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Proximity labeling in mammalian cells with TurboID and split-TurboID

Abstract

This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein–protein interactions and subcellular proteomes in live mammalian cells. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin–AMP, a reactive intermediate that covalently labels proximal proteins. Optimized using directed evolution, TurboID has substantially higher activity than previously described biotin ligase–related proximity labeling methods, such as BioID, enabling higher temporal resolution and broader application in vivo. Split-TurboID consists of two inactive fragments of TurboID that can be reconstituted through protein–protein interactions or organelle–organelle interactions, which can facilitate greater targeting specificity than full-length enzymes alone. Proteins biotinylated by TurboID or split-TurboID are then enriched with streptavidin beads and identified by mass spectrometry. Here, we describe fusion construct design and characterization (variable timing), proteomic sample preparation (5–7 d), mass spectrometric data acquisition (2 d), and proteomic data analysis (1 week).

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Fig. 1: Proximity-dependent biotinylation catalyzed by TurboID and split-TurboID.
Fig. 2: Workflow for performing a TurboID proteomic experiment.
Fig. 3: Example data characterizing ERM-targeted TurboID.
Fig. 4: Proteomic data analysis using the ratiometric approach.
Fig. 5: Example experimental design and analysis using split-TurboID for proteomic mapping of ER–mitochondria contacts.

Data availability

The data presented in this paper have been previously published, and associated raw data are provided in the original articles6,7.

References

  1. Huber, L. A., Pfaller, K. & Vietor, I. Organelle proteomics: implications for subcellular fractionation in proteomics. Circ. Res. 92, 962–968 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Stasyk, T. & Huber, L. A. Zooming in: fractionation strategies in proteomics. Proteomics 4, 3704–3716 (2004).

    Article  CAS  PubMed  Google Scholar 

  4. Lee, W. C. & Lee, K. H. Applications of affinity chromatography in proteomics. Anal. Biochem. 324, 1–10 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Gingras, A. C., Abe, K. T. & Raught, B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles. Curr. Opin. Chem. Biol. 48, 44–54 (2019).

    Article  CAS  PubMed  Google Scholar 

  6. Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880–887 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Cho, K. F. et al. Split-TurboID enables contact-dependent proximity labeling in cells. Proc. Natl Acad. Sci. USA 117, 12143–12154 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Udeshi, N. D. et al. Antibodies to biotin enable large-scale detection of biotinylation sites on proteins. Nat. Methods 14, 1167–1170 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Fazal, F. M. et al. Atlas of subcellular RNA localization revealed by APEX-Seq. Cell 178, 473–490.e26 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9-APEX-mediated proximity labeling. Nat. Methods 15, 437–439 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Michalski, A. et al. Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol. Cell. Proteom. 10, M111.011015 (2011).

    Article  Google Scholar 

  12. Eliuk, S. & Makarov, A. Evolution of Orbitrap mass spectrometry instrumentation. Annu. Rev. Anal. Chem. 8, 61–80 (2015).

    Article  Google Scholar 

  13. Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).

    Article  CAS  PubMed  Google Scholar 

  14. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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 

  16. Mortensen, A. & Skibsted, L. H. Importance of carotenoid structure in radical-scavenging reactions. J. Agric. Food Chem. 45, 2970–2977 (1997).

    Article  CAS  Google Scholar 

  17. Wishart, J. F. & Rao, B. S. M. Recent Trends in Radiation Chemistry (World Scientific, 2010). https://doi.org/10.1142/7413

  18. 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 

  19. Rodríguez-López, J. N. et al. Mechanism of reaction of hydrogen peroxide with horseradish peroxidase: identification of intermediates in the catalytic cycle. J. Am. Chem. Soc. 123, 11838–11847 (2001).

    Article  PubMed  CAS  Google Scholar 

  20. Loh, K. H. et al. Proteomic analysis of unbounded cellular compartments: synaptic clefts. Cell 166, 1295–1307.e21 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bar, D. Z. et al. Biotinylation by antibody recognition—a method for proximity labeling. Nat. Methods 15, 127–133 (2018).

