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An auxin-based degron system for the rapid depletion of proteins in nonplant cells

Abstract

Plants have evolved a unique system in which the plant hormone auxin directly induces rapid degradation of the AUX/IAA family of transcription repressors by a specific form of the SCF E3 ubiquitin ligase. Other eukaryotes lack the auxin response but share the SCF degradation pathway, allowing us to transplant the auxin-inducible degron (AID) system into nonplant cells and use a small molecule to conditionally control protein stability. The AID system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.

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Figure 1: Schematic illustration of the AID system.
Figure 2: A nuclear GFP fused with the aid degron can be controlled by the AID system in budding yeast.
Figure 3: Cell cycle–defective phenotype after Mcm4 depletion using the AID system.
Figure 4: Protein expression can be controlled by the AID system in mammalian cells.
Figure 5: Cell cycle phenotype of chicken DT40 cells after depletion of CENP-H mRNA or CENP-H protein.

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Acknowledgements

We thank K. Labib (Cancer Research UK) for critical reading of the manuscript, for support and for providing Myc and HA antibodies, yeast strains and tagging plasmids for budding yeast; S. Mimura (Nagoya University) for providing the cdc34-2 yeast strain; H. Kanazawa (Osaka University) for providing a GFP plasmid; T. Hori for help with flow cytometry analysis; and H. Araki, H. Masukata and K. Sugasawa for discussion. M.K. thanks Y. Tak for her support. This work was funded by a Challenging Exploratory Research and a Grant-in-Aid for Young Scientists (A) from the Ministry of Education, Science, Sports and Culture of Japan and by a Research Promotion Grant from the Uehara Memorial Foundation to M.K. K.N. is funded by a Japan Society for the Promotion of Science fellowship (DC2).

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Contributions

K.N. and M.K. designed and performed all experiments. T.F. supervised experiments using DT40. T.K. created the basic concept of AID to control protein levels in nonplant cells. M.K. and H.T. supervised and led this project. K.N. and M.K. wrote the manuscript. K.N., T.F., H.T., T.K. and M.K. discussed and checked the manuscript.

Corresponding author

Correspondence to Masato Kanemaki.

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Supplementary Figures 1–13 and Supplementary Table 1–3 (PDF 5386 kb)

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Nishimura, K., Fukagawa, T., Takisawa, H. et al. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6, 917–922 (2009). https://doi.org/10.1038/nmeth.1401

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