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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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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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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DOI: https://doi.org/10.1038/nprot.2010.141
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