Bimolecular Fluorescence Complementation (BiFC) Analysis: Advances and Recent Applications for Genome-Wide Interaction Studies

Abstract

Complex protein networks are involved in nearly all cellular processes. To uncover these vast networks of protein interactions, various high-throughput screening technologies have been developed. Over the last decade, bimolecular fluorescence complementation (BiFC) assay has been widely used to detect protein-protein interactions (PPIs) in living cells. This technique is based on the reconstitution of a fluorescent protein in vivo. Easy quantification of the BiFC signals allows effective cell-based high-throughput screenings for protein binding partners and drugs that modulate PPIs. Recently, with the development of large screening libraries, BiFC has been effectively applied for genome-wide PPI studies and has uncovered novel protein interactions, providing new insight into protein functions. In this review, we describe the development of reagents and methods used for BiFC-based screens in yeast, plants, and mammalian cells. We also discuss the advantages and drawbacks of these methods and highlight the application of BiFC in large-scale studies.

Keywords: bimolecular fluorescence complementation (BiFC) analysis; fluorescent proteins; genomics; proteomics.

Figure 1. Schematic representation of the BiFC analysis

The N-terminal and C-terminal fragments (YN and YC) of YFP are fused to two proteins of interest (A and B). The interaction between A and B allows formation of a bimolecular fluorescent complex. In contrast, b (a mutant form of B or a non-binding partner) cannot form a complex with YN-A, showing no fluorescence. The images show an example of BiFC complex formed on the vacuolar membrane of yeast cells (upper panel) and a control with a mutant that fails to form a complex. The image is reproduced from Singh et al. [85]. Copyright (2008) National Academy of Sciences, U.S.A.

Figure 2. Schematic diagram of the genome-wide BiFC analysis analysis in budding yeast

For the genome-wide BiFC analysis, each strain of the VN fusion library is mated with a MATα strain expressing a protein of interest tagged with VC, thus generating a diploid collection. Each strain of the obtained diploid collection expressing both the VN fusion and the VC fusion is analyzed by fluorescence microscopy, and the images are collected and quantitatively analyzed. Adapted from Sung et al. [90].