The study investigates the mechanism by which Toll-like receptor 4 (TLR4) couples endocytosis to the induction of interferon-β. TLR4 activates two distinct signaling pathways, TIRAP-MyD88 and TRAM-TRIF, which lead to the production of proinflammatory cytokines and type I interferons, respectively. The authors found that TLR4 sequentially activates these pathways, with the TIRAP-MyD88 pathway initiated at the plasma membrane and the TRAM-TRIF pathway activated in early endosomes after TLR4 endocytosis. They propose that TRAM functions as a sorting adaptor, similar to TIRAP, and that its bipartite localization motif controls its trafficking between the plasma membrane and endosomes. The study also suggests that the subcellular localization of signaling pathway components, such as TRAF3, may dictate the ability of receptors to induce type I interferon production.The study investigates the mechanism by which Toll-like receptor 4 (TLR4) couples endocytosis to the induction of interferon-β. TLR4 activates two distinct signaling pathways, TIRAP-MyD88 and TRAM-TRIF, which lead to the production of proinflammatory cytokines and type I interferons, respectively. The authors found that TLR4 sequentially activates these pathways, with the TIRAP-MyD88 pathway initiated at the plasma membrane and the TRAM-TRIF pathway activated in early endosomes after TLR4 endocytosis. They propose that TRAM functions as a sorting adaptor, similar to TIRAP, and that its bipartite localization motif controls its trafficking between the plasma membrane and endosomes. The study also suggests that the subcellular localization of signaling pathway components, such as TRAF3, may dictate the ability of receptors to induce type I interferon production.