Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Oct 1;319(Pt 1):149–158. doi: 10.1042/bj3190149

Association of insulin-degrading enzyme with a 70 kDa cytosolic protein in hepatoma cells.

F Authier 1, P H Cameron 1, V Taupin 1
PMCID: PMC1217748  PMID: 8870662

Abstract

We have investigated the biosynthesis, subcellular location and expression of insulin-degrading enzyme (IDE). a type-I peroxisomal protease, in semi-permeabilized hepatoma cells using pulse-chase experiments, non-denaturing immunoprecipitation protocols and Northern-blot analyses. In HcpG2 cell lysates prepared from cells radiolabelled with Tran[35S]-label, immunoprecipitated IDE was observed immediately after a 5 min pulse and subsequently declined during chase with t1/2 of approx. 33 h. In addition to the 110 kDa IDE protein, a protein of 70 kDa (p70) was identified in radiolabelled immunoprecipitates when using a monoclonal anti-IDE antibody 9B12 under non-denaturing conditions. This same antibody did not recognize p70 on Western blots of whole-cell lysates nor in sequential immunoprecipitates of immunocomplex-bead eluates from anti-IDE immunoprecipitations. Likewise, cross-linking studies performed on intact HepG2 and H35 hepatoma cells in vivo revealed the existence of a hetero-oligomeric complex of 180 kDa in which IDE and p70 were physically associated. Digitonin-permeabilization studies in normal and 35S-labelled HepG2 cells have defined a predominant association of IDE and its associated protein p70 with cytosol (supernatant); only a minor amount of the protein IDE was detected in peroxisomes (cellular pellet). Immunoprecipitation of IDE from 35S-labelled cell lysates of normal and stably transfected Chinese hamster ovary cells overexpressing IDE failed to detect p70. Treatment of HepG2 cells with clofibrate, a peroxisome proliferator, resulted in a dose-dependent increase of the two human IDE transcripts of 3.6 and 3.2 kb. This effect was not accompanied by a similar change at the protein level, nor by a change in the subcellular location of the proteins IDE and p70. Based on these findings we propose that in hepatoma cells: (1) IDE mainly exists in a stable cytoplasmic pool that is unchanged in cells undergoing peroxisomal proliferation; and (2) p70 binding to IDE may serve to maintain the dual cytosolic and peroxisomal pools of IDE in a stable equilibrium.

Full Text

The Full Text of this article is available as a PDF (471.3 KB).