    Article  CAS  PubMed  Google Scholar 

  22. Honke, K. & Kotani, N. The enzyme-mediated activation of radical source reaction: a new approach to identify partners of a given molecule in membrane microdomains. J. Neurochem 116, 690–695 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Li, X.-W. et al. New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay. J. Biol. Chem. 289, 14434–14447 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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 

  25. 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 

  26. 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 

  27. 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 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Choi-Rhee, E., Schulman, H. & Cronan, J. E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Sci. 13, 3043–3050 (2008).

    Article  CAS  Google Scholar 

  30. 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 

  31. Kido, K. et al. Airid, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions. eLife 9, e54983 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 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 

  33. 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 

  34. 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 

  35. Mair, A., Xu, S. L., Branon, T. C., Ting, A. Y. & Bergmann, D. C. Proximity labeling of protein complexes and cell type specific organellar proteomes in Arabidopsis enabled by TurboID. eLife 8, e47864 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhang, Y. et al. TurboID-based proximity labeling reveals that UBR7 is a regulator of N NLR immune receptor-mediated immunity. Nat. Commun. 10, 3252 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Larochelle, M., Bergeron, D., Arcand, B. & Bachand, F. Proximity-dependent biotinylation mediated by TurboID to identify protein-protein interaction networks in yeast. J. Cell Sci. 132, jcs232249 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Struk, S. et al. Exploring the protein–protein interaction landscape in plants. Plant Cell Environ. 42, 387–409 (2019).

    Article  CAS  PubMed  Google Scholar 

  39. 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. Proteom. 16, 2199–2218 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 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 

  42. Khan, M., Youn, J. Y., Gingras, A. C., Subramaniam, R. & Desveaux, D. In planta proximity dependent biotin identification (BioID). Sci. Rep. 8, 1123 (2018).

    Article  CAS  Google Scholar 

  43. Conlan, B., Stoll, T., Gorman, J. J., Saur, I. & Rathjen, J. P. Development of a rapid in planta bioid system as a probe for plasma membrane-associated immunity proteins. Front. Plant Sci. 9, 1882 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Roux, K. J., Kim, D. I., Burke, B. & May, D. G. BioID: a screen for protein-protein interactions. Curr. Protoc. Protein Sci. 91, 19.23.1–19.23.15 (2018).

    CAS  Google Scholar 

  45. May, D. G., Scott, K. L., Campos, A. R. & Roux, K. J. Comparative application of BioID and TurboID for protein-proximity biotinylation. Cells 9, 1070 (2020).

    Article  PubMed Central  CAS  Google Scholar 

  46. Chapman-Smith, A. & Cronan, J. E. Jr Molecular biology of biotin attachment to proteins. J. Nutr. 129, 477S–484S (1999).

    Article  CAS  PubMed  Google Scholar 

  47. Han, Y. et al. Directed evolution of split APEX2 peroxidase. ACS Chem. Biol. 14, 619–635 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 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 

  49. De Munter, S. et al. Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions. FEBS Lett. 591, 415–424 (2017).

    Article  PubMed  CAS  Google Scholar 

  50. Schopp, I. M. et al. Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes. Nat. Commun. 8, 15690 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Kwak, C. et al. Contact-ID, a new tool for profiling organelle contact site, reveals proteins of mitochondrial-associated membrane formation. Proc. Natl Acad. Sci. USA 117, 12109–12120 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. McClellan, D. et al. Growth factor independence 1B-mediated transcriptional repression and lineage allocation require lysine-specific demethylase 1-dependent recruitment of the BHC complex. Mol. Cell. Biol. 39, e00020-19 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Lambert, J. P. et al. Interactome rewiring following pharmacological targeting of BET bromodomains. Mol. Cell 73, 621–638.e17 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  55. Couzens, A. L. et al. Protein interaction network of the mammalian hippo pathway reveals mechanisms of kinase-phosphatase interactions. Sci. Signal. 6, rs15–rs15 (2013).

