Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1985 Feb;75(2):370–376. doi: 10.1172/JCI111709

Inhibitors of protein synthesis also inhibit lysosomal proteolysis. Studies using cystinotic fibroblasts.

J G Thoene, R Lemons, S Boskovich, K Borysko
PMCID: PMC423496  PMID: 3973010

Abstract

Cystine depleted cystinotic fibroblasts incubated in cystine-free medium accumulate lysosomal-free cystine from the degradation of cystine-containing intracellular and extracellular proteins. In this report we have used this characteristic of these cells to study lysosomal proteolysis. We find that inhibitors of protein synthesis (cycloheximide, emetine, and puromycin) inhibit cystine accumulation from endogenous proteins and therefore act to inhibit lysosomal proteolysis of these proteins. However, cycloheximide does not inhibit cystine accumulation derived from the degradation of the extracellular disulfide-rich proteins, albumin and RNase, but lysosomal cystine accumulation derived from insulin is inhibited by cycloheximide. We conclude that a rapidly turning over protein may be required for the lysosomal degradation of intracellular and some extracellular proteins.

Full text

PDF
370

Images in this article

374Image
on p.374

Selected References

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

  1. Baccino F. M., Tessitore L., Cecchini G., Messina M., Zuretti M. F., Bonelli G., Gabriel L., Amenta J. S. Control of cell protein catabolism in rat liver. Effects of starvation and administration of cycloheximide. Biochem J. 1982 Aug 15;206(2):395–405. doi: 10.1042/bj2060395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ballard F. J. Regulation of protein accumulation in cultured cells. Biochem J. 1982 Nov 15;208(2):275–287. doi: 10.1042/bj2080275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carpentier J. L., Gorden P., Freychet P., Le Cam A., Orci L. Lysosomal association of internalized 125I-insulin in isolated rat hepatocytes. Direct demonstration by quantitative electron microscopic autoradiography. J Clin Invest. 1979 Jun;63(6):1249–1261. doi: 10.1172/JCI109420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ciechanover A., Finley D., Varshavsky A. Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell. 1984 May;37(1):57–66. doi: 10.1016/0092-8674(84)90300-3. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Finley D., Ciechanover A., Varshavsky A. Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell. 1984 May;37(1):43–55. doi: 10.1016/0092-8674(84)90299-x. [DOI] [PubMed] [Google Scholar]
  7. Gahl W. A., Tietze F., Bashan N., Bernardini I., Raiford D., Schulman J. D. Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions. Biochem J. 1983 Nov 15;216(2):393–400. doi: 10.1042/bj2160393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gahl W. A., Tietze F., Bashan N., Steinherz R., Schulman J. D. Defective cystine exodus from isolated lysosome-rich fractions of cystinotic leucocytes. J Biol Chem. 1982 Aug 25;257(16):9570–9575. [PubMed] [Google Scholar]
  9. Gelehrter T., Dilworth V., Valka B., McDonald R., Schorry E. Insulin binding and insulin action in fibroblasts from patients with maturity-onset diabetes of the young. Diabetes. 1981 Nov;30(11):940–946. doi: 10.2337/diab.30.11.940. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Hershko A., Tomkins G. M. Studies on the degradation of tyrosine aminotransferase in hepatoma cells in culture. Influence of the composition of the medium and adenosine triphosphate dependence. J Biol Chem. 1971 Feb 10;246(3):710–714. [PubMed] [Google Scholar]
  13. Jonas A. J., Greene A. A., Smith M. L., Schneider J. A. Cystine accumulation and loss in normal, heterozygous, and cystinotic fibroblasts. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4442–4445. doi: 10.1073/pnas.79.14.4442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kadle R., Kalter V. G., Raizada M. K., Fellows R. E. Cycloheximide causes accumulation of insulin receptors at the cell surface of cultured fibroblasts. J Biol Chem. 1983 Nov 10;258(21):13116–13119. [PubMed] [Google Scholar]
  15. Knutson V. P., Ronnett G. V., Lane M. D. Rapid, reversible internalization of cell surface insulin receptors. Correlation with insulin-induced down-regulation. J Biol Chem. 1983 Oct 25;258(20):12139–12142. [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Marshall S., Olefsky J. M. Separate intracellular pathways for insulin receptor recycling and insulin degradation in isolated rat adipocytes. J Cell Physiol. 1983 Nov;117(2):195–203. doi: 10.1002/jcp.1041170210. [DOI] [PubMed] [Google Scholar]
  19. Oshima R. G., Willis R. C., Furlong C. E., Schneider J. A. Binding assays for amino acids. The utilization of a cystine binding protein from Escherichia coli for the determination of acid-soluble cystine in small physiological samples. J Biol Chem. 1974 Oct 10;249(19):6033–6039. [PubMed] [Google Scholar]
  20. PRESS E. M., PORTER R. R., CEBRA J. The isolation and properties of a proteolytic enzyme, cathepsin D, from bovine spleen. Biochem J. 1960 Mar;74:501–514. doi: 10.1042/bj0740501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rechler M. M., Podskalny J. M. Insulin receptors in cultured human fibroblasts. Diabetes. 1976 Apr;25(4):250–255. doi: 10.2337/diab.25.4.250. [DOI] [PubMed] [Google Scholar]
  22. Thoene J. G., Lemons R. M. Cystine accumulation in cystinotic fibroblasts from free and protein-linked cystine but not cysteine. Biochem J. 1982 Dec 15;208(3):823–830. doi: 10.1042/bj2080823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thoene J. G., Lemons R. Modulation of the intracellular cystine content of cystinotic fibroblasts by extracellular albumin. Pediatr Res. 1980 Jun;14(6):785–787. doi: 10.1203/00006450-198006000-00001. [DOI] [PubMed] [Google Scholar]
  24. Thoene J. G., Oshima R. G., Crawhall J. C., Olson D. L., Schneider J. A. Cystinosis. Intracellular cystine depletion by aminothiols in vitro and in vivo. J Clin Invest. 1976 Jul;58(1):180–189. doi: 10.1172/JCI108448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Thoene J. G., Oshima R. G., Ritchie D. G., Schneider J. A. Cystinotic fibroblasts accumulate cystine from intracellular protein degradation. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4505–4507. doi: 10.1073/pnas.74.10.4505. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES