The Cytoplasmic and N-terminal Transmembrane Domains of Cytochrome P450 Contain Independent Signals for Retention in the Endoplasmic Reticulum

doi:10.1074/jbc.270.41.24327

The Cytoplasmic and N-terminal Transmembrane Domains of Cytochrome P450 Contain Independent Signals for Retention in the Endoplasmic Reticulum (*)

From the (1)Departments of Molecular and Integrative Physiology and
(2) Cell and Structural Biology and College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

^¶ To whom correspondence should be addressed:
Dept. of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
Tel.: 217-333-1146; Fax: 217-333-1133.

↵^§ Present address: Dept. of Immunology, The Scripps Research Institute, La Jolla, California 92037.

Abstract

Microsomal cytochrome P450 is inserted into the membrane of the endoplasmic reticulum (ER) by its N-terminal signal/anchor sequence which also functions as an ER retention signal. To analyze further potential retention signals of cytochrome P450, topological domains of cytochrome P450 2C1 or 2C2, epidermal growth factor receptor, a plasma membrane protein, and bacterial alkaline phosphatase, a secreted protein were exchanged. The N-terminal signal/anchor of cytochrome P450 2C1 functioned as an ER retention signal when placed at the N terminus of several reporter proteins but not when fused at the C terminus of the extracellular domain of epidermal growth factor receptor, with or without a heterologous cytoplasmic domain. Chimeric proteins in which the cytoplasmic domain of cytochrome P450 2C2 was substituted for that of epidermal growth factor receptor were retained in the ER indicating that an independent retention signal is present in the cytoplasmic part of cytochrome P450 2C2. These chimeras were enzymatically active which argues against misfolding as the primary cause of retention. The ER retention signal of the cytoplasmic domain could not be localized to a single amino acid segment by deletion analysis. These results show that cytochrome P450 2C2 contains redundant, complex ER retention signals in its cytoplasmic and N-terminal hydrophobic domains and that the function of the N-terminal signal is context-dependent.

Footnotes

↵* This work was supported by Grant GM 35897 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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

P450

cytochrome P450

EGFR

epidermal growth factor receptor

endo H

endoglycosidase H

PCR

polymerase chain reaction

FITC

fluorescein isothiocyanate

CHO

Chinese hamster ovary.
↵²C. Chen and B. Kemper, unpublished result.
- Received July 5, 1995.