Engineered allostery in a light-regulated version of TurboID for precise spatiotemporal control of proximity labeling in living cells

ABSTRACT: Lee SY, Chea JS, Zhao BX, Xu C, Udeshi ND, Roh H, Kim C, Cho K, Carr SA, Ting A. 2022 The incorporation of light-responsive domains into engineered proteins has produced optogenetic tools with the ability to regulate protein localization, interactions, and function with light. We introduced this mode of regulation into proximity labeling (PL), which has been a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through a combination of structure-guided screening and yeast display directed evolution, we inserted the light sensitive LOV domain into a surface exposed loop of the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. We showed that "LOV-Turbo" works in multiple organelles and cell types and can dramatically reduce background labeling in biotin-rich environments such as living neurons. We utilized LOV-Turbo's reversibility to perform pulse-chase labeling followed by organelle fractionation to discover endogenous proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. Lastly, we showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from proximal luciferase in living cells, opening the door to chemical and/or interaction dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL and expands the scope of experimental questions that can be addressed using PL.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Steven A. Carr  

PROVIDER: MSV000090683 | MassIVE | Wed Nov 09 14:24:00 GMT 2022

REPOSITORIES: MassIVE