Chapter 15
Using Tripartite Split-sfGFP for the Study of Membrane
Protein–Protein Interactions
Tzu-Yin Liu
Abstract
The study of protein–protein interaction (PPI) is critical for understanding cellular processes within
biological systems. The conventional biomolecular fluorescence complementation (BiFC) or bipartite
split-fluorescent protein (FP) is a noninvasive fluorescent-based technique that enables direct visualization
of PPI in living cells once the two nonfluorescent fragments are brought into close vicinity. However, BiFC
can potentially lead to a high background noise arising from an inherent feature of the irreversible selfassembly of the nonfluorescent fragments. Recently, the newly developed tripartite split-sfGFP method was
demonstrated to detect membrane PPIs in plant cells without spurious background signals even when
fusion proteins are highly expressed and accessible to the compartments of interaction. Here we describe a
protocol for using the ß-Estradiol-inducible tripartite split-sfGFP assay for side-by-side analyses of in vivo
PPI along with in situ subcellular localization of fusion proteins in agroinfiltrated Nicotiana benthamiana
leaves.
Key words Tripartite split-sfGFP, Membrane protein–protein interactions, Bimolecular fluorescent
complementation (BiFC)
1 Introduction
Protein–protein interactions (PPIs) play a pivotal role in biological
systems through regulation of signal transduction, enzymatic activities, and gene expression. In particular, identifying protein interaction partners is a fundamental step forward in elucidating the
mechanisms underlying cascades of cellular processes at the molecular level. Over the past years, several techniques that rely on
genetically encoded reporters have emerged to directly image
PPIs in living cells at subcellular resolution [1]. Detection of
dynamic PPIs based on the principle of fluorescence/bioluminescence resonance energy transfer (FRET/BRET) is feasible when
two proteins of interests are each fused to different chromophores
[1, 2]. If they are in close proximity, a dipole-dipole resonancemediated energy is transferred from the donor to the acceptor
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
323
Using Tripartite Split-sfGFP for the Study of Membrane
Protein–Protein Interactions
Tzu-Yin Liu
Abstract
The study of protein–protein interaction (PPI) is critical for understanding cellular processes within
biological systems. The conventional biomolecular fluorescence complementation (BiFC) or bipartite
split-fluorescent protein (FP) is a noninvasive fluorescent-based technique that enables direct visualization
of PPI in living cells once the two nonfluorescent fragments are brought into close vicinity. However, BiFC
can potentially lead to a high background noise arising from an inherent feature of the irreversible selfassembly of the nonfluorescent fragments. Recently, the newly developed tripartite split-sfGFP method was
demonstrated to detect membrane PPIs in plant cells without spurious background signals even when
fusion proteins are highly expressed and accessible to the compartments of interaction. Here we describe a
protocol for using the ß-Estradiol-inducible tripartite split-sfGFP assay for side-by-side analyses of in vivo
PPI along with in situ subcellular localization of fusion proteins in agroinfiltrated Nicotiana benthamiana
leaves.
Key words Tripartite split-sfGFP, Membrane protein–protein interactions, Bimolecular fluorescent
complementation (BiFC)
1 Introduction
Protein–protein interactions (PPIs) play a pivotal role in biological
systems through regulation of signal transduction, enzymatic activities, and gene expression. In particular, identifying protein interaction partners is a fundamental step forward in elucidating the
mechanisms underlying cascades of cellular processes at the molecular level. Over the past years, several techniques that rely on
genetically encoded reporters have emerged to directly image
PPIs in living cells at subcellular resolution [1]. Detection of
dynamic PPIs based on the principle of fluorescence/bioluminescence resonance energy transfer (FRET/BRET) is feasible when
two proteins of interests are each fused to different chromophores
[1, 2]. If they are in close proximity, a dipole-dipole resonancemediated energy is transferred from the donor to the acceptor
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
323