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Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors

Protein networks and signaling cascades are key mechanisms for intra- and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The bimolecular fluorescence complementation (BiFC) assay has become a routine tool...

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Autores principales: Gookin, Timothy E, Assmann, Sarah M
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BlackWell Publishing Ltd 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260091/
https://www.ncbi.nlm.nih.gov/pubmed/25187041
http://dx.doi.org/10.1111/tpj.12639
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author Gookin, Timothy E
Assmann, Sarah M
author_facet Gookin, Timothy E
Assmann, Sarah M
author_sort Gookin, Timothy E
collection PubMed
description Protein networks and signaling cascades are key mechanisms for intra- and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The bimolecular fluorescence complementation (BiFC) assay has become a routine tool for in vivo analysis of protein–protein interactions and their subcellular location. Although the BiFC system has improved since its inception, the available options for in planta analysis are still subject to very low signal-to-noise ratios, and a systematic comparison of BiFC confounding background signals has been lacking. Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives. To overcome these problems, we performed an extensive in planta analysis of published BiFC fragments used in metazoa and plants, and then developed an optimized single vector BiFC system which utilizes monomeric Venus (mVenus) split at residue 210, and contains an integrated mTurquoise2 marker to precisely identify transformed cells in order to distinguish true negatives. Here we provide our streamlined double ORF expression (pDOE) BiFC system, and show that our advance in BiFC methodology functions even with an internally fused mVenus210 fragment. We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin-like (CML) protein, and by showing that heterotrimeric G-protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells. We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so-called PLDα1 ‘DRY’ motif abolishes both of these interactions.
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spelling pubmed-42600912014-12-11 Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors Gookin, Timothy E Assmann, Sarah M Plant J Technical Advance Protein networks and signaling cascades are key mechanisms for intra- and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The bimolecular fluorescence complementation (BiFC) assay has become a routine tool for in vivo analysis of protein–protein interactions and their subcellular location. Although the BiFC system has improved since its inception, the available options for in planta analysis are still subject to very low signal-to-noise ratios, and a systematic comparison of BiFC confounding background signals has been lacking. Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives. To overcome these problems, we performed an extensive in planta analysis of published BiFC fragments used in metazoa and plants, and then developed an optimized single vector BiFC system which utilizes monomeric Venus (mVenus) split at residue 210, and contains an integrated mTurquoise2 marker to precisely identify transformed cells in order to distinguish true negatives. Here we provide our streamlined double ORF expression (pDOE) BiFC system, and show that our advance in BiFC methodology functions even with an internally fused mVenus210 fragment. We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin-like (CML) protein, and by showing that heterotrimeric G-protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells. We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so-called PLDα1 ‘DRY’ motif abolishes both of these interactions. BlackWell Publishing Ltd 2014-11 2014-09-27 /pmc/articles/PMC4260091/ /pubmed/25187041 http://dx.doi.org/10.1111/tpj.12639 Text en © 2014 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd. http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open access article under the terms of the Creative CommonsAttribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
spellingShingle Technical Advance
Gookin, Timothy E
Assmann, Sarah M
Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors
title Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors
title_full Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors
title_fullStr Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors
title_full_unstemmed Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors
title_short Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors
title_sort significant reduction of bifc non-specific assembly facilitates in planta assessment of heterotrimeric g-protein interactors
topic Technical Advance
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260091/
https://www.ncbi.nlm.nih.gov/pubmed/25187041
http://dx.doi.org/10.1111/tpj.12639
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