title: Functional expression of bovine 17 alpha-hydroxylase in COS 1 cells is dependent upon the presence of an amino-terminal signal anchor sequence.
abstract: Microsomal forms of eukaryotic cytochrome P450 proteins are integral membrane proteins of the endoplasmic reticulum (ER) membrane which are targeted to the ER via the signal recognition particle pathway. A hydrophobic amino terminus serves as a combined signal sequence and major membrane anchor (signal-anchor sequence) for the microsomal P450s. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the ER of COS 1 cells in order to evaluate the role of membrane insertion of the amino-terminal signal-anchor of microsomal P450s as a functional determinant for these enzymes. Previously, we have shown that deletion of the hydrophobic amino terminus from P45017 alpha reduced membrane targeting and insertion by 5-fold compared with the wild-type protein, abolished enzymatic activity, and resulted in an aberrant CO difference spectrum. In the present study we have replaced the amino terminus of P45017 alpha with two heterologous signal-anchor sequences, one that is similar and one that is very different from the P45017 alpha sequence. The chimeric proteins were expressed in COS 1 cells. Immunoblot analysis of isolated microsomal membranes show that the heterologous signal-anchor sequences functioned to target the P45017 alpha protein to the ER. Enzymatic assays in intact COS 1 cells indicate that both the chimeric proteins are efficient 17 alpha-hydroxylase enzymes. The amino terminus of P45017 alpha was also replaced with a sequence that is not a signal-anchor, and the expressed protein was neither targeted to the ER nor was functional in COS 1 cells. In conclusion, both the structure and catalytic activity of P45017 alpha in COS 1 cells is dependent upon an amino-terminal sequence that functions as a signal-anchor sequence and not upon the precise sequence of the amino terminus.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: B J
authors.initials: BJ
authors.lastname: Clark
authors.forename: M R
authors.initials: MR
authors.lastname: Waterman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013254:Steroid 17-alpha-Hydroxylase / Q000096:biosynthesis* / Q000235:genetics*; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013254:Steroid 17-alpha-Hydroxylase