    Article  PubMed  CAS  Google Scholar 

  56. 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 

  57. Youn, J. Y. et al. High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies. Mol. Cell 69, 517–532.e11 (2018).

    Article  CAS  PubMed  Google Scholar 

  58. Firat-Karalar, E. N., Rauniyar, N., Yates, J. R. & Stearns, T. Proximity interactions among centrosome components identify regulators of centriole duplication. Curr. Biol. 24, 664–670 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Chou, C. C. et al. TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Nat. Neurosci. 21, 228–239 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kabeiseman, E. J., Cichos, K. H. & Moore, E. R. The eukaryotic signal sequence, YGRL, targets the chlamydial inclusion. Front. Cell. Infect. Microbiol. 4, 129 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Mojica, S. A. et al. SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors. Mol. Biol. Cell 26, 1918–1934 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Le Sage, V., Cinti, A., Valiente-Echeverría, F. & Mouland, A. J. Proteomic analysis of HIV-1 Gag interacting partners using proximity-dependent biotinylation. Virol. J. 12, 138 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Ritchie, C., Cylinder, I., Platt, E. J. & Barklis, E. Analysis of HIV-1 Gag protein interactions via biotin ligase tagging. J. Virol. 89, 3988–4001 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Kueck, T. et al. Serine phosphorylation of HIV-1 Vpu and its binding to tetherin regulates interaction with clathrin adaptors. PLoS Pathog. 11, e1005141 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Holthusen, K., Talaty, P. & Everly, D. N. Regulation of latent membrane protein 1 signaling through interaction with cytoskeletal proteins. J. Virol. 89, 7277–7290 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Coyaud, E. et al. Global interactomics uncovers extensive organellar targeting by Zika virus. Mol. Cell. Proteom. 17, 2242–2255 (2018).

    Article  CAS  Google Scholar 

  67. Rider, M. A. et al. The interactome of EBV LMP1 evaluated by proximity-based BioID approach. Virology 516, 55–70 (2018).

    Article  CAS  PubMed  Google Scholar 

  68. Cheerathodi, M. R. & Meckes, D. G. BioID combined with mass spectrometry to study herpesvirus protein–protein interaction networks. Methods Mol. Biol. 2060, 327–341 (2020).

    Article  CAS  PubMed  Google Scholar 

  69. Bradley, P. J., Rayatpisheh, S., Wohlschlegel, J. A. & Nadipuram, S. M. Using BioID for the identification of interacting and proximal proteins in subcellular compartments in Toxoplasma gondii. Methods Mol. Biol. 2071, 323–346 (2020).

    Article  CAS  PubMed  Google Scholar 

  70. Gillingham, A. K., Bertram, J., Begum, F. & Munro, S. In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation. eLife 8, e45916 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Hoyer, M. J. et al. A novel class of ER membrane proteins regulates ER-associated endosome fission. Cell 175, 254–265.e14 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. van Vliet, A. R. et al. The ER stress sensor PERK coordinates ER-plasma membrane contact site formation through interaction with filamin-A and F-actin remodeling. Mol. Cell 65, 885–899.e6 (2017).

    Article  PubMed  CAS  Google Scholar 

  73. Spence, E. F. et al. In vivo proximity proteomics of nascent synapses reveals a novel regulator of cytoskeleton-mediated synaptic maturation. Nat. Commun. 10, 386 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  74. Feng, W. et al. Identifying the cardiac dyad proteome in vivo by a BioID2 knock-in strategy. Circulation 141, 940–942 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  75. 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 

  76. Hung, V. et al. Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging. Mol. Cell 55, 332–341 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Han, S. et al. Proximity biotinylation as a method for mapping proteins associated with mtDNA in living cells. Cell Chem. Biol. 24, 404–414 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. 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 

  79. Mertins, P. et al. Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry. Nat. Protoc. 13, 1632–1661 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Li, J. et al. Cell-surface proteomic profiling in the fly brain uncovers wiring regulators. Cell 180, 373–386.e15 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Vandemoortele, G. et al. A well-controlled BioID design for endogenous bait proteins. J. Proteome Res. 18, 95–106 (2019).

