Engineered allostery in light-regulated lov-turbo enables precise spatiotemporal control of proximity labeling in living cells

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. Access through your institution Buy or subscribe This is a preview of subscription

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SCREENING IN MAMMALIAN CELLS Article Open access 02 June 2023 POT, AN OPTOGENETICS-BASED ENDOGENOUS PROTEIN DEGRADATION SYSTEM Article Open access 18 March 2025 PROXIMITY LABELING IN

MAMMALIAN CELLS WITH TURBOID AND SPLIT-TURBOID Article 02 November 2020 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

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Central  Google Scholar  Download references 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 * Christina K. Kim Present address: Center for Neuroscience and Department of Neurology, University of California, Davis, CA, USA

* Kelvin F. Cho Present address: Amgen Research, South San Francisco, CA, USA * These authors contributed equally: Song-Yi Lee, Joleen S. Cheah. AUTHORS AND AFFILIATIONS * Department of

Genetics, Stanford University, Stanford, CA, USA Song-Yi Lee, Boxuan Zhao, Christina K. Kim, Kelvin F. Cho & Alice Y. Ting * Department of Biology, Stanford University, Stanford, CA, USA

Joleen S. Cheah & Alice Y. Ting * Broad Institute of MIT and Harvard, Cambridge, MA, USA Charles Xu, Namrata D. Udeshi & Steven A. Carr * Department of Chemistry, Stanford

University, Stanford, CA, USA Heegwang Roh & Alice Y. Ting * Chan Zuckerberg Biohub–San Francisco, San Francisco, CA, USA Alice Y. Ting Authors * Song-Yi Lee View author publications You

can also search for this author inPubMed Google Scholar * Joleen S. Cheah View author publications You can also search for this author inPubMed Google Scholar * Boxuan Zhao View author

publications You can also search for this author inPubMed Google Scholar * Charles Xu View author publications You can also search for this author inPubMed Google Scholar * Heegwang Roh View

author publications You can also search for this author inPubMed Google Scholar * Christina K. Kim View author publications You can also search for this author inPubMed Google Scholar *

Kelvin F. Cho View author publications You can also search for this author inPubMed Google Scholar * Namrata D. Udeshi View author publications You can also search for this author inPubMed 

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author inPubMed Google Scholar 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. ETHICS DECLARATIONS 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. PEER REVIEW PEER REVIEW INFORMATION _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. ADDITIONAL

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

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applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 14 November 2022 *

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