Cell-free analysis of targeting of cytochrome P450 to microsomal membranes.
Analysis of P450 biosynthesis in cell-free systems has been useful in studying the mechanism of the targeting of P450 to the membrane. These techniques have allowed the demonstration that the N-terminal region of P450 is an uncleaved signal sequence for SRP-dependent membrane insertion. Lack of protection from protease indicated that most of the P450 is on the cytoplasmic side of the membrane. Substitution of basic amino acids at the P450 N terminus or replacing the N terminus of P450 with a secretory signal sequence results in partial or complete translocation across the membrane. This result indicates that at most one and probably none of the internal hydrophobic regions function as stop-transfer signals, which would be present if the protein spanned the membrane more than once.
Szczesna-Skorupa
E
E
Kemper
B
B
D000818:Animals; D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007425:Intracellular Membranes / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000378:metabolism*; D015183:Restriction Mapping; D014158:Transcription, Genetic
D021382:Protein Sorting Signals; D003577:Cytochrome P-450 Enzyme System