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.
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References
Drees, B. L. (1999) Progress and variations in two-hybrid and three-hybrid technologies. Curr. Opin. Chem. Biol. 3, 64–70.
Evangelista, C., Lockshon, D., and Fields, S. (1996) The yeast two-hybrid system—prospects for protein linkage maps. Trends Cell Biol. 6, 196–199.
Fields, S. and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.
Vidal, M. and Legrain, P. (1999) Yeast forward and reverse ‘n’-hybrid systems. Nucleic Acids Res. 27, 919–929.
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.
Uetz, P., Giot, L., Cagney, G., et al. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627.
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.
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.
Anfinsen, C. B. (1973). Principles that govern the folding of protein chains. Science 181, 223–230.
Gutte, B. and Merrifield, R. B. (1971) The synthesis of ribonuclease A. J. Biol. Chem. 246, 1922–1941.
Richards, F. M. (1958). On the enzymatic activity of subtilisin-modified ribo-nuclease. Proc. Natl. Acad. Sci. USA 44, 162–166.
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.
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.
Pelletier, J. N. and Michnick, S. W. (1997) A protein complementation assay for detection of protein-protein interactions in vivo. Protein Eng. 10, 89.
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.
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.
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.
Remy, I., Wilson, I. A., and Michnick, S. W. (1999) Erythropoietin receptor activation by a ligand-induced conformation change. Science 283, 990–993.
Remy, I. and Michnick, S. W. (2001) Visualization of biochemical networks in living cells. Proc. Natl. Acad. Sci. USA 98, 7678–7683.
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.
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.
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.
Kaufman, R. J. (1990) Selection and coamplification of heterologous genes in mammalian cells. Methods Enzymol. 185, 537–566.
O’Callaghan, C. and Morris, A. (1972) Inhibition of betalactamases by beta-lactam antibiotics. Antimicrob. Agents Chemother. 2, 442–448.
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.
Zlokarnik, G. (2000) Fusions to beta-lactamase as a reporter for gene expression in live. Methods Enzymol. 326, 221–244.
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Remy, I., Michnick, S.W. (2004). Mapping Biochemical Networks With Protein-Fragment Complementation Assays. In: Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 261. Humana Press. https://doi.org/10.1385/1-59259-762-9:411
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DOI: https://doi.org/10.1385/1-59259-762-9:411
Publisher Name: Humana Press
Print ISBN: 978-1-58829-120-2
Online ISBN: 978-1-59259-762-8
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