title: Critical amino-terminal segments in insertion of rat liver cytochrome P450 3A1 into the endoplasmic reticulum membrane.
abstract: An in vitro transcription-translation assay was used to study the membrane topology of rat liver cytochrome P450 3A1. N-terminus deletion mutants were constructed to assess the importance of N-terminal regions in the stable incorporation of the protein into the microsomal membranes. Wild-type nascent cytochrome P450 bound to microsomes as an integral membrane protein through its hydrophobic N-terminal segments, uncleaved by signal peptidase. Deletion of the most N-terminal hydrophobic segment (positions 7-26) had a dramatic effect on endoplasmic reticulum membrane integration. Confirming the essential role of this stretch in P450 3A1 membrane targeting, proteolysis-resistant membrane-associated peptides were observed in all the in vitro translated mutants containing that segment. It is concluded that the membrane topogenesis of P450 3A1 is determined mainly by the amino-terminal hydrophobic segment.
authors.affiliation: Department of Biochemistry, Gulbenkian Institute of Science, Oeiras, Portugal.
authors.forename: P J
authors.initials: PJ
authors.lastname: Van den Broek
authors.forename: M
authors.initials: M
authors.lastname: Barroso
authors.forename: M C
authors.initials: MC
authors.lastname: Lechner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A