Skip to main content
NCBI 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
. 1984 Jan;81(1):90–94. doi: 10.1073/pnas.81.1.90

Effects of temperature on the degradation of proteins in rabbit reticulocyte lysates and after injection into HeLa cells.

R Hough, M Rechsteiner
PMCID: PMC344616  PMID: 6364139

Abstract

Bovine serum albumin, pyruvate kinase, hemoglobin, and the Fc fragment of IgG were labeled and introduced into HeLa cells by erythrocyte-mediated microinjection. Degradation of the injected proteins was then measured in cells cultured at temperatures between 6 degrees C and 37 degrees C. Arrhenius plots revealed a constant Ea of 27 +/- 5 kcal/mol over this temperature interval. Similarly, the apparent Ea for the degradation of long-term endogenously labeled HeLa proteins was 22-26 kcal/mol. Both local protein unfolding and proteolysis by defined enzymes, such as trypsin or papain, proceed with EaS between 5 and 15 kcal/mol. The 2-fold higher values obtained in this study indicate that protein unfolding or simple proteolysis is not rate limiting in the degradation of injected or long-lived endogenous HeLa proteins. Moreover, the relatively uniform EaS suggest that a similar biochemical event is rate limiting in the degradation of a specific protein independent of its half-life. This event may involve a reaction in the ATP-dependent proteolytic pathway from rabbit reticulocyte lysates because we observed that EaS for ATP-dependent proteolysis in this system were also 27 +/- 5 kcal/mol.

