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. 1996 Sep 1;24(17):3341–3347. doi: 10.1093/nar/24.17.3341

The reverse two-hybrid system: a genetic scheme for selection against specific protein/protein interactions.

C A Leanna 1, M Hannink 1
PMCID: PMC146093  PMID: 8811088

Abstract

The yeast two-hybrid system is a powerful experimental approach for the characterization of protein/ protein interactions. A unique strength of the yeast two-hybrid system is the provision for genetic selection techniques that enable the identification of specific protein/protein interactions. We now report the development of a modified yeast two-hybrid system which enables genetic selection against a specific protein/protein interaction. This reverse two-hybrid system utilizes a yeast strain which is resistant to cycloheximide due to the presence of a mutant cyh2 gene. This strain also contains the wild-type CYH2 allele under the transcriptional control of the Gal1 promoter. Expression of the wild-type Gal4 protein is sufficient to restore growth sensitivity to cycloheximide. Growth sensitivity towards cycloheximide is also restored by the coexpression of the avian c-Rel protein and its I kappa B alpha counterpart, p40, as Gal4 fusion proteins. Restoration of growth sensitivity towards cycloheximide requires the association of c-Rel and p40 at the Gal1 promoter and correlates with the ability of the c-Rel/p40 interaction to activate expression from the Gal1 promoter. A genetic selection scheme against specific protein/protein interactions may be a valuable tool for the analysis of protein/protein interactions.

