Visualization and translocation of ternary Calcineurin-A/Calcineurin-B/Calmodulin-2 protein complexes by dual-color trimolecular fluorescence complementation.

Autor: Offenborn JN; Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 7, Münster, 48149, Germany., Waadt R; 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., Kudla J; Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 7, Münster, 48149, Germany.
Jazyk: angličtina
Zdroj: The New phytologist [New Phytol] 2015 Oct; Vol. 208 (1), pp. 269-79. Date of Electronic Publication: 2015 Apr 28.
DOI: 10.1111/nph.13439
Abstrakt: 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.
(© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)
Databáze: MEDLINE
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