Cytochrome P450 1A2 N‐terminal Site Directed Mutagenesis: Effects on Lipid Microdomain Localization

Abstract

The protein components of the cytochrome P450 monooxygenase system consist of cytochromes P450 (CYPs), NADPH‐dependent cytochrome P450 reductase (CPR), and cytochrome b5. They form functional complexes that catalyze the oxidation of exogenous and endogenous molecules, rendering them more water soluble. In eukaryotes, these membrane bound proteins are found in many tissues and are located primarily in the endoplasmic reticulum. The lipid milieu into which the P450 system proteins reside is heterogenous, consisting of ordered and disordered domains. Ordered domains have a higher content of saturated fatty acids, cholesterol, and sphingomyelin than disordered domains, whereas disordered domains have a higher content of phospholipids with more unsaturated fatty acid chains. When a bilayer membrane is treated with non‐ionic detergent such as Brij 98, the disordered domains readily solubilize whereas ordered regions remain intact. Our lab has shown that various P450 system proteins reside in different lipid microdomains. For example, despite their high level of amino acid sequence similarity, CYP1A2 and CYP1A1 reside in ordered and disordered regions, respectively. Furthermore, our lab has shown that the N‐terminal amino acid sequences of CYP1A2 and CYP1A1 contribute to their specific localization in the ER membrane. The goal of this study is to use site directed mutagenesis to identify which N‐terminal amino acids may be responsible in the lipid raft localization of CYP1A2 and how this effects CYP1A2 enzymatic activity. CYP1A2 N‐terminal amino acids were mutated to their CYP1A1 counterparts and expressed in HEK293‐T cells, and their microdomain localization determined. Although substitution of the NH2‐terminal amino acids of CYP1A2 with that of CYP1A1 leads to changes in their microdomain localization, the results show that Serine4Aspartic Acid and Proline5Phenylalanine mutations alone do not alter microdomain localization of the CYP1A proteins.Support or Funding InformationThis work was supported, in whole or in part, by National Institutes of Health, NIEHS, United StatesPublic Health Service Grants ES004344, and ES013648.