Reconstitution of Yeast Farnesyltransferase from Individually Purified Subunits

  • Jun Urano
  • Fuyuhiko Tamanoi
Part of the Methods in Molecular Biology book series (MIMB, volume 116)


Farnesyltransferase (FTase) is a heterodimeric enzyme consisting of an α- and a β-subunit involved in a crucial C-terminal modification of various proteins, including Ras (1, 2, 3). This modification by a 15-carbon isoprenyl (farnesyl) group is essential for proper localization and function of these proteins (4, 5, 6). Because the membrane localization is necessary for Ras function, inhibiting FTase will prevent this localization, which in turn will block the ability of Ras to transform cells. Therefore, the structure and function of FTase has recently been intensively studied. Many studies have characterized substrate recognition by FTase in hope of being able to design small molecules that can act as anticancer drugs by specifically inhibiting this enzyme (1, 2, 3, 4, 5, 6, 7). Recently, the structure of the rat FTase has been solved (8).


Scintillation Cocktail Maltose Binding Protein Individual Subunit Column Buffer Reconstitution Assay 
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  1. 1.
    Del Villar, K., Dorin, D., Sattler, I., Urano, J., Poullet, P., Robinson, N., et al. (1996) C-terminal motifs found in Ras-superfamily G-proteins: CAAX and C-seven motifs. Biochem. Soc. Trans. 24, 709–713.PubMedGoogle Scholar
  2. 2.
    Sattler, I. and Tamanoi, F. (1996) Prenylation of Ras and inhibitors of prenyltransferases, in Regulation of the RAS Signalling Network (Maruta, H. and Burgess, A. W., eds.) RG Landes, Austin, TX, pp. 95–137.Google Scholar
  3. 3.
    Zhang, F. L. and Casey, P. J. (1996) Protein prenylation: molecular mechanisms and functional consequences. Annu. Rev. Biochem. 65, 241–270.PubMedCrossRefGoogle Scholar
  4. 4.
    Gelb, M. H. (1997) Protein prenylation, et cetra: Signal transduction in two dimensions. Science 275, 1750–1751.PubMedCrossRefGoogle Scholar
  5. 5.
    Lowy, D. R. and Willumsen, B. M. (1993) Function and regulation of RAS. Annu. Rev. Biochem. 62, 851–891.PubMedCrossRefGoogle Scholar
  6. 6.
    Khosravi-Far, R., Cox, A. D., Kato, K., and Der, C. J. (1992) Protein prenylation: key to ras function and cancer prevention? Cell Growth Diff. 3, 461–469.PubMedGoogle Scholar
  7. 7.
    Gibbs, J. B., Oliff, A., and Kohl, N. E. (1994) Farnesyltransferase inhibitors: RAS research yields a potential cancer therapeutic. Cell 77, 175–178.PubMedCrossRefGoogle Scholar
  8. 8.
    Park, H. W., Boduluri, S. R., Moomaw, J. F., Casey, P. J., and Beese, L. S. (1997) Crystal structure of protein farnesyltransferase at 2.25 angstrom resolution. Science 275, 1800–1804.PubMedCrossRefGoogle Scholar
  9. 9.
    Chen, W. J., Moomaw, J. W., Overton, L., Kost, T. A., and Casey P. J. (1993) High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc. J. Biol. Chem. 268, 9675–9680.PubMedGoogle Scholar
  10. 10.
    He, B., Chen, P., Chen, S. Y., Vancura, K. L., Michaelis, S. and Powers, S. (1991) RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and RAS proteins. Proc. Natl. Acad. Sci. USA 88, 1373–1377.Google Scholar
  11. 11.
    Omer, C. A., Diehl, R. E. and Kral, A. M. (1995) Bacterial expression and purification of human protein prenyltransferases using epitope-tagged, translationally coupled systems. Methods Enzymol. 250, 3–12.PubMedCrossRefGoogle Scholar
