The spectral characteristics of bimolecular fluorescent complexes formed by fragments of YFP were virtually identical to those of intact YFP 1
. We reasoned that fragments of other GFP variants might support bimolecular fluorescent complex formation, and that such complexes might have distinct spectral characteristics. To identify such complexes, we investigated fluorescence complementation using the corresponding fragments of the enhanced GFP and cyan fluorescent protein (CFP) fused to the bZIP domains of Fos and Jun (bFos and bJun) (). Each pair of fusion proteins was expressed in mammalian cells and the cells were examined by fluorescence microscopy (). No complementation was detected when fragments of GFP (GN155 and GC155) fused to bFos and bJun were expressed in mammalian cells. However, fragments of CFP (CN155 and CC155) exhibited fluorescence complementation when fused to bFos and bJun. (). All of the fusion proteins were expressed at comparable levels as determined by Western analysis (Supplementary Fig. 1A online).
Figure 1 Visualization of bimolecular fluorescence complementation between fragments of different fluorescent proteins fused to bFos and bJun. (A) Diagram of amino acid substitutions among enhanced green fluorescent protein variants and the positions where they (more ...)
To examine the selectivity of bimolecular complex formation, we tested fluorescence complementation between all 9 combinations of fragments (Supplementary Fig. 2 online). YN155 exhibited fluorescence complementation with YC155 and CC155, whereas CN155 exhibited fluorescence complementation only with CC155 when fused to bFos and bJun (). Significantly, the fluorescence spectrum of cells expressing YN155 and CC155 fusions was distinct from those of cells expressing either YN155 and YC155 or CN155 and CC155. GN155 and GC155 did not exhibit detectable fluorescence complementation with any of the other fragments. YC155 and CC155 differ from GC155 by single amino acid residues whereas YN155 and CN155 differ from GN155 by four and three amino acid residues respectively (). These amino acid substitutions determined the selectivity of bimolecular fluorescence complementation among these fragments.
We used a genetic screen in E. coli 1
to identify a second pair of YFP fragments (YN173 and YC173) that exhibit bimolecular fluorescence complementation when fused to bFos and bJun. We examined fluorescence complementation between these fragments and the corresponding fragments of GFP, CFP and the enhanced blue fluorescent protein (BFP) fused to bFos and bJun. The sequences of the C-terminal fragments of GFP, CFP and BFP are identical (), and thus only YC173 and GC173 were tested. YC173 exhibited fluorescence complementation with YN173, GN173 and CN173 when fused to bFos and bJun (), whereas GC173 did not exhibit detectable fluorescence complementation with any of the fragments tested. The two positions of fragmentation (155 and 173) enabled complementation between distinct combinations of fluorescent protein fragments (Supplementary Fig. 2 online). Thus, a small number of amino acid substitutions can influence the positions where proteins must be fragmented to support bimolecular complementation.
GFP is a β barrel structure containing 11 strands surrounding a central α helix10
.The two sites of fragmentation in YFP that support BiFC are separated by one strand of the β barrel that surrounds the fluorophore10
. We examined fluorescence complementation between all 24 combinations of fragments truncated at these positions fused to bFos and bJun (Supplementary Fig. 2 online). None of the N-terminal fragments truncated at residue 155 exhibited fluorescence complementation with C-terminal fragments truncated at residue 173. In contrast, all of the N-terminal fragments truncated at residue 173 (YN173, GN173 and CN173) formed bimolecular fluorescent complexes with CC155 and YC155 (, data not shown). The corresponding N-terminal fragment of BFP (BN173) also exhibited fluorescence complementation with CC155 (). Duplication of the segment separating the points of truncation therefore facilitated bimolecular fluorescence complementation. This duplication had no detectable effect on the spectra of the bimolecular complexes ().
Figure 2 Excitation and emission spectra of cells expressing fragments of different fluorescent proteins fused to bFos and bJun. Solid lines correspond to bimolecular fluorescent complexes and dashed lines to intact fluorescent proteins (A) The excitation spectra (more ...)
