Decoding microprotein interactome via a minimal genetically encoded photocatalytic proximity labeling system

Microproteins (typically <100 amino acids) regulate diverse cellular processes, yet their small size and low abundance make interactome mapping challenging and particularly sensitive to perturbation by conventional enzyme-based proximity-labeling tags. Here, we developed FlashID, an ultra-compact genetically encoded proximity labeling (PL) system that combines a six-amino-acid tetracysteine tag (CCXXCC, <700 Da) with the turn-on biarsenical photocatalyst FlAsH. Upon tag binding and visible-light irradiation, FlAsH generates singlet oxygen to enable spatially and temporally controlled oxidative labeling of proximal proteins. In side-by-side analyses of the bacterial microprotein AzuC, FlashID recovered the validated interactor GlpD and identified additional physiologically relevant partners that were not captured by TurboID or IP-MS under the conditions tested. FlashID also resolved compartment-specific interactomes of diverse microproteins, including RP11-469A, which we identified as a mitochondrial intermembrane protein that interacts with PHB2 to regulate proliferation. Finally, the extension of FlashID to E. coli, mammalian cells, C. elegans and Arabidopsis thaliana establishes FlashID as a complementary, minimally perturbative platform for decoding microprotein interaction networks in vivo and advancing photocatalytic proximity labeling technologies. Shi, Yan, and colleagues present FlashID, an ultra-compact light-activated proximity-labeling system that maps microprotein interactions with minimal perturbation across bacteria, mammalian cells, worms and plants.