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Received for publication May 18, 2005.
Revised June 21, 2005.
Accepted for publication June 22, 2005.
Interactions between cytochromes P450 (P450s) and P450 reductase are required for enzymatic activity, and homo- or hetero-oligomerization of P450s may also be functionally important. Bimolecular fluorescence complementation (BiFC) was used to examine P450 interactions in a natural membrane context within living cells. BiFC detects protein interactions in living cells by reconstitution of a fluorescent protein from two fragments that are fused to the two interacting proteins. Non-specific protein-protein interactions were detected if proteins were expressed at high levels. At low protein expression levels, homooligomerization of P450 2C2, but not P450 2E1, and interactions of these P450s with P450 reductase were detected by BiFC consistent with interactions detected previously by fluorescence resonance emission transfer. Weak interaction of P450 2C2 with P450 2E1 and homooligomerization of P450 reductase were also detected by BiFC. Homooligomerization of the N-terminal P450 2C1 signal anchor sequence and interactions between the signal anchor and full length P450 2C2 were detected suggesting that homooligomerization of P450 2C2 is mediated by the signal anchor. However, interactions between the signal anchor and either P450 2E1 or P450 reductase were not detected by BiFC. Although high concentrations of the substrate lauric acid increased BiFC for both P450 2E1 and P450 2C2 with P450 reductase, the concentration-dependence did not correlate with reported Km's. These results demonstrate that BiFC is an effective method to study the complex protein interactions that occur within the microsomal P450 system in living cells.
Key words:
cytochrome P450, cytochrome P450 function, endoplasmic reticulum, NADPH cytochrome P450 reductase, protein-protein interactions
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