<em>In Vivo</em> Application of TurboID-based Proximity Labeling in <em>Drosophila melanogaster</em>

Proximity labeling (PL) is a powerful method for mapping protein-protein interaction networks. Advances in labeling enzymes, especially TurboID, have broadened its use across biological systems. TurboID is particularly advantageous for in vivo applications due to its robustness, non-toxicity, and ease of use. This enzyme promiscuously biotinylates nearby biomolecules, including proteins and nucleic acids, within an approximately 10 nm range in the presence of biotin. We applied this method to investigate the interactome of Zucchini (Zuc), a key factor in piwi-interacting RNA (piRNA) biogenesis on the outer mitochondrial membrane (OMM) in germline cells of the Drosophila melanogaster ovary. By expressing Zuc-TurboID in germ cells and supplementing with biotin, we induced the biotinylation of Zuc-proximal proteins. Mass spectrometry analysis following the enrichment of biotinylated peptides identified proteins in close proximity to Zuc, including well-known interactors in piRNA biogenesis. Interestingly, we discovered novel interactors of Zuc involved in protein folding, membrane organization, and vesicle trafficking.