Quantitative analysis of dynamic protein–protein interactions
Dynamic protein–protein interactions are essential in all cellular and developmental processes. Protein‐fragment complementation assays allow such protein–protein interactions to be investigated in vivo. In contrast to other protein‐fragment complementation assays, the split‐luciferase (split‐LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein–protein interaction of investigated proteins is reversible. Here, we describe the development of a floated‐leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split‐LUC‐labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway‐compatible split‐LUC destination vectors, enabling fast, and almost fail‐safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well‐established homodimerization of the 14‐3‐3 regulator proteins. Quantitative interaction analyses of the molybdenum co‐factor biosynthesis proteins CNX6 and CNX7 show that the luciferase‐based protein‐fragment complementation assay allows direct real‐time monitoring of absolute values of protein complex assembly. Furthermore, the split‐LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway‐compatible split‐LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.
Document Type: Research Article
Affiliations: 1: Institut für Pflanzenbiologie, Technische Universität Braunschweig, Humboldtstraße 1, D-38106 Braunschweig, Germany 2: Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 4, D-48149 Münster, Germany
Publication date: August 1, 2011