Negative feedback regulation of STING signaling by TAX1BP1-directed Golgiphagy

Domain: neuroscience
Published: 2026-02-11
Editorial outcome: major_revision

Abstract
The cGAS-STING pathway is a critical regulator of type I Interferon (IFN) and inflammation upon cytosolic DNA-sensing. cGAS-STING signaling termination is regulated by lysosomal-mediated degradation of STING; however, the mechanisms controlling the inhibitory targeting of STING are incompletely understood. Here, we identify the selective autophagy receptor TAX1BP1 as a negative regulator of the cGAS-STING pathway. TAX1BP1-deficient macrophages activated by cGAS or STING agonists accumulate higher-order STING aggregates, exhibit heightened STING signaling, and increased production of type I IFN and proinflammatory cytokines. Mechanistically, TAX1BP1 promotes STING degradation through microautophagy by facilitating the interaction of STING with the ESCRT-0 protein HGS. Furthermore, STING activation is associated with the swelling and fragmentation of the Golgi apparatus, and TAX1BP1 and p62/SQSTM1 are essential for the autophagic degradation of fragmented Golgi (Golgiphagy). Our findings suggest that STING activation at the Golgi is coupled to its downregulation by Golgiphagy to restrict innate immune responses.

Introduction
The cGAS-STING pathway is an ancient DNA-sensing pathway critical for inflammatory responses to bacteria, DNA viruses and retroviruses1. Cyclic GMP-AMP synthase (cGAS) is a DNA sensor that binds double-stranded DNA and catalyzes the cyclic dinucleotide and second messenger 2’3’-cyclic GMP-AMP (2’3’-cGAMP; hereafter referred to as cGAMP), which binds to and activates Stimulator of Interferon Genes (STING) at the endoplasmic reticulum (ER)2–4. STING then translocates from the ER to the Golgi via coat protein complex II (COPII) vesicles5. At the trans-Golgi, oligomerized STING activates the kinase TBK1 and transcription factor IRF3, leading to the induction of type I interferon (IFN)6.

The activation of the cGAS-STING pathway is tightly regulated to prevent overexuberant or chronic inflammation. Upon activation at the trans-Golgi, STING is targeted for degradation in lysosomes. The selective autophagy receptor p62/SQSTM1 was previously shown to promote STING degradation7; however, the underlying mechanisms are not well understood. STING also has a primordial function, inducing autophagy independent of TBK1 activation and the induction of type I IFN8,9. STING can interact with LC3 and induce autophagy dependent on ATG5 but independent of ULK1 and Beclin18. STING-induced autophagy plays a role in the clearance of viruses and DNA from the cytoplasm. Recent studies have indicated that microautophagy is the main STING degradation pathway that terminates STING signaling. STING exits the trans-Golgi network (TGN) by adaptor protein complex 1 (AP-1)-mediated sorting into clathrin-coated vesicles and is ubiquitinated by lysine 63 (K63)-linked polyubiquitin (polyUb) chains, which are sensed by the endosomal sorting complexes required for transport (ESCRT) machinery10–13. STING vesicles are then directly encapsulated into LAMP1-positive lysosomes, resulting in STING degradation11. STING degradation is critical to prevent prolonged activation and induction of type I IFN and proinflammatory cytokines; however, the mechanisms underlying STING downregulation after its activation at the trans-Golgi are incompletely understood.

Tax1 Binding Protein 1 (TAX1BP1) was initially identified as an interacting protein of the human T-cell leukemia virus 1 (HTLV-1) Tax protein, and the NF-κB regulatory proteins A20 (also known as TNFAIP3) and TRAF614,15. TAX1BP1 was shown to inhibit NF-κB signaling by serving as an adaptor for A20 and its substrates targeted for deubiquitination16. In addition to regulating NF-κB, TAX1BP1 also inhibits the RIG-I-like receptor (RLR) pathway and the induction of type I IFN triggered by RNA virus infection17,18. TAX1BP1 also blocks RLR-mediated apoptosis by interacting with MAVS and promoting its degradation19. In the absence of TAX1BP1, MAVS aggregates spontaneously form and accumulate further with RNA virus infection20. In addition, TAX1BP1 suppresses the Toll-like receptor (TLR) 3/4 pathways by targeting the adaptor TRIF for degradation21,22. TAX1BP1 inhibits NF-κB and innate immune signaling pathways to quell inflammation and prevent autoimmunity since TAX1BP1 expression was shown to be downregulated in patients with systemic lupus erythematosus (SLE)23. TAX1BP1 functions as a selective autophagy receptor that contains two LC3 interaction regions (LIR) in the amino (N)-terminus and a ubiquitin binding zinc finger domain 2 (ZnF2) in the carboxy (C)-terminus24–27. TAX1BP1 detects ubiquitinated cargo via its ZnF2 domain, which is then recruited to developing autophagosomes via its LIR domains. TAX1BP1 also interacts with myosin VI, a cytoskeletal actin-based motor protein regulating vesicular transport, to induce autophagosome maturation26. Therefore, TAX1BP1 exerts multiple roles in autophagy, including cargo selection and autophagosome maturation. Although TAX1BP1 has been shown to inhibit NF-κB, RLR and TLR signaling, a potential role of TAX1BP1 in the regulation of the cGAS-STING pathway has not yet been investigated.

In this study, we identify TAX1BP1 as a negative regulator of the cGAS-STING pathway by targeting STING for degradation through autophagy of the Golgi apparatus (Golgiphagy) and ESCRT-mediated microautophagy. Loss of TAX1BP1 impairs STING degradation, leading to the accumulation of fragmented Golgi and STING aggregates and increased production of type I IFN and proinflammatory cytokines. Mice lacking TAX1BP1 in myeloid cells exhibit enhanced STING-mediated inflammatory responses and are resistant to poxvirus infection. These findings indicate that TAX1BP1 targets STING by Golgiphagy and microautophagy to limit the magnitude and duration of innate immune activation.

Results
TAX1BP1 inhibits cGAS-STING-mediated type I IFN and cytokine activation
We previously generated TAX1BP1-deficient mice using a gene trapping strategy but these mice were embryonic lethal, thus precluding in vivo studies16. To understand the physiological roles of TAX1BP1 we generated “floxed” conditional knockout mice on a C57BL/6 background. Embryonic stem (ES) cells were obtained with exon 4 of Tax1bp1 flanked by LoxP sites to facilitate excision by Cre recombinase (Supplementary Fig. 1). Tax1bp1fl/fl mice were crossed with LysM-Cre mice to delete TAX1BP1 in myeloid cells. We generated bone marrow-derived macrophage (BMDM) cells from control LysM-Cre mice and Tax1bp1fl/fl LysM-Cre mice (Tax1bp1–/–). Tax1bp1 mRNA and protein were undetectable in Tax1bp1–/– BMDMs as shown by real-time qRT-PCR and immunoblotting (Supplementary Fig. 2A, B). We also deleted TAX1BP1 in the human monocytic cell line, THP-1, using CRISPR-Cas9, and confirmed knockout by real-time qRT-PCR and immunoblotting (Supplementary Fig. 2C, D). We next sought to determine if the cGAS-STING pathway was dysregulated in TAX1BP1-KO THP-1 cells and Tax1bp1–/– BMDMs.

TAX1BP1-KO THP-1 cells were transfected with G3-YSD, a Y-form 26-mer DNA sequence from the HIV-1 genome, and cGAS-dependent agonist. TAX1BP1-KO THP-1 cells transfected with G3-YSD expressed significantly more IFN-β and IL-6 mRNA and protein compared to control THP-1 cells expressing a control nontargeting gRNA as shown by real-time qRT-PCR and ELISA (Fig. 1A-D). To determine if TAX1BP1 inhibits the pathway at or downstream of STING, we next permeabilized control and TAX1BP1-KO THP-1 cells and stimulated with the STING agonist cGAMP. TAX1BP1-KO THP-1 cells expressed significantly more IFN-β and IL-6 mRNA and protein compared to control cells after cGAMP stimulation (Fig. 1E-H). Tax1bp1–/– BMDMs similarly exhibited heightened IFN-β and IL-6 induction with both G3-YSD transfection and cGAMP stimulation (Fig. 1I-L). Therefore, TAX1BP1 is a negative regulator of the cGAS-STING pathway that can inhibit STING induction of downstream target genes.