Full text

PDF
90

Selected References

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

  1. Amenta J. S., Sargus M. J., Venkatesan S., Shinozuka H. Role of the vacuolar apparatus in augmented protein degradation in cultured fibroblasts. J Cell Physiol. 1978 Jan;94(1):77–86. doi: 10.1002/jcp.1040940110. [DOI] [PubMed] [Google Scholar]
  2. Auld D. S., Vallee B. L. Kinetics of carboxypeptidase A, pH and Temperature dependence of tripeptide hydrolysis. Biochemistry. 1971 Jul 20;10(15):2892–2897. doi: 10.1021/bi00791a015. [DOI] [PubMed] [Google Scholar]
  3. Bates P. J., Coetzee G. A., Van der Westhuyzen D. R. The degradation of endogenous and exogenous proteins in cultured smooth muscle cells. Biochim Biophys Acta. 1982 Nov 24;719(2):377–387. doi: 10.1016/0304-4165(82)90113-1. [DOI] [PubMed] [Google Scholar]
  4. Bigelow S., Hough R., Rechsteiner M. The selective degradation of injected proteins occurs principally in the cytosol rather than in lysosomes. Cell. 1981 Jul;25(1):83–93. doi: 10.1016/0092-8674(81)90233-6. [DOI] [PubMed] [Google Scholar]
  5. Brahm J. Temperature-dependent changes of chloride transport kinetics in human red cells. J Gen Physiol. 1977 Sep;70(3):283–306. doi: 10.1085/jgp.70.3.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bukhari A. I., Zipser D. Mutants of Escherichia coli with a defect in the degradation of nonsense fragments. Nat New Biol. 1973 Jun 20;243(129):238–241. doi: 10.1038/newbio243238a0. [DOI] [PubMed] [Google Scholar]
  7. Charette M. F., Henderson G. W., Markovitz A. ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4728–4732. doi: 10.1073/pnas.78.8.4728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chin D. T., Kuehl L., Rechsteiner M. Conjugation of ubiquitin to denatured hemoglobin is proportional to the rate of hemoglobin degradation in HeLa cells. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5857–5861. doi: 10.1073/pnas.79.19.5857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chung C. H., Goldberg A. L. The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4931–4935. doi: 10.1073/pnas.78.8.4931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ciehanover A., Hod Y., Hershko A. A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem Biophys Res Commun. 1978 Apr 28;81(4):1100–1105. doi: 10.1016/0006-291x(78)91249-4. [DOI] [PubMed] [Google Scholar]
  11. Craig N., Fahrman C. Regulation of protein synthesis by temperature in mammalian cells. Non-involvement of the plasma membrane. Biochim Biophys Acta. 1977 Feb 3;474(3):478–490. doi: 10.1016/0005-2787(77)90276-3. [DOI] [PubMed] [Google Scholar]
  12. Cupo P., El-Deiry W., Whitney P. L., Awad W. M., Jr Stabilization of proteins by guanidination. J Biol Chem. 1980 Nov 25;255(22):10828–10833. [PubMed] [Google Scholar]
  13. Dunn W. A., Hubbard A. L., Aronson N. N., Jr Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver. J Biol Chem. 1980 Jun 25;255(12):5971–5978. [PubMed] [Google Scholar]
  14. Funk J., Wunderlich F., Kreutz W. Thermotropic 'two-stage' liquid crystalline equilibrium crystalline lipid phase separation in microsomal membranes. Biochim Biophys Acta. 1982 Sep 9;690(2):306–309. doi: 10.1016/0005-2736(82)90336-4. [DOI] [PubMed] [Google Scholar]
  15. Gottesman S., Zipser D. Deg phenotype of Escherichia coli lon mutants. J Bacteriol. 1978 Feb;133(2):844–851. doi: 10.1128/jb.133.2.844-851.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haas A. L., Rose I. A. Hemin inhibits ATP-dependent ubiquitin-dependent proteolysis: role of hemin in regulating ubiquitin conjugate degradation. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6845–6848. doi: 10.1073/pnas.78.11.6845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Kielian M. C., Cohn Z. A. Phagosome-lysosome fusion. Characterization of intracellular membrane fusion in mouse macrophages. J Cell Biol. 1980 Jun;85(3):754–765. doi: 10.1083/jcb.85.3.754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Levy H. M., Sharon N., Koshland D. E. PURIFIED MUSCLE PROTEINS AND THE WALKING RATE OF ANTS. Proc Natl Acad Sci U S A. 1959 Jun;45(6):785–791. doi: 10.1073/pnas.45.6.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lin S., Zabin I. Beta-galactosidase. Rates of synthesis and degradation of incomplete chains. J Biol Chem. 1972 Apr 10;247(7):2205–2211. [PubMed] [Google Scholar]
  22. McGarry T., Hough R., Rogers S., Rechsteiner M. Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells. J Cell Biol. 1983 Feb;96(2):338–346. doi: 10.1083/jcb.96.2.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Okada C. Y., Rechsteiner M. Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell. 1982 May;29(1):33–41. doi: 10.1016/0092-8674(82)90087-3. [DOI] [PubMed] [Google Scholar]
  25. Pace C. N. The stability of globular proteins. CRC Crit Rev Biochem. 1975 May;3(1):1–43. doi: 10.3109/10409237509102551. [DOI] [PubMed] [Google Scholar]
  26. Poole B., Wibo M. Protein degradation in cultured cells. The effect of fresh medium, fluoride, and iodoacetate on the digestion of cellular protein of rat fibroblasts. J Biol Chem. 1973 Sep 10;248(17):6221–6226. [PubMed] [Google Scholar]
  27. Rote K. V., Rechsteiner M. Degradation of microinjected proteins: effects of lysosomotropic agents and inhibitors of autophagy. J Cell Physiol. 1983 Jul;116(1):103–110. doi: 10.1002/jcp.1041160116. [DOI] [PubMed] [Google Scholar]
  28. SIMPSON M. V. The release of labeled amino acids from the proteins of rat liver slices. J Biol Chem. 1953 Mar;201(1):143–154. [PubMed] [Google Scholar]
  29. Steinman R. M., Silver J. M., Cohn Z. A. Pinocytosis in fibroblasts. Quantitative studies in vitro. J Cell Biol. 1974 Dec;63(3):949–969. doi: 10.1083/jcb.63.3.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Waxman L., Goldberg A. L. Protease La from Escherichia coli hydrolyzes ATP and proteins in a linked fashion. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4883–4887. doi: 10.1073/pnas.79.16.4883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yang C. S., Strickhart F. S., Kicha L. P. The effect of temperature on monoxygenase reactions in the microsomal membrane. Biochim Biophys Acta. 1977 Mar 1;465(2):362–370. doi: 10.1016/0005-2736(77)90085-2. [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