Protein–Protein Interactions: Yeast Two-Hybrid System

Part of the Methods in Molecular Biology book series (MIMB, volume 1615)


The yeast two-hybrid system is a powerful and commonly used genetic tool to investigate interactions between artificial fusion proteins inside the nucleus of yeast. Here we describe how to use the Matchmaker GAL4-based yeast two-hybrid system to detect the interaction of the Agrobacterium type VI secretion system (T6SS) sheath components TssB and TssC41. The bait and prey gene are expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD) and GAL4 activation domain (AD, prey/library fusion protein) respectively. When bait and prey fusion proteins interact in yeast nucleus, the DNA-BD and AD are brought into proximity, thereby activating the transcription of reporter genes. This technology can be widely used to identify interacting partners, confirm suspected interactions, and define interacting domains.

Key words

Protein–protein interaction Yeast two-hybrid Gal4 transcriptional activation domain (AD) Gal4 DNA-binding domain (BD) Saccharomyces cerevisiae AH109 Type VI secretion system TssB TssC 



This work was supported by a research grant from the Ministry of Science and Technology (MOST 104-2311-B-001-025 -MY3) to E.M. Lai. J. S. Lin is the recipient of postdoctoral fellowships from Academia Sinica.


  1. 1.
    Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340:245–246CrossRefGoogle Scholar
  2. 2.
    Stasi M, De Luca M, Bucci C (2015) Two-hybrid-based systems: powerful tools for investigation of membrane traffic machineries. J Biotechnol 202:105–117CrossRefGoogle Scholar
  3. 3.
    Clontech (2007) Matchmaker™ GAL4 two-hybrid system 3 & libraries user manual. Scholar
  4. 4.
    Chien CT, Bartel PL, Sternglanz R, Fields S (1991) The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A 88:9578–9582CrossRefGoogle Scholar
  5. 5.
    Causier B, Davies B (2002) Analysing protein–protein interactions with the yeast two-hybrid system. Plant Mol Biol 50:855–870CrossRefGoogle Scholar
  6. 6.
    Petschnigg J, Groisman B, Kotlyar M, Taipale M, Zheng Y et al (2014) The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane–protein interactions in human cells. Nat Methods 11:585–592CrossRefGoogle Scholar
  7. 7.
    Reece-Hoyes JS, Barutcu AR, McCord RP, Jeong JS, Jiang L et al (2011) Yeast one-hybrid assays for gene-centered human gene regulatory network mapping. Nat Methods 8:1050–1052CrossRefGoogle Scholar
  8. 8.
    Reece-Hoyes JS, Marian Walhout AJ (2012) Yeast one-hybrid assays: a historical and technical perspective. Methods 57:441–447CrossRefGoogle Scholar
  9. 9.
    Tsai YL, Chiang YR, Narberhaus F, Baron C, Lai EM (2010) The small heat-shock protein HspL is a VirB8 chaperone promoting type IV secretion-mediated DNA transfer. J Biol Chem 285:19757–19766CrossRefGoogle Scholar
  10. 10.
    Baron C, Thorstenson YR, Zambryski PC (1997) The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens. J Bacteriol 179:1211–1218CrossRefGoogle Scholar
  11. 11.
    Das A, Anderson LB, Xie YH (1997) Delineation of the interaction domains of Agrobacterium tumefaciens VirB7 and VirB9 by use of the yeast two-hybrid assay. J Bacteriol 179:3404–3409CrossRefGoogle Scholar
  12. 12.
    Das A, Xie YH (2000) The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and VirB10 interact with one another. J Bacteriol 182:758–763CrossRefGoogle Scholar
  13. 13.
    Ma LS, Lin JS, Lai EM (2009) An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens. J Bacteriol 191:4316–4329CrossRefGoogle Scholar
  14. 14.
    Lin JS, Ma LS, Lai EM (2013) Systematic dissection of the Agrobacterium type VI secretion system reveals machinery and secreted components for subcomplex formation. PLoS One 8:e67647CrossRefGoogle Scholar
  15. 15.
    Bonemann G, Pietrosiuk A, Diemand A, Zentgraf H, Mogk A (2009) Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J 28:315–325CrossRefGoogle Scholar
  16. 16.
    Lossi NS, Manoli E, Forster A, Dajani R, Pape T et al (2013) The HsiB1C1 (TssB-TssC) complex of the Pseudomonas aeruginosa type VI secretion system forms a bacteriophage tail sheathlike structure. J Biol Chem 288:7536–7548CrossRefGoogle Scholar
  17. 17.
    Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168PubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Institute of Plant and Microbial BiologyAcademia SinicaTaipeiTaiwan
  2. 2.Institute of Plant and Microbial BiologyAcademia SinicaTaipeiTaiwan

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