To examine type I IFN responses, Tax1bp1fl/fl LysM-Cre mice were crossed with interferon-stimulated response element (ISRE) reporter mice (Mx1-GFP)28, which have GFP knocked into the Mx1 locus, and thus GFP expression can be used to monitor type I IFN responses at the single-cell level. BMDMs were generated from Tax1bp1fl/fl LysM-Cre-Mx1-GFP (Tax1bp1–/– Mx1-GFP) and control LysM-Cre-Mx1-GFP mice and were either transfected with G3-YSD to activate cGAS or stimulated with the STING agonist diABZI (compound 3). Incucyte S3 live-cell imaging was used to detect GFP expression in real-time after stimulation. Significantly more GFP was detected in Tax1bp1–/– Mx1-GFP BMDMs than control BMDMs transfected with G3-YSD or stimulated with diABZI (Fig. 1M, N). We also examined the expression of IFN-α4 and interferon-stimulated genes (ISG) by real-time qRT-PCR in TAX1BP1-KO THP-1 cells and Tax1bp1–/– BMDMs after cGAS-STING activation. TAX1BP1-KO THP-1 cells expressed significantly more Mx1, Isg15, Irf7 and Ifna4 mRNAs compared to control cells after transfection with G3-YSD, herring testes DNA (HT-DNA) or stimulation with cGAMP (Supplementary Fig. 3A-N). Tax1bp1–/– BMDMs similarly expressed more Mx1, Isg15, Irf7 and Ifna4 mRNAs compared to control BMDMs after stimulation with G3-YSD or cGAMP (Supplementary Fig. 3O-V).

TAX1BP1-deficient macrophages have heightened expression of HSV-1-induced type I IFN and ISGs
The cGAS-STING pathway is activated in response to herpes simplex virus type 1 (HSV-1) infection to induce type I IFN, although HSV-1 can antagonize cGAS-STING activation29,30. Control and TAX1BP1-KO THP-1 cells were infected with HSV-1 expressing yellow fluorescent protein (HSV-1-YFP) and subjected to Incucyte S3 live-cell imaging to monitor infection in real-time. TAX1BP1-KO THP-1 cells exhibited significantly decreased yellow fluorescence compared to control cells indicating that TAX1BP1-KO THP-1 cells are more resistant to HSV-1 infection (Fig. 2A). TAX1BP1-KO THP-1 cells infected with HSV-1 expressed more Ifnβ mRNA and protein (Fig. 2B, C) compared to control cells, which is likely mediating the increased resistance. HSV-1-infected TAX1BP1-KO THP-1 cells also expressed more Ifnα4, Mx1, Isg15 and Irf7 mRNAs compared to control HSV-1-infected cells (Fig. 2D-G).

Enhanced STING signaling and impaired STING degradation in TAX1BP1-deficient macrophages
STING activation induces the expression of type I IFN and proinflammatory cytokines via activation of TBK1-IRF3 and IKK-NF-κB pathways31–33. Therefore, we next examined the activation of TBK1-IRF3 and IKK-NF-κB downstream of STING by immunoblotting with phospho-specific antibodies after stimulation with diABZI. There was enhanced and prolonged diABZI-induced TBK1 and IRF3 activation in TAX1BP1-KO THP-1 cells compared to control cells (Fig. 3A, C, D). Similarly, there was more robust diABZI-induced IKKα/β phosphorylation in TAX1BP1-KO THP-1 cells compared to control cells (Fig. 3A, G). After binding to cGAMP, STING is phosphorylated by TBK1 on Ser366 to promote activation of IRF3 and induction of type I IFN6. STING phosphorylation was stronger and more persistent in diABZI-stimulated TAX1BP1-KO THP-1 cells compared to control cells (Fig. 3A, E). Interestingly, STING degradation was partially impaired in TAX1BP1-KO THP-1 cells treated with diABZI (Fig. 3A, F). We next examined STING signaling induced by HSV-1 infection of control and TAX1BP1-KO THP-1 cells. There was enhanced HSV-1-induced TBK1, IRF3, STING and IKKα/β phosphorylation in TAX1BP1-KO THP-1 cells compared to control cells (Fig. 3B, H-L). Therefore, TAX1BP1 inhibits cGAS-STING-mediated signaling induced by diABZI stimulation and HSV-1 infection.

STING degradation is a critical mechanism that downregulates its activation to ensure a transient type I IFN response. STING degradation mainly occurs through ESCRT-mediated microautophagy where clathrin-coated STING vesicles egress from the trans-Golgi and become directly encapsulated in lysosomes to degrade STING11–13. We next examined STING degradation in Tax1bp1–/– BMDMs treated with diABZI. As expected, in control BMDMs, STING was degraded beginning at 2 h post-stimulation and was almost completely degraded by 6 h (Fig. 3M). However, in Tax1bp1–/– BMDMs treated with diABZI, STING degradation was partially impaired (Fig. 3M). Lipidation of Atg8 family members (i.e., LC3B) is a hallmark of autophagosome biogenesis and lipidated LC3 (LC3-II) migrates faster compared to unlipidated (LC3-I) on SDS-PAGE gels. STING can induce the lipidation of ATG8 family members onto single-membrane vesicles in a process known as conjugation of ATG8 to single membranes (CASM)34. However, STING-induced LC3 lipidation was comparable in WT and Tax1bp1–/– BMDMs (Fig. 3M) and WT and TAX1BP1-KO THP-1 cells treated with diABZI (Fig. 3N). Therefore, loss of TAX1BP1 has no effect on STING-mediated LC3B lipidation.

To further examine the role of TAX1BP1 in STING degradation, we next performed a quantitative STING degradation flow cytometry assay using control THP-1 and TAX1BP1-KO THP-1 cells stably expressing STING-eGFP (Supplementary Fig. 4). Treatment of control THP-1 STING-eGFP cells with diABZI for 0, 2, 4, 6 and 8 h led to a progressive loss of eGFP fluorescence over time due to STING-eGFP degradation (Fig. 4A, B). However, diABZI-induced STING-eGFP degradation was significantly impaired in TAX1BP1-KO THP-1 cells (Fig. 4A, B). To determine the contribution of microautophagy to STING degradation in these cells, we used siRNA to knock down the ESCRT-I protein TSG101, which was previously shown to be important for STING degradation11. As expected, knockdown of TSG101 largely impaired diABZI-induced STING degradation in control THP-1 cells (Fig. 4C, D). STING degradation was also impaired in TAX1BP1-KO THP-1 cells, and TSG101 knockdown completely blocked STING degradation in these cells (Fig. 4C, D). Knockdown of TSG101 and degradation of STING-eGFP were confirmed by immunoblotting (Fig. 4E). These data suggest that the residual degradation of STING in the absence of TAX1BP1 occurs through microautophagy.

We next sought to determine if TAX1BP1 played a role in STING regulation by microautophagy. Given that p62/SQSTM1 was previously shown to colocalize with STING and the ESCRT-0 protein HGS12, we hypothesized that TAX1BP1, STING and HGS were in the same complex and TAX1BP1 linked ubiquitinated STING with the ESCRT machinery. Control HeLa and HeLa TAX1BP1-KO cells stably expressing STING-eGFP were generated for imaging studies (Supplementary Fig. 5C). We performed immunofluorescence microscopy using control HeLa-STING-eGFP and HeLa TAX1BP1-KO STING-eGFP cells stained with anti-HGS. There was no colocalization of STING and HGS in unstimulated cells, but the STING-HGS colocalization was strongly induced by diABZI stimulation (Fig. 4F, G). However, in TAX1BP1-KO HeLa cells the STING-HGS colocalization was partially impaired (Fig. 4F, G). Furthermore, a diABZI-induced STING-HGS interaction was detected by a co-IP assay, and the interaction was decreased in TAX1BP1-KO THP-1 cells (Fig. 4H). These data suggest that TAX1BP1 promotes an interaction of STING with the ESCRT machinery.

TAX1BP1 interacts with STING
We next examined whether endogenous TAX1BP1 could interact with STING. A co-immunoprecipitation (co-IP) assay was performed using WT THP-1 cells treated with diABZI in a time-course experiment. We immunoprecipitated with anti-TAX1BP1 and immunoblotted for STING. TAX1BP1 interacted with STING under basal conditions and the interaction further increased upon diABZI stimulation (Fig. 5A). The reciprocal IP was also performed (IP: anti-STING; IB: anti-TAX1BP1) and TAX1BP1 was detected in STING immunoprecipitates which was further enhanced with diABZI treatment (Fig. 5A). A proximity ligation assay (PLA) also confirmed basal TAX1BP1-STING interaction that was significantly increased by diABZI stimulation (Fig. 5B, C). Immunofluorescence and confocal microscopy were next performed to determine the cellular location of TAX1BP1-STING interaction. Since STING-induced TBK1 activation occurs at the trans-Golgi we stained cells with a marker of the trans-Golgi, TGN46. TAX1BP1 colocalized with STING, predominantly at the trans-Golgi, after diABZI stimulation (Fig. 5D, E). Together, these results suggest that TAX1BP1 interacts with STING basally, and this interaction is enhanced upon STING activation. To determine if the interaction between TAX1BP1 and STING was direct we next performed a NanoBiT complementation assay. In this assay, the luminescent protein NanoLuc is split into two subunits, Small BiT (SmB) and Large BiT (LgB) which interact very weakly. The interaction of target proteins tagged with the subunits brings SmB and LgB into an active luminescent complex35. Fusion of TAX1BP1 with LgB and STING with SmBiT resulted in luciferase activity (Fig. 5F, G), suggesting that TAX1BP1 and STING can directly interact.

