Association of insulin-degrading enzyme with a 70 kDa cytosolic protein in hepatoma cells.
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.
Authier
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F
Institut National de la Santé et de la Recherche Médicale U30, Hôpital des Enfants Malades, Paris, France.
Cameron
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PH
Taupin
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D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D002478:Cells, Cultured; D002994:Clofibrate / Q000494:pharmacology; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D007339:Insulysin / Q000096:biosynthesis* / Q000737:chemistry; D008830:Microbodies / Q000187:drug effects; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology
D009363:Neoplasm Proteins; D012333:RNA, Messenger; D014212:Tretinoin; D007339:Insulysin; D002994:Clofibrate