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TurboID-EV: a proximity-labeling proteomic approach to dissect the uptake mechanism of extracellular vesicles

ABSTRACT: Extracellular vesicles (EVs), including exosomes, have been recognized as key mediators of intercellular communications through donor EV and recipient cell interaction. Until now, most studies have focused on the development of analytical tools to separate EVs and their applications for molecular profiling of EV cargo. However, we lack a complete picture of the mechanism of uptake of EVs by recipient cells. Here, we developed the TurboID-EV system with the engineered biotin ligase, TurboID, tethered to the EV membrane, which allowed us to track the footprints of EVs during and after EV uptake by proximity-dependent biotinylation of recipient cellular proteins. To enrich and analyze biotinylated recipient proteins from low amounts of input cells (corresponding to ~10 µg proteins), we developed an integrated proteomic workflow that combined SILAC labeling, fluorescence-activated cell sorting, spintip-based streptavidin affinity purification, and mass spectrometry. Using the method, we successfully identified 456 biotinylated recipient proteins, including not only well-known proteins involved in endocytosis and macropinocytosis but also previously unappreciated membrane-associated proteins such as DSP and JUP. The TurboID-EV system should be readily applicable to various EV subtypes and recipient cell types, providing a promising tool to dissect the specificity of EV uptake mechanisms on a proteome-wide scale.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD040569 | JPOST Repository | Mon Aug 21 00:00:00 BST 2023

REPOSITORIES: jPOST

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