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

• Published in: Experientia Vol. 52; no. 9; p. 851
• Main Authors: Van den Broek, P J, Barroso, M, Lechner, M C
• Format: Journal Article
• Language: English
• Published: Switzerland 15.09.1996
• Subjects: Amino Acid Sequence; Animals; Cell Compartmentation; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Intracellular Membranes - metabolism; Microsomes, Liver - enzymology; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Rats; Solubility; Structure-Activity Relationship
• ISSN: 0014-4754
• DOI: 10.1007/BF01938869

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.