ROTATION AND MEMBRANE TOPOLOGY OF GENETICALLY EXPRESSED METHYLCHOLANTHRENE-INDUCIBLE CYTOCHROME P-450IA1 LACKING THE N-TERMINAL HYDROPHOBICSEGMENT IN YEAST MICROSOMES

A modified rat liver cytochrome P-450IA1, lacking amino acids 2-30, a proposed membrane anchor for cytochrome P-450, was expressed genetical ly in yeast microsomal membranes. This truncated cytochrome is practic ally active in the deethylation of 7-ethoxycoumarin. A full-length cyt ochrome P-450IA1 was also expressed in yeast microsomes. Rotational di ffusion of P-450IA1 was examined by observing the flash-induced absorp tion anisotropy r(t) of the P-450.CO complex. The anisotropy decayed t o a time-independent value within a 2-ms time range. Since the decay c urve has the characteristics of a slow rotation of membrane-embedded c ytochrome, the theoretical analysis of r(t) was performed based on a ' 'rotation-about-membrane normal'' model. 41% of the shortened P-450IA1 was rotating with the rotational relaxation time phi of 1020 mu s, wh ereas 27% of the full length P-450IA1 was mobile with phi = 1101 mu s. The high salt treatment did not remove the shortened cytochrome from the membrane and also did not drastically weaken the interactions of t he cytochrome with the membrane, as judged from the slow rotation char acteristics (phi = 830 mu s). These results demonstrate that the N-ter minal shortened P-450IA1 is incorporated properly into the yeast micro somal membrane and that the N-terminal hydrophobic segment is not sole ly responsible for attachment to the membrane, providing evidence that additional segments of P-450IA1 are involved in the membrane binding