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Making the Right Choice: Critical Parameters of the Y2H Systems

  • Jitender Mehla
  • J. Harry Caufield
  • Peter UetzEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1794)

Abstract

Two-hybrid methods remain among the most preferred choices for detecting protein–protein interactions (PPIs) and much of the PPI data in databases have been produced using yeast two-hybrid (Y2H) screens. The Y2H methods are extensively used to detect PPIs because of their scalability and accessibility. Several variants of Y2H methods have been developed and used by different research groups, increasing the accessibility of these methods and their applications in detecting different types of PPIs. However, the availability of variations on the same core methodology emphasizes the need to have a systematic comparison of available Y2H methods in the context of their applicability, coverage and efficiency. In this chapter, we discuss the key parameters of Y2H methods, namely proteins of interest, vectors, libraries, screening strategies, data analysis, and provide a flowchart that should help to decide which Y2H strategy is most appropriate for a protein interaction screen.

Key words

High-throughput Protein–protein interactions Yeast two-hybrid screening Y2H strategy and approaches 

Notes

Acknowledgments

This work was supported by National Institutes of Health grant R01GM109895.

References

  1. 1.
    Bartel PL, Roecklein JA, SenGupta D, Fields S (1996) A protein linkage map of Escherichia coli bacteriophage T7. Nat Genet 12(1):72–77.  https://doi.org/10.1038/ng0196-72 CrossRefPubMedGoogle Scholar
  2. 2.
    Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci U S A 98(8):4569–4574.  https://doi.org/10.1073/pnas.061034498 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403(6770):623–627.  https://doi.org/10.1038/35001009 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP, Vidal M (2005) Towards a proteome-scale map of the human protein-protein interaction network. Nature 437(7062):1173–1178.  https://doi.org/10.1038/nature04209 CrossRefPubMedGoogle Scholar
  5. 5.
    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksoz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H, Wanker EE (2005) A human protein-protein interaction network: a resource for annotating the proteome. Cell 122(6):957–968.  https://doi.org/10.1016/j.cell.2005.08.029 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340(6230):245–246.  https://doi.org/10.1038/340245a0 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Karimova G, Pidoux J, Ullmann A, Ladant D (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 95(10):5752–5756CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Luo Y, Batalao A, Zhou H, Zhu L (1997) Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system. BioTechniques 22(2):350–352PubMedCrossRefGoogle Scholar
  9. 9.
    Snider J, Kittanakom S, Damjanovic D, Curak J, Wong V, Stagljar I (2010) Detecting interactions with membrane proteins using a membrane two-hybrid assay in yeast. Nat Protoc 5(7):1281–1293.  https://doi.org/10.1038/nprot.2010.83 CrossRefPubMedGoogle Scholar
  10. 10.
    Wang Y, Cui T, Zhang C, Yang M, Huang Y, Li W, Zhang L, Gao C, He Y, Li Y, Huang F, Zeng J, Huang C, Yang Q, Tian Y, Zhao C, Chen H, Zhang H, He ZG (2010) Global protein-protein interaction network in the human pathogen mycobacterium tuberculosis H37Rv. J Proteome Res 9(12):6665–6677.  https://doi.org/10.1021/pr100808n CrossRefPubMedGoogle Scholar
  11. 11.
    Mehla J, Caufield JH, Sakhawalkar N, Uetz P (2017) A comparison of two-hybrid approaches for detecting protein-protein interactions. Methods Enzymol 586:333–358.  https://doi.org/10.1016/bs.mie.2016.10.020 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Caufield JH, Sakhawalkar N, Uetz P (2012) A comparison and optimization of yeast two-hybrid systems. Methods 58(4):317–324.  https://doi.org/10.1016/j.ymeth.2012.12.001 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Mehla J, Caufield JH, Uetz P (2015) The yeast two-hybrid system: a tool for mapping protein-protein interactions. Cold Spring Harb Protoc 2015(5):425–430.  https://doi.org/10.1101/pdb.top083345 CrossRefPubMedGoogle Scholar
  14. 14.