To establish whether bimolecular fluorescence complementation between fragments of different fluorescent proteins fused to bFos and bJun required the leucine zipper dimerization interface, we examined complementation by bFos fusions in which the carboxy-terminal half of the leucine zipper was deleted (bFosΔZIPYC155 and bFosΔZIPCC155). We compared the complementation efficiencies of the wild type and mutant fusions with bJun fused to fragments of different fluorescent proteins (, Supplementary Table 1 online). Mutation of the leucine zipper resulted in a more than 10-fold reduction in the efficiencies of fluorescence complementation between all fragments of fluorescent proteins tested. Cells expressing fragments of different fluorescent proteins fused to wild type bFos and bJun produced different intensities of fluorescence emissions at different times following transfection. Cells expressing the same fragments, but with a deletion in the leucine zipper of Fos, produced either undetectable or lower fluorescence emissions at all times following transfection (Supplementary Table 1 online). Similar results were obtained when the fluorescence emissions of the cell populations were measured using fluorescence spectroscopy (data not shown). The mutant proteins were expressed at the same levels and were localized to the same subnuclear sites as the wild type proteins. Thus, efficient fluorescence complementation between all fragments of fluorescent proteins examined required a specific interaction interface.
Figure 3 Effects of a deletion in the leucine zipper on the efficiencies of complementation between fragments of different fluorescent proteins fused to the bZIP domains of Fos and Jun. The efficiencies of fluorescence complementation were determined in individual (more ...)
Multicolor BiFC allows comparison of the efficiencies of complex formation between alternative interaction partners, providing that the fragments do not influence the selectivity of protein interactions. To compare the effects of fragments of different fluorescent proteins on dimerization efficiency, we examined the competition between proteins in which bJun was fused to fragments of different fluorescent proteins for dimerization with bFosYC173 in vitro (). The ratio of bimolecular fluorescent complexes formed by the alternative interaction partners was calculated by fitting the spectrum of the mixture to the weighted sum of the spectra of the two complexes. This ratio exhibited a perfect correspondence with the ratio of bJun fusion proteins that was added to each reaction. Thus, the bZIP domains of Fos and Jun have identical dimerization efficiencies when fused to fragments of different fluorescent proteins, suggesting that the fusions do not have differential effects on interactions between bFos and bJun in vitro.
Figure 4 Comparison of the efficiencies of bimolecular complex formation by fragments of different fluorescent proteins fused to bFos and bJun. We examined the competition between different ratios of bJun fused to fragments of different fluorescent proteins for (more ...)
The spectral differences between bimolecular complexes formed by fragments of different fluorescent proteins enable comparison of the subcellular sites of interactions between different proteins in the same cell providing that the fragments do not differentially affect complex localization. To compare the subcellular locations of bFos-bJun heterodimers fused to fragments of different fluorescent proteins, we co-expressed bJunCN173 and bJunYN173 with bFosYC173 in COS-1 cells and imaged the cells using filters optimized for the detection of CN173-YC173 and YN173-YC173 complexes respectively (). Both complexes were localized preferentially to the nucleoli, and exhibited perfect co-localization. Similar results were obtained when bJunCN173 and bJunYN173 were co-expressed with bFosYC155 or with bFosCC155. Although the efficiency of nucleolar localization of the complexes varied between different cells in the population, the efficiencies of nucleolar localization of complexes containing fragments of different fluorescent proteins were identical in individual cells. Thus, fragments of different fluorescent proteins did not have differential effects on the subcellular sites of interactions between bFos and bJun.
Figure 5 Multicolor BiFC analysis of the subcellular sites of interactions between different proteins in the same cell. (A) Co-localization of bJunCN173-bFosYC173 and bJunYN173-bFosYC173 bimolecular fluorescent complexes. (B) Differential localization of bJunCN173-bFosYC155 (more ...)
To visualize the subcellular sites of interactions between different proteins in the same cell, we co-expressed bJunCN173 and JunYN155 with bFosYC155. bJunCN173-bFosYC155 exhibited nucleolar localization, but JunYN155-bFosYC155 was localized to the nucleoplasm (). The two complexes exhibited distinct distributions in the same nucleus, indicating that regions outside the bZIP domain of Jun affected the subcellular localization of the complex. Similar results were obtained when bJunCN173 and JunYN155 were co-expressed with bFosCC155. Thus, multicolor BiFC can be used to compare the subcellular sites of interactions between different proteins in the same cell.