TAX1BP1 promotes STING localization to lysosomes
Because STING degradation was partially impaired in both TAX1BP1-KO THP-1 cells and Tax1bp1–/– BMDMs treated with diABZI (Figs. 3, 4), we hypothesized that TAX1BP1 regulated STING targeting to lysosomes. Immunofluorescence and confocal microscopy experiments were next performed using control HeLa STING-eGFP and HeLa TAX1BP1-KO STING-eGFP cells to examine STING colocalization with the trans-Golgi marker Golgin-97 and the lysosomal marker LAMP2. STING trafficking proceeded as expected in control HeLa cells as STING colocalization with Golgin-97 increased 30 min post-diABZI treatment followed by a subsequent increase in colocalization with the lysosome (LAMP2) at later times (Fig. 6A, C, D, Supplementary Fig. 5A). However, in HeLa TAX1BP1-KO STING-eGFP cells, STING colocalization with Golgin-97 was enhanced at 1 and 2 h post-stimulation with diABZI, but STING colocalization with LAMP2 was diminished in TAX1BP1-KO HeLa cells treated with diABZI (Fig. 6A, C, D, Supplementary Fig. 5A).

Upon activation at the Golgi, STING is sorted into clathrin-coated vesicles derived from the recycling endosome (RE)10,11. To further define the block in STING trafficking in TAX1BP1-KO cells, we next examined STING trafficking to REs using transferrin receptor (TfnR) as a marker11. As expected, in control HeLa STING-eGFP cells STING colocalization with TfnR increased with 1 and 2 h diABZI treatment (Fig. 6B, E, Supplementary Fig. 5B). However, in HeLa TAX1BP1-KO STING-eGFP cells, STING colocalization with TfnR after 1 and 2 h diABZI stimulation was significantly decreased (Fig. 6B, E). Taken together, TAX1BP1 regulates the targeting of STING from the trans-Golgi to REs and lysosomes, and in the absence of TAX1BP1, STING accumulates at the trans-Golgi.

TAX1BP1 clears STING aggregates
TAX1BP1 functions as an aggrephagy receptor and clears protein aggregates together with p62/SQSTM136. To examine protein aggregates in TAX1BP1-deficient macrophages, we used PROTEOSTAT dye which emits red fluorescence upon binding to protein aggregates37. Control and TAX1BP1-KO THP-1 cells were incubated with PROTEOSTAT and subjected to flow cytometry to quantify fluorescence. As a positive control, cells were treated with the proteasome inhibitor MG132 to promote the accumulation of protein aggregates. Interestingly, there was more PROTEOSTAT staining in TAX1BP1-KO THP-1 cells after MG132 treatment suggesting more protein aggregates in the absence of TAX1BP1 (Fig. 7A, B, Supplementary Fig. 6). There were also more protein aggregates in TAX1BP1-KO THP-1 cells infected with HSV-1 or stimulated with cGAMP (Fig. 7A, B). We next examined STING aggregates using an SDD-AGE assay, an agarose gel-based electrophoresis assay that enables detection of large molecular weight protein complexes. SDD-AGE has previously been used to examine STING aggregates38,39. Indeed, STING aggregates were increased in TAX1BP1-KO THP-1 cells infected with HSV-1 compared to control cells (Fig. 7C). Control and TAX1BP1-KO THP-1 cells were also treated with diABZI, and STING aggregates were examined by SDD-AGE. STING aggregates were also increased in TAX1BP1-KO THP-1 cells upon diABZI stimulation (Fig. 7D). Together, these data suggest that TAX1BP1 functions as an aggrephagy receptor in the cGAS-STING pathway, and STING aggregates accumulate in the absence of TAX1BP1.

TAX1BP1 requires LC3, FIP200/TBK1 and Ub binding to inhibit STING
TAX1BP1 contains two N-terminal LIRs, three central coiled-coil domains and two C-terminal ZnF domains, of which ZnF2 binds to polyUb chains (Supplementary Fig. 7A). A recent study demonstrated that a TAX1BP1 point mutant (A114Q) was impaired in the interaction with the ULK1 scaffold protein FIP200 as well as TBK140. Point mutations were introduced in TAX1BP1 expressed in a lentiviral vector to disrupt the two LIRs (W49A, V143S), FIP200/TBK1 binding (A114Q) and polyUb binding (C760A). Silent mutations were also made in the cDNA to render TAX1BP1 resistant to the gRNA expressed in the CRISPR knockout THP-1 cells. Lentiviruses expressing WT TAX1BP1 or the indicated mutants were used to transduce TAX1BP1-KO THP-1 cells. Cells were then treated with diABZI and the expression of IFN-β and IL-6 was examined by real-time qRT-PCR. WT TAX1BP1 fully rescued the inhibition of STING-induced IFN-β and IL-6, which was comparable to control THP-1 cells (Supplementary Fig. 7B-D). The TAX1BP1 FIP200/TBK1 (A114Q), LIR (W49A, V143S) and ubiquitin binding (C760A) mutants were all impaired to varying degrees for the inhibition of STING-induced IFN-β and IL-6 (Supplementary Fig. 7B-D). Thus, TAX1BP1 interaction with LC3, Ub, FIP200 and/or TBK1 contributes to the inhibition of STING.

STING induces TAX1BP1-dependent Golgiphagy
We noticed that the Golgi appeared more fragmented in TAX1BP1-KO cells compared to control cells treated with diABZI (Fig. 6); therefore, we hypothesized that STING activation triggered Golgi stress and fragmentation which then elicited TAX1BP1-dependent autophagy of the Golgi apparatus (Golgiphagy). To test this hypothesis, we performed immunoblotting using trans-Golgi (Golgin-97) and cis-Golgi (GM130) markers. In control THP-1 cells treated with diABZI, there was degradation of both Golgin-97 and GM130; however, this degradation was not evident in TAX1BP1-KO THP-1 cells (Fig. 8A). Similar results were obtained in WT and Tax1bp1–/– BMDMs treated with diABZI (Fig. 8B). However, Golgiphagy induction by golgicide A, an inhibitor of cis-Golgi ADP ribosylation factor guanine nucleotide exchange factor (ARFGEF) GBF1 and Golgi dispersal agent, promoted the degradation of Golgi proteins independently of TAX1BP1 (Fig. 8A, B). These results indicate that TAX1BP1 is specific for STING-induced Golgiphagy and is not required for Golgiphagy induced by golgicide A.

To determine if diABZI-induced degradation of Golgi proteins was mediated by LC3-dependent autophagy, we performed immunofluorescence and confocal microscopy experiments using control HeLa STING-eGFP and HeLa TAX1BP1-KO STING-eGFP cells. Cells were treated with diABZI and bafilomycin A1 (BafA1) to block autophagic flux and then stained with anti-Golgin-97 and anti-LC3 to visualize Golgi and LC3+ autophagosomes, respectively. As expected, STING colocalized with Golgin-97 in control cells treated with diABZI, and this colocalization was enhanced in TAX1BP1-KO cells (Supplementary Fig. 8A). However, LC3 did not colocalize with Golgin-97 in control or TAX1BP1-KO cells, and there was only partial colocalization of LC3 with STING in control cells that was comparable to TAX1BP1-KO cells (Supplementary Fig. 8A-C). Therefore, TAX1BP1 does not appear to target Golgi or STING to LC3+ autophagosomes.

We next generated a Golgiphagy tandem fluorescence reporter which expresses mCherry and EGFP targeted to the Golgi by a minimal Golgi targeting sequence and alternative open reading frame (altORF) within the mRNA of the centromere protein CENP-R (Supplementary Fig. 8D). Under steady state conditions, Golgi will fluoresce both red and green; however, during late stages of Golgiphagy, fragmented Golgi are delivered to lysosomes where mCherry fluorescence is stable, but GFP fluorescence becomes quenched by the acidic microenvironment. Therefore, cells undergoing Golgiphagy will exhibit mCherry+ EGFP– foci. Control and TAX1BP1-KO HeLa cells were generated that stably express the Golgiphagy reporter. First, we confirmed that mCherry and GFP fluorescence were localized to the Golgi by staining with anti-Golgin-97. As expected, mCherry and GFP puncta were perinuclear and colocalized with Golgin-97 (Supplementary Fig. 8E). We next utilized the Golgiphagy reporter to determine if STING activation induced Golgiphagy. Immunofluorescence and confocal microscopy experiments revealed that Golgiphagy occurred in control HeLa cells treated with diABZI as revealed by an increase of mCherry+ EGFP– foci; however, mCherry+ EGFP– foci were significantly decreased in TAX1BP1-KO HeLa cells (Fig. 8C, D). These data suggest that TAX1BP1 is critical for STING-induced Golgiphagy.

