Split-TurboID enables contact-dependent proximity labeling in cells Kelvin F. Choa, Tess C. Branonb,c,d,e, Sanjana Rajeevb, Tanya Svinkinaf, Namrata D. Udeshif, Themis Thoudamg, Chulhwan Kwakh,i, Hyun-Woo Rheeh,j, In-Kyu Leeg,k,l, Steven A. Carrf, and Alice Y. Tingb,c,d,m,1 aCancer Biology Program, Stanford University, Stanford, CA 94305; bDepartment of Genetics, Stanford University, Stanford, CA 94305; cDepartment of Biology, Stanford University, Stanford, CA 94305; dDepartment of Chemistry, Stanford University, Stanford, CA 94305; eDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; fBroad Institute of MIT and Harvard, Cambridge, MA 02142; gResearch Institute of Aging and Metabolism, Kyungpook National University, 37224 Daegu, South Korea; hDepartment of Chemistry, Seoul National University, 08826 Seoul, South Korea; iDepartment of Chemistry, Ulsan National Institute of Science and Technology, 44919 Ulsan, South Korea; jSchool of Biological Sciences, Seoul National University, 08826 Seoul, South Korea; kDepartment of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, 41944 Daegu, South Korea; lLeading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University, 41944 Daegu, South Korea; and mChan Zuckerberg Biohub, San Francisco, CA 94158 Edited by Tony Hunter, The Salk Institute for Biological Studies, La Jolla, CA, and approved April 7, 2020 (received for review November 7, 2019) Proximity labeling catalyzed by promiscuous enzymes, such as Split forms of APEX (18) and BioID (19–21) have previously TurboID, have enabled the proteomic analysis of subcellular regions been reported. However, split-APEX (developed by us) has not difficult or impossible to access by conventional fractionation-based ap- been used for proteomics, and the requirement for exogenous proaches. Yet some cellular regions, such as organelle contact sites, re- H2O2 and heme addition limits its utility in vivo. Split-BioID was main out of reach for current PL methods. To address this limitation, we first reported by De Munter et al. (19), followed by more active split the enzyme TurboID into two inactive fragments that recombine versions from Schopp et al. (20) and Kwak et al. (21). All are when driven together by a protein–protein interaction or membrane– derived from the parental enzyme BioID, which requires 18 to membrane apposition. At endoplasmic reticulum–mitochondria contact 24 h of biotin labeling. We show below that the Schopp et al. (20) sites, reconstituted TurboID catalyzed spatially restricted biotinyla- split-BioID does not produce detectable activity, while the Kwak > tion, enabling the enrichment and identification of 100 endoge- et al. (21) split-BioID requires 16+ h of labeling to generate nous proteins, including many not previously linked to endoplasmic sufficient signal. – CELL BIOLOGY reticulum mitochondria contacts. We validated eight candidates by Hence we sought to develop an improved, more active split PL biochemical fractionation and overexpression imaging. Overall, enzyme by starting from TurboID. In contrast to APEX, Tur- split-TurboID is a versatile tool for conditional and spatially specific boID does not require any cofactors or cooxidants; just biotin proximity labeling in cells. addition initiates labeling in cells or animals. TurboID is also >100-fold faster than BioID, requiring only 1 to 10 min of la- proximity labeling | ER–mitochondria contacts | split-TurboID beling time (7). We performed a screen of 14 different TurboID split sites to identify optimal fragments for high-affinity and low- roximity labeling (PL) has been shown to be a valuable tool affinity reconstitution. We converged upon TurboID split at L73/ Pfor studying protein localization and interactions in living G74, which gave rapamycin-dependent reconstitution when – cells (1 3). In PL, a promiscuous enzyme such as APEX (4, 5), fused to FRB and FKBP in multiple subcellular organelles. We BioID (6), or TurboID (7) is genetically targeted to an organelle or protein complex of interest. Addition of a biotin-derived small- Significance molecule substrate then initiates biotinylation of endogenous proteins within a few nanometers of the promiscuous enzyme, via Most of the thousands of proteins that comprise a human cell a diffusible radical intermediate in the case of APEX, or an ac- have specific subcellular localization patterns essential for their tivated biotin adenylate intermediate in the case of BioID and function. “Proximity labeling” (PL) is a method for mapping the TurboID. After cell lysis, biotinylated proteins are harvested using localization of endogenous cellular proteins on a proteome- streptavidin beads and identified by mass spectrometry. wide