Networks of protein interactions coordinate cellular functions. We describe a bimolecular fluorescence complementation (BiFC) assay for determination of the locations of protein interactions in living cells. This approach is based on complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are brought together by interactions between proteins fused to each fragment. BiFC analysis was used to investigate interactions among bZIP and Rel family transcription factors. Regions outside the bZIP domains determined the locations of bZIP protein interactions. The subcellular sites of protein interactions were regulated by signaling. Cross-family interactions between bZIP and Rel proteins affected their subcellular localization and modulated transcription activation. These results attest to the general applicability of the BiFC assay for studies of protein interactions.

The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified twelve new bimolecular fluorescent complexes that correspond to seven different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency or subcellular sites of interactions between the bZIP domains of Fos and Jun. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation among alternative interaction partners.Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of developmental programs. Each protein typically has a large number of alternative interaction partners, and the selectivity of these interactions determines the developmental potential of the cell and its responses to extracellular stimuli. We recently described a new approach for the visualization of protein interactions in living cells designated bimolecular fluorescence complementation (BiFC) analysis 1 . The BiFC approach is based on the formation of a fluorescent complex by fragments of the yellow fluorescent protein (YFP) brought together by the association of two interaction partners fused to the fragments. This approach enables visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment.Molecular engineering of the green fluorescent protein (GFP) has produced several variants with altered spectral characteristics 2 . These variants allow simultaneous visualization of the distributions of multiple proteins in living cells. Moreover, fluorescence resonance energy transfer (FRET) between different variants enables analysis of interactions between individual pairs of proteins in living cells 3, 4 . Thus far, it has not been possible to visualize multiple interactions in the same cell.Selected fragments of many proteins can associate to produce functional bimolecular complexes. Such bimolecular complementation provides a convenient approach for detection of protein interactions in cells if the protein fragments can associate only when they are brought together by interaction partners fused to the fragments 1, 5-9 . The unique characteristic of the BiFC approach is that the bright intrinsic fluorescence of the bimolecular complex allows direct * To whom correspondence should be addressed:Tel: (734) FAX: (734) RESULTSThe spectral characteristics of bimolecular fluorescent complexes formed by fragments of YFP were virtually identical t...

Fos and Jun family proteins regulate the expression of a myriad of genes in a variety of tissues and cell types. This functional versatility emerges from their interactions with related bZIP proteins and with structurally unrelated transcription factors. These interactions at composite regulatory elements produce nucleoprotein complexes with high sequence-speci®city and regulatory selectivity. Several general principles including binding cooperativity and conformational adaptability have emerged from studies of regulatory complexes containing Fos-Jun family proteins. The structural properties of Fos-Jun family proteins including opposite orientations of heterodimer binding and the ability to bend DNA can contribute to the assembly and functions of such complexes. The cooperative recruitment of transcription factors, coactivators and chromatin remodeling factors to promoter and enhancer regions generates multiprotein transcription regulatory complexes with cell-and stimulus-speci®c transcriptional activities. The genespeci®c architecture of these complexes can mediate the selective control of transcriptional activity. Oncogene (2001) 20, 2438 ± 2452.