Potential lipid microdomains of the endoplasmic reticulum: how does this affect the P450 system

Abstract

Little is known about the organization of NADPH‐cytochrome P450 reductase (CPR) and multiple P450 enzymes in the endoplasmic reticulum (ER). Although these proteins are known to form a 1:1 molar complex in the ER, in most tissues the P450 concentration exceeds that of CPR by about a 20:1. The goal of this study is to examine how components of the P450 system are organized, and to determine if they are heterogeneously distributed in lipid microdomains of the ER. Liver microsomes from untreated rabbits and those pretreated with P450 inducing agents were partially solubilized with 1% Brij 98 at 4°C, and centrifuged, separating the detergent‐resistant (pellet) and ‐soluble (supernatant) membranes. The supernatants and pellets were then examined by immunoblotting to determine the regional distribution of CPR and P450s in the membrane. Both CPR (∼90%) and CYP1A2 (∼50%) remained in a region of the membrane that is resistant to solubilization, whereas CYP2E1 and CYP2B4 were located in a more detergent‐sensitive region. Lipid analysis of these samples illustrated that the pellet was enriched with saturated and mono‐unsaturated fatty acids of phosphatidylcholine and sphingomyelin while the supernatant was enriched with polyunsaturated fatty acids. These results suggest that the proteins of the ER are not randomly distributed in the membrane, but appear to exist in specific lipid microdomains. (supported by NIEHS ES00434)