TAX1BP1 clears protein aggregates together with p62/SQSTM141, which has also been implicated in STING degradation7. To determine if p62/SQSTM1 was involved in STING-induced Golgiphagy we generated p62/SQSTM1 knockout (p62-KO) THP-1 cells using CRISPR/Cas9. Knockout of p62/SQSTM1 was validated by immunoblotting (Fig. 8E). We treated control and p62-KO THP-1 cells with diABZI and examined the expression of Golgin-97, GM130 and STING. Interestingly, diABZI-mediated degradation of Golgi proteins (Golgin-97 and GM130) and STING (Fig. 8F) was significantly impaired in p62-KO THP-1 cells. Reconstitution of Flag-p62 into p62-KO THP-1 cells using a lentiviral vector expressing Flag-p62 restored diABZI-induced Golgin-97 and STING degradation (Supplementary Fig. 9A). Therefore, p62/SQSTM1 and TAX1BP1 are both required for STING-induced Golgiphagy.

We next examined a potential role of macroautophagy in STING-induced Golgiphagy by generating ATG3 knockout (ATG3-KO) THP-1 cells using CRISPR/Cas9. ATG3 functions as an E2-like enzyme for LC3 lipidation that is essential for macroautophagy42. We also generated FIP200 (also known as RB1CC1) knockout (FIP200-KO) THP-1 cells by CRISPR/Cas9. FIP200 is a scaffold protein for the ULK1 complex required for the initial stages of autophagosome formation43. FIP200 has also been implicated in TAX1BP1-mediated noncanonical autophagy, whereby TAX1BP1 clusters FIP200 around cargo to form autophagosomes independent of LC3 lipidation40. ATG3-KO and FIP200-KO THP-1 cells were validated by immunoblotting (Supplementary Fig. 9B, D). Control, ATG3-KO and FIP200-KO THP-1 cells were treated with diABZI and immunoblotting was performed to examine the degradation of Golgin-97 and STING. Golgin-97 degradation seemed to be enhanced in diABZI-stimulated ATG3-KO and FIP200-KO THP-1 cells compared to control cells, and there was no significant difference in STING degradation in ATG3-KO and FIP200-KO THP-1 cells compared to control cells (Supplementary Fig. 9C, E). These data suggest that macroautophagy and FIP200-mediated noncanonical autophagy are not involved in STING degradation and STING-induced Golgiphagy.

To determine if microautophagy played a role in STING-induced Golgiphagy we used siRNA to knockdown the ESCRT-0 protein TSG101. The knockdown of TSG101 in THP-1 cells was confirmed by immunoblotting (Supplementary Fig. 9F). However, knockdown of TSG101 had no effect on STING-induced Golgin-97 degradation compared to cells transfected with nontargeting control siRNA (Supplementary Fig. 9F). VPS4A and VPS4B function as AAA ATPases that are essential for the function of the ESCRT machinery and are required for STING degradation by microautophagy11. Knockdown of VPS4A/B had no effect on Golgin-97 degradation induced by diABZI stimulation (Supplementary Fig. 9G) providing further evidence that this Golgi degradation pathway is independent of microautophagy.

To determine if STING was required for diABZI-induced Golgi degradation, we treated control THP-1 and STING-KO THP-1 cells with diABZI and examined the expression of Golgin-97 by immunoblotting. Interestingly, basal levels of Golgin-97 were elevated in STING-KO cells (Fig. 8G) suggesting that tonic activation of STING may promote Golgiphagy. Furthermore, diABZI-induced Golgin-97 and GM130 degradation was completely impaired in the absence of STING (Fig. 8G).

Fragmented Golgi accumulate in TAX1BP1-deficient macrophages upon STING activation
To examine Golgi with higher resolution, we next performed transmission electron microscopy (TEM) using WT and Tax1bp1–/– BMDMs treated with diABZI (Fig. 9, Supplementary Fig. 10). In WT BMDMs, intact Golgi stacks and cisternae were readily detected; however, after diABZI treatment the Golgi became swollen and fragmented consistent with a recent study44. Interestingly, in WT BMDMs we could detect phagophore formation around fragmented Golgi, and fragmented Golgi localized in autophagosomes and autolysosomes (Fig. 9A-D). In Tax1bp1–/– BMDMs, fragmented Golgi accumulated in diABZI-treated Tax1bp1–/– BMDMs and these were not found in autophagosomes or autolysosomes (Fig. 9A-H, Supplementary Fig. 10). Tax1bp1–/– BMDMs treated with diABZI exhibited a significant decrease in the number of autophagic vesicles (Fig. 9I). Furthermore, small electron dense structures in Golgi that may represent protein aggregates (potentially STING) were observed in WT BMDMs treated with diABZI, and these were increased in diABZI-treated Tax1bp1–/– BMDMs (Fig. 9A-H). Therefore, STING activation induces Golgi stress and fragmentation that triggers Golgiphagy mediated by TAX1BP1.

Recent studies have demonstrated that STING exerts proton channel activity upon trafficking to the Golgi44,45. The STING proton channel activity increases the pH in the Golgi, leading to LC3B lipidation and inflammasome activation, functions distinct from IRF3 activation and type I IFN induction. We hypothesized that STING proton channel activity promoted Golgi stress and fragmentation which then triggered Golgiphagy. To assess the importance of STING proton channel function in STING-induced Golgiphagy, we used the STING agonist C53, which was identified as an inhibitor of its proton channel function and downstream LC3 lipidation45. We treated control and TAX1BP1-KO THP-1 cells with diABZI for 30 min to allow for STING to traffic to the Golgi and then added compound C53 to inhibit STING proton channel function. Treatment of control and TAX1BP1-KO THP1 cells with C53 did not block the degradation of Golgin-97 induced by diABZI, although C53 suppressed diABZI-induced LC3 lipidation as expected (Fig. 9J). Therefore, STING-mediated Golgiphagy does not appear to depend on its proton channel activity.

Tax1bp1fl/fl LysM-Cre mice exhibit heightened STING activation
To understand the physiological relevance of TAX1BP1 inhibition of cGAS-STING, Tax1bp1fl/fl LysM-Cre and LysM-Cre mice were injected intraperitoneally (i.p.) with diABZI and serum levels of type I IFN and inflammatory cytokines were quantified by the LEGENDplexTM mouse inflammation panel using flow cytometry. Serum levels of IFN-β, IL-6 and TNF were significantly increased in Tax1bp1fl/fl LysM-Cre mice (Fig. 10A-C). The other cytokines in the inflammation panel were not significantly changed in Tax1bp1fl/fl LysM-Cre mice injected with diABZI (Supplementary Fig. 11). Tax1bp1fl/fl LysM-Cre and LysM-Cre mice were next infected with Ectromelia virus (ECTV), a mouse DNA virus and orthopoxvirus that causes mousepox and to which the innate immune response is dependent upon STING46 and cGAS47. Mice were infected in the footpad and viral loads in the draining lymph node and liver quantified by plaque assays. ECTV viral loads were significantly diminished in the draining lymph node of Tax1bp1fl/fl LysM-Cre mice (Fig. 10D); there was also a trend of lower viral load in the liver of Tax1bp1fl/fl LysM-Cre mice (although not statistically significant) (Fig. 10E). Therefore, deletion of TAX1BP1 in myeloid cells promotes resistance to ECTV replication and/or spread in infected mice.

