{"date_published":"2022-09-01T00:00:00Z","publication_status":"published","issue":"9","department":[{"_id":"MaDe"}],"abstract":[{"text":"Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.","lang":"eng"}],"type":"journal_article","file":[{"relation":"main_file","checksum":"e726c7b9315230e6710e0b1f1d1677e9","file_name":"2022_JBC_Artan.pdf","creator":"dernst","access_level":"open_access","content_type":"application/pdf","date_updated":"2022-09-12T08:14:50Z","date_created":"2022-09-12T08:14:50Z","file_size":2101656,"file_id":"12092","success":1}],"day":"01","month":"09","article_processing_charge":"No","date_updated":"2023-08-03T13:56:46Z","pmid":1,"tmp":{"image":"/images/cc_by.png","name":"Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)","short":"CC BY (4.0)","legal_code_url":"https://creativecommons.org/licenses/by/4.0/legalcode"},"title":"Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans","quality_controlled":"1","status":"public","oa_version":"Published Version","oa":1,"date_created":"2022-09-11T22:01:55Z","user_id":"4359f0d1-fa6c-11eb-b949-802e58b17ae8","publisher":"Elsevier","scopus_import":"1","article_type":"original","citation":{"mla":"Artan, Murat, et al. “Depletion of Endogenously Biotinylated Carboxylases Enhances the Sensitivity of TurboID-Mediated Proximity Labeling in Caenorhabditis Elegans.” <i>Journal of Biological Chemistry</i>, vol. 298, no. 9, 102343, Elsevier, 2022, doi:<a href=\"https://doi.org/10.1016/j.jbc.2022.102343\">10.1016/j.jbc.2022.102343</a>.","ieee":"M. Artan, M. Hartl, W. Chen, and M. de Bono, “Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans,” <i>Journal of Biological Chemistry</i>, vol. 298, no. 9. Elsevier, 2022.","ama":"Artan M, Hartl M, Chen W, de Bono M. Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans. <i>Journal of Biological Chemistry</i>. 2022;298(9). doi:<a href=\"https://doi.org/10.1016/j.jbc.2022.102343\">10.1016/j.jbc.2022.102343</a>","ista":"Artan M, Hartl M, Chen W, de Bono M. 2022. Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans. Journal of Biological Chemistry. 298(9), 102343.","apa":"Artan, M., Hartl, M., Chen, W., &#38; de Bono, M. (2022). Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans. <i>Journal of Biological Chemistry</i>. Elsevier. <a href=\"https://doi.org/10.1016/j.jbc.2022.102343\">https://doi.org/10.1016/j.jbc.2022.102343</a>","chicago":"Artan, Murat, Markus Hartl, Weiqiang Chen, and Mario de Bono. “Depletion of Endogenously Biotinylated Carboxylases Enhances the Sensitivity of TurboID-Mediated Proximity Labeling in Caenorhabditis Elegans.” <i>Journal of Biological Chemistry</i>. Elsevier, 2022. <a href=\"https://doi.org/10.1016/j.jbc.2022.102343\">https://doi.org/10.1016/j.jbc.2022.102343</a>.","short":"M. Artan, M. Hartl, W. Chen, M. de Bono, Journal of Biological Chemistry 298 (2022)."},"ec_funded":1,"article_number":"102343","_id":"12082","language":[{"iso":"eng"}],"has_accepted_license":"1","publication":"Journal of Biological Chemistry","author":[{"id":"C407B586-6052-11E9-B3AE-7006E6697425","full_name":"Artan, Murat","last_name":"Artan","first_name":"Murat"},{"first_name":"Markus","last_name":"Hartl","full_name":"Hartl, Markus"},{"full_name":"Chen, Weiqiang","last_name":"Chen","first_name":"Weiqiang"},{"full_name":"De Bono, Mario","id":"4E3FF80E-F248-11E8-B48F-1D18A9856A87","last_name":"De Bono","first_name":"Mario","orcid":"0000-0001-8347-0443"}],"doi":"10.1016/j.jbc.2022.102343","external_id":{"isi":["000884241800011"],"pmid":["35933017"]},"publication_identifier":{"issn":["0021-9258"],"eissn":["1083-351X"]},"isi":1,"acknowledgement":"We thank de Bono laboratory members for helpful comments on the article and the Mass Spec Facilities at IST Austria and Max Perutz Labs for invaluable discussions and comments on how to optimize mass spec analyses of worm samples. We are grateful to Ekaterina Lashmanova for designing the degron knock-in constructs and preparing the injection mixes for CRISPR/Cas9-mediated genome editing. All LC–MS/MS analyses were performed on instruments of the Vienna BioCenter Core Facilities instrument pool.\r\nThis work was supported by a Wellcome Investigator Award (grant no.: 209504/Z/17/Z ) to M.d.B. and an ISTplus Fellowship to M.A. (Marie Sklodowska-Curie agreement no.: 754411).","file_date_updated":"2022-09-12T08:14:50Z","project":[{"name":"Molecular mechanisms of neural circuit function","_id":"23870BE8-32DE-11EA-91FC-C7463DDC885E","grant_number":"209504/A/17/Z"},{"call_identifier":"H2020","name":"ISTplus - Postdoctoral Fellowships","_id":"260C2330-B435-11E9-9278-68D0E5697425","grant_number":"754411"}],"ddc":["570"],"intvolume":"       298","volume":298,"year":"2022","acknowledged_ssus":[{"_id":"Bio"}]}