Protein-Protein Interactions pp 411-426

Part of the Methods in Molecular Biology book series (MIMB, volume 261)

Mapping Biochemical Networks With Protein-Fragment Complementation Assays

  • Ingrid Remy
  • Stephen W. Michnick

Abstract

Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. Although our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells. The experimental strategy is based on protein-fragment complementation assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, and so on). In this chapter we review some of the essential principles of PCA and provide details and protocols for applications of PCA, particularly in mammalian cells, based on three PCA reporters, dihydrofolate reductase, green fluorescent protein, and β-lactamase.

Key Words

Protein fragment complementation assays dihydrofolate reductase green fluorescent protein TEM β-lactamase two-hybrid protein-protein interactions methotrexate CCF2/AM nitrocefin fluorescein flow cytometry CHO COS HEK 293 cells 

References

  1. 1.
    Drees, B. L. (1999) Progress and variations in two-hybrid and three-hybrid technologies. Curr. Opin. Chem. Biol. 3, 64–70.PubMedCrossRefGoogle Scholar
  2. 2.
    Evangelista, C., Lockshon, D., and Fields, S. (1996) The yeast two-hybrid system—prospects for protein linkage maps. Trends Cell Biol. 6, 196–199.PubMedCrossRefGoogle Scholar
  3. 3.
    Fields, S. and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.PubMedCrossRefGoogle Scholar
  4. 4.
    Vidal, M. and Legrain, P. (1999) Yeast forward and reverse ‘n’-hybrid systems. Nucleic Acids Res. 27, 919–929.PubMedCrossRefGoogle Scholar
  5. 5.
    Walhout, A.J., Sordella, R., Lu, X., et al. (2000) Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287, 116–122.PubMedCrossRefGoogle Scholar
  6. 6.
    Uetz, P., Giot, L., Cagney, G., et al. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627.PubMedCrossRefGoogle Scholar
  7. 7.
    Michnick, S. W., Remy, I., Campbell-Valois, F.-X., V.-Belisle, A., and Pelletier, J. N. (2000) Detection of protein-protein interactions by protein fragment complementation strategies, in Methods in Enzymology, Vol. 328, (Abelson, J. N., Emr, S. D., and Thorner, J., eds.), Academic Press, New York, NY, pp. 208–230.Google Scholar
  8. 8.
    Anfinsen, C. B., Haber, E., Sela, M., and White Jr., F. H. (1961) The kinetics of formation of native ribonuclease during oxidation of the reduced polypeptide chain. Proc. Natl. Acad. Sci. USA 47, 1309–1314.PubMedCrossRefGoogle Scholar
  9. 9.
    Anfinsen, C. B. (1973). Principles that govern the folding of protein chains. Science 181, 223–230.PubMedCrossRefGoogle Scholar
  10. 10.
    Gutte, B. and Merrifield, R. B. (1971) The synthesis of ribonuclease A. J. Biol. Chem. 246, 1922–1941.PubMedGoogle Scholar
  11. 11.
    Richards, F. M. (1958). On the enzymatic activity of subtilisin-modified ribo-nuclease. Proc. Natl. Acad. Sci. USA 44, 162–166.PubMedCrossRefGoogle Scholar
  12. 12.
    Taniuchi, H. and Anfinsen, C. B. (1971) Simultanious formation of two alternative enzymically active structures by complementation of two overlapping fragments of staphylococcal nuclease. J. Biol. Chem. 216, 2291–2301.Google Scholar
  13. 13.
    Pelletier, J. N., Campbell-Valois, F., and Michnick, S. W. (1998). Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl. Acad. Sci. USA 95, 12,141–12,146.PubMedCrossRefGoogle Scholar
  14. 14.
    Pelletier, J. N. and Michnick, S. W. (1997) A protein complementation assay for detection of protein-protein interactions in vivo. Protein Eng. 10, 89.CrossRefGoogle Scholar
  15. 15.
    Johnsson, N. and Varshavsky, A. (1994) Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. USA 91, 10,340–10,344.PubMedCrossRefGoogle Scholar
  16. 16.
    Rossi, F., Charlton, C. A., and Blau, H. M. (1997) Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc. Natl. Acad. Sci. USA 94, 8405–8410.PubMedCrossRefGoogle Scholar
  17. 17.
    Remy, I. and Michnick, S. W. (1999) Clonal selection and in vivo quantitation of protein interactions with protein fragment complementation assays. Proc. Natl. Acad. Sci. USA 96, 5394–5399.PubMedCrossRefGoogle Scholar
  18. 18.
    Remy, I., Wilson, I. A., and Michnick, S. W. (1999) Erythropoietin receptor activation by a ligand-induced conformation change. Science 283, 990–993.PubMedCrossRefGoogle Scholar
  19. 19.
    Remy, I. and Michnick, S. W. (2001) Visualization of biochemical networks in living cells. Proc. Natl. Acad. Sci. USA 98, 7678–7683.PubMedCrossRefGoogle Scholar
  20. 20.
    Galarneau, A., Primeau, M., Trudeau, L. E., and Michnick, S. W. (2002) beta-Lactamase protein fragment complementation assays as in vivo and in vitro. Nat. Biotechnol. 20, 619–622.PubMedCrossRefGoogle Scholar
  21. 21.
    Israel, D. I. and Kaufman, R. J. (1993) Dexamethasone negatively regulates the activity of a chimeric dihydrofolate reductase/glucocorticoid receptor protein. Proc. Natl. Acad. Sci. USA 90, 4290–4294.PubMedCrossRefGoogle Scholar
  22. 22.
    Kaufman, R. J., Bertino, J. R., and Schimke, R. T. (1978) Quantitation of dihydrofolate reductase in individual parental and methotrexate-resistant murine cells. Use of a fluorescence activated cell sorter. J. Biol. Chem. 253, 5852–5860.PubMedGoogle Scholar
  23. 23.
    Kaufman, R. J. (1990) Selection and coamplification of heterologous genes in mammalian cells. Methods Enzymol. 185, 537–566.PubMedCrossRefGoogle Scholar
  24. 24.
    O’Callaghan, C. and Morris, A. (1972) Inhibition of betalactamases by beta-lactam antibiotics. Antimicrob. Agents Chemother. 2, 442–448.Google Scholar
  25. 25.
    Zlokarnik, G., Negulescu, P. A., Knapp, T.E., et al. (1998). Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279, 84–88.PubMedCrossRefGoogle Scholar
  26. 26.
    Zlokarnik, G. (2000) Fusions to beta-lactamase as a reporter for gene expression in live. Methods Enzymol. 326, 221–244.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Ingrid Remy
    • 1
  • Stephen W. Michnick
    • 1
  1. 1.Département de BiochimieUniversité de MontréalMontréalCanada

Personalised recommendations