Methods
Tax1bp1 knockout mice
An embryonic stem (ES) cell clone (clone ID: HEPD0680_3_H08) with exon 4 of the Tax1bp1 gene flanked by LoxP sites was obtained from the European Conditional Mouse Mutagenesis Program (EUCOMM). The LacZ/Neo cassettes were flanked by FRT sites to facilitate excision by flippase (Flp) recombinase prior to deletion of exon 4 by Cre recombinase. A C57BL/6 embryonic stem cell (ES) clone with a correctly targeted Tax1bp1 allele was microinjected into mouse blastocysts to create chimeric mice. Male chimeras were crossed with female C57BL/6 mice and pups were screened for germline transmission of the mutant allele by PCR genotyping. Germline transmission of the LoxP flanked Tax1bp1 allele was confirmed by PCR genotyping. Heterozygous mice were then crossed with transgenic mice expressing Flp under the control of the universal Pgk1 promoter and deletion of the LacZ/Neo cassettes was confirmed by PCR. Tax1bp1fl/fl mice were crossed with LysM-Cre mice (Jackson Laboratory; strain #004781) to delete TAX1BP1 in myeloid cells. Tax1bp1fl/fl LysM Cre mice were also crossed with Mx1-GFP mice28 (a gift from Adolfo García-Sastre). Animals were housed in a specific pathogen-free (SPF) barrier facility kept at 22°C with 40–60% humidity and maintained on a 12 h light/12 h dark cycle with ad libitum access to food and water. Experimental animals were bred separately and age- and sex-matched mice were used for each experiment. Animals were euthanized by CO2 inhalation. All animal experiments were approved and performed in accordance with the Penn State College of Medicine Institutional Animal Care and Use Guidelines (Protocol Number 1854).

Cell culture, plasmids, and reagents
THP-1 cells were purchased from ATCC (TIB-202) and cultured in RPMI media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). THP-1 Dual KO-STING cells were purchased from InvivoGen (thpd-kostg). BMDMs were generated by culturing bone marrow cells isolated from LysM-Cre and Tax1bp1fl/fl LysM-Cre mouse femurs in DMEM medium supplemented with 30% L929 conditioned medium, 20% FBS and 1% P/S for 7 days as described previously60. TAX1BP1-KO HeLa cells were provided by Richard Youle61. Lenti-X 293T cells were purchased from Takara (632180) and cultured in DMEM medium supplemented with 10% FBS and 1% P/S. Plasmid transfection was performed using GenJet version II (SignaGen Laboratories; SL100489), and G3-YSD transfection was performed using Lipofectamine 3000 reagent (ThermoFisher Scientific; L3000008) following the manufacturer’s instructions. LentiCRISPRv2 was a gift from Feng Zhang (Addgene plasmid #52691; 422 http://n2t.net/addgene:52691; RRID:Addgene_52691). pUltra-Hot was a gift from Malcolm Moore (Addgene plasmid # 24130; http://n2t.net/addgene:24130; RRID:Addgene_24130). psPAX2 was a gift from Didier Trono (Addgene plasmid #12260; http://n2t.net/addgene:12260; RRID:Addgene_12260). pHR-SFFV-FLAG-SQSTM1 (p62) was a gift from Benjamin Wolozin (Addgene plasmid #223715; http://n2t.net/addgene:223715; RRID:Addgene_223715). The STING-eGFP plasmid (pMRX-hSTING-eGFP) was a gift from Nan Yan62. PMA (12-011) was purchased from Fisher Scientific. G3-YSD (tlrl-ydna), cGAMP (tlrl-nacga23) and diABZI compound 3 (tlrl-diabzi) were purchased from InvivoGen. HT-DNA (D6898) was purchased from Millipore-Sigma.

Antibodies used for immunoblotting
p-TBK1 (5483S), TBK1 (3504S), p-IRF3 (4947S), IRF3 (4302S), p-STING (50907S), p-IKKα/β (2697S), IKKβ (8943S), TSG101 (72312S) and HGS (15087S) antibodies were purchased from Cell Signaling Technology. TAX1BP1 antibodies were purchased from Abcam (ab176572) and Bethyl (A303-792A). STING antibody was purchased from Cell Signaling Technology (13647S). GFP (B-2) (sc-9996), vinculin (sc-73614) and β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology. Golgin-97 polyclonal antibody (12640-1-AP), TGN46 monoclonal antibody (66477-1-1g), VPS4A polyclonal antibody (14272-1-AP), VPS4B monoclonal antibody (68443-1-Ig), FIP200 (RB1CC1) polyclonal antibody (17250-1-AP), ATG3 polyclonal antibody (11262-2-AP) and p62/SQSTM1 polyclonal antibody (18420-1-AP) were purchased from Proteintech. GM130 polyclonal antibody (PA5-95727) and p-IKKα/β (37515) antibody were purchased from ThermoFisher Scientific. Flag M2 monoclonal antibody (F3165) was purchased from Millipore-Sigma.

Antibodies used for immunofluorescence assays
TAX1BP1 (Ab176572) and Golgin-97 (Ab169287) antibodies were purchased from Abcam. Golgin-97 (12640-1-AP) and CoraLite 594-conjugated TGN46 antibodies were purchased from Proteintech (CL594-13573). LAMP2 antibody (A15464) and Alexa Fluor 647-conjugated donkey anti-mouse IgG (A-31573) were purchased from ThermoFisher Scientific. Transferrin receptor (TfnR)/CD71 antibody (46222) was purchased from Cell Signaling Technology.

Nucleic acid manipulation
pUltra-Hot-TAX1BP1 was generated by subcloning TAX1BP1 PCR product digested with Nhe1-HF (New England Biolabs; R3131S) and BamH1-HF (New England Biolabs; R3136S) into the pUltra-Hot vector. Silent mutations were introduced into the TAX1BP1 cDNA by PCR site-directed mutagenesis to render TAX1BP1 resistant to the TAX1BP1 gRNA. Point mutations for TAX1BP1 LIR double mutant (W49A and V143S), ZnF2 mutant (C760A) and FIP200/TBK1 mutant (A114Q) were introduced by PCR site-directed mutagenesis in the pUltra-Hot-TAX1BP1 plasmid. Site-directed mutagenesis was performed with the QuikChange II mutagenesis kit (Agilent; 200523) using the primers listed in Supplementary Table 1. The Golgiphagy reporter was generated by adding a minimal Golgi targeting sequence (FLWRIFCFRK)56 to the C-terminus of the tandem fusion of fluorescence proteins, mCherry and EGFP, as described previously63. All constructs generated in this study were verified by sequencing.

CRISPR-Cas9-mediated generation of knockout cells
CRISPR-Cas9-mediated knockout of TAX1BP1, ATG3, FIP200 and p62/SQSTM1 was performed as described previously19,64 by cloning control nontargeting gRNA and gRNAs for Tax1bp1, Atg3, Fip200 and p62/SQSTM120,61 (Supplementary Table 2) into the pLentiCRISPRv2 plasmid. THP-1 cells were transduced with lentiviruses expressing Cas9 and control nontargeting gRNA or Tax1bp1, Atg3, Fip200 or p62/SQSTM1 gRNAs to generate control and knockout cell lines. Bulk population, puromycin-resistant THP-1 control-gRNA and knockout cells were validated by immunoblotting and qRT-PCR (for TAX1BP1-KO).

Knockdown of gene expression with siRNAs
THP-1 cells were transfected with control nontargeting (D-001810-01-05) or OnTargetPlus human TSG101 (L-003549-00-0005), VPS4A (L-013092-00-0005) or VPS4B (L-013119-00-0005) SMARTpool siRNAs (Horizon Discovery) using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific; 13778075). Cells were harvested after 48 h and knockdowns were confirmed by immunoblotting.

Virus infections
HSV-1-YFP was a gift from Drs. John Wills and Lynn Enquist65. Cells were starved for 1 h in serum-free RPMI and inoculated with HSV-1-YFP for 1 h at a multiplicity of infection=1 (MOI=1) in serum-free RPMI and further incubated in complete DRPMI for the indicated times.

Live-cell imaging
For HSV-1 replication, 3×105 control or TAX1BP1-KO THP-1 cells were differentiated with PMA in 24-well plates and then infected with HSV-1-YFP (MOI=1). For Mx1-GFP expression, 3×105 BMDMs prepared from LysM-Cre-Mx1-GFP and Tax1bp1fl/fl LysM-Cre-Mx1-GFP mice were seeded in 24-well plates and transfected with either 1 μg G3-YSD or stimulated with 1 μM diABZI. Live-cell imaging was performed with an IncuCyte S3 imaging system (Sartorius). Images were acquired every 2 h using phase contrast and green channels with a 10× objective in triplicate. GFP fluorescence was quantified as green object count per image normalized to phase area confluence.

Real-time quantitative reverse transcription PCR
2×106 BMDMs or PMA-differentiated THP-1 cells in 6-well plates were transfected with either 1 μg G3-YSD or 1 μg HT-DNA, infected with HSV-1 (MOI=1) or stimulated with either 50 nM cGAMP or 1 μM diABZI. Cells were harvested by gentle scraping and processed for RNA isolation using the RNA Spin II Kit (Macherey-Nagel; 740955) using the manufacturer’s instructions. cDNA was synthesized using M-MLV Reverse Transcriptase (ThermoFisher Scientific; 28025-013) and Oligo(dT) (ThermoFisher Scientific; 18418-012), and qRT–PCR (ThermoFisher Scientific; QuantStudio 3) was performed using Power SYBR green PCR master mix (ThermoFisher Scientific; A25742) according to the manufacturer’s protocol. β-Actin was used for normalization of the assay, and fold change in expression was calculated by the 2−ΔΔCT method. Primers used for real-time qRT-PCR are listed in Supplementary Table 3.

