YEAST EXPRESSED CYTOCHROME-P450 2D6 (CYP2D6) EXPOSED ON THE EXTERNAL FACE OF PLASMA-MEMBRANE IS FUNCTIONALLY COMPETENT

CYP2D6, a xenobiotic metabolizing cytochrome P450 (P450), was found to be present in significant amount on the outer face of cell plasma mem brane in addition to the regular microsomal location. Present work dem onstrates that this external P450 is catalytically competent and that activity is supported by NADPH-P450 reductase present on the inner fac e of plasma membrane. Purified plasma membranes from yeast expressing CYP2D6 sustained NADPH- and cumene hydroperoxide-dependent dextrometho rphan demethylation and NADPH-cytochrome c activity confirming previou s observations in human hepatocytes. CYP2D6 found on the outside of pl asma membrane (by differential immune-inhibition and acidic shift assa ys on transformed spheroplasts) was catalytically competent at the cel l surface for NADPH-supported activities. Anti-yeast P450-reductase an tibodies inhibited neither CYP2D6 nor P450-reductase activities upon i ncubation with intact spheroplasts. In contrast, both activities were inhibited on isolated plasma membrane fragments. This highly suggested a cytosolic-orientation of the plasma membrane P450-reductase. This f inding was confirmed by immunostaining in confocal microscopy. Finally , gene deletion of P450-reductase caused a complete loss of plasma mem brane NADPH-supported CYP2D6 activity, which suggests that the reducta se participates to some degree in the transmembrane electron transfer chain. This work illustrates that the outside-exposed plasma membrane CYP2D6 is active and may play an important metabolic role.