SI Appendix 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, USA bDepartment of Genetics, Stanford University, Stanford, CA, USA cDepartment of Biology, Stanford University, Stanford, CA, USA dDepartment of Chemistry, Stanford University, Stanford, CA, USA eDepartment of Chemistry, MIT, Cambridge, MA, USA fBroad Institute of MIT and Harvard, Cambridge, MA, USA gResearch Institute of Aging and Metabolism, Kyungpook National University, Daegu, South Korea hDepartment of Chemistry, Seoul National University, Seoul, Korea iDepartment of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Korea jSchool of Biological Sciences, Seoul National University, Seoul, Korea kDepartment of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu, South Korea lLeading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University, Daegu, South Korea mChan Zuckerberg Biohub, San Francisco, CA, USA 1Correspondence should be addressed to Alice Y. Ting ([email protected]) www.pnas.org/cgi/doi/10.1073/pnas.1919528117 1 Table S1. Table of plasmids used in this study. Plasmid Plasmid Promoter Features Variants Details Used for Used in name vector P1-P18 pLX304 CMV BstBI- N-term 10 aa linkers: Transient Figure 1, 2; FKBP- fragments: 71, GSGSGGSGSG, expression Figure S1, 10aa 72, 73, 74, 75, KGSGSTSGSG; in whole S2 linker-V5- 78, 98, 101, V5: cell 10aa 112, 212, 256, GKPIPNPLLGL linker- 258, 270, 273, DST AgeI-N- 78 (BioID), term 256 (BioID), fragment- TurboID (FL), Stop-NheI BioID (FL) P19-P34 pLX304 CMV BstBI- C-term HA: Transient Figure 1, 2; HA-Halo- fragments: YPYDVPDY; 10 expression Figure S1, NheI-10aa 72, 73, 74, 75, aa linkers: in whole S2 linker- 76, 79, 99, TKGSGGTGGS, cell FRB- 102, 113, 213, KGSGSTSGSG BamHI- 257, 259, 271, 10aa 274, 79 linker-C- (BioID), 257 term (BioID fragment- Stop-NheI P35 pLX304 CMV BstBI- N-term 10 aa linkers: Transient Figure 2 FKBP- fragment: L73 GSGSGGSGSG, expression 10aa KGSGSTSGSG; in cytosol linker-V5- V5: 10aa GKPIPNPLLGL linker- DST; NES: AgeI-L73- LQLPPLERLTL NES- D Stop-NheI P36 pLX304 CMV BstBI- C-term HA: Transient Figure 2 HA-10aa fragment: G74 YPYDVPDY; 10 expression linker- aa linkers: in cytosol FRB- TKGSGGTGGS, BamHI- KGSGSTSGSG; 10aa NES: linker- LQLPPLERLTL G74-NES- D Stop-NheI P37 pCDNA3 CMV NotI- N-term 10 aa linkers: Transient Figure 2 FKBP- fragment: L73 GSGSGGSGSG, expression 10aa KGSGSTSGSG; in nucleus linker-V5- V5: 10aa GKPIPNPLLGL linker- DST; NLS: AgeI-L73- SRADPKKKRK EcoRI- VDPKKKRKVD NLS- PKKKRKV Stop-XhoI P38 pCDNA3 CMV NotI-HA- C-term HA: Transient Figure 2 10aa fragment: G74 YPYDVPDY; 10 expression linker- aa linkers: in nucleus FRB- TKGSGGTGGS, 2 BamHI- KGSGSTSGSG; 10aa NLS: linker- SRADPKKKRK G74- VDPKKKRKVD EcoRI- PKKKRKV NLS- Stop-XhoI P39 pLX304 CMV BstBI- N-term MTS (COX4): Transient Figure 2; MTS- fragment L73 MLATRVFSLV expression Figure S3 BamHI- GKRAISTSVCV in FKBP- RAH; 10 aa mitochond 10aa linkers: rial matrix linker-V5- GSGSGGSGSG, 10aa KGSGSTSGSG; linker- V5: AgeI-L73- GKPIPNPLLGL Stop-NheI DST P40 pLX304 CMV BstBI- C-term MTS (COX4): Transient Figure 2; MTS- fragment: G74 MLATRVFSLV expression Figure S3 BamHI- GKRAISTSVCV in HA-10aa RAH; HA: mitochond linker- YPYDVPDY; 10 rial matrix FRB- aa linkers: BamHI- TKGSGGTGGS, 