IMPORTANCE OF THE PROLINE-RICH REGION FOLLOWING SIGNAL-ANCHOR SEQUENCE IN THE FORMATION OF CORRECT CONFORMATION OF MICROSOMAL CYTOCHROME P-450S

A proline-rich region is present following the signal-anchor sequence in the aminoterminal portion of all known microsomal cytochrome P-450s . To assess the functional significance of the proline residues in thi s region, we systematically altered these residues of cytochrome P450( M1) (P450 2C11); one, two, and three proline residues out of the five in the region were exchanged for alanine residues. The wild-type and t he mutated proteins were expressed in the fission yeast Schizosaccharo myces pombe under the control of nmt1 promoter. The wild-type and the mutated proteins were all highly expressed in the yeast cells (5-9% of the total membrane protein). The expressed wild-type P450(M1) showed a typical carbon monoxide difference spectrum of P-450 and the activit y of testosterone hydroxylation, whereas all the mutated proteins cons tructed in the present study showed no characteristic P-450 spectrum, suggesting that the substitution of the proline residues in this regio n resulted in a defect of proper heme incorporation. Furthermore, the mutated proteins in which more than one proline residues had been exch anged were more sensitive to trypsin digestion than the wild type. Fro m these results, we propose that the proline residues in the proline-r ich region are crucial for the formation of the correct conformation o f microsomal P-450 molecules.