Determination of membrane‐bound fragments of cytochrome P‐450 2B4

Abstract

Membrane-bound sites of cytochrome P-450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar 'head' were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N-terminal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N-terminus but also to the segments 273-314 and 427-491. Epitope mapping of the domain next to the N-terminus (residues 21-119) of the isolated hemoprotein was performed with the help of a peptide-scanning method, a programmable peptide synthesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P-450 2B4. This domain was shown to possess a considerable density of sites with high antigenic activity. No membrane-penetrating part of this domain was found except for the fragment 1-21. A model of structure of P-450 2B4 was computed by comparison with the structure of cytochrome P-450,, on the basis of an alignment of 47 cytochromes P-450 with the former hemoprotein. Major parts of the protein sequences photoreacting with the phospholipid probes, but not the antibody-reactive epitopes of the region 21 -119, are located at the membrane-facing side in this model Though cytochromes P-450 are the most thoroughly studied membrane enzymes, there is no agreement on their topology in the lipid bilayer. At present there are several models of the membrane orientation of microsomal cytochrome P-450 [l-61 constructed on the basis of various methods of polypeptide chain-structure prediction or some experimental data [7-111. The aim of our investigation was to examine these models by analysis of the interaction of photoreactive phospholipids bearing the photolabel at different distances from their polar 'heads' with cytochrome P-450 2B4 in proteoliposomes and by surface epitope mapping of the 119 N-terminal amino acid residues in the solubilized protein with the help of the peptide-scanning technique (PEPSCAN) [ 121. MATERIALS AND METHODS MaterialsCytochrome P-450 2B4 was obtained from phenobarbital-induced rat liver microsomes [ 131. Proteoliposomes were prepared as described earlier [14]. Photoreactive lipid was added to the solution of egg phosphatidylcholine, a phosphoCorrespondence to V. Y. Uvarov, Institute Biomedical Chemis-

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

“…The 160 -200 and the C-terminal regions contain two segments of high hydrophobicity within P450 so that peripheral interactions with the membrane may be important in the retention. This view is consistent with evidence that regions in the cytoplasmic domain of P450s are associated with the membrane, in addition to the N-terminal transmembrane domain (17,18). A second mechanism for retention of ER proteins may be the formation of networks that are excluded from the transport vesicles (10,12).…”

“…In addition to the N terminus, some other sequences of the protein may also interact with, but not span, the membrane (17,18). The 29 N-terminal amino acids of P450 2C1 and 2C2 can induce ER retention when fused to the N terminus of reporter proteins that normally are cytoplasmic or secreted (19).…”

Critical amino-terminal segments in insertion of rat liver cytochrome P450 3A1 into the endoplasmic reticulum membrane

“…membrane integration being mainly mediated by the first N-terminal hydrophobic segment. A similar conclusion has been drawn for P450 members from different families, such as P450 2B1 [6,26,27] and P450 2B4 [28,29], in spite of the relatively low degree of structural homology with P450 3A1. Thus, most evidence from various P450s now favors a topological model where the protein is inserted into the Ell membrane by a single transmembrane peptide anchor localized at the N-terminus of the protein.…”

Targeting of heterologous membrane proteins into proliferated internal membranes in Saccharomyces cerevisiae

“…The P450 molecules are largely exposed on the cytoplasmic surface of the ER membrane to which they are anchored by a hydrophobic amino-terminal segment (Ahn et al, 1993;Monier et al, 1988;Nelson and Strobel, 1988;Sakaguchi et al, 1987;Szczesna-Skorupa et al, 1988). Based on sequence information and direct biochemical analysis, this membrane association is mediated by one (Edwards et al, 1991;Hsu et al, 1993;Lemos-Chiarandini et al, 1987;Shimozawa et al, 1993;Szczesna-Skorupa and Kemper, 1993;Vergeres et al, 1991;Black et al, 1994;Uvarov et al, 1994) or two (Cullin, 1992;Larson et al, 1991;Nelson and Strobel, 1988;Ruan et al, 1993Ruan et al, , 1994 membrane spanning domains. Localization of P450 enzymes in a phospholipid environment is important for full catalytic activity (French et al, 1980;Ingelman-Sundberg, 1986).…”