    CAS  PubMed  Google Scholar 

  82. Bian, Y. et al. Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS. Nat. Commun. 11, 157 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. 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 

  84. Ashburner, M. et al. Gene ontology: tool for the unification of biology. Nat. Genet. 25, 25–29 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

  86. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    Article  CAS  PubMed  Google Scholar 

  87. Bateman, A. et al. UniProt: the universal protein knowledgebase. Nucleic Acids Res 45, D158–D169 (2017).

    Article  CAS  Google Scholar 

  88. Lee, S. Y. et al. APEX fingerprinting reveals the subcellular localization of proteins of interest. Cell Rep. 15, 1837–1847 (2016).

    Article  CAS  PubMed  Google Scholar 

  89. Cho, I. T. et al. Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum–mitochondrial contacts. J. Biol. Chem. 292, 16382–16392 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Cao, Q. et al. PAQR3 regulates endoplasmic reticulum-to-Golgi trafficking of COPII vesicle via interaction with Sec13/Sec31 coat proteins. iScience 9, 382–398 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Le Guerroué, F. et al. Autophagosomal content profiling reveals an LC3C-dependent piecemeal mitophagy pathway. Mol. Cell 68, 786–796.e6 (2017).

    Article  PubMed  CAS  Google Scholar 

  92. Mick, D. U. et al. Proteomics of primary cilia by proximity labeling. Dev. Cell 35, 497–512 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Santin, Y. G. et al. In vivo TssA proximity labelling during type VI secretion biogenesis reveals TagA as a protein that stops and holds the sheath. Nat. Microbiol. 3, 1304–1313 (2018).

    Article  CAS  PubMed  Google Scholar 

  94. Mannix, K. M., Starble, R. M., Kaufman, R. S. & Cooley, L. Proximity labeling reveals novel interactomes in live Drosophila tissue. Development 146, dev176644 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Liu, G. et al. Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics. Nature 577, 695–700 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Chojnowski, A. et al. Progerin reduces LAP2α-telomere association in Hutchinson-Gilford progeria. eLife 4, 1–21 (2015).

    Article  Google Scholar 

  97. Cross, S. H. et al. The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification. PLoS Genet 16, e1008583 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Pagac, M. et al. SEIPIN regulates lipid droplet expansion and adipocyte development by modulating the activity of glycerol-3-phosphate acyltransferase. Cell Rep. 17, 1546–1559 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Cole, A. et al. Inhibition of the mitochondrial protease ClpP as a therapeutic strategy for human acute myeloid leukemia. Cancer Cell 27, 864–876 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Janer, A. et al. SLC 25A46 is required for mitochondrial lipid homeostasis and cristae maintenance and is responsible for Leigh syndrome. EMBO Mol. Med. 8, 1019–1038 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Antonicka, H. et al. A pseudouridine synthase module is essential for mitochondrial protein synthesis and cell viability. EMBO Rep. 18, 28–38 (2017).

    Article  CAS  PubMed  Google Scholar 

  102. Van Itallie, C. M. et al. Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion. J. Cell Sci. 127, 885–895 (2014).

    PubMed  PubMed Central  Google Scholar 

  103. Guo, Z. et al. E-cadherin interactome complexity and robustness resolved by quantitative proteomics. Sci. Signal. 7, rs7 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  104. Hua, R. et al. VAPs and ACBD5 tether peroxisomes to the ER for peroxisome maintenance and lipid homeostasis. J. Cell Biol. 216, 367–377 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Chan, C. J. et al. BioID performed on Golgi enriched fractions identify C10orf76 as a GBF1 binding protein essential for Golgi maintenance and secretion. Mol. Cell. Proteom. 18, 2285–2297 (2019).

    Article  CAS  Google Scholar 

  106. Opitz, N. et al. Capturing the Asc1p/ R eceptor for A ctivated C K inase 1 (RACK1) microenvironment at the head region of the 40S ribosome with quantitative BioID in yeast. Mol. Cell. Proteom. 16, 2199–2218 (2017).

