Abstract
Interactions between cytochromes P450 (P450s) and P450 reductase are required for enzymatic activity, and homo- or heterooligomerization 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. Nonspecific protein-protein interactions were detected if proteins were expressed at high levels. At low protein expression levels, homo-oligomerization 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 was also detected by BiFC. Homo-oligomerization of the N-terminal P450 2C1 signal anchor sequence and interactions between the signal anchor and full-length P450 2C2 were detected, suggesting that homo-oligomerization 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 values. 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.
Footnotes
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This work was supported by Grant GM35897 from the National Institutes of Health.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.005538.
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ABBREVIATIONS: P450, cytochrome P450; ER, endoplasmic reticulum; FRET, fluorescence resonance energy transfer; BiFC, bimolecular fluorescence complementation; YN, N-terminal YFP fragment; YC, C-terminal YFP fragment; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; PCR, polymerase chain reaction; HEK, human embryonic kidney; GFP, green fluorescent protein.
- Received May 17, 2005.
- Accepted June 22, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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