A systematic in planta survey of 14-3-3 protein interactions by bimolecular fluorescence complementation

Proteins of the highly conserved eukaryotic 14-3-3 family act as key regulators of a wide range of biological processes, such as metabolic enzyme regulation, cell cycle control, ion transport, gene expression, protein assembly and translocation. Such regulation is achieved by interactions of 14-3-3 homo- and heterodimers with target proteins. To date, more than one hundred 14-3-3 binding targets have been identified, mainly through far-Western analysis, immuno-precipitation, yeast two-hybrid screening, proteomic approaches or in silico predictions. Currently, the literature proposes two mechanisms that could contribute to 14-3-3 isoform-target protein specificity. Firstly, it is suggested that the specificity is mediated via interaction of the 14-3-3 protein’s carboxy-terminal ends with target proteins. On the other hand, the variability of the amino-terminal ends of 14-3-3 proteins gave rise to the idea that dimerisation is selective and can therefore contribute to 14-3-3 specificity. Analysing whether 14-3-3s can dimerise freely with each other would further our understanding of the role the amino termini play during dimerisation and whether specificity is associated with this. Bimolecular fluorescence complementation (BiFC) enables the visualisation of protein-protein interaction in planta. The technique is based on the interaction of two candidate proteins translationally fused to non-fluorescent fragments of the yellow fluorescent protein (YFP). Interaction of the candidate proteins leads to functional YFP reconstitution and fluorescence. In this study, ten out of thirteen expressed Arabidopsis 14-3-3 isoforms were analysed for dimerisation using a BiFC approach in transiently transformed Nicotiana benthamiana leaf epidermal cells. This approach demonstrated that all tested 14-3-3 isoforms were able to freely form homo- and heterodimers with each other.