    Article  CAS  Google Scholar 

  107. Domsch, K. et al. The hox transcription factor ubx stabilizes lineage commitment by suppressing cellular plasticity in Drosophila. eLife 8, e42675 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  108. Bagchi, P., Torres, M., Qi, L. & Tsai, B. Selective EMC subunits act as molecular tethers of intracellular organelles exploited during viral entry. Nat. Commun. 11, 1127 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Yoshinaka, T. et al. Structural basis of mitochondrial scaffolds by prohibitin complexes: insight into a role of the coiled-coil region. iScience 19, 1065–1078 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Callegari, S. et al. A MICOS–TIM22 association promotes carrier import into human mitochondria. J. Mol. Biol. 431, 2835–2851 (2019).

    Article  CAS  PubMed  Google Scholar 

  111. Chen, Z. et al. Global phosphoproteomic analysis reveals ARMC10 as an AMPK substrate that regulates mitochondrial dynamics. Nat. Commun. 10, 104 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  112. Liu, L., Doray, B. & Kornfeld, S. Recycling of Golgi glycosyltransferases requires direct binding to coatomer. Proc. Natl Acad. Sci. USA. 115, 8984–8989 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Mirza, A. N. et al. LAP2 proteins chaperone GLI1 movement between the lamina and chromatin to regulate transcription. Cell 176, 198–212.e15 (2019).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by NIH R01-DK121409 (to A.Y.T. and S.A.C.) and the Stanford Wu Tsai Neurosciences Institute Big Ideas Initiative (to A.Y.T.). K.F.C. was supported by NIH Training Grant 2T32CA009302-41 and the Blavatnik Graduate Fellowship. T.C.B. is a Robert Black Fellow of the Damon Runyon Cancer Research Foundation (DRG-2391-20). A.Y.T. is an investigator of the Chan Zuckerberg Biohub.

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Contributions

K.F.C., T.C.B, N.D.U., S.A.M., S.A.C., and A.Y.T. contributed to the writing and editing of the manuscript.

Corresponding author

Correspondence to Alice Y. Ting.

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

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Related links

Key references using this protocol:

Branon, T. C. et al. Nat. Biotechnol. 36, 880–887 (2018): https://www.nature.com/articles/nbt.4201

Cho, K. F. et al. Proc. Natl Acad. Sci. USA 117, 12143–12154 (2020): https://www.pnas.org/content/117/22/12143

Supplementary information

Reporting Summary

Supplementary Table 1

Human proteome of proteins, annotated by whether each protein was previously detected in a PL proteomic experiment from our lab (regions include: mitochondrial matrix6,15, mitochondrial intermembrane space76, mitochondrial nucleoid77, ER membrane6,7,78, outer mitochondrial membrane7,78, ER-mitochondria contact sites7,78, nucleus6, synaptic cleft20, and cytosol6,7,78). For each protein, the compartment(s) in which they were detected are listed.

Supplementary Table 2

Compilation of data from previous PL proteomic mapping experiments performed by our lab, categorized by organelle/region of interest (each tab is a different subcellular compartment). In each tab, the relevant studies and corresponding enrichment ratios (SILAC, TMT, or iTRAQ) for proteins detected above the respective cutoffs are provided. Data are included for the mitochondrial matrix6,15 (Tab 1), mitochondrial intermembrane space76 (Tab 2), mitochondrial nucleoid77 (Tab 3), ER membrane6,7,78 (Tab 4), outer mitochondrial membrane7,78 (Tab 5), ER-mitochondria contact sites7,78 (Tab 6), nucleus6 (Tab 7), synaptic cleft20 (Tab 8), and cytosol6,7,78 (Tab 9).

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Cho, K.F., Branon, T.C., Udeshi, N.D. et al. Proximity labeling in mammalian cells with TurboID and split-TurboID. Nat Protoc 15, 3971–3999 (2020). https://doi.org/10.1038/s41596-020-0399-0

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