Abstract
Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.
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Acknowledgments
We thank Dr. Akinobu Nakamura (ExCELLS, National Institutes of Natural Sciences) for his contribution to the development and application of the mDcTMP/DHFRiK6-based SLIPT system. This work was supported by JSPS Grants-in-Aid for Scientific Research (KAKENHI): grant nos. 15H03835, 15H05949 “Resonance Bio,” 18H02086, and 18H04546 and 20H04706 “Chemistry for Multimolecular Crowding Biosystems” (to S.T.). S.S. acknowledges scholarship support from the Hirota Scholarship Society and the SUNBOR Scholarship from the Suntory Foundation for Life Sciences.
Conflicts of Interest: S.S., T.Y., and S.T. are coinventors on a patent application related to this work. Y.H. declares no competing interests.
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Suzuki, S., Hatano, Y., Yoshii, T., Tsukiji, S. (2021). Chemogenetic Control of Protein Localization and Mammalian Cell Signaling by SLIPT. In: Kojima, R. (eds) Mammalian Cell Engineering. Methods in Molecular Biology, vol 2312. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1441-9_14
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DOI: https://doi.org/10.1007/978-1-0716-1441-9_14
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