    Mehla J, Dedrick RM, Caufield JH, Siefring R, Mair M, Johnson A, Hatfull GF, Uetz P (2015) The protein interactome of mycobacteriophage giles predicts functions for unknown proteins. J Bacteriol 197(15):2508–2516.  https://doi.org/10.1128/jb.00164-15 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Xin X, Rual JF, Hirozane-Kishikawa T, Hill DE, Vidal M, Boone C, Thierry-Mieg N (2009) Shifted transversal design smart-pooling for high coverage interactome mapping. Genome Res 19(7):1262–1269.  https://doi.org/10.1101/gr.090019.108 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Rajagopala SV, Casjens S, Uetz P (2011) The protein interaction map of bacteriophage lambda. BMC Microbiol 11:213.  https://doi.org/10.1186/1471-2180-11-213 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Stellberger T, Hauser R, Baiker A, Pothineni VR, Haas J, Uetz P (2010) Improving the yeast two-hybrid system with permutated fusions proteins: the varicella zoster virus interactome. Proteome Sci 8:8.  https://doi.org/10.1186/1477-5956-8-8 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Dohmen RJ, Strasser AW, Honer CB, Hollenberg CP (1991) An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7(7):691–692.  https://doi.org/10.1002/yea.320070704 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hayama Y, Fukuda Y, Kawai S, Hashimoto W, Murata K (2002) Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells. J Biosci Bioeng 94(2):166–171CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Walhout AJ, Vidal M (2001) High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 24(3):297–306.  https://doi.org/10.1006/meth.2001.1190 CrossRefPubMedGoogle Scholar
  21. 21.
    Meng X, Smith RM, Giesecke AV, Joung JK, Wolfe SA (2006) Counter-selectable marker for bacterial-based interaction trap systems. BioTechniques 40(2):179–184CrossRefPubMedGoogle Scholar
  22. 22.
    Titz B, Thomas S, Rajagopala SV, Chiba T, Ito T, Uetz P (2006) Transcriptional activators in yeast. Nucleic Acids Res 34(3):955–967CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Keskin O, Tuncbag N, Gursoy A (2016) Predicting protein-protein interactions from the molecular to the proteome level. Chem Rev 116(8):4884–4909.  https://doi.org/10.1021/acs.chemrev.5b00683 CrossRefPubMedGoogle Scholar
  24. 24.
    Kotlyar M, Pastrello C, Pivetta F, Lo Sardo A, Cumbaa C, Li H, Naranian T, Niu Y, Ding Z, Vafaee F, Broackes-Carter F, Petschnigg J, Mills GB, Jurisicova A, Stagljar I, Maestro R, Jurisica I (2015) In silico prediction of physical protein interactions and characterization of interactome orphans. Nat Methods 12(1):79–84.  https://doi.org/10.1038/nmeth.3178 CrossRefPubMedGoogle Scholar
  25. 25.
    Stumpf MP, Thorne T, de Silva E, Stewart R, An HJ, Lappe M, Wiuf C (2008) Estimating the size of the human interactome. Proc Natl Acad Sci U S A 105(19):6959–6964.  https://doi.org/10.1073/pnas.0708078105 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Goldberg DS, Roth FP (2003) Assessing experimentally derived interactions in a small world. Proc Natl Acad Sci U S A 100(8):4372–4376.  https://doi.org/10.1073/pnas.0735871100 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Sprinzak E, Sattath S, Margalit H (2003) How reliable are experimental protein-protein interaction data? J Mol Biol 327(5):919–923CrossRefPubMedGoogle Scholar
  28. 28.
    Rajagopala SV, Hughes KT, Uetz P (2009) Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins. Proteomics 9(23):5296–5302.  https://doi.org/10.1002/pmic.200900282 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Taipale M, Tucker G, Peng J, Krykbaeva I, Lin ZY, Larsen B, Choi H, Berger B, Gingras AC, Lindquist S (2014) A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways. Cell 158(2):434–448.  https://doi.org/10.1016/j.cell.2014.05.039 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Rajagopala SV, Titz B, Goll J, Parrish JR, Wohlbold K, McKevitt MT, Palzkill T, Mori H, Finley RL Jr, Uetz P (2007) The protein network of bacterial motility. Mol Syst Biol 3:128.  https://doi.org/10.1038/msb4100166 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Chen YC, Rajagopala SV, Stellberger T, Uetz P (2010) Exhaustive benchmarking of the yeast two-hybrid system. Nat Methods 7(9):667–668.; author reply 668.  https://doi.org/10.1038/nmeth0910-667 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Bader JS, Chaudhuri A, Rothberg JM, Chant J (2004) Gaining confidence in high-throughput protein interaction networks. Nat Biotechnol 22(1):78–85.  https://doi.org/10.1038/nbt924 CrossRefPubMedGoogle Scholar
  33. 33.
    Titz B, Rajagopala SV, Goll J, Hauser R, McKevitt MT, Palzkill T, Uetz P (2008) The binary protein interactome of Treponema pallidum--the syphilis spirochete. PLoS One 3(5):e2292CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Jitender Mehla
    • 1
  • J. Harry Caufield
    • 2
  • Peter Uetz
    • 1
    Email author
  1. 1.VCU Life Sciences, Center for the Study of Biological ComplexityVirginia Commonwealth UniversityRichmondUSA
  2. 2.NIH BD2K Center of Excellence at UCLAUniversity of California, Los AngelesLos AngelesUSA

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