Directed evolution of TurboID for efficient proximity labeling in living cells and organisms
Posted on: 22 March 2018
Preprint posted on 2 October 2017
Article now published in Nature Biotechnology at http://dx.doi.org/10.1038/nbt.4201
Categories: biochemistry, cell biology
Context
How do we study spatially compartmentalized protein networks, macromolecular complexes and organelles in living cells? Classical methods include immunoprecipitation and biochemical fractionation. In recent years, proximity labeling that uses engineered APEX1 and BirA (BioID)2,3 enzymes to tag neighboring proteins with biotin in living cells has gained a substantial impact in the field of proteomics. APEX labeling is fast (<1 min), but requires the use of H2O2, which is toxic to cells, whereas BioID labeling is simple and non-toxic, but has slow kinetics (>18 h). These disadvantages limit APEX and BioID proximity labeling applications in vivo and in certain contexts, such as the analysis of protein complexes in the ER lumen, or for the analysis of fast cellular processes.
Key findings
The Ting lab used yeast display coupled to fluorescence-activated cell sorting to engineer two mutants of the biotin ligase BioID, termed TurboID and miniTurbo, with much greater catalytic efficiency than BioID. These variants not only allow shorter proximity labeling times (as little as 10 min), but do also enable biotinylation in a variety of different cellular organelles and in vivo in yeast, Drosophila and C. elegans. TurboID and miniTurbo have unique properties and adjustments, and show strength in distinct experimental context (maximization of biotinylation yield vs restriction of promiscuous biotinylation): TurboID is more active than miniTurbo, but uses low levels of biotin present in cells and organisms that are grown in typical biotin-containing media, and therefore yields a higher background in the absence of exogenous biotin.
Why I chose it
What I love about this preprint is that it shows the systematic continuous development of BioID with TurboID and miniTurbo and thereby increases our proximity labeling toolbox. I have used BioID to identify and study protein-protein interaction, in parallel to immunoprecipitation, and particularly liked its simplicity. These new tools further increase the scope of applications and processes that can be analyzed.
References
1) Rhee, H.-W. et al. Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging. Science 339, 1328–1331 (2013)
2) Roux, K.J., et al. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–810 (2012)
3) Kim, D.I. et al. An improved smaller biotin ligase for BioID proximity labeling. Mol. Biol. Cell 27, 1188–1196 (2016)
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