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 fr...
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| Main Authors: | , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
28 April 2015
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| In: |
The new phytologist
Year: 2015, Volume: 208, Issue: 1, Pages: 269-279 |
| ISSN: | 1469-8137 |
| DOI: | 10.1111/nph.13439 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1111/nph.13439 Verlag, lizenzpflichtig, Volltext: https://nph.onlinelibrary.wiley.com/doi/abs/10.1111/nph.13439 
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| Author Notes: | Jan Niklas Offenborn, Rainer Waadt and Jörg Kudla |
| Summary: | 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. |
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| Item Description: | Gesehen am 19.06.2020 |
| Physical Description: | Online Resource |
| ISSN: | 1469-8137 |
| DOI: | 10.1111/nph.13439 |