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PreprintCited by 118Biotin and Related StudiesAdvanced Fluorescence Microscopy TechniquesProtein Degradation and Inhibitors

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

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Lee, Song-Yi, Cheah, Joleen S., Zhao, Boxuan, Xu, Charles, Roh, Heegwang, Kim, Christina K., Cho, Kelvin F., Udeshi, Namrata D., Carr, Steven A., Ting, Alice Y.·2023·Nature Methods
DOI 10.1101/2023.03.09.531939PMC PMC10539039

Preprint — published as 10.1038/s41592-023-01880-5 · Paper page

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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 (PL), 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 PL 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 traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by BRET from luciferase, enabling interaction-dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL, expanding the scope of experimental questions that can be addressed with PL.

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Metadata

Authors
Lee, S.-Y., Cheah, J. S., Zhao, B., Xu, C., Roh, H., Kim, C. K., Cho, K. F., Udeshi, N. D., Carr, S. A., & Ting, A. Y.
Affiliations
  • Stanford University (US)
  • Broad Institute (US)
  • University of California, Davis (US)
  • Amgen (United States) (US)
  • Chan Zuckerberg Biohub San Francisco (US)
Corresponding author
Ting, Alice Y.
ORCID
Venue
Nature Methods
Publisher
openRxiv
Published
2023-03-09
Volume
20
Issue
6
Pages
908-917
ISSN
1548-7091 (ISSN-L)
DOI
10.1101/2023.03.09.531939
PMC
PMC10539039
OpenAlex
W4376618234
Cited by
118
References
1
Citations by year
2026: 32 · 2025: 45 · 2024: 29 · 2023: 12
FWCI
17.01
Type
journal article
Language
en
Open access
Locations
  • Nature Methods (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • PubMed Central (submittedVersion)
Funders
  • National Science Foundation (2014862, T32 GM007276, DGE-165651 8, 1656518, 1019469)
  • U.S. Department of Health and Human Services (R01-OD026223, RC2DK129964, T32 GM007276)
  • Burroughs Wellcome Fund (1019469, CASI 1019469)
  • National Research Foundation
  • National Research Foundation of Korea (NRF-2019R1A6A3A03033677)
  • National Institutes of Health (R01-OD026223)
  • Wu Tsai Neurosciences Institute, Stanford University
  • National Institute on Drug Abuse (R01DA026223)
  • National Institute of General Medical Sciences (T32GM007276)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK121409, RC2DK129964)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Biotin and Related Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
Keywords
TurboID, LOV domain, optogenetics, allosteric regulation, spatiotemporal control, bioluminescence resonance energy transfer, pulse-chase labeling, protein trafficking, endoplasmic reticulum, mitochondria, nuclear proteins, cellular stress, protein-protein interaction

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