Identification of sequences responsible for intracellular targeting and membrane binding of rat CYP2E1 in yeast.
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.
Neve
Etienne P A
EP
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Hidestrand
Mats
M
Ingelman-Sundberg
Magnus
M
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D005822:Genetic Vectors; D007424:Intracellular Fluid / Q000201:enzymology* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D017384:Sequence Deletion; D012995:Solubility; D055672:Static Electricity
D010446:Peptide Fragments; D019392:Cytochrome P-450 CYP2E1