IntroductionΒ-catenin is a highly conserved scaffold protein with critical functions in cell adhesion and cell signalling1. In the plasma membrane, β-catenin acts as an adapter molecule that connects E-cadherin to α-catenin in adherens junctions. Additionally, cytoplasmic β-catenin is an essential transcription co-factor of, among others, TCF/LEF family transcription factors in the Wnt/β-catenin signalling pathway. Wnt/β-catenin signalling promotes stem cell maintenance and turn-over and is limited in differentiated cells through the constitutive proteasomal degradation of β-catenin mediated by a multi-protein destruction complex. Spontaneous mutations that increase the stability of β-catenin are causatively involved in the pathogenesis of many types of cancer, especially colorectal cancer, in which most tumours are initiated by aberrant Wnt/β-catenin pathway activation2,3. Because of its prominent roles in human physiology and pathology, β-catenin has been studied extensively for several decades, and its function and regulation are generally well-understood. However, some aspects of its biology, such as the tissue-specific regulation of β-catenin-dependent gene transcription4 or the precise mode of its subcellular trafficking5, remain incompletely resolved and under active investigation.Considering that β-catenin requires various interaction partners to exert its functions, affinity purification/mass spectrometry (AP-MS)-based proteomics assays have been crucial in identifying important β-catenin interactors in different contexts and continue facilitating the discovery of new interactors to this day6,7,8,9,10,11,12. In this study, we generated and tested expression constructs of β-catenin fused to BioID13 or its derivative TurboID14 for proximity labelling. BioID and TurboID, which has a substantially faster reaction time, are based on the Escherichia coli biotin ligase BirA that allows for stable labelling of vicinal proteins of proteins-of-interest in living cells upon addition of biotin. Biotin-labelled proteins can be easily isolated by streptavidin pull-down, thus enabling convenient analysis of interacting proteins by mass spectrometry (i.e., proximity proteomics). AP-MS and proximity proteomics are complementary methods that capture largely discrete yet biologically meaningful sets of interactors15, which means that proximity proteomics may increase our understanding of the interactome of β-catenin. Using this approach, we identify numerous known as well as new potential β-catenin interactors in 293 T cells, and highlight ABCF2 as a putative functional β-catenin interactor. We conclude that proximity proteomics are a useful tool for the further exploration of β-catenin biology.Materials and methodsCell culture and plasmid constructionAuthenticated 293 T and HCT116 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). DLD1 cells were a kind gift from Dr. Xiao-Feng Sun (Linköping University). The cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1% penicillin/streptomycin. Cells were maintained in a 5% CO2 incubator at 37 °C and routinely tested negative for mycoplasma contamination by analytical qPCR (Eurofins Genomics, Ebersberg, Germany). BioID and TurboID tagged β-catenin constructs were generated using standard enzymatic restriction digestion and ligation with T4 DNA ligase. Plasmids were validated by Sanger sequencing (Eurofins Genomics). Stable cell lines were generated by cloning TurboID in place of GFP in pMaCTag-P05 plasmid (Addgene plasmid # 120,016, a gift from Michael Knop) and performing gene editing using a CRISPR-Cas12a-based method16. After puromycin selection, clonal cell lines were derived by limiting dilution. All newly generated constructs have been submitted to the Addgene repository (Addgene plasmid # 242,995, 242,996, 242,998, 242,999). mCherry-Beta-Catenin-20 (mCherry-CTNNB1; Addgene plasmid # 55,001, a gift from Michael Davidson) was used for overexpression of wild-type β-catenin.Luciferase reporter assayThe dual luciferase assay was conducted as described previously17. Briefly, cells were seeded on a 96-well plate and transfected with 50 ng of TOPFlash reporter (Addgene plasmid # 12,456, a gift from Randall Moon18), 5 ng of Renilla luciferase control (Addgene plasmid # 12,179, a gift from David Bartel) and 10 ng of β-catenin plasmids. After 24 h of transfection, cells were lysed in a passive lysis buffer (25 mM Tris, 2 mM DTT, 2 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, pH 7.8) by shaking for 10 min and then the lysates were transferred to a flat-bottomed 96-well luminescence assay plate. First, luciferin containing firefly luciferase buffer was added to the plate and luminescence was measured within 10 min using a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices). Next, coelenterazine-h (Promega) containing Renilla