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Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization

Abstract

We present here a protocol to conditionally control the nuclear trafficking of target proteins in yeast. In this system, rapamycin is used to heterodimerize two chimeric proteins. One chimera consists of a FK506-binding protein (FKBP12) fused to a cellular 'address' (nuclear localization signal or nuclear export sequence). The second chimera consists of a target protein fused to a fluorescent protein and the FKBP12-rapamycin-binding (FRB) domain from FKBP-12-rapamycin associated protein 1 (FRAP1, also known as mTor). Rapamycin induces dimerization of the FKBP12- and FRB-containing chimeras; these interactions selectively place the target protein under control of the cell address, thereby directing the protein into or out of the nucleus. By chemical-induced dimerization, protein mislocalization is reversible and enables the identification of conditional loss-of-function and gain-of-function phenotypes, in contrast to other systems that require permanent modification of the targeted protein. Yeast strains for this analysis can be constructed in 1 week, and the technique allows protein mislocalization within 15 min after drug treatment.

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Figure 1: Overview of this approach using chemical-induced dimerization to modulate the nuclear import and/or export of target proteins in yeast.
Figure 2: Workflow for application of the drug-directed nuclear trafficking system in yeast.
Figure 3: Coding sequence for the FRBPLF, FKBP12-NES and FKBP12-NLS fusions.
Figure 4: Sample quantified results from applications of chemical-induced mislocalization.
Figure 5: Sample microscope images indicating successful and unsuccessful applications of this system for drug-dependent control of nuclear protein localization in yeast.

References

  1. Shaw, J.P. et al. Identification of a putative regulator of early T cell activation genes. Science 241, 202–205 (1988).

    Article  CAS  PubMed  Google Scholar 

  2. Matheos, D.P., Kingsbury, T.J., Ahsan, U.S. & Cunningham, K.W. Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae. Genes Dev. 11, 3445–3458 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Stathopoulos, A.M. & Cyert, M.S. Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. Genes Dev. 11, 3432–3444 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Graef, I.A., Chen, F. & Crabtree, G.R. NFAT signaling in vertebrate development. Curr. Opin. Genet. Dev. 11, 505–512 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. O'Neill, E.M., Kaffman, A., Jolly, E.R. & O′Shea, E.K. Regulation of PHO4 nuclear localization by the PHO80-PHO85 cyclin-CDK complex. Science 271, 209–212 (1996).

    Article  CAS  PubMed  Google Scholar 

  6. Htun, H., Barsony, J., Renyi, I., Gould, D.L. & Hager, G.L. Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera. Proc. Natl Acad. Sci. USA 93, 4845–4850 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bharucha, N. et al. Analysis of the yeast kinome reveals a network of regulated protein localization during filamentous growth. Mol. Biol. Cell 19, 2708–2717 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Spencer, D.M., Wandless, T.J., Schreiber, S.L. & Crabtree, G. Controlling signal transduction with synthetic ligands. Science 262, 1019–1024 (1993).

    Article  CAS  PubMed  Google Scholar 

  9. Belshaw, P.J., Ho, S.N., Crabtree, G.R. & Schreiber, S.L. Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins. Proc. Natl Acad. Sci. USA 93, 4604–4607 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Liberles, S.D., Diver, S.T., Austin, D.J. & Schreiber, S.L. Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen. Proc. Natl Acad. Sci. USA 94, 7825–7830 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Harding, W.M., Galat, A., Uehling, D.E. & Schreiber, S.L. A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature 341, 758–760 (1989).

    Article  CAS  PubMed  Google Scholar 

  12. Bierer, B.E. et al. Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. Proc. Natl Acad. Sci. USA 87, 9231–9235 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Brown, E.J. et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369, 756–758 (1994).

    Article  CAS  PubMed  Google Scholar 

  14. Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S.H. RAFT1: a mammalian protein that binds FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78, 35–43 (1994).

    Article  CAS  PubMed  Google Scholar 

  15. Gestwicki, J.E. & Marinec, P.S. Chemical Control Over Protein-Protein Interactions: Beyond Inhibitors. Comb. Chem. High Throughput Screen 10, 667–675 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Lee, W.-C. & Melese, T. Identification and characterization of a nuclear localization sequence-binding protein in yeast. Proc. Natl Acad. Sci. USA 86, 8808–8812 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nelson, M. & Silver, P. Context affects nuclear protein localization in Saccharomyces cerevisiae. Mol. Cell. Biol. 9, 384–389 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Murphy, R. & Wente, S.R. An RNA-export mediator with an essential nuclear export signal. Nature 383, 357–360 (1996).

