TurboID-mediated proximity labelling of cytoophidium proteome in Drosophila

Abstract

Proximity-based biotinylation combined with mass spectrometry has emerged as a powerful approach to study protein interaction networks and protein subcellular compartmentation. However, low kinetics and the requirement of toxic chemicals limit the broad utilisation of current proximity labelling methods in living organisms. TurboID, the newly engineered promiscuous ligase, has been reported to label bait proteins effectively in various species. Here, we systematically demonstrated the application of TurboID-mediated biotinylation in a wide range of developmental stages and tissues, and we also verified the feasibility of TurboID-mediated labelling in desired cells via cell-type-specific GAL4 driver in Drosophila. Furthermore, using TurboID-mediated biotinylation coupled with mass spectrometry, we characterized the proximate proteome of the cytoophidium, a newly identified filamentous structure containing the metabolic enzyme CTP synthase (CTPS) in Drosophila. Our study demonstrates a referable tool and resource for research in subcellular compartments of metabolic enzymes in vivo.