The bZIP domains of Fos, Jun and ATF2 can interact with each other in all pairwise combinations in vitro
, and activate different genes in cells 11-14
. To investigate the competition between alternative interaction partners in cells, we examined the relative efficiencies of bJun interactions with bFos and bATF2. We expressed bJunCC155 with bFosCN173 and bATF2YN155 separately as well as together, and compared the ratio between the fluorescence emissions of the complexes (, Supplementary Table 2 online). Cells expressing the two pairs of proteins separately exhibited cyan and yellow fluorescence respectively. Co-expression of a limiting amount of bJunCC155 with bFosCN173 and bATF2YN155 together resulted in cyan fluorescence that was comparable to that observed in cells transfected with bJunCC155 and bFosCN173, but yellow fluorescence that was 100-fold lower than that of cells transfected with the same amounts of bJunCC155 and bATF2YN155 (Supplementary Table 2). The ratio between yellow and cyan fluorescence was therefore comparable for cells expressing all three proteins and those expressing only bJunCC155 and bFosCN173 (). Similar results were obtained when bJunCC155 was expressed with bFosCN155 and bATF2YN155 (data not shown). When excess bJunCC155 was expressed with bFosCN173 and bATF2YN155, both cyan and yellow fluorescence was observed in the cells. It is therefore likely that the inhibition of bimolecular fluorescence complementation between bATF2YN155 and bJunCC155 in the presence of either bFosCN155 or bFosCN173 was caused by a higher efficiency of complex formation by bJunCC155 with either bFosCN155 or bFosCN173 than with bATF2YN155.
Figure 6 Multicolor BiFC analysis of the competition between alternative interaction partners in cells. Two combinations of interaction partners producing bimolecular complexes with different spectra were transfected into different cell populations (cyan and yellow (more ...)
To confirm that the identity of the fragments fused to bJun, bFos and bATF2 did not affect the preference for bimolecular fluorescent complex formation, we exchanged the fragments between bFos and bATF2 (). Cells expressing a limiting amount of bJunCC155 with bFosYN155 and bATF2CN173 produced yellow fluorescence comparable to that of cells transfected with bJunCC155 and bFosYN155, but cyan fluorescence that was 10-fold lower than that of cells transfected with the same amounts of bJunCC155 and bATF2CN173 (Supplementary Table 2). Again, the ratio between yellow and cyan fluorescence for cells expressing all three proteins was similar to that of cells expressing only bJunCC155 and bFosYN173 (). The fluorescent protein fragments were fused to the same positions in bJun and bATF2 relative to the leucine zipper, and the same linker sequences were used for all fusions. The competing proteins were expressed at comparable levels (Supplementary Fig. 1B online). Consequently, it is likely that the higher efficiency of complementation between bJun and bFos fusions reflected preferential heterodimerization of bJun with bFos than with bATF2 in cells.
To examine the relative efficiencies of bFos interactions with bJun and bATF2 in cells, we expressed limiting bFosCC155 with bJunCN173 and bATF2YN155. The cells exhibited cyan fluorescence comparable to cells transfected with bFosCC155 and bJunCN173, but markedly lower yellow fluorescence than cells transfected with the same amounts of bFosCC155 and bATF2YN155 (data not shown). A similar inhibition of bFos-bATF2 and bJun-bATF2 heterodimer formation was observed in cells transfected using a limiting concentration of bATF2YC155 with bFosYN155 and bJunCN173 (data not shown). The higher efficiency of bFos-bJun heterodimer formation therefore did not require bimolecular complex formation since bFosYN155-bJunCN173 heterodimers could not form a bimolecular fluorescent complex. Thus, the bZIP domains of Fos and Jun favor dimerization with each other over dimerization with the bZIP domain of ATF2 in cells.
To examine the relative efficiencies of Fos-Jun heterodimerization and Jun homodimerization in cells, we expressed bJunCC155 with bFosYN155 and bJunCN173 (). The cells produced on average 60% of the cyan fluorescence and 35% of the yellow fluorescence produced by the control cells. The fluorescence intensities of individual cells were normally distributed and exhibited a moderate (r=0.83) correlation between cyan and yellow fluorescence. Thus, Fos-Jun heterodimers and Jun homodimers can co-exist in cells, consistent with the similar thermodynamic stabilities of these dimers in vitro 15