TOPOLOGY INVERSION OF CYP2D6 IN THE ENDOPLASMIC-RETICULUM IS NOT REQUIRED FOR PLASMA-MEMBRANE TRANSPORT

The presence of CYP2D6 at the surface of isolated rat and human hepato cytes and its recognition by autoantibodies were reported recently. We wondered whether the unexpected outside orientation at the plasma mem brane could be related to topological inversion (luminal-oriented form ) of cytochrome P450 in the endoplasmic reticulum. To examine the pote ntial role of cDNA polymorphism, a CYP2D6 variant carrying three posit ive charges at the amino terminus (2D6ext) was constructed and express ed in yeast. Immunoblotting, flow cytometry, and electron microscopy s howed that wild-type CYP2D6 expressed in yeast was present on the oute r face of the cell plasma membrane in addition to the regular microsom al location. This location reproduces the hepatocyte situation. 2D6ext expressed in yeast and COS7 cells seemed to be partially N-glycosylat ed and was located at the plasma membrane surface. Nevertheless, the g lycosylated form was not enriched in the plasma membranes compared wit h microsomes. The relationship between CYP2D6 and 2D6ext topologies an d catalytic competence was tested. Cumene hydroperoxide-dependent dext romethorphan demethylation was performed on microsomal vesicles after combined proteolysis and immunoinhibition experiments. CYP2D6 activity was completely abolished, whereas the glycosylated and luminal-orient ed fraction of 2D6ext remained active. This suggests that a luminal-or iented glycosylated form is not involved in cytochrome P450 transport to the plasma membrane. Yeast thus reproduces the unusual CYP2D6 plasm a membrane location and orientation, which do not require sequence alt eration, glycosylation, or even an inverted endoluminal orientation.