Enzyme-linked immunosorbent assay
3×106 PMA-differentiated THP-1 cells in six-well plates were transfected with 1 μg G3-YSD, infected with HSV-1 (MOI=1) or stimulated with 50 nM cGAMP and the culture supernatants were collected at the indicated time points. The concentration of IFN-β and IL-6 in the cell culture supernatants was measured using the Human IFN-β ELISA kit (PBL Assay Science; 41410) and Human IL-6 Quantikine ELISA kit (R&D; D6050) according to the manufacturers’ instructions.

Immunoblotting and co-immunoprecipitation assays
Whole cell lysates were generated by lysing cells in RIPA buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, Pierce Protease and Phosphatase Inhibitor) on ice, followed by centrifugation. Lysates were separated using SDS–polyacrylamide gels, transferred onto a nitrocellulose membrane and blocked for 1 h in 5% skim milk followed by incubation with the indicated primary antibodies overnight at 4°C followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Bio-Rad; 34580) and the Bio-Rad ChemiDoc Imaging System. Western blot images were processed using Image Lab software (Bio-Rad Laboratories). The Dynabeads Protein G Immunoprecipitation Kit (ThermoFisher Scientific; 10007D) was used for co-IP assays according to the manufacturer’s recommendations.

Protein aggregation detection assay
The PROTEOSTAT Aggresome detection kit (Enzo Life Sciences; ENZ-51035-0025) was used to detect total protein aggregates according to the manufacturer’s instructions. 1×106 control or TAX1BP1-KO THP-1 cells were differentiated with PMA in 6-well plates and either infected with HSV-1-YFP (MOI=0.1) or stimulated with 50 nM cGAMP. As a positive control, cells were treated with MG132 for 16 h. Cells were then harvested by gentle scraping and centrifuged at 500 g for 5 min followed by fixation with 4% formaldehyde for 30 min at RT, permeabilized with 0.5% Triton X-100 for 30 min on ice, and stained with the PROTEOSTAT dye for 30 min at RT. Samples were then acquired on a BD LSRFortessa flow cytometer and data analysis was performed using FlowJo v10 software. The gating strategy for these experiments is shown in Supplementary Fig. 6.

Semi-denaturing detergent agarose gel electrophoresis
SDD-AGE was performed as previously described35. Briefly, whole cell lysates were resuspended in 2× sample buffer (0.5x Tris-borate-EDTA [TAE], 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) followed by incubation at room temperature for 15 min and loaded onto a 1.5% agarose gel. Electrophoresis was performed in the running buffer (1× TAE and 0.1% SDS) for 35 min with a constant voltage of 100 V at 4°C, proteins were transferred to a nitrocellulose membrane for immunoblotting.

Immunofluorescence microscopy
Control HeLa and TAX1BP1-KO HeLa cells stably expressing STING-eGFP plasmid were generated with a recombinant retrovirus expressing STING-eGFP. Cells were treated with 10 mM leupeptin and 1 μM diABZI for the indicated time points. Immunofluorescence/confocal microscopy was performed as previously described64. Images were acquired with using a Leica SP8 confocal microscope equipped with a 63× oil objective. Images were processed, analyzed and Manders’ coefficient analysis was performed using Fiji image analysis software (NCBI).

STING quantitative flow cytometry degradation assay
Control THP-1 and TAX1BP1-KO THP-1 cells stably expressing STING-eGFP plasmid were generated with a recombinant retrovirus expressing STING-eGFP. Cells were sorted for high GFP expression. In certain experiments, cells were first transfected with control nontargeting or OnTargetPlus human TSG101 SMARTpool siRNAs (Horizon Discovery) using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific). Cells were treated with 1 μM diABZI and then harvested by gentle scraping and centrifuged at 500 g for 5 min followed by staining with SYTOX Blue. Samples were then acquired on a BD LSRFortessa flow cytometer. Data analysis was performed using FlowJo v10 software. The gating strategy for these experiments is shown in Supplementary Fig. 4.

Transmission electron microscopy
WT and Tax1bp1–/– BMDMs (3×106) were seeded in 60 mm cell culture dishes (174888; Nalgene Nunc) and treated with 1 μM diABZI for 4 h. Cells were then gently washed with PBS followed by fixation with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and further fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 h. Samples were dehydrated in a graduated ethanol series, acetone, and embedded in LX-112 (Ladd Research, Williston, VT). Thin sections (65 nm) were stained with uranyl acetate and lead citrate and viewed in a JEOL JEM 1400 Transmission Electron Microscope (JEOL USA Inc., Peabody, MA, USA) located at the Penn State College of Medicine TEM Facility (RRID Number: SCR_021200).

Proximity ligation assay
PLA was performed using the DuoLink In Situ Red Starter Kit Mouse/Rabbit (Millipore-Sigma; DUO92101) as recommended by the manufacturer. 5×104 PMA-differentiated THP-1 cells were seeded in 8-well chamber slides (Corning; 354118) and stimulated with 1 μM diABZI for the indicated time points. Cells were then fixed, permeabilized, and incubated with primary antibodies anti-rabbit TAX1BP1 (Abcam; ab176572) and anti-mouse STING (Proteintech; 66680-1-Ig). The slides were incubated with Duolink PLA probes, ligated, amplified, and washed. Images were acquired using a Leica SP8 confocal microscope equipped with a 63× oil objective. Images were processed, analyzed, and quantified using Fiji image analysis software (NCBI).

Protein fragment complementation NanoBiT assay
293T cells were transfected with the NanoBiT binary plasmids (Promega; N2014) expressing TAX1BP1 and STING for 24 h, harvested and resuspended at 5×105 cells/ml in Opti-MEM I medium. Next, 50 µl of cell suspension was transferred to a 96-well opaque plate in triplicate and treated with 25 µl of Opti-MEM I medium supplemented with 1% furimazine (Promega; N1110) for 5 min as described previously35. Luminescence intensities were measured using a GloMax 96 microplate luminometer (Promega).

In vivo diABZI treatment
8–10-week-old Tax1bp1fl/fl LysM-Cre and LysM-Cre mice (n=10; male n=5 and female n=5), were intraperitoneally administered with 2.5 mg/kg diABZI compound-3 (InvivoGen; tlrl-diabzi) for 3 h. Serum cytokines were measured by the LEGENDplexTM mouse inflammation panel (BioLegend; 740446) and acquired on a BD LSRFortessa flow cytometer according to the manufacturer’s protocol. Data analysis was performed using LEGENDplexTM data analysis software.

Ectromelia virus infection and titration
All experiments used WT ECTV (Moscow strain)66 propagated in L929 cells. 8–10-week-old Tax1bp1fl/fl LysM-Cre and LysM-Cre mice (n=10; male n=5 and female n=5) were injected with 3×103 plaque-forming units (PFU) of ECTV into one hind footpad or mock-infected with HBSS/0.1% BSA. Mice were monitored daily for morbidity and mortality. Infected footpad, draining lymph node, spleen, and liver were harvested at 7 d.p.i. and frozen in HBSS/0.1% BSA at −80°C. Tissues were homogenized using a Tissue Lyser II, and titers were determined by standard plaque assay under carboxymethylcellulose overlay on WT-19 cells at 3 d.p.i. For weighed tissues, PFU per gram tissue was calculated.

