BiFC can be used to determine subcellular localization of protein complexes. A, Under physiological conditions reconstitution of a fluorescent YFP molecule can only take place following interaction between proteins or peptides that are fused to YN and YC fragments. B, A BiFC assay showing that complexes of AtROP6 and the ROP-interacting coiled-coil scaffold protein ICR1 are localized in the plasma membrane of N. benthamiana leaf epidermal cells. C, A BiFC assay showing that the complexes between the Atrop6mS mutant, in which the prenyl-acceptor Cys was changed to Ser, and the ROP-interacting coiled-coil scaffold protein ICR1 is localized in the cytoplasm and nuclei. D, Possible configurations for testing BiFC. YN, The N-terminal fragment of YFP; YC, the C-terminal fragment of YFP. Yellow-shaded squares symbolize potential fluorescence resulting from protein-protein interaction leading to YFP reconstitution.

Protein immunoblots reveal expression of split YFP fragments in the BiFC assay. Protein immunoblots decorated with anti-hemagglutinin (HA) tag (left section) and anti-GFP (right section) monoclonal Abs were used for detection of YC and YN AP1 fusion proteins, respectively. The weaker bands observed with the anti-GFP Abs were due to the lower specificity of these Abs toward the YN moiety. Comparable expression levels of the YN and YC AP1 fusion proteins were detected in blots decorated with anti-AP1 Abs (K. Shichrur and S. Yalovsky, unpublished data).

Fluorescence emission spectra of intact, BiFC reconstituted YFP and autofluorescence of plant tissues. Samples of YFP (yellow line) and ROP-ICR1 (green line) were excited at 488 nm and the fluorescent spectra were determined using the spectral scanning module of a Leica TCS-SL CLSM. Fluorescence intensities are shown in arbitrary units relative to the maximal fluorescence, which received a value of 1. Autofluo, Autofluorescence (red line).

Different uses of BiFC. A, Interactions can occur only if two proteins are localized in the same compartment. Thus, how one protein affects the subcellular localization of the complex can be tested, as exemplified in Figure 1. The stability of the bimolecular complexes formed in BiFC should enable tracking of subcellular targeting pathways of proteins that may otherwise be difficult to observe. B, Protein interaction may depend on the presence of a cofactor or another protein(s) (Ueguchi-Tanaka et al., 2007). Screening cDNA vector libraries or chemical libraries can be used to identify protein and nonprotein mediators. Expression in relevant mutant backgrounds and sequestration or removal of cofactors could be used as controls. C, Some protein-protein interactions depend on posttranslational modifications. For example, a ubiquitin-based BiFC system has been described and proteins showed different localizations upon interaction with ubiquitin or the related Sumo protein (Fang and Kerppola, 2004). The use of mutated interacting proteins or mutated genetic backgrounds can aid in analyzing requirements for posttranslational modifications. In addition, site-directed mutagenesis could be used to remove the modification site and to map the domain mediating the interaction. D, Protein interactions depend on expression times and patterns. Using endogenous promoters to express a particular BiFC enables analysis of the temporal and spatial interaction pattern, as has been demonstrated in C. elegans (Zhang et al., 2004). Coexpression patterns of genes could be studied by expressing known interacting protein partners under respective promoters. In this case, the YFP fluorescence should only be detected in the cells where the two promoters are active.