Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Efficient proximity labeling in living cells and organisms with TurboID

Nature Biotechnology volume 36, pages 880–887 (2018)Cite this article

Subjects

An Author Correction to this article was published on 20 November 2019

Abstract

Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, current PL methods require over 18 h of labeling time or utilize chemicals with limited cell permeability or high toxicity. We used yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which catalyze PL with much greater efficiency than BioID or BioID2, and enable 10-min PL in cells with non-toxic and easily deliverable biotin. Furthermore, TurboID extends biotin-based PL to flies and worms.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Directed evolution of TurboID.
Figure 2: Characterization of TurboID and miniTurbo in mammalian cells.
Figure 3: TurboID and miniTurbo in flies, worms, and other species.

References

  1. Kim, D.I. & Roux, K.J. Filling the void: proximity-based labeling of proteins in living cells. Trends Cell Biol. 26, 804–817 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Rhee, H.-W. et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Choi-Rhee, E., Schulman, H. & Cronan, J.E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Sci. 13, 3043–3050 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Roux, K.J., Kim, D.I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–810 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Paek, J. et al. Multidimensional tracking of GPCR signaling via peroxidase-catalyzed proximity labeling. Cell 169, 338–349.e11 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lobingier, B.T. et al. An approach to spatiotemporally resolve protein interaction networks in living cells. Cell 169, 350–360.e12 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kaewsapsak, P., Shechner, D.M., Mallard, W., Rinn, J.L. & Ting, A.Y. Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking. eLife 6, e29224 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Martell, J.D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gupta, G.D. et al. a dynamic protein interaction landscape of the human centrosome-cilium interface. Cell 163, 1484–1499 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kim, D.I. et al. Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc. Natl. Acad. Sci. USA 111, E2453–E2461 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lin, Q. et al. Screening of proximal and interacting proteins in rice protoplasts by proximity-dependent biotinylation. Front. Plant Sci. 8, 749 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Morriswood, B. et al. Novel bilobe components in Trypanosoma brucei identified using proximity-dependent biotinylation. Eukaryot. Cell 12, 356–367 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chen, A.L. et al. Novel components of the Toxoplasma inner membrane complex revealed by BioID. MBio 6, e02357–e14 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Nadipuram, S.M. et al. In vivo biotinylation of the toxoplasma parasitophorous vacuole reveals novel dense granule proteins important for parasite growth and pathogenesis. MBio 7, e00808–16 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Chen, A.L. et al. Novel insights into the composition and function of the Toxoplasma IMC sutures. Cell. Microbiol. 19, e12678 (2017).

    Article  CAS  Google Scholar 

  17. Long, S. et al. Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii. PLoS Pathog. 13, e1006379 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Zhou, Q., Hu, H. & Li, Z. An EF-hand-containing protein in Trypanosoma brucei regulates cytokinesis initiation by maintaining the stability of the cytokinesis initiation factor CIF1. J. Biol. Chem. 291, 14395–14409 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dang, H.Q. et al. Proximity interactions among basal body components in Trypanosoma brucei identify novel regulators of basal body biogenesis and inheritance. MBio 8, e02120–16 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kehrer, J., Frischknecht, F. & Mair, G.R. Proteomic analysis of the Plasmodium berghei gametocyte egressome and vesicular bioID of osmiophilic body proteins identifies merozoite TRAP-like protein (MTRAP) as an essential factor for parasite transmission. Mol. Cell. Proteomics 15, 2852–2862 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gaji, R.Y. et al. Phosphorylation of a myosin motor by TgCDPK3 facilitates rapid initiation of motility during Toxoplasma gondii egress. PLoS Pathog. 11, e1005268 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Batsios, P., Ren, X., Baumann, O., Larochelle, D.A. & Gräf, R. Src1 is a protein of the inner nuclear membrane interacting with the Dictyostelium Lamin NE81. Cells 5, 13 (2016).

    Article  PubMed Central  CAS  Google Scholar 

  23. Meyer, I. et al. CP39, CP75 and CP91 are major structural components of the Dictyostelium centrosome's core structure. Eur. J. Cell Biol. 96, 119–130 (2017).

