A single amino acid mutation (Ser180----Cys) determines the polymorphism in cytochrome P450g (P4502C13) by altering protein stability.

M B Faletto,P Linko,J A Goldstein

doi:10.1016/s0021-9258(18)46049-8

Abstract

Cytochrome P450g is polymorphic in the male rat. This polymorphism is characterized by a 20-40-fold difference in the hepatic content of P450g in the two phenotypes. Sequencing of cDNAs from high (+g) and low (-g) phenotype rats has shown that the low phenotype is due to a defective mRNA containing nine base mutations encoding 7 amino acid substitutions. To determine the role of these structural changes in the phenotypic expression of P450g, we altered each of these residues by site-directed mutagenesis in the present studies and expressed the normal and mutant cDNAs in Saccharomyces cerevisiae. P450+g protein was expressed at a level 4-6-fold higher than that of P450-g in yeast cells, despite the presence of identical mRNA levels. This difference in protein expression approaches the difference seen in the rat. A single amino acid change from Ser180 in P450+g to Cys in P450-g, in a highly conserved region in the P4502C subfamily, was found to be solely responsible for the phenotypic differences in expression of P450g. Protein half-life studies demonstrated that this mutation increases the degradation of P450g. This is the first example of a single amino acid substitution which alters the phenotypic expression of a P450 protein by affecting its stability.

Highlights

This polymorphism is characterized by a 20-40-fold difference in the hepatic content of P450g in the two phenotypes
This is thefirst example of a single amino acid substitution which alters the phenotypic expression of a P450 protein by affecting its stability
Hybridization studies using oligonucleotide probes specific for +g and -g sequences demonstrated that thephenotypic differences in hepatic levels of P450g are due to the expression of the variant mRNA with nine base mutations in the low phenotype

Summary

THEJOURNAOLF BIOLOGICACLHEMISTRY

2032-2037,1992 Printed in U.S.A. A Single Amino Acid Mutation(SerlS0--?) Cys) Determines the Polymorphism in Cytochrome P450g (P4502C13)by Altering Protein Stability*. Protein half-life studies demonstrated that this mutation increases the degradation of P450g This is thefirst example of a single amino acid substitution which alters the phenotypic expression of a P450 protein by affecting its stability. Hybridization studies using oligonucleotide probes specific for +g and -g sequences demonstrated that thephenotypic differences in hepatic levels of P450g are due to the expression of the variant mRNA with nine base mutations in the low phenotype. The cDNA sequences for P450+g and P450-g were inserted into the vector pAAH5 for expression in Saccharomyces cereuisiae, and site-directed mutagenesis studies were performed to assess the role of the individual amino acid changes in the phenotypic expression of P450g. Essentially identical amounts of mRNA for P450+g and P450-g were transcribed under controlof the ADHI promoter in the yeast protein precipitated

RESULTS

DISCUSSION

Findings

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