Fluorescence resonance energy transfer analysis of cytochromes P450 2C2 and 2E1 molecular interactions in living cells.
The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.
Szczesna-Skorupa
Elzbieta
E
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Mallah
Bahar
B
Kemper
Byron
B
D000818:Animals; D019556:COS Cells; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry* / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004579:Electron Transport; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D018613:Microscopy, Confocal; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D014162:Transfection
D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; C084930:cytochrome P-450 2C2; D019392:Cytochrome P-450 CYP2E1; D009251:NADPH-Ferrihemoprotein Reductase