Visualization and translocation of ternary Calcineurin-A/Calcineurin-B/Calmodulin-2 protein complexes by dual-color trimolecular fluorescence complementation.
Fluorescence complementation (FC) techniques are expedient for analyzing bimolecular protein-protein interactions. Here we aimed to develop a method for visualization of ternary protein complexes using dual-color trimolecular fluorescence complementation (TriFC). Dual-color TriFC combines protein fragments of mCherry and mVenus, in which a scaffold protein is bilaterally fused to C-terminal fragments of both fluorescent proteins and combined with potential interacting proteins fused to an N-terminal fluorescent protein fragment. For efficient visual verification of ternary complex formation, TriFC was combined with a cytoplasm to plasma membrane translocation assay. Modular vector sets were designed which are fully compatible with previously reported bimolecular fluorescence complementation (BiFC) vectors. As a proof-of-principle, the ternary complex formation of the PP2B protein phosphatase Calcineurin-A/Calcineurin-B with Calmodulin-2 was investigated in transiently transformed Nicotiana benthamiana leaf epidermal cells. The results indicate a Calcineurin-B-induced interaction of Calmodulin-2 with Calcineurin-A. TriFC and the translocation of TriFC complexes provide a novel tool to investigate ternary complex formations with the simplicity of a BiFC approach. The robustness of FC applications and the opportunity to quantify fluorescence complementation render this assay suitable for a broad range of interaction analyses.
Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 7, Münster, 48149, Germany.
Jan Niklas
JN
Offenborn
Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 7, Münster, 48149, Germany.|Plant Developmental Biology, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, Heidelberg, 69120, Germany.
Rainer
R
Waadt
Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 7, Münster, 48149, Germany.
Jörg
J
Kudla
D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D059828:Plant Cells / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D002147:Calmodulin; D008164:Luminescent Proteins; D010940:Plant Proteins; D019703:Calcineurin
Calcineurin; Calmodulin; Nicotiana benthamiana (tobacco); bimolecular fluorescence complementation (BiFC); fluorescent protein; interaction analyses; protein fragment complementation; trimolecular fluorescence complementation (TriFC)