PhagoID: Quantitative in situ proximity labeling defines the proteomes of bead- and microbe-containing phagosome lumina across species, maturation, and immune activation.

The phagosome is an immune organelle that integrates signals from diverse cargos to drive pathogen killing, tissue homeostasis, and antigen presentation. How this compartment coordinates such diverse functions remains incompletely understood. A comprehensive view of phagosomal composition across cell states and perturbations could accelerate insight into its regulation. Here, we establish PhagoID, a modular proximity labeling approach that enables in situ proteomic profiling of the phagosome lumen in living cells. Applying PhagoID to murine and human macrophages, we define conserved and species-specific phagosomal proteomes that include both canonical lysosomal components and unexpected contributors from nuclear, mitochondrial, and endoplasmic reticulum (ER) compartments. Integrating these proteomes with 25 genome-scale CRISPR screens of phagosomal phenotypes, we identify a network of lumenal proteins that regulate cargo uptake, acidification, antigen presentation, and microbial control highlighting previously unrecognized phagosomal effectors. We next adapt PhagoID to profile the microbe-containing phagosome and compare the sterile bead-containing phagosome to the Escherichia coli-containing phagosome, demonstrating a modest E. coli signature of phagosome remodeling. Comparative analyses further reveal that loss of LAMP1 enhances recruitment of ER-derived proteins to the phagosome, whereas loss of TFEB minimally perturbs the lumenal proteome despite its role in lysosomal biogenesis. Last, cytokine stimulation remodels the phagosome lumen to enrich for proteins involved in antigen presentation and antimicrobial activity in response to type I and II interferons. These data demonstrate that PhagoID delivers a high-confidence, quantitative, and integrative map of the phagosome lumen, revealing previously unidentified axes of organellar communication and regulatory control in innate immunity.