatalytic proteinase. Nature. 1988 Jan 14;331(6152):190–192. doi: 10.1038/331190a0. [DOI] [PubMed] [Google Scholar]
  24. Floyd R. A., Lewis C. A. Hydroxyl free radical formation from hydrogen peroxide by ferrous iron-nucleotide complexes. Biochemistry. 1983 May 24;22(11):2645–2649. doi: 10.1021/bi00280a008. [DOI] [PubMed] [Google Scholar]
  25. Ganoth D., Leshinsky E., Eytan E., Hershko A. A multicomponent system that degrades proteins conjugated to ubiquitin. Resolution of factors and evidence for ATP-dependent complex formation. J Biol Chem. 1988 Sep 5;263(25):12412–12419. [PubMed] [Google Scholar]
  26. Goldberg A. L., Boches F. S. Oxidized proteins in erythrocytes are rapidly degraded by the adenosine triphosphate-dependent proteolytic system. Science. 1982 Feb 26;215(4536):1107–1109. doi: 10.1126/science.7038874. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Hershko A., Ciechanover A. Mechanisms of intracellular protein breakdown. Annu Rev Biochem. 1982;51:335–364. doi: 10.1146/annurev.bi.51.070182.002003. [DOI] [PubMed] [Google Scholar]
  29. Hershko A., Heller H., Elias S., Ciechanover A. Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem. 1983 Jul 10;258(13):8206–8214. [PubMed] [Google Scholar]
  30. Hough R., Pratt G., Rechsteiner M. Purification of two high molecular weight proteases from rabbit reticulocyte lysate. J Biol Chem. 1987 Jun 15;262(17):8303–8313. [PubMed] [Google Scholar]
  31. Hough R., Pratt G., Rechsteiner M. Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates. J Biol Chem. 1986 Feb 15;261(5):2400–2408. [PubMed] [Google Scholar]
  32. Ishiura S., Sano M., Kamakura K., Sugita H. Isolation of two forms of the high-molecular-mass serine protease, ingensin, from porcine skeletal muscle. FEBS Lett. 1985 Sep 9;189(1):119–123. doi: 10.1016/0014-5793(85)80854-1. [DOI] [PubMed] [Google Scholar]
  33. Ismail F., Gevers W. A high-molecular-weight cysteine endopeptidase from rat skeletal muscle. Biochim Biophys Acta. 1983 Jan 26;742(2):399–408. doi: 10.1016/0167-4838(83)90327-8. [DOI] [PubMed] [Google Scholar]
  34. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  35. Martins de Sa C., Grossi de Sa M. F., Akhayat O., Broders F., Scherrer K., Horsch A., Schmid H. P. Prosomes. Ubiquity and inter-species structural variation. J Mol Biol. 1986 Feb 20;187(4):479–493. doi: 10.1016/0022-2836(86)90328-1. [DOI] [PubMed] [Google Scholar]
  36. McGuire M. J., Croall D. E., DeMartino G. N. ATP-stimulated proteolysis in soluble extracts of BHK 21/C13 cells. Evidence for multiple pathways and a role for an enzyme related to the high-molecular-weight protease, macropain. Arch Biochem Biophys. 1988 Apr;262(1):273–285. doi: 10.1016/0003-9861(88)90189-0. [DOI] [PubMed] [Google Scholar]
  37. McGuire M. J., Croall D. E., DeMartino G. N. ATP-stimulated proteolysis in soluble extracts of BHK 21/C13 cells. Evidence for multiple pathways and a role for an enzyme related to the high-molecular-weight protease, macropain. Arch Biochem Biophys. 1988 Apr;262(1):273–285. doi: 10.1016/0003-9861(88)90189-0. [DOI] [PubMed] [Google Scholar]
  38. Müller M., Dubiel W., Rathmann J., Rapoport S. Determination and characteristics of energy-dependent proteolysis in rabbit reticulocytes. Eur J Biochem. 1980 Aug;109(2):405–410. doi: 10.1111/j.1432-1033.1980.tb04808.x. [DOI] [PubMed] [Google Scholar]
  39. Narayan K. S., Rounds D. E. Minute ring-shaped particles in cultured cells of malignant origin. Nat New Biol. 1973 May 30;243(126):146–150. doi: 10.1038/newbio243146a0. [DOI] [PubMed] [Google Scholar]
