Michael Waterman
Faculty Member
Last active: 2/12/2015

The hydrophobic amino-terminal sequence of bovine 17 alpha-hydroxylase is required for the expression of a functional hemoprotein in COS 1 cells.

Clark BJ, Waterman MR
J Biol Chem. 1991 266 (9): 5898-904

PMID: 2005126

The endoplasmic reticulum is a major site of localization for eukaryotic cytochrome P-450 mixed-function oxidase complexes. Previous studies have shown that the microsomal forms of P-450 insert into the membrane via their hydrophobic amino terminus through the signal recognition particle-dependent pathway. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the endoplasmic reticulum of COS 1 cells to evaluate the functional role of its hydrophobic amino-terminal sequence and membrane insertion. An NH2-terminal truncated protein, P450 delta 2-17, which lacked amino acids 2-17 was expressed in COS 1 cells, subcellular fractions were isolated, and P450 delta 2-17 was localized by immunoblot analysis. Compared to the full-length P45017 alpha, the NH2-terminal truncation resulted in a 2.5-fold decrease in P45017 alpha protein recovered with the microsomal fraction, 50% of which was an integral membrane protein as defined by resistance to Na2CO3 extraction. Despite correct membrane localization, P450 delta 2-17 was not a functional enzyme in COS 1 cells. A CO difference spectrum of microsomes containing P450 delta 2-17 did not give a typical 450 nm absorbance. We conclude that the hydrophobic amino terminus is required for the expression of a functionally competent P45017 alpha in COS 1 cells and suggest that the insertion of the amino terminus into the membrane is necessary for the folding of this protein into its correct structural form.

MeSH Terms (13)

Amino Acid Sequence Animals Base Sequence Blotting, Western Cattle Cell Fractionation Cell Line Endoplasmic Reticulum Gene Expression Regulation, Enzymologic Hemeproteins Molecular Sequence Data Steroid 17-alpha-Hydroxylase Transfection

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