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J. Biol. Chem., Vol. 275, Issue 22, 17130-17135, June 2, 2000
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From the Division of Molecular Toxicology, National Institute of
Environmental Medicine, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden
Endoplasmic reticulum-resident cytochrome P450
enzymes that face the cytosol are present on the plasma membrane of
hepatocytes, but the molecular origin for their transport to this
compartment has until now remained unknown. The molecular basis for the
transport of rat ethanol-inducible cytochrome P450 2E1 (CYP2E1) to the
plasma membrane was investigated by transfection of several different mutant cDNAs into mouse H2.35 hepatoma cells. Two
NH2-terminal CYP2E1 mutants were constructed:
N++2E1, which carried two positive charges in the
NH2 terminus, and 2C-2E1, in which the transmembrane domain
of CYP2E1 was replaced with that of CYP2C1, which was previously
described to cause retention of CYP2C1 in the endoplasmic reticulum, as
well as CYP2E1 COOH-terminally tagged with the vesicular stomatitis
virus G protein (VSV-G) epitope (2E1-VSV-G). Immunofluorescent
microscopy and cell surface biotinylation experiments revealed that all
CYP2E1 variants were present on the extracellular side of the plasma
membrane. The VSV-G epitope on CYP2E1 was detected on the outside of
the plasma membrane using VSV-G-specific antibodies, indicating that
the large COOH-terminal part of CYP2E1 is indeed exposed on the outside
of the plasma membrane. The relative levels of CYP2E1, 2C-2E1, and
2E1-VSV-G on the cell surface were found to be about 2% of total
cellular enzyme, whereas twice this amount of N++2E1 was
recovered at the cell surface. Protease protection experiments performed on microsomes isolated from cDNA transfected cells
revealed that a small fraction of CYP2E1 and all variant proteins was
found to be located in the lumen of the endoplasmic reticulum (type II
orientation), whereas the majority of the proteins were in the expected
cytosolic or type I orientation. It is concluded that the
NH2-terminal transmembrane domain of CYP2E1 plays a
critical role in directing the protein to the cell surface and that
topological inversion of a small fraction of CYP2E1 in the endoplasmic
reticulum directs the protein to the plasma membrane.
Molecular Basis for the Transport of Cytochrome P450 2E1 to
the Plasma Membrane*
and
*
This study was supported by grants from the Swedish Medical
Research Council and AstraZeneca.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.
To whom correspondence should be addressed. Tel.: 46-8-7287762;
Fax: 46-8-337327; E-mail etienne.neve@imm.ki.se.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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