10aa KGSGSTSGSG linker- G74-Stop- NheI P41 pDisplay CMV EcoRV- N-term SS (Ig kappa Transient Figure 2; SS-BglII- fragment L73 chain): expression Figure S3 FKBP- METDTLLLWV in ER 10aa LLLWVPGSTG lumen linker-V5- D; 10 aa linkers: 10aa GSGSGGSGSG, linker- KGSGSTSGSG; AgeI-L73- V5: 3aa GKPIPNPLLGL spacer- DST; 3aa spacer: KDEL- GSG; KDEL: Stop-AscI KDEL P42 pDisplay CMV EcoRV- C-term SS (Ig kappa Transient Figure 2; SS-BglII- fragment: G74 chain): expression Figure S3 HA-10aa METDTLLLWV in ER linker- LLLWVPGSTG lumen FRB- D; HA: BamHI- YPYDVPDY; 10 10aa aa linkers: linker- TKGSGGTGGS, G74-3aa KGSGSTSGSG; spacer- 3aa spacer: GSG; KDEL- KDEL: KDEL Stop-AscI P43 pVSVG Lentiviral Producing Figure 3; envelope plasmid lentivirus Figure S4 P44 D8.9 Lentiviral helper Producing Figure 3; plasmid lentivirus Figure S4 P45 pLX304 CMV BstBI- N-term OMM (Tom20 Stable Figure 3; OMM-12 fragment L73 anchor): outer Figure S4 3 aa linker- MVGRNSAIAA mitochond FKBP- GVCGALFIGY rial 10aa CIYFDRKRRSD membrane linker-V5- PNF; 12 aa expression 10aa linker: linker- GSGASGSAGG AgeI-L73- GV; 10 aa Stop-NheI linkers: GSGSGGSGSG, KGSGSTSGSG; V5: GKPIPNPLLGL DST P46 pLX208 CMV BamHI- C-term 7aa linker: Stable ER Figure 3; G74-7aa fragment: G74 GSGGSTG; HA: membrane Figure S4 linker- YPYDVPDY; 5 expression HA-5aa aa linker: linker- GSGSG; spacer: FRB- SAGGSAGGSA spacer- GGSAGGSAGG ERM- PRAQASNSSA Stop-MluI GG; ERM (Cb5 anchor): ITTVESNSSW WTNWVIPAIS ALVVALMYRL YMAED P47(7) pLX304 CMV BstBI-V5- TurboID (FL) V5: Stable Figure 3; NheI- GKPIPNPLLGL outer Figure S5 TurboID- DST; OMM mitochond EcoRI- (MAVS anchor): rial OMM- RPSPGALWLQ membrane Stop-NheI VAVTGVLVVT expression LLVVLYRRRL H P48(7) pLX304 CMV BstBI- TurboID (FL) ERM Stable ER Figure 3; ERM-4aa (cytochrome membrane Figure S5 linker- P450 anchor): expression NotI-19aa MDPVVVLGLC linker- LSCLLLLSLW TurboID- KQSYGGG; 4 aa NheI-V5- linker: GSGS; Stop-NheI 19aa linker: GSGSGSGSGS GSGSGSGSG; V5: GKPIPNPLLGL DST P49(7) pLX304 CMV BstBI-V5- TurboID (FL) V5: Stable Figure 3; NheI- GKPIPNPLLGL cytosol Figure S5 TurboID- DST; NES: expression NES- LQLPPLERLTL Stop-NheI D P50-P54 pCDNA3 CMV HindIII- GOI: V5: Transient Figure 6; GOI-V5- SYNJ2BP, GKPIPNPLLGL expression Figure S9 Stop-XhoI FUNDC2, DST MTFR1, 4 OCIAD1, USP30 P55 pCDNA3 CMV HindIII- GOI: LBR V5: Transient Figure 6; V5-GOI- GKPIPNPLLGL expression Figure S9 Stop-XhoI DST P56 pCDNA3 CMV HindIII- C-term 12 aa linker: Transient Figure 7 G74-12 aa fragment: G74 GSGGSTGGSG expression linker- SG in whole Arrestin- cell Stop-XhoI P57 pAAV CMV HindIII- N-term GCGR Transient Figure 7 GCGR- fragment L73 (Glucagon expression SpeI- receptor); NES: at cell NES- ELAEKLAGLDI surface NheI-V5- N; V5: 10aa GKPIPNPLLGL linker- DST; 10 aa AgeI-L73- linker: Stop- KGSGSTSGSG EcoRI P58 pCAG CAG EcoRI- Glucagon TSP (trypsin Transient Figure 7 TSP- peptide signal peptide): expression Glucagon- MSALLILALV at cell KpnI-18 GAAVA; 18 aa surface aa linker- linker: SpeI- GNGNGNGNG ICAM1 NGNGNGNGN; (EC)- 3xHA:AAVYPY AgeI-3x DVPDYAGPYP HA-AgeI- DVPDYAGSYP pre- YDVPDYAPAA mGRASP -Stop- XhoI 5 Table S2. Table of antibodies used in this study. Antibody Source Vendor Catalog Number Dilution(s) Streptavidin-HRP - Thermo Fisher S911 WB: 