  40. Ray K., Harris H. Purification of neutral lens endopeptidase: close similarity to a neutral proteinase in pituitary. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7545–7549. doi: 10.1073/pnas.82.22.7545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Rivett A. J., Levine R. L. Enhanced proteolytic susceptibility of oxidized proteins. Biochem Soc Trans. 1987 Oct;15(5):816–818. doi: 10.1042/bst0150816. [DOI] [PubMed] [Google Scholar]
  43. Rivett A. J. Preferential degradation of the oxidatively modified form of glutamine synthetase by intracellular mammalian proteases. J Biol Chem. 1985 Jan 10;260(1):300–305. [PubMed] [Google Scholar]
  44. Rivett A. J. Purification of a liver alkaline protease which degrades oxidatively modified glutamine synthetase. Characterization as a high molecular weight cysteine proteinase. J Biol Chem. 1985 Oct 15;260(23):12600–12606. [PubMed] [Google Scholar]
  45. Rivett A. J. The multicatalytic proteinase of mammalian cells. Arch Biochem Biophys. 1989 Jan;268(1):1–8. doi: 10.1016/0003-9861(89)90558-4. [DOI] [PubMed] [Google Scholar]
  46. Rose I. A., Warms J. V., Hershko A. A high molecular weight protease in liver cytosol. J Biol Chem. 1979 Sep 10;254(17):8135–8138. [PubMed] [Google Scholar]
  47. Schmid H. P., Akhayat O., Martins De Sa C., Puvion F., Koehler K., Scherrer K. The prosome: an ubiquitous morphologically distinct RNP particle associated with repressed mRNPs and containing specific ScRNA and a characteristic set of proteins. EMBO J. 1984 Jan;3(1):29–34. doi: 10.1002/j.1460-2075.1984.tb01757.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Speiser S., Etlinger J. D. ATP stimulates proteolysis in reticulocyte extracts by repressing an endogenous protease inhibitor. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3577–3580. doi: 10.1073/pnas.80.12.3577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tanaka K., Ichihara A. Involvement of proteasomes (multicatalytic proteinase) in ATP-dependent proteolysis in rat reticulocyte extracts. FEBS Lett. 1988 Aug 15;236(1):159–162. doi: 10.1016/0014-5793(88)80306-5. [DOI] [PubMed] [Google Scholar]
  50. Tanaka K., Ii K., Ichihara A., Waxman L., Goldberg A. L. A high molecular weight protease in the cytosol of rat liver. I. Purification, enzymological properties, and tissue distribution. J Biol Chem. 1986 Nov 15;261(32):15197–15203. [PubMed] [Google Scholar]
  51. Tanaka K., Waxman L., Goldberg A. L. ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin. J Cell Biol. 1983 Jun;96(6):1580–1585. doi: 10.1083/jcb.96.6.1580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tanaka K., Yoshimura T., Kumatori A., Ichihara A., Ikai A., Nishigai M., Kameyama K., Takagi T. Proteasomes (multi-protease complexes) as 20 S ring-shaped particles in a variety of eukaryotic cells. J Biol Chem. 1988 Nov 5;263(31):16209–16217. [PubMed] [Google Scholar]
  53. Waxman L., Fagan J. M., Goldberg A. L. Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes, one of which degrades ubiquitin conjugates. J Biol Chem. 1987 Feb 25;262(6):2451–2457. [PubMed] [Google Scholar]
  54. Wilk S., Orlowski M. Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme. J Neurochem. 1980 Nov;35(5):1172–1182. doi: 10.1111/j.1471-4159.1980.tb07873.x. [DOI] [PubMed] [Google Scholar]
  55. Wilk S., Orlowski M. Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex. J Neurochem. 1983 Mar;40(3):842–849. doi: 10.1111/j.1471-4159.1983.tb08056.x. [DOI] [PubMed] [Google Scholar]
  56. Wold F. In vivo chemical modification of proteins (post-translational modification). Annu Rev Biochem. 1981;50:783–814. doi: 10.1146/annurev.bi.50.070181.004031. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES