Covalent flavin installation enables peptide-scale proximity proteomics of microproteins in living cells
Abstract
Microproteins often derive their functions from context-dependent molecular interactions, yet their small size makes proximity mapping particularly susceptible to perturbation by protein-sized labeling enzymes. Here we develop flavin-mediated peptide-tag proximity labeling (FPPL), which decouples photocatalyst installation from the protein of interest. In FPPL, ApbE covalently installs FMN onto a 12-amino-acid ApbE acceptor peptide (AAP) fused to the target protein, generating a light-responsive photocatalytic center in living cells. AAP fusion largely preserved the localization of microprotein NUPR1 and BANF1 compared with direct TurboID fusion, while supporting spatially selective proteome mapping across distinct subcellular compartments. Applying FPPL to BANF1 revealed a functionally connected nuclear homeostatic network, as depletion of multiple FPPL-identified proteins induced nuclear reporter leakage, focal BANF1 recruitment and local lamina remodeling. FPPL profiling of GNG4 unexpectedly uncovered a virus-associated proximal network and identified GNG4 as a positive regulator of Sendai-virus-induced IFNB1 expression and downstream STAT1 signaling. Several GNG4-proximal proteins further modulated this response, with ARF6 and MTCH2 partially restoring antiviral signaling in GNG4-depleted cells. Together, FPPL establishes enzymatic covalent flavin installation as a distinct architecture for peptide-scale proximity proteomics and enables minimally perturbative discovery of functional microprotein networks in living cells.
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