Selected References

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

  1. Adames N., Blundell K., Ashby M. N., Boone C. Role of yeast insulin-degrading enzyme homologs in propheromone processing and bud site selection. Science. 1995 Oct 20;270(5235):464–467. doi: 10.1126/science.270.5235.464. [DOI] [PubMed] [Google Scholar]
  2. Affholter J. A., Fried V. A., Roth R. A. Human insulin-degrading enzyme shares structural and functional homologies with E. coli protease III. Science. 1988 Dec 9;242(4884):1415–1418. doi: 10.1126/science.3059494. [DOI] [PubMed] [Google Scholar]
  3. Affholter J. A., Hsieh C. L., Francke U., Roth R. A. Insulin-degrading enzyme: stable expression of the human complementary DNA, characterization of its protein product, and chromosomal mapping of the human and mouse genes. Mol Endocrinol. 1990 Aug;4(8):1125–1135. doi: 10.1210/mend-4-8-1125. [DOI] [PubMed] [Google Scholar]
  4. Alexson S. E., Fujiki Y., Shio H., Lazarow P. B. Partial disassembly of peroxisomes. J Cell Biol. 1985 Jul;101(1):294–304. doi: 10.1083/jcb.101.1.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Authier F., Bergeron J. J., Ou W. J., Rachubinski R. A., Posner B. I., Walton P. A. Degradation of the cleaved leader peptide of thiolase by a peroxisomal proteinase. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3859–3863. doi: 10.1073/pnas.92.9.3859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Authier F., Desbuquois B. Degradation of glucagon in isolated liver endosomes. ATP-dependence and partial characterization of degradation products. Biochem J. 1991 Nov 15;280(Pt 1):211–218. doi: 10.1042/bj2800211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Authier F., Janicot M., Lederer F., Desbuquois B. Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment. Biochem J. 1990 Dec 15;272(3):703–712. doi: 10.1042/bj2720703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Authier F., Mort J. S., Bell A. W., Posner B. I., Bergeron J. J. Proteolysis of glucagon within hepatic endosomes by membrane-associated cathepsins B and D. J Biol Chem. 1995 Jun 30;270(26):15798–15807. doi: 10.1074/jbc.270.26.15798. [DOI] [PubMed] [Google Scholar]
  9. Authier F., Posner B. I., Bergeron J. J. Endosomal proteolysis of internalized proteins. FEBS Lett. 1996 Jun 24;389(1):55–60. doi: 10.1016/0014-5793(96)00368-7. [DOI] [PubMed] [Google Scholar]
  10. Authier F., Posner B. I., Bergeron J. J. Insulin-degrading enzyme. Clin Invest Med. 1996 Jun;19(3):149–160. [PubMed] [Google Scholar]
  11. Authier F., Rachubinski R. A., Posner B. I., Bergeron J. J. Endosomal proteolysis of insulin by an acidic thiol metalloprotease unrelated to insulin degrading enzyme. J Biol Chem. 1994 Jan 28;269(4):3010–3016. [PubMed] [Google Scholar]
  12. Becker A. B., Roth R. A. Insulysin and pitrilysin: insulin-degrading enzymes of mammals and bacteria. Methods Enzymol. 1995;248:693–703. doi: 10.1016/0076-6879(95)48046-3. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Braun H. P., Schmitz U. K. Are the 'core' proteins of the mitochondrial bc1 complex evolutionary relics of a processing protease? Trends Biochem Sci. 1995 May;20(5):171–175. doi: 10.1016/s0968-0004(00)88999-9. [DOI] [PubMed] [Google Scholar]
  15. Doherty J. J., 2nd, Kay D. G., Lai W. H., Posner B. I., Bergeron J. J. Selective degradation of insulin within rat liver endosomes. J Cell Biol. 1990 Jan;110(1):35–42. doi: 10.1083/jcb.110.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hertz R., Bar-Tana J. Induction of peroxisomal beta-oxidation genes by retinoic acid in cultured rat hepatocytes. Biochem J. 1992 Jan 1;281(Pt 1):41–43. doi: 10.1042/bj2810041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Issemann I., Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature. 1990 Oct 18;347(6294):645–650. doi: 10.1038/347645a0. [DOI] [PubMed] [Google Scholar]
  18. Knehr M., Thomas H., Arand M., Gebel T., Zeller H. D., Oesch F. Isolation and characterization of a cDNA encoding rat liver cytosolic epoxide hydrolase and its functional expression in Escherichia coli. J Biol Chem. 1993 Aug 15;268(23):17623–17627. [PubMed] [Google Scholar]
  19. Kuo W. L., Gehm B. D., Rosner M. R., Li W., Keller G. Inducible expression and cellular localization of insulin-degrading enzyme in a stably transfected cell line. J Biol Chem. 1994 Sep 9;269(36):22599–22606. [PubMed] [Google Scholar]
  20. Ossendorp B. C., Wirtz K. W. The non-specific lipid-transfer protein (sterol carrier protein 2) and its relationship to peroxisomes. Biochimie. 1993;75(3-4):191–200. doi: 10.1016/0300-9084(93)90077-6. [DOI] [PubMed] [Google Scholar]
  21. Ou W. J., Cameron P. H., Thomas D. Y., Bergeron J. J. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature. 1993 Aug 26;364(6440):771–776. doi: 10.1038/364771a0. [DOI] [PubMed] [Google Scholar]
  22. Pierotti A. R., Prat A., Chesneau V., Gaudoux F., Leseney A. M., Foulon T., Cohen P. N-arginine dibasic convertase, a metalloendopeptidase as a prototype of a class of processing enzymes. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6078–6082. doi: 10.1073/pnas.91.13.6078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rachubinski R. A., Fujiki Y., Mortensen R. M., Lazarow P. B. Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver. J Cell Biol. 1984 Dec;99(6):2241–2246. doi: 10.1083/jcb.99.6.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rachubinski R. A., Subramani S. How proteins penetrate peroxisomes. Cell. 1995 Nov 17;83(4):525–528. doi: 10.1016/0092-8674(95)90091-8. [DOI] [PubMed] [Google Scholar]
  25. Rawlings N. D., Barrett A. J. Evolutionary families of peptidases. Biochem J. 1993 Feb 15;290(Pt 1):205–218. doi: 10.1042/bj2900205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shii K., Roth R. A. Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4147–4151. doi: 10.1073/pnas.83.12.4147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. VanderVere P. S., Bennett T. M., Oblong J. E., Lamppa G. K. A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7177–7181. doi: 10.1073/pnas.92.16.7177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Walton P. A., Wendland M., Subramani S., Rachubinski R. A., Welch W. J. Involvement of 70-kD heat-shock proteins in peroxisomal import. J Cell Biol. 1994 Jun;125(5):1037–1046. doi: 10.1083/jcb.125.5.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wendland M., Subramani S. Cytosol-dependent peroxisomal protein import in a permeabilized cell system. J Cell Biol. 1993 Feb;120(3):675–685. doi: 10.1083/jcb.120.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zhang B., Marcus S. L., Sajjadi F. G., Alvares K., Reddy J. K., Subramani S., Rachubinski R. A., Capone J. P. Identification of a peroxisome proliferator-responsive element upstream of the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7541–7545. doi: 10.1073/pnas.89.16.7541. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES