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Detection of Membrane Protein Interactions by Cell-based Tango Assays   

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Abstract

The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea et al., 2008). Association between the two interaction partners leads to an efficient cleavage of the transcription factor, allowing it to translocate to the nucleus and activate a luciferase reporter gene as measurement of the interactions. In this modified assay, we fused one copy of the membrane-spanning amyloid precursor protein (APP) C99 region to TEV site-rTA (C99-TEV site-rTA) and a second copy to TEV protease (C99-TEV) to analyze intramembrane C99-C99 interaction in live cells.

Keywords: Tango assay, γ-Secretase, Cleavage, Activity, Interaction, C99

Background

The amyloid precursor protein (APP) has three dimerization domains in its N-terminal extracellular domain. In addition, APP can also form dimers through the membrane-bound C99 (C-terminal 99 amino acid fragment) region. Importantly, C99 dimerization has been linked to Aβ production in Alzheimer’s disease (AD) pathology. The Tango assay described here and schematically shown as cartoon in Figure 1 is a fast and sensitive method for investigating homodimerization of C99 and other membrane proteins (Yan et al., 2017).


Figure 1. Cartoon illustration of the Tango interaction assay. Upon membrane cleavage of the C99 hybrid protein by TEV protease, the rTA transactivator protein is released from the membrane into the cytoplasm. This allows rTA to enter the nucleus and bind the tetO DNA-binding site upstream of an integrated luciferase reporter gene to stimulate luciferase reporter gene activity as measured by luminescence. (Yan et al., 2017).

Here we use the Dual-luciferase reporter assay kit. The stably integrated luciferase-Firefly reads represent the γ-secretase cleavage activity, while the transfected Renilla luciferase reads serve as normalization standard.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yan, Y., Xu, T., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017). Detection of Membrane Protein Interactions by Cell-based Tango Assays. Bio-protocol 7(22): e2903. DOI: 10.21769/BioProtoc.2903.
  2. Yan, Y., Xu, T. H., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017a). Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the gamma-secretase cleavage sites. J Biol Chem, 292: 15826-15837.
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