    Article  CAS  PubMed  Google Scholar 

  24. Uezu, A. et al. Identification of an elaborate complex mediating postsynaptic inhibition. Science 353, 1123–1129 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Opitz, N. et al. Capturing the Asc1p/receptor for activated C kinase 1 (RACK1) microenvironment at the head region of the 40S ribosome with quantitative BioID in yeast. Mol. Cell. Proteomics 16, 2199–2218 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kim, D.I. et al. An improved smaller biotin ligase for BioID proximity labeling. Mol. Biol. Cell 27, 1188–1196 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ramanathan, M. et al. RNA-protein interaction detection in living cells. Nat. Methods 15, 207–212 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Birendra, K.C. et al. VRK2A is an A-type lamin-dependent nuclear envelope kinase that phosphorylates BAF. Mol. Biol. Cell 28, 2241–2250 (2017).

    Article  CAS  PubMed Central  Google Scholar 

  29. Redwine, W.B. et al. The human cytoplasmic dynein interactome reveals novel activators of motility. eLife 6, e28257 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Jung, E.M. et al. Arid1b haploinsufficiency disrupts cortical interneuron development and mouse behavior. Nat. Neurosci. 20, 1694–1707 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Martell, J.D. et al. A split horseradish peroxidase for the detection of intercellular protein-protein interactions and sensitive visualization of synapses. Nat. Biotechnol. 34, 774–780 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bobrow, M.N., Harris, T.D., Shaughnessy, K.J. & Litt, G.J. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. Immunol. Methods 125, 279–285 (1989).

    Article  CAS  PubMed  Google Scholar 

  33. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Dingar, D. et al. BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors. J. Proteomics 118, 95–111 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. Reinke, A.W., Balla, K.M., Bennett, E.J. & Troemel, E.R. Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins. Nat. Commun. 8, 14023 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Reinke, A.W., Mak, R., Troemel, E.R. & Bennett, E.J. In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans. Sci. Adv. 3, e1602426 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Chen, C.-L. Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase. Proc. Natl. Acad. Sci. USA 112, 12093–12098 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Weaver, L.H., Kwon, K., Beckett, D. & Matthews, B.W. Corepressor-induced organization and assembly of the biotin repressor: a model for allosteric activation of a transcriptional regulator. Proc. Natl. Acad. Sci. USA 98, 6045–6050 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  40. Jan, C.H., Williams, C.C. & Weissman, J.S. LOCAL TRANSLATION. response to comment on “Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling”. Science 348, 1217 (2015).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  42. Ausubel, F.M. et al. Current protocols in molecular biology. Mol. Biol. 1, 13.2.1 (2003).

    Google Scholar 

  43. 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).

    Article  CAS  PubMed  Google Scholar 

  44. Xu, Y. & Beckett, D. Evidence for interdomain interaction in the Escherichia coli repressor of biotin biosynthesis from studies of an N-terminal domain deletion mutant. Biochemistry 35, 1783–1792 (1996).

    Article  CAS  PubMed  Google Scholar 

  45. Eginton, C., Cressman, W.J., Bachas, S., Wade, H. & Beckett, D. Allosteric coupling via distant disorder-to-order transitions. J. Mol. Biol. 427, 1695–1704 (2015).

    Article  CAS  PubMed  Google Scholar 

  46. Hung, V. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat. Protoc. 11, 456–475 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Apweiler, R. et al. UniProt: the universal protein knowledgebase. Nucleic Acids Res. 45, D158–D169 (2017).

    Article  CAS  Google Scholar 

  48. Käll, L., Krogh, A. & Sonnhammer, E.L.L. A combined transmembrane topology and signal peptide prediction method. J. Mol. Biol. 338, 1027–1036 (2004).

    Article  PubMed  CAS  Google Scholar 

  49. Gene Ontology Consortium. Gene Ontology Consortium: going forward. Nucleic Acids Res. 43, D1049–D1056 (2015).

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Thul, P.J. et al. A subcellular map of the human proteome. Science 356, eaal3321 (2017).

    Article  PubMed  CAS  Google Scholar 

  52. Muthusamy, B. et al. Plasma Proteome Database as a resource for proteomics research. Proteomics 5, 3531–3536 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E.L.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001).

    Article  CAS  PubMed  Google Scholar 

  54. Calvo, S.E., Clauser, K.R. & Mootha, V.K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 44, D1251–D1257 (2016).

    Article  CAS  PubMed  Google Scholar 

  55. Pagliarini, D.J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112–123 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Sultan, M. et al. A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science 321, 956–960 (2008).

