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Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells

Nature Methods volume 20, pages 908–917 (2023)Cite this article

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Abstract

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. ‘LOV-Turbo’ works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

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Fig. 1: Design and directed evolution of LOV-Turbo.
Fig. 2: Characterization of LOV-Turbo.
Fig. 3: LOV-Turbo is reversible and works in multiple cell types and in the rodent brain.
Fig. 4: Applications of LOV-Turbo.
Fig. 5: Mapping proteome dynamics with LOV-Turbo by mass spectrometry.

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Data availability

The data associated with this study are available in the article and the Supplementary Information. The original mass spectra, spectral library and the protein sequence database used for searches have been deposited in the public proteomics repository MassIVE (http://massive.ucsd.edu) and are accessible at ftp://massive.ucsd.edu/MSV000090683/. Additional data beyond that provided in the figures and Supplementary Information are available from the corresponding author on request.

References

  1. Bruder, M., Polo, G. & Trivella, D. B. B. Natural allosteric modulators and their biological targets: molecular signatures and mechanisms. Nat. Prod. Rep. 37, 488–514 (2020).

    CAS  PubMed  Google Scholar 

  2. Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Sanchez, M. I., Nguyen, Q.-A., Wang, W., Soltesz, I. & Ting, A. Y. Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease. Proc. Natl Acad. Sci. USA 117, 33186–33196 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Kimberly, R. & Ranganathan, R. Hot spots for allosteric regulation on protein surfaces. Cell 147, 1564–1575 (2011).

    Google Scholar 

  5. Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880–887 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Qin, W., Cho, K. F., Cavanagh, P. E. & Ting, A. Y. Deciphering molecular interactions by proximity labeling. Nat. Methods 18, 133–143 (2021).

    CAS  PubMed  Google Scholar 

  7. Cho, K. F. et al. Proximity labeling in mammalian cells with TurboID and split-TurboID. Nat. Protoc. 15, 3971–3999 (2020).

    CAS  PubMed  Google Scholar 

  8. Wood, Z. A., Weaver, L. H., Brown, P. H., Beckett, D. & Matthews, B. W. Co-repressor induced order and biotin repressor dimerization: a case for divergent followed by convergent evolution. J. Mol. Biol. 357, 509–523 (2006).

    CAS  PubMed  Google Scholar 

  9. Kim, M. W. et al. Time-gated detection of protein-protein interactions with transcriptional readout. eLife 6, e30233 (2017).

    PubMed  PubMed Central  Google Scholar 

  10. Kim, C. K. et al. A molecular calcium integrator reveals a striatal cell type driving aversion. Cell 183, 2003–2019.e2016 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

    CAS  PubMed  Google Scholar 

  12. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Guntas, G. et al. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc. Natl Acad. Sci. USA 112, 112–117 (2015).

    CAS  PubMed  Google Scholar 

  14. Lee, D. et al. Temporally precise labeling and control of neuromodulatory circuits in the mammalian brain. Nat. Methods 14, 495–503 (2017).

    CAS  PubMed  Google Scholar 

  15. Kawano, F., Aono, Y., Suzuki, H. & Sato, M. Fluorescence imaging-based high-throughput screening of fast- and slow-cycling LOV proteins. PLoS ONE 8, e82693 (2013).

    PubMed  PubMed Central  Google Scholar 

  16. Wang, W. et al. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons. Nat. Biotechnol. 35, 864–871 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Eitoku, T., Nakasone, Y., Matsuoka, D., Tokutomi, S. & Terazima, M. Conformational dynamics of phototropin 2 LOV2 domain with the linker upon photoexcitation. J. Am. Chem. Soc. 127, 13238–13244 (2005).

    CAS  PubMed  Google Scholar 

  18. Welch, W. J. & Feramisco, J. R. Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells. J. Biol. Chem. 259, 4501–4513 (1984).

    CAS  PubMed  Google Scholar 

  19. Miyamoto, Y. et al. Cellular stresses induce the nuclear accumulation of importin α and cause a conventional nuclear import block. J. Cell Biol. 165, 617–623 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kim, C. K., Cho, K. F., Kim, M. W. & Ting, A. Y. Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions. eLife 8, e43826 (2019).

    PubMed  PubMed Central  Google Scholar 

  21. Hall, M. P. et al. Engineered Luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. Am. Chem. Soc. Chem. Biol. 7, 1848–1857 (2012).

