Selective fast degradation of cytochrome P-450 2E1 in serum-deprived hepatoma cells by a mechanism sensitive to inhibitors of vesicular transport.
Cytochrome P-450 2E1 (CYP2E1) is characterized by a rapid turnover in the liver and some cell lines and the ability of substrates and heme iron ligands to inhibit significantly enzyme degradation. In the Fao hepatoma cell line, CYP2E1 was found to be fairly stable (half-life of 26 h), but serum withdrawal resulted in its rapid disappearance from the microsomal fraction (half-life of about 7 h) as evaluated using cycloheximide chase. The effect of serum withdrawal could be partially reversed by the addition of albumin to the culture medium, whereas insulin and the insulin-like growth factor IGF-I had no additional effect. The effect of serum withdrawal was specific for CYP2E1 since (a) no concomitant fast degradation of CYP2B1 and NADPH-cytochrome P-450 reductase was observed and (b) the CYP2E1 ligands ethanol and imidazole prevented the fast degradation of the enzyme. The lysosomotropic agent ammonium chloride and the inhibitor of autophagocytosis 3-methyladenine slowed down CYP2E1 degradation by about 30%, while leupeptin had no effect. Under the same conditions, the degradation of total long-lived cell protein showed the same sensitivity to ammonium chloride, but was significantly less sensitive to 3-methyladenine and serum and not sensitive to ethanol and imidazole. CYP2E1 degradation was inhibited by combined treatment with brefeldin A and nocodazole, which blocks both anterograde and retrograde vesicular transport between endoplasmic reticulum and the Golgi apparatus. The data point to the existence of a selective mechanism for the degradation of membrane proteins in serum-deprived cells in addition to nonselective autophagocytosis. The selective degradation of CYP2E1 may be attained by means of its selective vesicular transport to an acidic post-endoplasmic reticulum compartment.
Zhukov
A
A
Institute of Environmental Medicine, Division of Molecular Toxicology, Karolinska Institutet, Stockholm, Sweden.
Ingelman-Sundberg
M
M
D000818:Animals; D001692:Biological Transport; D001790:Blood Physiological Phenomena; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D003513:Cycloheximide / Q000494:pharmacology; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D003513:Cycloheximide; D019392:Cytochrome P-450 CYP2E1