Statistical analysis
Data are presented as mean ± standard deviation from a representative experiment with triplicate samples. Statistical analysis was performed in GraphPad Prism 10.1.2 and indicated in the Figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Suklabaidya and colleagues identify new mechanisms through which TAX1BP1 regulates the cGAS-\nSTING pathway. The data presented supports an important role for TAX1BP1 in modulating inflammation by degrading\nactivated STING aggregates through the process of Golgiphagy.\nIn TAX1BP1-deficient macrophages, cGAS or STING agonists trigger stronger STING signaling, leading to increased\nproduction of type I IFN and proinflammatory cytokines. Conditional knockout mice lacking TAX1BP1 in myeloid cells exhibit\nheightened inflammatory responses upon STING activation and show increased resistance to poxvirus infection.\nThis research underlines the significance of TAX1BP1 in modulating cGAS-STING-mediated inflammation by selectively\ndegrading fragmented Golgi structures, which may have implications for controlling immune responses in infectious\ndiseases and inflammatory conditions.\nThe experiments are well-designed and executed, and the imaging figures are clear and illustrative. The study can be\nconsidered for publication, pending clarification of the following questions and suggested areas for improvement:\nSpecific comments:\n1) In the “TAX1BP1 promotes STING localization to lysosomes” section (Figure 5), why were HeLa cells chosen for the\ncolocalization experiments? Most of the study was conducted in cells of the myeloid lineage, specifically BMDMs or THP-1\ncells. Although the HeLa cell line is a useful and convenient tool, it originates from cervical cancer, raising concerns about\nwhether the signaling and localization observed with the lysosome marker LAMP2 are comparable to those in innate\nimmune cells. It would be preferable for the authors to provide colocalization data in BMDMs, THP-1 cells, or other myeloid-\nderived cell lines.\n2) In Supplementary Figure 3, G3-YSD-induced Mx-1, Isg15, Irf7, and Ifna4 mRNA data are presented in THP-1 cells, but not\nin BMDMs. It would be relevant to include these data for BMDMs as well.\n3) In Figure 9, the LEGENDplex mouse inflammation panel was utilized to investigate the physiological relevance of\nTAX1BP1 inhibition of cGAS-STING in vivo. Serum levels of IFN-β and IL-6 are presented. Does the panel include other\ninflammatory cytokines? If so, these data should be included and discussed.\n4) Have the authors investigated the mechanism of TAX1BP1-regulated STING Golgiphagy in other cell lineages important\nfor innate immunity, such as B cells? Understanding the lineage specificity of the TAX1BP1-STING interaction would be of\nsignificant interest to the community, and its disease relevance should be addressed in the Discussion section. Additionally,\nhas TAX1BP1 been associated with STING-related diseases, such as autoimmune or degenerative conditions? Including\nthese implications in the Discussion section would enhance its value for readers from various research backgrounds.\nMinor comments:\n1) TAX1BP1 is an abbreviation and should be spelled out in full when first introduced in the text.\n2) In Supplementary Figure 4 and Figure 6, it would be more illustrative to include a merged figure to show the colocalization\nof different markers.", "reviewer_2": "In the present manuscript from the group of Edward Harhaj entitled “Negative feedback regulation of STING signaling 1 by\nTAX1BP1-directed Golgiphagy” the authors have identified the selective autophagy receptor TAX1BP1 as a negative\nregulator of the cGAS-STING pathway. They present evidence to suggest that TAX1BP1 targets STING for degradation via\na Golgiphagy pathway.\nThe paper is clearly interesting, but several of the important conclusions made are not justified based on the data presented.\nMore experiments are required to justify the conclusions made.\nMajor points:\n1.Particularly, overexpression of STING in some vital experiments raise significant doubt about the interpretation of the\nresults since the results may be based on a non-physiological model system with artefacts affecting the results. The\nfluorescence microscopy imaging data need to be improved. The imaging data in this paper will also be very much improved\nto obtain more secure conclusions if the authors used correlative light and electron microscopy to determine the fate of\nSTING from the Golgi to the lysosome. Another improvement would be to use Halo-tagged STING to follow the lysosomal\nturnover of STING in a quantitative manner to enable comparison with other pathways like ESCRT-dependent\nmicroautophagy (ref. 11) and STING-dependent macroautophagy.\n2. As a continuation of point 1: How important is this pathway described here relative to ESCRT-dependent degradation of\nSTING by microautophagy? Can the authors determine the fraction of STING that is degraded by the Golgi-fragmentation\npathway aided by TAX1BP1? What about KO of p62 and other SLRs to determine if they have a role together with\nTAX1BP1?\n3. Could it be that TAX1BP1 is primarily important here as an autophagy receptor for degradation of fragmented Golgi and\nthat STING is simply degraded as a bystander? Do the authors provoke Golgi fragmentation and thereby TAX1BP1-\ndependent Golgiphagy by overexpressing STING too much?\n4. KO of TAX1BP1 in human monocytic THP-1 cells led to increased resistance to Herpes Simplex Virus type 1 (HSV-1)\ninfection and increased expression of mRNAs for Ifn-b, Ifna4, Mx1, Isg15 and Irf7 (Fig. 2B, D-G). However, I find the increase\nin the IFN-b protein level to be relatively small in Fig.2C with no change at 12h and a small increase at 24h. Can the authors\ncomment on this?\n5. In Figure 3A and -B, the blots for pTBK1 (S172), pIRF3 (S396), pSTING (S366) as well as pIKKalpha/beta (S176/180 and\npIkBalpha (S32/36) need quantifications with statistics.\n6. Importantly, also the total levels of STING should be quantified in Fig. 3A and 3B from the blots shown. From just visual\ninspection there does not seem to be a profound turnover of STING, but this may of course be balanced by increased\nexpression of STING?\n7. Lines 168-169: The authors write that “However, in Tax1bp1–/– BMDMs treated with diABZI, STING degradation was\nlargely impaired (Fig. 3C).” From the top blot in Figure 3C this clearly seems an overstatement and the authors need to\nquantify the STING levels from the experiments in Fig. 3C.\n8. In Fig. 4A the band of IPed TAX1BP1 (IPed with a TAX1BP1 antibody) migrates below the 75 kDa MW marker while the\nband of TAX1BP1 IPed with STING (IPed with a STING antiboy) migrates clearly above the 75 kDa MW marker. How can\nthe authors explain these seemingly very different molecular weights of TAX1BP1 observed in the two IP experiments?\n9. Does TAX1BP1 bind directly to STING? Importantly, the authors need to show that STING and TAX1BP1 can directly\ninteract with each other. IP from cells and PLA suggest that STING and TAX1BP1 may be in a complex but assays for direct\ninteractions are needed such as for example GST pulldowns of recombinant proteins\nand/or bimolecular fluorescence\ncomplementation assays.\n10. The imaging shown in Figure 4D needs quantifications of colocalizations and an estimate of how relevant the\nfluorescence signal distributions shown are as only single cells are shown in the top and bottom panels.\n11. The resolution of the images in Fig 4D should be significantly improved. This is especially important for the lower panel\ndiABZI (2h). See for example the imaging shown in reference 11 (Kuchitsu et al. 2023).\n12. The quality and resolution of the imaging shown in Fig. 5A and D need to be improved.\n13 (Related to point 1 and 3). Importantly, the overexpression of STING affects the experiment a lot since Golgi looks\ncompletely fragmented in the TGN46 staining in Fig 5A. The impression that STING is degraded as a bystander by\nTAX1BP1 mediated Golgiphagy caused by a non-physiological level of STING due to overexpression becomes strong from\nlooking at these data.\n14. (Related to 1, 3 and 13). In Fig.7C STING is highly overexpressed completely compromising the imaging data making it\nimpossible to trust the conclusions made.\n15. The imaging data in Supplementary Figure 7B are far from publication quality and the experiments need quantification of\nthe number of red only dots per cells and fraction of red to yellow dots.\nMinor comments:\nLine 56 in the Introduction: The authors refer to the “ULK-Beclin kinase complex”. This needs to be corrected since these are\ntwo complexes with the ULK1/2 kinase complex being distinct from the PI3K class III complex 1 containing Beclin1.", "reviewer_3": "The authors identified a novel mechanism by which TAX1BP1 regulates anti-viral response pathways through STING\ninhibition. This regulatory mechanism involves TAX1BP1 facilitating the degradation of STING via Golgi autophagy\n(Golgiphagy), thereby downregulating the cGAS-STING pathway and suggesting its role in modulating inflammatory\nresponses. Although the negative regulation of the cGAS-STING pathway through the Golgi is highly intriguing, there are\nsignificant gaps that need to be addressed before accepting the authors’ claims. The primary concern is that the\nsuppression mechanism of the cGAS-STING pathway by TAX1BP1 appears to be highly limited in humans or mice, making the\ninterpretation of the data unclear. I strongly recommend that the authors conduct a more detailed analysis in both humans\nand mice.