1:3000 Scientific Anti-V5 Mouse Thermo Fisher R96025 WB: 1:10000; IF: 1:1000 Scientific Anti-HA Rabbit Cell Signaling C29F4 WB: 1:5000; IF: 1:1000 Technology Anti-mouse-HRP Goat BioRad 170-6516 WB: match dilution of primary antibody Anti-rabbit-HRP Goat BioRad 170-6515 WB: match dilution of primary antibody Neutravidin- - Thermo Fisher A2666; A20006 IF: 1:1000 AlexaFluor647 Scientific DAPI - Enzo Life AP402-0010 IF: 1 µg/mL final Sciences concentration Anti-mouse- Goat Thermo Fisher A11029 IF: 1:1000 AlexaFluor488 Scientific Anti-mouse- Goat Thermo Fisher A11031 IF: 1:1000 AlexaFluor568 Scientific Anti-mouse- Goat Thermo Fisher A21236 IF: 1:1000 AlexaFluor647 Scientific Anti-rabbit- Goat Thermo Fisher A11008 IF: 1:1000 AlexaFluor488 Scientific Anti-rabbit- Goat Thermo Fisher A11036 IF: 1:1000 AlexaFluor568 Scientific Anti-rabbit- Goat Thermo Fisher A21245 IF: 1:1000 AlexaFluor647 Scientific MitoTracker - Thermo Fisher M7512 IF: 500 nM final Scientific concentration Anti-Mouse- Goat LI-COR 92632210 WB: match dilution of IRDye 800 primary antibody Anti-Rabbit- Goat Li-COR 92568071 WB: match dilution of IRDye 680 primary antibody Anti-Tom20 Rabbit Abcam Ab186735 IF: 1:1000 Anti-Calnexin Rabbit Thermo Fisher PA5-34754 IF: 1:500 Scientific Anti-GAPDH- Mouse Santa Cruz Sc-47724 WB: 1:3000 HRP Biotechnology Anti-FACL4 Rabbit Abnova PAB2504 WB: 1:500 Anti-Mff Rabbit Proteintech 17090-1-AP WB: 1:500 Group Anti-ABCD3 Mouse Santa Cruz Sc-514728 WB: 1:500 Biotechnology Anti-EXD2 Rabbit Atlas Antibodies HPA005848 WB: 1:500 Anti-OCIAD1 Rabbit Abcam Ab91574 WB: 1:500 Anti-NEK4 Rabbit Bethyl A302-673A WB: 1:500 Laboratories Anti-LBR Rabbit Abcam Ab32535 WB: 1:500 Anti-MLF2 Mouse Santa Cruz Sc-166874 WB: 1:500 Biotechnology Anti-GRP75 Rabbit Santa Cruz Sc-133137 WB: 1:500 Biotechnology Anti-IP3R1 Rabbit Santa Cruz Sc-28614 WB: 1:500 Biotechnology 6 Anti-PDI Mouse Santa Cruz Sc-74551 WB: 1:500 Biotechnology Anti-VDAC1 Mouse Abcam Ab14734 WB: 1:500 Anti-COX4 Rabbit Abcam Ab16056 WB: 1:500 Anti-b-Tubulin Mouse Applied G098 WB: 1:500 Biological Materials Inc. Anti-PCNA Mouse BD Biosciences 610664 WB: 1:500 7 Figure S1. Screening of TurboID split sites. Related to Figure 1. (A) Comparison of split sites in HEK293T cells. Cells transiently transfected with the indicated constructs were treated with 50 µM biotin and 100 nM rapamycin for 24 hours, and whole cell lysates were analyzed by streptavidin blotting. For each construct pair, lanes are shown with both fragments present (B), N- terminal fragment only (N), or C-terminal fragment only (C). Anti-V5 and anti-HA staining show 8 expression levels of N- and C-terminal fragments, respectively. Full-length TurboID (30 minutes labeling) and full-length BioID were included for comparison. Dashed lines indicate separate blots performed at the same time and developed simultaneously. Quantification of biotinylation activity is shown in Figure 1C. (B) Testing of additional split sites around the most promising split (73/74) from the first round of screening in HEK293T cells. Cells transiently transfected with the indicated constructs were treated with 50 µM biotin and 100 nM rapamycin for 1 hour, and whole cell lysates were analyzed by streptavidin blotting.