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When less is more - a fast TurboID knock-in approach for high-sensitivity endogenous interactome mapping.
Stockhammer, Alexander; Spalt, Carissa; Klemt, Antonia; Benz, Laila S; Harel, Shelly; Natalia, Vini; Wiench, Lukas; Freund, Christian; Kuropka, Benno; Bottanelli, Francesca.
Affiliation
  • Stockhammer A; Membrane Trafficking Laboratory, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • Spalt C; Membrane Trafficking Laboratory, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • Klemt A; Membrane Trafficking Laboratory, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • Benz LS; Membrane Trafficking Laboratory, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • Harel S; Membrane Trafficking Laboratory, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • Natalia V; Membrane Trafficking Laboratory, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • Wiench L; Membrane Trafficking Laboratory, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • Freund C; Laboratory of Protein Biochemistry, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • Kuropka B; Laboratory of Protein Biochemistry, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • Bottanelli F; Membrane Trafficking Laboratory, Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
J Cell Sci ; 137(16)2024 08 15.
Article in En | MEDLINE | ID: mdl-39056144
ABSTRACT
In recent years, proximity labeling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Although physiological expression of labeling enzymes is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labeling enzymes when endogenously expressed. Endogenous tagging of the µ subunit of the adaptor protein (AP)-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labeling enzyme fusion protein could not reveal. We used the KI strategy to compare the interactome of the different AP complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labeling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: CRISPR-Cas Systems Limits: Humans Language: En Journal: J Cell Sci Year: 2024 Document type: Article Affiliation country: Germany Country of publication: United kingdom

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: CRISPR-Cas Systems Limits: Humans Language: En Journal: J Cell Sci Year: 2024 Document type: Article Affiliation country: Germany Country of publication: United kingdom