Identification of a Disordered Region‐Targeting Motif in Cytochromes P450

Abstract

Microsomal cytochromes P450 are enzymes involved in metabolism of thousands of substrates. P450s intimately interact with ER lipids. Like other membranous organelles, the ER is organized into ordered regions enriched in cholesterol and sphingomyelin, and disordered microdomains that are more mobile. Different P450s have been shown to differentially localize into these domains. Despite their sequence similarity, CYP1A1 and CYP1A2 localize into disordered and ordered microdomains, respectively. Previously, a CYP1A2 chimera that contained the first 109 amino acids of CYP1A1 showed altered microdomain localization and catalytic activity. The goal of this study was to specifically identify the residues responsible for the microdomain localization of the CYP1As. It was hypothesized that chimeric CYP1A2 containing 3-5 amino acids from the N-terminus of CYP1A1 would govern membrane localization and potentially alter catalytic activity. To test this, CYP1A1-CYP1A2 chimeras were generated by substitution of homologous residues from CYP1A1 into the CYP1A2 molecule. The wild type and chimeric proteins were transfected and expressed in HEK293T/17 cells, and the microdomain localization and stability were examined. Substitution of amino acids 1-30 of CYP1A1 into CYP1A2 caused its relocalization to the disordered region; however, a smaller chimera containing residues 1-28 showed reduced relocalization, suggesting that the residues in this region direct localization. Smaller constructs were then examined, and showing that three amino acids at position 28-30 of CYP1A1 changed the localization of the chimera. The chimera exhibited similar thermolysin resistance as wild-type proteins, suggesting that the protein folded normally. These findings demonstrate that three residues in the CYP1A N-terminus are responsible for the microdomain targeting of these proteins.