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

J Biol Chem. 1991 Mar 25;266(9):5898-904.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

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

Substances

  • Hemeproteins
  • Steroid 17-alpha-Hydroxylase

Associated data

  • GENBANK/M12547
  • GENBANK/M38179
  • GENBANK/M59039
  • GENBANK/M59494
  • GENBANK/M59495
  • GENBANK/M59496
  • GENBANK/M59497
  • GENBANK/M59498
  • GENBANK/M59499
  • GENBANK/M61698