Abstract
The in vivo analysis of protein–protein interactions (PPIs) is a critical factor for gaining insights into cellular mechanisms and their biological functions. To that end, a constantly growing number of genetic tools has been established, some of which are using baker’s yeast (Saccharomyces cerevisiae) as a model organism. Here, we provide a detailed protocol for the yeast mating-based split-ubiquitin system (mbSUS) to study binary interactions among or with full-length membrane proteins in their native subcellular environment. The system is based on the reassembly of two autonomously non-functional ubiquitin moieties attached to proteins of interest (POIs) into a native-like molecule followed by the release of a transcription factor. Upon its nuclear import, the activation of reporter gene expression gives a visual output via growth on interaction-selective media. Additionally, we apply a modification of the classical split-ubiquitin technique called CytoSUS that detects interactions of non-membrane/soluble proteins in their full-length form via translational fusion of an ER membrane anchor.
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References
Xing S, Wallmeroth N, Berendzen KW, Grefen C (2016) Techniques for the analysis of protein-protein interactions in vivo. Plant Physiol 171(2):727–758
Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340(6230):245–246
Stynen B, Tournu H, Tavernier J, Van Dijck P (2012) Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system. Microbiol Mol Biol Rev 76(2):331–382
Johnsson N, Varshavsky A (1994) Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A 91(22):10340–10344
Stagljar I, Korostensky C, Johnsson N, te Heesen S (1998) A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 95(9):5187–5192
Obrdlik P et al (2004) K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions. Proc Natl Acad Sci U S A 101(33):12242–12247
Grefen C et al (2008) Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene receptor family members. Mol Plant 1(2):308–320
Grefen C et al (2010) A novel motif essential for SNARE interaction with the K(+) channel KC1 and channel gating in Arabidopsis. Plant Cell 22(9):3076–3092
Grefen C, Blatt MR (2012) Do calcineurin B-like proteins interact independently of the serine threonine kinase CIPK23 with the K+ channel AKT1? Lessons learned from a menage a trois. Plant Physiol 159(3):915–919
Xing S et al (2017) Loss of GET pathway orthologs in Arabidopsis thaliana causes root hair growth defects and affects SNARE abundance. Proc Natl Acad Sci U S A 114(8):E1544–E1553
Grefen C, Lalonde S, Obrdlik P (2007) Split-ubiquitin system for identifying protein-protein interactions in membrane and full-length proteins. Curr Protoc Neurosci. Chapter 5:Unit 5 27
Möckli N et al (2007) Yeast split-ubiquitin-based cytosolic screening system to detect interactions between transcriptionally active proteins. BioTechniques 42(6):725–730
Karnik R et al (2015) Binding of SEC11 indicates its role in SNARE recycling after vesicle fusion and identifies two pathways for vesicular traffic to the plasma membrane. Plant Cell 27(3):675–694
Grefen C, Obrdlik P, Harter K (2009) The determination of protein-protein interactions by the mating-based split-ubiquitin system (mbSUS). Methods Mol Biol 479:217–233
Ludewig U et al (2003) Homo- and hetero-oligomerization of ammonium transporter-1 NH4 uniporters. J Biol Chem 278(46):45603–45610
Raquet X, Eckert JH, Muller S, Johnsson N (2001) Detection of altered protein conformations in living cells. J Mol Biol 305(4):927–938
Grefen C (2014) The split-ubiquitin system for the analysis of three-component interactions. Methods Mol Biol 1062:659–678
Jones AM et al (2014) Border control--a membrane-linked interactome of Arabidopsis. Science 344(6185):711–716
Karnik A, Karnik R, Grefen C (2013) SDM-assist software to design site-directed mutagenesis primers introducing “silent” restriction sites. BMC Bioinformatics 14:105
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Asseck, L.Y., Grefen, C. (2018). Detecting Interactions of Membrane Proteins: The Split-Ubiquitin System. In: Oñate-Sánchez, L. (eds) Two-Hybrid Systems. Methods in Molecular Biology, vol 1794. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7871-7_4
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DOI: https://doi.org/10.1007/978-1-4939-7871-7_4
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