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Stimulator of interferon genes (STING) triggers the type I interferon and inflammatory responses against a variety of DNA pathogens, which is essential to limiting viral infection and replication. STING activates the downstream kinase TBK1 at the trans-Golgi network (TGN) and is degraded at lysosomes through a process called lysosomal microautophagy. Impaired STING targeting to lysosomes results in the prolonged inflammatory signal, which may be associated with a variety of neurodegenerative and autoinflammatory diseases. Thus, development of methods to quantify STING degradation helps understand the mechanism of lysosomal microautophagy and its related diseases. Here we report a quantitative method to monitor STING degradation with two luciferases, firefly luciferase (FLuc) and Nanoluciferase (NLuc). The expression plasmid is composed of FLuc, a P2A self-cleavage site, and NLuc-tagged STING. FLuc intensity reflects the total amount of translated protein, serving as an internal control, while NLuc intensity corresponds to the amount of STING. Comparison of the NLuc/FLuc ratios at different time points after STING stimulation revealed the kinetics of decay of STING levels in live cells. This method should provide a useful complement to western blotting and fluorescence-activated cell sorter (FACS) analysis presently used to monitor STING degradation.Key words: innate immunity, STING, membrane traffic, lysosomal degradation, luciferase

Stimulator of interferon genes (STING) is critical for the type I interferon responses to pathogen- or self-derived cytosolic DNA. STING signalling is terminated by ESCRT-driven lysosomal microautophagy. How STING is directly encapsulated by lysosomes has not yet been understood. Here we show that two lysosomal components, a phosphoinositide PI(3,5)P 2 and CHMP4B (a subunit of ESCRT-III subcomplex) are essential for STING encapsulation by lysosomes. Liposome sedimentation assay reveals that CHMP4B binds to PI(3,5)P 2 . The forced recruitment of the catalytic core of Pikfyve (a lipid kinase generating PI(3,5)P 2 ) to early endosomes, recruits a fraction of CHMP4B to early endosomes. CHMP4B mutant, defective in the binding to PI(3,5)P 2 , cannot restore the microautophagic degradation of STING or the resolution of the STING signalling in cells depleted of Chmp4b . Our results reveal a molecular mechanism that terminates innate immune signalling at the lysosomal membrane. Inhibition of Pikfyve, a kinase generating PI(3,5)P2, abolishes CHMP4B (an ESCRT-III subunit) recruitment to lysosomes, and leads to an accumulation of STING vesicles and sustained signaling. Our results reveal a lysosomal PI(3,5)P2/CHMP4B axis that terminates innate immune signaling.

Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs. Kuchitsu et al. show that STING-positive vesicles of a recycling endosome origin are encapsulated into lysosomes after STING ubiquitination and this process requires ESCRT proteins Tsg101 and Vps4.

Stimulator of interferon genes (STING) triggers the type I interferon and inflammatory responses against a variety of DNA pathogens, which is essential to limiting viral infection and replication. STING activates the downstream kinase TBK1 at the trans-Golgi network (TGN) and is degraded at lysosomes through a process called lysosomal microautophagy. Impaired STING targeting to lysosomes results in the prolonged inflammatory signal, which may be associated with a variety of neurodegenerative and autoinflammatory diseases. Thus, development of methods to quantify STING degradation helps understand the mechanism of lysosomal microautophagy and its related diseases. Here we report a quantitative method to monitor STING degradation with two luciferases, firefly luciferase (FLuc) and Nanoluciferase (NLuc). The expression plasmid is composed of FLuc, a P2A self-cleavage site, and NLuc-tagged STING. FLuc intensity reflects the total amount of translated protein, serving as an internal control, while NLuc intensity corresponds to the amount of STING. Comparison of the NLuc/FLuc ratio after STING stimulation reported the kinetics of decay of STING levels in live cells. This method should provide a useful complement to western blotting and fluorescence- activated cell sorter (FACS) analysis presently used to monitor STING degradation.Key words: Innate immunity, STING, membrane traffic, lysosomal degradation, luciferase

Stimulator of interferon genes (STING) is critical for the type I interferon responses to pathogen- or self-derived cytosolic DNA. STING signalling is terminated by ESCRT-driven lysosomal microautophagy. How STING is directly encapsulated by lysosomes has not yet been understood. Here we show that two lysosomal components, a phosphoinositide PI(3,5)P and CHMP4B (a subunit of ESCRT-III subcomplex) are essential for STING encapsulation by lysosomes. Liposome sedimentation assay reveals that CHMP4B binds to PI(3,5)P . The forced recruitment of the catalytic core of Pikfyve (a lipid kinase generating PI(3,5)P ) to early endosomes, recruits a fraction of CHMP4B to early endosomes. CHMP4B mutant, defective in the binding to PI(3,5)P , cannot restore the microautophagic degradation of STING or the resolution of the STING signalling in cells depleted of Chmp4b. Our results reveal a molecular mechanism that terminates innate immune signalling at the lysosomal membrane.

Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived cytosolic DNA. STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven lysosomal microautophagy (LMA), the impairment of which leads to sustained inflammatory responses. It has been unknown how ESCRT targets STING directly to lysosomes. Here, through kinase inhibitor screening and knockdown experiments of all the individual components of ESCRT, we show that STING degradation requires PIKfyve (a lipid kinase that generates PI(3,5)P2) and CHMP4B/C (components of ESCRT-III subcomplex). Knockdown of Pikfyve or Chmp4b/c results in the accumulation of STING vesicles of a recycling endosomal origin in the cytosol, leading to sustained type I interferon response. CHMP4B/C localize at lysosomes and their lysosomal localization is abolished by interference with PIKfyve activity. Our results identify lysosomal ESCRT-III as a PI(3,5)P2 effector, reveal a role of the less characterized phosphoinositide PI(3,5)P2 in lysosomal biology, and provide insights into the molecular framework that distinguishes LMA from other cellular processes engaged with ESCRT.