luciferase buffer was added to the plate and luminescence was measured immediately. Each experiment was performed in triplicate and data were normalised to the Renilla luminescence values.Antibodies and reagentsThe following primary antibodies were used: rabbit anti-Histone H3 (PA5-16,183, Invitrogen), mouse anti-Flag M2 (F3165, Sigma Aldrich), mouse anti-β-catenin (sc-133240, Santa Cruz), rabbit anti-ABCF2 (10,226–1-1AP, Proteintech), rabbit anti-HSP70 (AF1663, R&D Systems), and rabbit anti-HA (NB600-363, Novus Biologicals). Near infrared (NIR) fluorophore-labelled secondary antibodies and IRDye 800CW-linked Streptavidin were purchased from LI-COR. Non-immune IgG control antibodies were from Invitrogen. Dicer-ready siRNAs (DsiRNA) against ABCF2 (hs.Ri.ABCF2.13.1 and hs.Ri.ABCF2.13.2) were purchased from Integrated DNA Technologies. DAPI (4’,6-diamidino-2-phenylindole) was obtained from Thermo Fisher Scientific.Immunoblotting and immunoprecipitationThe cells were harvested in PBS and lysed with 1% NP40 in TBS supplemented with a 1 × Complete protease inhibitor cocktail (Pierce) on ice for 10 min. Then, the lysates were sonicated and centrifuged at 13,000 rpm for 10 min to remove cell debris. Subcellular fractionation was performed with the REAP method19. Protein lysates were boiled with 4 × Laemmli sample buffer with 50 mM DTT, separated on 10% polyacrylamide gel, and transferred to nitrocellulose membranes. The membrane was incubated in a blocking buffer for 1 h and blotted with primary antibodies. The primary antibodies were detected by Near-infrared (NIR)-labelled secondary antibodies. Blots were visualised using the LI-COR CLx imaging system (LI-COR) controlled through ImageStudio software.For immunoprecipitation, DLD1 whole cell lysates were pre-cleared with Protein A/G Plus agarose beads (Santa Cruz) for 1 h at 4 °C, and incubated with 2 µg primary antibodies overnight at 4 °C with end-over-end rotation. Following repeated washes with TBS/0.1% Tween-20, the beads were boiled in 4 × Laemmli sample buffer with 50 mM DTT.Uncropped images of all immunoblots presented in this manuscript can be found in the supplemental information.Immunofluorescence microscopyHEK293T cells and endogenously CTNNB1-TurboID-tagged cells were incubated for 21 h and then treated with or without 500 µM Biotin for 3 h. Cells were fixed with 4% w/v paraformaldehyde for 15 min, then permeabilised with 0.25% v/v Triton X-100 for 5 min and blocked with 5% w/v bovine serum albumin for 1 h. The cells were incubated with rabbit anti-HA for the detection of TurboID-tagged CTNNB1. The cells were then washed and incubated with anti-rabbit DY488 secondary antibody for the detection of the primary antibody. For the detection of biotinylated proteins, Alexa Fluor 488-conjugated streptavidin antibody (S11223, Thermo Fisher Scientific) was used. Cells were mounted with ProLong Glass Antifade Mountant with NucBlue (Invitrogen). Images were acquired using an LSM700 confocal laser scanning microscope controlled through ZEN software (Carl Zeiss, Jena, Germany), and post-processed using ImageJ 1.52p (NIH, Bethesda, USA).Proximity labelling mass spectrometryThe BioID and TurboID samples for mass spectrometry were performed basically as described previously13,14,20,21. Briefly, BioID- and TurboID-tagged β-catenin and control plasmids were transfected into 293 T cells using Lipofectamine 2000 (Thermo Fisher Scientific). For BioID labelling, after 6 h of transfection, cells were treated with 50 μM biotin, and incubated for 18 h at 37 °C, 5% CO2. For TurboID, cells were treated with 500 μM biotin after 21 h of transfection and incubated for 30 min and 3 h at 37 °C, 5% CO2. For endogenous beta-catenin TurboID experiments, a successfully tagged clone was cultured for 21 h and then incubated with 500 µM biotin for 3 h at 37 °C, 5% CO2. The cells were surface washed with PBS before harvesting and cell pellets were washed three times with PBS to remove any remaining biotin.For BioID, cells were lysed in RIPA buffer (Thermo Fisher Scientific) for 1 h at 4 °C with end-over-end rotation, then sonicated and centrifuged at 13,000 rpm for 30 min at 4 °C. Pre-washed streptavidin beads (GE Healthcare, USA) were added to the cell lysate and incubated for 3 h at 4 °C with end-over-end rotation. The beads were collected by centrifuging at 2000 rpm for 2 min and then washed four times with 50 mM ammonium bicarbonate (NH4HCO3). On-bead digestion was performed by adding 100 μl of freshly prepared 1 µg of trypsin (Thermo Fisher Scientific) in 50 mM NH4HCO3 to the beads followed by overnight at 37 °C with end-over-end rotation.For proximity labelling by TurboID, cells were lysed in RIPA buffer containing 1 × Complete protease inhibitor cocktail for 5 min on ice. Then the samples were sonicated, and cell debris was removed by centrifuging at 10,000 