Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation

Abstract

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.

An improved mRFP1 adds red to bimolecular fluorescence complementation

“…The recently developed BiFC assay is a comparably fast and simple noninvasive technology to study protein interactions inside living cells. BiFC is based on the reconstitution of the yellow fluorescent protein (YFP) from two nonfluorescent fragments when they are brought into close proximity by a physical interaction between proteins fused to each fragment 1 . This approach has proven to be robust and versatile, and multicolor versions using spectral variants of GFP further increase the potential of this technology 2 .…”

The variety of complexes formed by EcR and Usp nuclear receptors in the nuclei of living cells

“…Initially, we applied the technique of fluorescence recovery after photobleaching (FRAP) (Reits and Neefjes, 2001). FRAP relies on the ability of a laser scanning instrument to rapidly and irreversibly photobleach a small region of interest ( To test Usp-EcR interaction in a more direct manner, we decided to apply the BiFC assay (Hu et al, 2002), which has been proven to be an invaluable tool to monitor protein-protein interactions in vivo and in real time (Kerppola, 2006). To the best of our knowledge the BiFC technique has never been applied to the investigation of nuclear receptors.…”

“…To the best of our knowledge the BiFC technique has never been applied to the investigation of nuclear receptors. The BiFC assay is (Hu et al, 2002). To investigate the interaction between EcR and Usp, the YN fragment was attached to the N-terminus of EcR (YN-EcR), whereas the YC fragment to the N-terminus of Usp (YC-Usp).…”

“…An essential criterion for using the fusion proteins in the BiFC analysis is that the fluorescentprotein fragments should not associate with each other in the absence of interaction between the proteins which are fused to these fragments (Hu et al, 2002). To test if this criterion is fulfilled, we examined YN-EcR coexpressed with YC or YC-Usp with YN or YN with YC.…”

“…Our experimental approach relies on the new technique of bimolecular fluorescence complementation (BiFC) (Hu et al, 2002). This technique which allows monitoring proteinprotein interactions in vivo and in real time has been recently used for the successful visualization of interactions among a wide range of proteins in many cell types and organisms (Kerppola, 2006).…”