    Article  CAS  PubMed  Google Scholar 

  57. Perkins, L.A. et al. The transgenic RNAi project at Harvard medical school: resources and validation. Genetics 201, 843–852 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Markstein, M., Pitsouli, C., Villalta, C., Celniker, S.E. & Perrimon, N. Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat. Genet. 40, 476–483 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Ortega-Cuellar, D. et al. Biotin starvation with adequate glucose provision causes paradoxical changes in fuel metabolism gene expression similar in rat (Rattus norvegicus), nematode (Caenorhabditis elegans) and yeast (Saccharomycces cerevisiae). J. Nutrigenet. Nutrigenomics 3, 18–30 (2010).

    Article  CAS  PubMed  Google Scholar 

  61. Leung, B., Hermann, G.J. & Priess, J.R. Organogenesis of the Caenorhabditis elegans intestine. Dev. Biol. 216, 114–134 (1999).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

FACS was performed at the Koch Institute Flow Cytometry Core (MIT) and Stanford Shared FACS Facility. S. Han (Stanford) synthesized neutravidin-AlexaFluor647. S. Ax (Stanford) cloned the cell surface TurboID and miniTurbo constructs. We are grateful to I. Droujinine (Harvard) for advice on biotin labeling in D. melanogaster. Biotin auxotrophic E. coli MG1655bioB:kan was kindly donated by J. Cronan (University of Illinois). This work was supported by NIH R01-CA186568 (to A.Y.T.), Howard Hughes Medical Institute Collaborative Innovation Award (to A.Y.T., S.C., and N.P.), and NIH New Innovator Award DP2GM119136 (to J.L.F.). T.C.B. was supported by Dow Graduate Research and Lester Wolfe Fellowships. J.A.B. was supported by a Damon Runyon Post-Doctoral Fellowship. A.D.S. was supported by NIH Training Grant 2T32GM007276.

Author information

Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Tess C Branon & Alice Y Ting

  2. Departments of Genetics, Stanford University, Stanford, California, USA

    Tess C Branon & Alice Y Ting

  3. Department of Chemistry, Stanford University, Stanford, California, USA

    Tess C Branon & Alice Y Ting

  4. Department of Biology, Stanford University, Stanford, California, USA

    Tess C Branon, Ariana D Sanchez, Jessica L Feldman & Alice Y Ting

  5. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Justin A Bosch & Norbert Perrimon

  6. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Namrata D Udeshi, Tanya Svinkina & Steven A Carr

  7. Howard Hughes Medical Institute, Boston, Massachusetts, USA

    Norbert Perrimon

  8. Chan Zuckerberg Biohub, San Francisco, California, USA

    Alice Y Ting

Authors
  1. Tess C Branon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Justin A Bosch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ariana D Sanchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Namrata D Udeshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tanya Svinkina
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Steven A Carr
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jessica L Feldman
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Norbert Perrimon
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alice Y Ting
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.C.B. and A.Y.T. designed the research and analyzed all the data except those noted. T.C.B. performed all experiments except those noted. T.C.B., A.Y.T., N.D.U., and S.A.C. designed the proteomics experiments. T.C.B. prepared the proteomic samples. N.D.U. and T.S. processed the proteomic samples and performed mass spectrometry. J.A.B. performed D. melanogaster experiments. J.A.B. and N.P. analyzed D. melanogaster data. T.C.B., A.Y.T., A.D.S., and J.L.F. designed the C. elegans experiments. A.D.S. performed C. elegans experiments. A.D.S. and J.L.F. analyzed C. elegans data.

Corresponding author

Correspondence to Alice Y Ting.

Ethics declarations

Competing interests

A.Y.T. and T.C.B. have filed a patent application covering some aspects of this work.

Supplementary information

Supplementary Figures, Tables, and Notes

Supplementary Figures 1–16, Supplementary Table 1 and 8–9, Supplementary Notes 1–5 (PDF 7844 kb)

Reporting Summary (PDF 1346 kb)

Supplementary Table 2

True positive and false positive lists used to filter and analyze ERM datasets (XLSX 1721 kb)

Supplementary Table 3

True positive and false positive lists used to filter and analyse nuclear proteomic datasets. (XLSX 1038 kb)

Supplementary Table 4

True positive and false positive lists used to filter and analyse mitochondrial matrix proteomic datasets. (XLSX 377 kb)

Supplementary Table 5

ER membrane (ERM) proteomic data. (XLSX 1331 kb)

Supplementary Table 6

Nuclear proteomic data (XLSX 1393 kb)

Supplementary Table 7

Mitochondrial matrix proteomic data. (XLSX 1064 kb)

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Branon, T., Bosch, J., Sanchez, A. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol 36, 880–887 (2018). https://doi.org/10.1038/nbt.4201

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.4201

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research