rpm for 10 min. Prewashed streptavidin beads (GE Healthcare) were added to the cell lysate and incubated for 1 h at RT then overnight at 4 °C with end-over-end rotation. The beads were washed twice with 1 ml of RIPA buffer, once with 1 ml of 1 M KCl, once with 1 ml of 0.1 M Na2CO3, once with 1 ml of 2 M urea in 50 mM Tris–HCl, pH 7.5, and twice with 1 ml RIPA lysis buffer. The beads were then transferred to a new Eppendorf tube and washed twice with 50 mM Tris–HCl buffer, pH 7.5 and 2 M urea/50 mM Tris–HCl, pH 7.5 buffer. Beads were incubated with 0.4 μg of trypsin (Thermo Fisher Scientific) in 2 M urea/50 mM Tris–HCl, pH 7.5,1 mM DTT buffer for 1 h at RT with end-over-end rotation. After incubation, the supernatant was collected, and the beads were washed twice with 60 μl of 2 M urea/50 mM Tris–HCl buffer, pH 7.5 and the washes were combined with the collected supernatant. The supernatant was first reduced with 4 mM DTT for 30 min at RT, then alkylated with 10 mM iodoacetamide for 45 min in the dark at RT with end-over-end rotation. An additional 0.5 μg of trypsin was added to allow the complete digestion of the samples and incubated overnight under the same conditions. The digested samples were desalted with Pierce C18 pipette tips (Thermo Fisher Scientific) and then dried with a vacuum centrifuge.BioID and samples were analysed by mass spectrometry, using an Easy nano LC II HPLC interfaced with a nanoEasy spray ion source (Thermo Fisher) connected to an LTQ Orbitrap Velos Pro hybrid mass spectrometer (Thermo Fisher). The peptides were loaded on an NS-MP-10 Biosphere C18 column, 2 cm (100 μm inner diameter, packed with 5 μm resin), and the chromatographic separation was performed at RT on a 10.1 cm (75 μm inner diameter) NS-AC-10-C18 column packed with 5 μm of resin (NanoSeperations, Netherlands). The nanoHPLC was operating at 300 nl/min flow rate with a linear gradient of solvent B (0.1% (v/v) formic acid in acetonitrile) in solvent A (0.1% (v/v) formic acid in water) for 60 min. Full MS scans (380–2000 m/z) were recorded at a resolution of 30,000. The top 20 most intense multiple charged ions were selected with an isolation window of 2.0 and fragmented in the linear ion trap by Collision-induced dissociation.TurboID-labelled samples and endogenous TurboID-tagged samples were analysed as previously described20 using an EASY nLC 1200 system interfaced with a nanoEasy spray ion source (Thermo Scientific) connected to a Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific).Raw data were processed by Proteome Discover 2.0 (Thermo Fisher Scientific) searching against the Homo sapiens UniProt database with the Sequest HT search engine. The search parameters were as follows: enzymes: trypsin with two missed cleavages, no variable modifications (BioID and TurboID data); fixed modification: Carbamidomethylation (TurboID data); Peptide Mass Tolerance, 10 ppm; MS/MS Fragment Tolerance, 0.5 Da (BioID data), 0.02 Da (TurboID data). Quantification of the analyzed data was performed using Scaffold 5.1.0 (Proteome Software Inc, https://www.proteomesoftware.com/products/scaffold-5) using total spectral counts. Protein and peptide identifications were accepted if they could be established at greater than 95 and 90% probability, respectively and if protein identification contained at least two identified peptides.Proximity ligation assay (PLA)PLA was performed using a commercially available NaveniBright—MR, HRP kit (Navinci Diagnostics) according to the manufacturer’s instructions. Briefly, cells were fixed, permeabilized, and incubated with primary antibodies against β-catenin and ABCF2, followed by incubation with species-specific PLA probes. Ligation and amplification reactions were carried out to generate detectable signals. The PLA signal was visualized through an HRP-mediated chromogenic reaction that produces a brown precipitate at sites of protein proximity. Nuclear staining was performed using the NaveniBright nuclear stain, resulting in a blue counterstain.PLA images were acquired using an Olympus BX51 microscope equipped with an Olympus XC30 camera and cellSens software. Brightfield images were captured using a 60 × oil-immersion objective. Exposure was controlled automatically by the acquisition software. Images were acquired at a resolution of 2080 × 1544 in full-frame mode. All images were acquired using identical acquisition settings across all experimental groups to ensure comparability and represent single focal planes. Multiple fields of view were examined for each condition. No post-acquisition adjustments were applied.Data analysisProteomics data were processed in R v4.4.0 essentially as described previously20. BioID and TurboID data were further analysed in SAINTexpress22 v3.6.3, where significance was assumed at a Bayes false discovery rate (BFDR) of 0.65 and Bayes FDR