Faculty Collaboration Database

Identification of sequences responsible for intracellular targeting and membrane binding of rat CYP2E1 in yeast. Biochemistry 2003 Dec 16;42(49):14566-75

Date

12/10/2003

Pubmed ID

14661969

DOI

10.1021/bi035193s

Scopus ID

2-s2.0-0347481139 (requires institutional sign-in at Scopus site)   12 Citations

Abstract

The role of the hydrophobic NH(2)-terminal domain of rat CYP2E1 for intracellular targeting and membrane binding was investigated in Saccharomyces cerevisiae as a model system. Several different CYP2E1 variants with deletions and mutations were expressed in yeast, and their intracellular localization and membrane-binding properties were analyzed. We found that an amino acid stretch including the B-helix from glycine 82 to asparagine 95 is responsible for mitochondrial association of CYP2E1 in yeast. Furthermore, we investigated the membrane-binding properties of the variants and concluded that the same region in the B-helix is responsible for membrane interactions of CYP2E1 by electrostatic interactions. A soluble variant of CYP2E1 lacking the first 82 amino acids and containing leucine to aspartate amino acid exchanges at positions 90 and 91, which disrupted the amphipathic nature of the B-helix, was expressed at relatively high levels in the yeast and was found to be catalytically active toward chlorzoxazone in cumene hydroperoxide-supported reactions. We suggest that these amino acid changes at positions 90 and 91 abolish the electrostatic interaction between the negatively charged membrane and the positively charged B-helix, thereby producing a soluble product.

Author List

Neve EP, Hidestrand M, Ingelman-Sundberg M

MESH terms used to index this publication - Major topics in bold

Amino Acid Motifs
Amino Acid Sequence
Animals
Catalysis
Cell Membrane
Cytochrome P-450 CYP2E1
Genetic Vectors
Intracellular Fluid
Mitochondria
Molecular Sequence Data
Mutagenesis, Site-Directed
Peptide Fragments
Protein Binding
Protein Processing, Post-Translational
Protein Transport
Rats
Saccharomyces cerevisiae
Sequence Deletion
Solubility
Static Electricity