scale. To improve the specificity and versatility of PL, PL has been applied in many cell types and species to map the we developed split-TurboID, a promiscuous biotinylating en- proteome composition of organelles, including mitochondria (5, zyme split into two inactive fragments. The fragments are 8–10), synapses (11, 12), stress granules (13), and primary cilia coexpressed in cells and brought together by a drug, protein– (14). However, to increase the versatility of PL, new enzyme protein interaction, or organelle contact to reconstitute Tur- variants are needed. In particular, split enzymes could enable boID enzymatic activity. We used split-TurboID to map the greater spatial specificity in the targeting of biotinylation activity, protein composition of endoplasmic reticulum–mitochondria as well as PL activity that is conditional on a specific input, such contact sites, which are essential for mitochondrial fission, lipid as drug, calcium, or cell–cell contact. For example, contact sites biosynthesis, and calcium signaling. For conditional or higher- between mitochondria and the endoplasmic reticulum (ER) me- specificity PL, split-TurboID may be a valuable tool for diate diverse biology, from lipid biosynthesis and Ca+2 signaling to biological discovery. regulation of mitochondrial fission (15). There is great interest in – Author contributions: K.F.C., S.A.C., and A.Y.T. designed research; K.F.C., T.C.B., S.R., T.S., probing the proteomic composition of ER mitochondria contacts. N.D.U., T.T., C.K., H.-W.R., and I.-K.L. performed research; K.F.C. and A.Y.T. analyzed data; However, direct fusion of a PL enzyme to one of the known and K.F.C. and A.Y.T. wrote the paper. ER–mitochondria contact resident proteins (e.g., Drp1 or Mff) The authors declare no competing interest. would generate PL activity outside of ER–mitochondria con- This article is a PNAS Direct Submission. tacts as well, because these proteins also reside in other sub- Published under the PNAS license. cellular locations (16, 17). On the other hand, use of a split PL 1To whom correspondence may be addressed. Email: [email protected]. enzyme, with one fragment targeted to the mitochondria and This article contains supporting information online at https://www.pnas.org/lookup/suppl/ the other targeted to the ER, would restrict biotinylation ac- doi:10.1073/pnas.1919528117/-/DCSupplemental. tivity to ER–mitochondria contact sites specifically. www.pnas.org/cgi/doi/10.1073/pnas.1919528117 PNAS Latest Articles | 1of12 Downloaded by guest on September 30, 2021 then used this split-TurboID to perform proteomic mapping of best high-affinity pair (78/79). The discrepancy between the rapamycin- ER–mitochondria contact sites in mammalian cells. The result- dependence of Contact-ID and the rapamycin-independence of ing proteome of 101 proteins is highly specific and identifies high-affinity split-TurboID is likely explained by their different many new ER–mitochondria contact site candidates, eight of which regimes of activity; Contact-ID labeling may not be detectable in we validated by biochemical fractionation or overexpression the omit-rapamycin condition because the intrinsic activity is imaging. so low. In our hands, the previously reported split-BioID from Schopp Results et al. (20) did not give any detectable signal over background Development of a Split Promiscuous Biotin Ligase with High Activity. after 24 h of biotin incubation. Interestingly, TurboID split at the We started with TurboID, for the reasons given above, and same position (256/257) did show some labeling (Fig. 1C), but sought to design split protein fragments with no detectable ac- this activity was also observed with the N-terminal fragment tivity on their own, but high reconstituted activity. Given the alone (SI Appendix, Fig. S1A), suggesting that this cut site may diversity of ways in which split proteins are used, we envisioned not yield a true protein complementation system. Notably, we engineering both a low-affinity fragment pair, whose reconstitution found that the activity of split-TurboID is even greater than that couldbedrivenbyaprotein–protein or membrane–membrane as- of full-length BioID (Fig. 1D; side-by-side comparison using 24 h sociation (Fig. 1A), and a high-affinity pair that would spontane- of biotin incubation), suggesting that split-TurboID’s activity ously reconstitute upon cocompartmentalization of fragments. level should be adequate for a wide range of applications. Previously, we developed split enzymes [split-APEX (18) and split- By referencing the protein structure of E. coli biotin ligase HRP (22)] by manually selecting cut sites in exposed loops, guided (PDB ID 2EWN) (25), from which