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Selected References

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  1. Arenzana-Seisdedos F., Thompson J., Rodriguez M. S., Bachelerie F., Thomas D., Hay R. T. Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B. Mol Cell Biol. 1995 May;15(5):2689–2696. doi: 10.1128/mcb.15.5.2689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brockman J. A., Scherer D. C., McKinsey T. A., Hall S. M., Qi X., Lee W. Y., Ballard D. W. Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation. Mol Cell Biol. 1995 May;15(5):2809–2818. doi: 10.1128/mcb.15.5.2809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown K., Gerstberger S., Carlson L., Franzoso G., Siebenlist U. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science. 1995 Mar 10;267(5203):1485–1488. doi: 10.1126/science.7878466. [DOI] [PubMed] [Google Scholar]
  4. Brown K., Park S., Kanno T., Franzoso G., Siebenlist U. Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2532–2536. doi: 10.1073/pnas.90.6.2532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen Z. J., Parent L., Maniatis T. Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity. Cell. 1996 Mar 22;84(6):853–862. doi: 10.1016/s0092-8674(00)81064-8. [DOI] [PubMed] [Google Scholar]
  6. Cordle S. R., Donald R., Read M. A., Hawiger J. Lipopolysaccharide induces phosphorylation of MAD3 and activation of c-Rel and related NF-kappa B proteins in human monocytic THP-1 cells. J Biol Chem. 1993 Jun 5;268(16):11803–11810. [PubMed] [Google Scholar]
  7. Cressman D. E., Taub R. I kappa B alpha can localize in the nucleus but shows no direct transactivation potential. Oncogene. 1993 Sep;8(9):2567–2573. [PubMed] [Google Scholar]
  8. Diehl J. A., McKinsey T. A., Hannink M. Differential pp40I kappa B-beta inhibition of DNA binding by rel proteins. Mol Cell Biol. 1993 Mar;13(3):1769–1778. doi: 10.1128/mcb.13.3.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  10. Ernst M. K., Dunn L. L., Rice N. R. The PEST-like sequence of I kappa B alpha is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers. Mol Cell Biol. 1995 Feb;15(2):872–882. doi: 10.1128/mcb.15.2.872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  12. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hannink M., Temin H. M. Transactivation of gene expression by nuclear and cytoplasmic rel proteins. Mol Cell Biol. 1989 Oct;9(10):4323–4336. doi: 10.1128/mcb.9.10.4323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henkel T., Machleidt T., Alkalay I., Krönke M., Ben-Neriah Y., Baeuerle P. A. Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B. Nature. 1993 Sep 9;365(6442):182–185. doi: 10.1038/365182a0. [DOI] [PubMed] [Google Scholar]
  15. Inoue J., Kerr L. D., Ransone L. J., Bengal E., Hunter T., Verma I. M. c-rel activates but v-rel suppresses transcription from kappa B sites. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3715–3719. doi: 10.1073/pnas.88.9.3715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ito C. Y., Kazantsev A. G., Baldwin A. S., Jr Three NF-kappa B sites in the I kappa B-alpha promoter are required for induction of gene expression by TNF alpha. Nucleic Acids Res. 1994 Sep 11;22(18):3787–3792. doi: 10.1093/nar/22.18.3787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Käufer N. F., Fried H. M., Schwindinger W. F., Jasin M., Warner J. R. Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res. 1983 May 25;11(10):3123–3135. doi: 10.1093/nar/11.10.3123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Le Bail O., Schmidt-Ullrich R., Israël A. Promoter analysis of the gene encoding the I kappa B-alpha/MAD3 inhibitor of NF-kappa B: positive regulation by members of the rel/NF-kappa B family. EMBO J. 1993 Dec 15;12(13):5043–5049. doi: 10.1002/j.1460-2075.1993.tb06197.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lehming N., McGuire S., Brickman J. M., Ptashne M. Interactions of a Rel protein with its inhibitor. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10242–10246. doi: 10.1073/pnas.92.22.10242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ma J., Ptashne M. A new class of yeast transcriptional activators. Cell. 1987 Oct 9;51(1):113–119. doi: 10.1016/0092-8674(87)90015-8. [DOI] [PubMed] [Google Scholar]
  21. Mellits K. H., Hay R. T., Goodbourn S. Proteolytic degradation of MAD3 (I kappa B alpha) and enhanced processing of the NF-kappa B precursor p105 are obligatory steps in the activation of NF-kappa B. Nucleic Acids Res. 1993 Nov 11;21(22):5059–5066. doi: 10.1093/nar/21.22.5059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Richardson P. M., Gilmore T. D. vRel is an inactive member of the Rel family of transcriptional activating proteins. J Virol. 1991 Jun;65(6):3122–3130. doi: 10.1128/jvi.65.6.3122-3130.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rottjakob E. M., Sachdev S., Leanna C. A., McKinsey T. A., Hannink M. PEST-dependent cytoplasmic retention of v-Rel by I(kappa)B-alpha: evidence that I(kappa)B-alpha regulates cellular localization of c-Rel and v-Rel by distinct mechanisms. J Virol. 1996 May;70(5):3176–3188. doi: 10.1128/jvi.70.5.3176-3188.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sachdev S., Rottjakob E. M., Diehl J. A., Hannink M. I kappa B-alpha-mediated inhibition of nuclear transport and DNA-binding by Rel proteins are separable functions: phosphorylation of C-terminal serine residues of I kappa B-alpha is specifically required for inhibition of DNA-binding. Oncogene. 1995 Sep 7;11(5):811–823. [PubMed] [Google Scholar]
  25. Schatzle J. D., Kralova J., Bose H. R., Jr Avian I kappa B alpha is transcriptionally induced by c-Rel and v-Rel with different kinetics. J Virol. 1995 Sep;69(9):5383–5390. doi: 10.1128/jvi.69.9.5383-5390.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sikorski R. S., Boeke J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. doi: 10.1016/0076-6879(91)94023-6. [DOI] [PubMed] [Google Scholar]
  27. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sun S. C., Ganchi P. A., Ballard D. W., Greene W. C. NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science. 1993 Mar 26;259(5103):1912–1915. doi: 10.1126/science.8096091. [DOI] [PubMed] [Google Scholar]
  29. Sun S., Elwood J., Greene W. C. Both amino- and carboxyl-terminal sequences within I kappa B alpha regulate its inducible degradation. Mol Cell Biol. 1996 Mar;16(3):1058–1065. doi: 10.1128/mcb.16.3.1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Traenckner E. B., Pahl H. L., Henkel T., Schmidt K. N., Wilk S., Baeuerle P. A. Phosphorylation of human I kappa B-alpha on serines 32 and 36 controls I kappa B-alpha proteolysis and NF-kappa B activation in response to diverse stimuli. EMBO J. 1995 Jun 15;14(12):2876–2883. doi: 10.1002/j.1460-2075.1995.tb07287.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Xu X., Prorock C., Ishikawa H., Maldonado E., Ito Y., Gélinas C. Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor IIB. Mol Cell Biol. 1993 Nov;13(11):6733–6741. doi: 10.1128/mcb.13.11.6733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yocum R. R., Hanley S., West R., Jr, Ptashne M. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Oct;4(10):1985–1998. doi: 10.1128/mcb.4.10.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zabel U., Henkel T., Silva M. S., Baeuerle P. A. Nuclear uptake control of NF-kappa B by MAD-3, an I kappa B protein present in the nucleus. EMBO J. 1993 Jan;12(1):201–211. doi: 10.1002/j.1460-2075.1993.tb05646.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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