    Article  CAS  PubMed  Google Scholar 

  19. Geda, P. et al. A small molecule-directed approach to control protein localization and function. Yeast 25, 577–594 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Patury, S., Geda, P., Dobry, C.J., Kumar, A. & Gestwicki, J.E. Conditional nuclear import and export of yeast proteins using a chemical inducer of dimerization. Cell Biochem. Biophys. (2009).

  21. Briesewitz, R., Ray, G.T., Wandless, T.J. & Crabtree, G.R. Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces. Proc. Natl Acad. Sci. USA 96, 1953–1958 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Spencer, D.M., Graef, I., Austin, D.J., Schreiber, S.L. & Crabtree, G. A general strategy for producing conditional alleles of Src-like tyrosine kinases. Proc. Natl Acad. Sci. USA 92, 9805–9809 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Klemm, J.D., Beals, C.R. & Crabtree, G.R. Rapid targeting of nuclear proteins to the cytoplasm. Curr. Biol. 7, 638–644 (1997).

    Article  CAS  PubMed  Google Scholar 

  24. Stankunas, K. et al. Conditional protein alleles using knockin mice and a chemical inducer of dimerization. Mol. Cell 12, 1615–1624 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Liu, K.J., Arron, J.R., Stankunas, K., Crabtree, G.R. & Longaker, M.T. Chemical rescue of cleft palate and midline defects in conditional GSK-3beta mice. Nature 446, 79–82 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Komatsu, T. et al. Organelle-specific, rapid induction of molecular activities and membrane tethering. Nat. Methods 7, 206–208.

  27. Haruki, H., Nishikawa, J. & Laemmli, U.K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 31, 925–932 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Busch, A., Kiel, T. & Hubner, S. Quantification of nuclear protein transport using induced heterodimerization. Traffic 10, 1221–1227 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Costanzo, M.C. et al. New mutant phenotype data curation system in the Saccharomyces Genome Database. Database (Oxford) 2009, bap001 (2009).

    Article  Google Scholar 

  30. Siekierka, J.J., Hung, S.H.Y., Poe, M., Lin, C.S. & Sigal, N.H. A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature 341, 755–757 (1989).

    Article  CAS  PubMed  Google Scholar 

  31. Helliwell, S.B. et al. TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol. Biol. Cell 5, 105–118 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lorenz, M.C. & Heitman, J. TOR mutations confer rapamycin resistance by preventing interaction with FKBP12-rapamycin. J. Biol. Chem. 270, 27531–27537 (1995).

    Article  CAS  PubMed  Google Scholar 

  33. Stan, R. et al. Interaction between FKBP12-rapamycin and TOR involves a conserved serine residue. J. Biol. Chem. 269, 32027–32030 (1994).

    CAS  PubMed  Google Scholar 

  34. Bayle, J.H. et al. Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem. Biol. 13, 99–107 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Stankunas, K. et al. Rescue of degradation-prone mutants of the FK506-rapamycin binding (FRB) protein with chemical ligands. Chembiochem 8, 1162–1169 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Kumar, A. et al. Subcellular localization of the yeast proteome. Genes Dev. 16, 707–719 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Xu, T. et al. A profile of differentially abundant proteins at the yeast cell periphery during pseudohyphal growth. J. Biol. Chem. 285, 15476–15488.

  40. Luengo, J.I. et al. Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface. Chem. Biol. 2, 471–481 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. Hagen, G. & Mayr, H. Kinetics of the reactions of allylsilanes, allylgermanes, and allylstannanes with carbenium ions. J. Am. Chem. Soc. 113, 4954–4961 (1991).

    Article  CAS  Google Scholar 

  42. Jin, R., Dobry, C.J., McCown, P.J. & Kumar, A. Large-scale analysis of yeast filamentous growth by systematic gene disruption and overexpression. Mol. Biol. Cell 19, 284–296 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kumar, A. Multipurpose transposon-insertion libraries for large-scale analysis of gene function in yeast. Methods Mol. Biol. 416, 117–129 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Goldstein, A.L. & McCusker, J.H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553 (1999).

    Article  CAS  PubMed  Google Scholar 

  45. Keogh, M.C. et al. Cotranscriptional set2 methylation of histone H3 lysine 36 recruits a repressive Rpd3 complex. Cell 123, 593–605 (2005).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by grants from the NIH (to J.E.G.) and by grants from the American Cancer Society and NIH (to A.K.).

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A.K. and J.E.G. designed the research; all authors conducted experiments and contributed to the writing of the paper.

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Correspondence to Anuj Kumar.

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Xu, T., Johnson, C., Gestwicki, J. et al. Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization. Nat Protoc 5, 1831–1843 (2010). https://doi.org/10.1038/nprot.2010.141

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