    CAS  Google Scholar 

  22. Hedrick, M. N., Lonsdorf, A. S., Hwang, S. T. & Farber, J. M. CCR6 as a possible therapeutic target in psoriasis. Expert Opin. Ther. Targets 14, 911–922 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Olden, K., Pratt, R. M., Jaworski, C. & Yamada, K. M. Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin. Proc. Natl Acad. Sci. USA 76, 791–795 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Lytton, J., Westlin, M. & Hanley, M. R. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J. Biol. Chem. 266, 17067–17071 (1991).

    CAS  PubMed  Google Scholar 

  25. Haze, K., Yoshida, H., Yanagi, H., Yura, T. & Mori, K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol. Biol. Cell 10, 3787–3799 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Ye, J. et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol. Cell 6, 1355–1364 (2000).

    CAS  PubMed  Google Scholar 

  27. Berthel, E., Foray, N. & Ferlazzo, M. L. The nucleoshuttling of the ATM protein: a unified model to describe the individual response to high- and low-dose of radiation? Cancers 11, 905 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhao, S., Aviles, E. R. Jr & Fujikawa, D. G. Nuclear translocation of mitochondrial cytochrome c, lysosomal cathepsins B and D, and three other death-promoting proteins within the first 60 minutes of generalized seizures. J. Neurosci. Res. 88, 1727–1737 (2010).

    CAS  PubMed  Google Scholar 

  29. Billing, A. M. et al. Proteomic profiling of rapid non-genomic and concomitant genomic effects of acute restraint stress on rat thymocytes. J. Proteom. 75, 2064–2079 (2012).

    CAS  Google Scholar 

  30. Hotokezaka, Y., Katayama, I. & Nakamura, T. ATM-associated signalling triggers the unfolded protein response and cell death in response to stress. Commun. Biol. 3, 378 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Lee, J.-H. & Paull, T. T. Mitochondria at the crossroads of ATM-mediated stress signaling and regulation of reactive oxygen species. Redox Biol. 32, 101511 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Burman, J. L. et al. Scyl1, mutated in a recessive form of spinocerebellar neurodegeneration, regulates COPI-mediated retrograde traffic. J. Biol. Chem. 283, 22774–22786 (2008).

    CAS  PubMed  Google Scholar 

  33. Tang, Z. et al. Molecular cloning and characterization of a human gene involved in transcriptional regulation of hTERT. Biochem. Biophys. Res. Commun. 324, 1324–1332 (2004).

    CAS  PubMed  Google Scholar 

  34. Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Gene Ontology Consortium. The Gene Ontology resource: enriching a GOld mine. Nucleic Acids Res. 49, D325–d334 (2021).

    Google Scholar 

  36. Hung, V. et al. Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation. eLife 6, e24463 (2017).

    PubMed  PubMed Central  Google Scholar 

  37. Amodio, G. et al. Proteomic signatures in thapsigargin-treated hepatoma cells. Chem. Res. Toxicol. 24, 1215–1222 (2011).

    CAS  PubMed  Google Scholar 

  38. Cho, K. F. et al. Split-TurboID enables contact-dependent proximity labeling in cells. Proc. Natl Acad. Sci. USA 117, 12143–12154 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Hamasaki, M. et al. Autophagosomes form at ER–mitochondria contact sites. Nature 495, 389–393 (2013).

    CAS  PubMed  Google Scholar 

  40. Zabolotny, J. M. et al. PTP1B regulates leptin signal transduction in vivo. Dev. Cell 2, 489–495 (2002).

    CAS  PubMed  Google Scholar 

  41. Galic, S. et al. Coordinated regulation of insulin signaling by the protein tyrosine phosphatases PTP1B and TCPTP. Mol. Cell. Biol. 25, 819–829 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Kornicka-Garbowska, K., Bourebaba, L., Röcken, M. & Marycz, K. Inhibition of protein tyrosine phosphatase improves mitochondrial bioenergetics and dynamics, reduces oxidative stress, and enhances adipogenic differentiation potential in metabolically impaired progenitor stem cells. Cell Commun. Signal. 19, 106 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Kennedy, M. J. et al. Rapid blue-light–mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Kim, W.-Y. et al. ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light. Nature 449, 356–360 (2007).

    CAS  PubMed  Google Scholar 

  45. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Liu, Q. et al. A photoactivatable botulinum neurotoxin for inducible control of neurotransmission. Neuron 101, 863–875.e866 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Yazawa, M., Sadaghiani, A. M., Hsueh, B. & Dolmetsch, R. E. Induction of protein-protein interactions in live cells using light. Nat. Biotechnol. 27, 941–945 (2009).