  12. 12.
    Mayer M. P., Prestwich, G. D., Dolence, J. M., Bond, P. D., Wu, H. Y., and Poulter, C. D. (1993) Protein farnesyltransferase: production in Escherichia coli and immunoaffinity purification of the heterodimer from Saccharomyces cerevisiae. Gene 132, 1–47.CrossRefGoogle Scholar
  13. 13.
    Goodman, L. E., Judd, S. E., Farnsworth, C. C., Powers, S., Gelb, M. H., Glomset, J. A., et al. (1990) Mutants of Saccharomyces cerevisiae defective in the farnesylation of ras proteins. Proc. Natl. Acad. Sci. USA 90, 2281–2285.Google Scholar
  14. 14.
    Gomez, R., Goodman, L. E., Tripathy, S. K., O’Rourke, E., Manne, V., and Tamanoi, F. (1992) Purified yeast farnesyltransferase is structurally and functionally similar to its mammalian counterpart. Biochem. J. 289, 25–31.Google Scholar
  15. 15.
    Finegold, A. A., Johnson, D. I., Farnsworth, C. C., Gelb, M. H., Judd, S. R., Glomset, J. A., et al. (1991) Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product, but not the DPR1 gene product. Proc. Natl. Acad. Sci. USA 88, 4448–4452.PubMedCrossRefGoogle Scholar
  16. 16.
    Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., et al., (eds.) (1990) Expression and purification of Glutathione S-Transferase fusion proteins, in Current Protocols in Molecular Biology. John Wiley, New York, pp. 16.7.1–16.7.6.Google Scholar
  17. 17.
    Maina, C. V., Riggs, P. D., Grandea, A. G. III, Slatko, B. E., Moran, L. S., Tagliamonte, J. A., et al. (1988) An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene 74, 365–373.PubMedCrossRefGoogle Scholar
  18. 18.
    Reiss, Y. (1995) Substrate interactions of proteins prenyltransferases. Methods Enzymol. 250, 21–30.PubMedCrossRefGoogle Scholar
  19. 19.
    Del Villar, K., Mitsuzawa, H., Yang, W., Sattler, I., and Tamanoi, F. (1997) Amino acid substitutions that convert the protein substrate specificity of farnesyltransferase to that of geranylgeranyltransferase type I. J. Biol. Chem. 272, 680–687.PubMedCrossRefGoogle Scholar
  20. 20.
    Miyazono, K. (1997) TGF-β receptors and signal transduction. Int. J. Hematol. 65, 97–104.PubMedCrossRefGoogle Scholar
  21. 21.
    Ventura, F., Liu, F., Doody, J., and Massague, J. (1996) Interaction of transforming growth factor-β receptor I with farnesyl-protein transferase-α in yeast and mammalian cells. J. Biol. Chem. 271, 13,931–13,934.PubMedCrossRefGoogle Scholar
  22. 22.
    Wang, T., Danielson, P. D., Li, B. Y., Shah, P. C., Kim, S. D., and Donahoe, P.K. (1996) The p21(RAS) farnesyltransferase a subunit in TGF-β and activin signaling. Science 271, 1120–1122.PubMedCrossRefGoogle Scholar
  23. 23.
    Kawabata, M., Imamura, T., Miyazono, K., Engel, M. E., and Moses, H. L. (1995) Interaction of the transforming growth factor-β type I receptor with farnesyl-protein transferase-α. J. Biol. Chem. 270, 29,628–29,631.PubMedCrossRefGoogle Scholar
  24. 24.
    Goalstone, M.L. and Draznin, B. J. (1996) Effect of insulin on farnesyltransferase activity in 3T3-L1 adipocytes. J. Biol. Chem. 271, 27,585–27,589.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1998

Authors and Affiliations

  • Jun Urano
    • 1
  • Fuyuhiko Tamanoi
    • 1
  1. 1.Department of Microbiology and Molecular GeneticsUCLALos Angeles

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