Evidence of Proteasome-mediated Cytochrome P-450 Degradation

doi:10.1074/jbc.272.15.9771

Evidence of Proteasome-mediated Cytochrome P-450 Degradation*

Ben J. Roberts^‡

From the Laboratory of Clinical Studies, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892-1256

^‡ To whom correspondence should be addressed:
Laboratory of Clinical Studies, National Institute on Alcohol Abuse and Alcoholism, 10 Center Dr., MSC-1256, Bethesda, MD 20892-1256.
Tel.: 301-496-4936; Fax: 301-402-0445; E-mail: roberts{at}clinpharm.niaaa.nih.gov

Abstract

The cytochrome P-450 family of enzymes performs an incredibly diverse range of detoxification and oxidation reactions within the cell and constitutes between 5 and 10% of protein in hepatic endoplasmic reticulum. In this report it is demonstrated that constitutively expressed membranous P-450s are targeted for destruction by the proteasome, in a process which is ubiquitin-independent and is demonstrated in vitro to require prior labilization of the enzyme. This process was specific for P-450s CYP1A2, CYP2E1, CYP3A, and CYP4A and was not demonstrated to be involved in the turnover of CYP1A1, CYP2B1/2, or NADPH reductase. In reconstitution experiments using purified proteasomes and microsomal fractions, labilized P-450 conformations are protected from 20 S proteasome degradation by substrate addition, with proteolysis occurring while P-450s are still attached to the endoplasmic reticulum.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

CYP1A1

methylcholanthrene-inducible cytochrome P-450_1A1

CYP1A2

isosafrole-inducible cytochrome P-450_1A2

CYP2B1 and CYP2B2

phenobarbital-inducible P-450_2B1/2

CYP2E1

ethanol-inducible cytochrome P-450_2E1

CYP3A

dexamethasone/troleandomycin-inducible cytochrome P-450_3A

CYP4A

clofibrate-inducible cytochrome P-450_4A

ALLN

N-acetyl-leucine-leucine-norleucinal

PAGE

polyacrylamide gel electrophoresis

SLLVT-AMC

succinyl-Leu-Leu-Val-Tyr 7-amino-4-methylcoumarin

DDEP

3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine.
↵² Data are not shown, but summarized in Table II.
↵³ This is an unsurprising observation given the general requirements of the 26 S proteasome for ATP and ubiquitination.
- Received July 19, 1996.
- Revision received December 9, 1996.