    CAS  PubMed  Google Scholar 

  48. Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Liu, Y. et al. Spatiotemporally resolved subcellular phosphoproteomics. Proc. Natl Acad. Sci. USA 118, e2025299118 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Hananya, N., Ye, X., Koren, S. & Muir, T. W. A genetically encoded photoproximity labeling approach for mapping protein territories. Proc. Natl Acad. Sci. USA 120, e2219339120 (2023).

    CAS  PubMed  Google Scholar 

  51. Zhai, Y. et al. Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling. Nat. Commun. 13, 4906 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Chao, G. et al. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1, 755–768 (2006).

    CAS  PubMed  Google Scholar 

  53. Colby, D. W. et al. Engineering antibody affinity by yeast surface display. Methods Enzymol. 388, 348–358 (2004).

  54. Gagnon, K. T., Li, L., Janowski, B. A. & Corey, D. R. Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading. Nat. Protoc. 9, 2045–2060 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Senichkin, V. V., Prokhorova, E. A., Zhivotovsky, B. & Kopeina, G. S. Simple and efficient protocol for subcellular fractionation of normal and apoptotic cells. Cells 10, 852 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Rat cortical neurons were a kind gift from M. Lin (Stanford University). We thank J. Reinstein (Max Planck Institute) for helpful feedback. This work was supported by the NIH (grant nos. R01-DK121409 and RC2DK129964 to A.Y.T., R01-OD026223 to A.Y.T. and S.A.C. and T32GM007276 to J.S.C.), the Stanford Wu Tsai Neurosciences Institute (A.Y.T.), the National Science Foundation (NeuroNex grant no. 2014862 to A.Y.T. and GRFP DGE-1656518 to J.S.C.), the National Research Foundation of Korea grant no. NRF-2019R1A6A3A03033677 (S.-Y.L.), the Stanford Gerald J. Lieberman Fellowship (J.S.C.) and the Burroughs Wellcome Fund grant no. CASI 1019469 (C.K.K.). A.Y.T. is a Chan Zuckerberg Biohub – San Francisco Investigator.

Author information

Author notes

  1. Christina K. Kim

    Present address: Center for Neuroscience and Department of Neurology, University of California, Davis, CA, USA

  2. Kelvin F. Cho

    Present address: Amgen Research, South San Francisco, CA, USA

  3. These authors contributed equally: Song-Yi Lee, Joleen S. Cheah.

Authors and Affiliations

  1. Department of Genetics, Stanford University, Stanford, CA, USA

    Song-Yi Lee, Boxuan Zhao, Christina K. Kim, Kelvin F. Cho & Alice Y. Ting

  2. Department of Biology, Stanford University, Stanford, CA, USA

    Joleen S. Cheah & Alice Y. Ting

  3. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Charles Xu, Namrata D. Udeshi & Steven A. Carr

  4. Department of Chemistry, Stanford University, Stanford, CA, USA

    Heegwang Roh & Alice Y. Ting

  5. Chan Zuckerberg Biohub–San Francisco, San Francisco, CA, USA

    Alice Y. Ting

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Contributions

S.-Y.L. and A.Y.T. conceived this project. S.-Y.L., J.S.C. and A.Y.T. designed experiments and analyzed all the data except those noted. S.-Y.L. and J.S.C. performed all experiments, unless otherwise noted. N.D.U., C.X. and S.A.C. performed post-streptavidin-enrichment sample processing, mass spectrometry, and initial data analysis. B.Z. and S.-Y.L. performed the mouse brain experiments. C.K.K., K.F.C. and S.-Y.L. performed cultured rat cortical neuron experiments. H.R. and S.-Y.L. performed BRET experiments. S.-Y.L., J.S.C. and A.Y.T. wrote the paper with input from all authors.

Corresponding author

Correspondence to Alice Y. Ting.

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Competing interests

S.-Y.L., J.S.C. and A.Y.T. have filed a patent application covering some aspects of this work (US Provisional Patent Application No. 63/488,940; CZ SF ref. CZB-273S-P1; Stanford ref. S22-487; KT ref. 110221-1361830-009500PR). The remaining authors declare no competing interests.

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Nature Methods thanks Angelos Constantinou, Tatsuya Sawasaki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Table 1, Legends of Tables 2–4, Note 1, Methods, Antibodies list, Genetic constructs list and References.

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Supplementary Table 2–4

Table 2: Proteomic data for ERM to nucleus pulse-chase experiment. Table 3: Proteomic data for ERM to mitochondria pulse-chase experiment. Table 4: Proteomic data at peptide level for ERM to nucleus pulse-chase experiment.

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Lee, SY., Cheah, J.S., Zhao, B. et al. Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells. Nat Methods 20, 908–917 (2023). https://doi.org/10.1038/s41592-023-01880-5

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