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.