\nAs stated by the authors in the Introduction, TAX1BP1 has been previously shown to inhibit IFN induction through the RLRs\npathway and some TLR pathways. Therefore, the authors should investigate whether similar phenomena are observed in\nTAX1BP1 KO THP-1 cells and BMDMs. At the very least, experiments using PolyIC stimulation of RLRs, TLR stimulation\nwith agents like LPS, and RNA virus infection experiments should be performed. Additionally, further induction of IFN\nthrough the cGAS-STING pathway should be conducted using TAX1BP1 KO THP-1 cells and BMDMs. The authors’ current\nexperiments are limited to using G3-YSD, cGAMP, and HSV-1; hence, they should assess IFN induction through the cGAS-\nSTING pathway using ligands known to activate this pathway, such as PolydAdT, ISD, and several DNA viruses.\nUnderstanding the suppression mechanism of the cGAS-STING pathway by TAX1BP1 in humans and mice is crucial and\nreveals an extremely interesting phenomenon. However, despite using TAX1BP1 KO THP-1 cells and BMDMs in some\nexperiments, not all experiments were conducted using these cells. This is insufficient as supporting data for the authors'\nclaims. The authors should conduct experiments using TAX1BP1 KO THP-1 cells and BMDMs in all experimental setups,\nand discuss each result to comprehensively examine the suppression mechanism of the cGAS-STING pathway by\nTAX1BP1 in both humans and mice.\nThe suppression mechanism of the cGAS-STING pathway is critical, as its breakdown is reported to be associated with\nautoimmune diseases like COPA syndrome and SAVI. Previous studies have also reported that TAX1BP1 negatively\nregulates IFN induction, and the relationship between its malfunction and disease is an important issue. Nevertheless, the\nauthors have not provided sufficient results related to these matters, conducting only infection experiments with\nTAX1BP1flox/flox LysM-Cre mice. The authors should analyze the relationship between diseases related to the cGAS-\nSTING pathway and TAX1BP1 by performing scRNA-seq on samples obtained from patients. This approach could elucidate\nthe biological functions of TAX1BP1 more effectively. Additionally, a comprehensive analysis using samples from patients\nwith neurodegenerative diseases caused by Golgi abnormalities should be conducted.\nThere are several errors and inconsistencies in the figure legends and the main text that the authors should review and\ncorrect. For example, there is inconsistent terminology between \"Real-time qRT-PCR\" in L104 and \"qRT-PCR\" in L102, and\nan error in L786 where \"yellow arrow\" should be \"yellow arrowhead.\" Additionally, in Fig. 3A, the comparison targets are not\nshown on the same membrane, making the comparison invalid and unacceptable. Furthermore, in the case of\nimmunofluorescence staining, while some quantifications are provided, most figures show only a single cell in one field of\nview, and many lack quantification altogether. As a solution, the authors must provide quantitative data for all these\nexperiments to address these issues comprehensively.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "In this manuscript, the authors suggested that TAX1BP1 was involved in STING degradation and the degradation was\nmediated by Golgiphagy. They exploited several approaches to address the in vivo significance, including generation of KO\nmice and virus infections, and convincingly showed that TAX1BP1 was a negative regulator of STING signalling. In contrast,\nthe mechanistic insight of the study is weak as described below.\nThe authors might find the following specific comments useful to improve the study.\nCritiques:\n(1) Many studies excluded the contribution of macroautophagy to STING degradation (For example, Nature (2019) 567, 262-\n266, Extended Fig.6). Furthermore, recent papers suggested that microautophagy, which does not require Atg genes and\nthus isolation membrane, is the critical process that mediates STING degradation.\nThe data in Figure 3C, in which the blot of LC3 was shown, did not address which pathway was involved in STING\ndegradation. The authors should be cautious about that the emergence of LC3-II does not necessarily indicate that\nmacroautophagy is in process. In particular in the case of STING activation, CASM (conjugation of Atg8 to single membrane)\noccurs at the TGN/endosomes, which is not relevant to STING degradation.\nTherefore, the authors should test if knockout or knockdown of the Atg genes impacts STING degradation in BMDMs.\n(2) The degradation of STING did not appear to be significantly affected by TAX1BP1-KO (Figure 3). The STING becomes\nubiquitinated and thus upshifted (or smeared) in SDS-PAGE, which sometimes makes interpretation of the results difficult.\nThis reviewer suggests to use fluorescence protein-tagged STING to validate the degradation rate of STING by FACS (For\nexample, Nat Cell Biol (2023) 25, 453-466).\n(3) In related to the critique (2), WB needs quantitation throughout the manuscript.\n(4) In the Nat Cell Biol paper, STING was shown to move to recycling endosomes after its exit from the Golgi/TGN,\nwhere STING becomes ubiquitinated. Therefore, the authors should investigate carefully the site of STING arrest in TAX1BP1\nKO (the Golgi vs recycling endosomes). Also, the authors should investigate the ubiquitinated status of STING in TAX1BP1 KO.\n(5) The STING-stimulation-dependent degradation of Golgin97 is impressive. The authors should examine if the degradation\nof Golgin97 requires the function of Atg genes. It is possible that STING degradation proceeds with microautophagy,\nwhereas Golgin97 degradation does with macroautophagy, both processes requiring TAX1BP1. The authors would like to\ncheck if the degradation of Golgin 97 requires the function of ESCRT.\n(6) If TAX1BP1 is involved in microautophagic degradation of STING, the authors should provide the clear mechanistic\ninsights."}, "author_response": "We thank the reviewers for their constructive comments which have helped us to\nsignificantly improve the manuscript which we hope is now acceptable for\npublication in Nature Communications. The following is a point-by-point response\nto the reviewers’ comments:\nReviewer 1:\nIn this manuscript, Suklabaidya and colleagues identify new mechanisms through which\nTAX1BP1 regulates the cGAS-STING pathway. The data presented supports an\nimportant role for TAX1BP1 in modulating inflammation by degrading activated STING\naggregates through the process of Golgiphagy.\nIn TAX1BP1-deficient macrophages, cGAS or STING agonists trigger stronger STING\nsignaling, leading to increased production of type I IFN and proinflammatory cytokines.\nConditional knockout mice lacking TAX1BP1 in myeloid cells exhibit heightened\ninflammatory responses upon STING activation and show increased resistance to\npoxvirus infection.\nThis research underlines the significance of TAX1BP1 in modulating cGAS-STING-\nmediated inflammation by selectively degrading fragmented Golgi structures, which may\nhave implications for controlling immune responses in infectious diseases and\ninflammatory conditions.\nThe experiments are well-designed and executed, and the imaging figures are clear and\nillustrative. The study can be considered for publication, pending clarification of the\nfollowing questions and suggested areas for improvement:\nSpecific comments:\n1) In the “TAX1BP1 promotes STING localization to lysosomes” section (Figure 5), why\nwere HeLa cells chosen for the colocalization experiments? Most of the study was\nconducted in cells of the myeloid lineage, specifically BMDMs or THP-1 cells. Although\nthe HeLa cell line is a useful and convenient tool, it originates from cervical cancer,\nraising concerns about whether the signaling and localization observed with the\nlysosome marker LAMP2 are comparable to those in innate immune cells. It would be\npreferable for the authors to provide colocalization data in BMDMs, THP-1 cells, or\nother myeloid-derived cell lines.\nWe initially attempted confocal/immunofluorescence experiments in BMDMs and THP-1\ncells but found that STING staining and resolution of the Golgi were suboptimal in these\ncells. Many papers, including high profile studies in Nature, Mol. Cell and others (see\nPMID: 36261523, 37086726, 33201170, 39578541, 35649354) have used HeLa cells\nstably expressing STING-GFP for studying STING trafficking and localization owing to\ntheir relatively large cytoplasm and uniform morphology.\n2) In Supplementary Figure 3, G3-YSD-induced Mx-1, Isg15, Irf7, and Ifna4 mRNA data\nare presented in THP-1 cells, but not in BMDMs. It would be relevant to include these\ndata for BMDMs as well.\nThank you for this suggestion. In the revised manuscript, we now show G3-YSD-\ninduced ISGs in BMDMs (Supplementary Fig. 3O-R).\n3) In Figure 9, the LEGENDplex mouse inflammation panel was utilized to investigate\nthe physiological relevance of TAX1BP1 inhibition of cGAS-STING in vivo. Serum levels\nof IFN-β and IL-6 are presented. Does the panel include other inflammatory cytokines?\nIf so, these data should be included and discussed.\nThere were no significant differences in the other cytokines in the inflammation panel.\nNevertheless, in the revised manuscript we now show these cytokines in\nSupplementary Fig. 11 and discuss the results.\n4) Have the authors investigated the mechanism of TAX1BP1-regulated STING\nGolgiphagy in other cell lineages important for innate immunity, such as B cells?\nUnderstanding the lineage specificity of the TAX1BP1-STING interaction would be of\nsignificant interest to the community, and its disease relevance should be addressed in\nthe Discussion section. Additionally, has TAX1BP1 been associated with STING-related\ndiseases, such as autoimmune or degenerative conditions? Including these implications\nin the Discussion section would enhance its value for readers from various research\nbackgrounds.\nAs recommended, we examined TAX1BP1-STING regulation in B cells. We generated\nTAX1BP1 knockout BJAB cells (Burkitt lymphoma B cell line that is EBV-negative) using\nCRISPR-Cas9. We validated TAX1BP1 knockout by western blotting (see below). Our\ndata suggests that TAX1BP1 regulates basal and diABZI-induced STING degradation in\nthis cell line. For reasons that are currently unclear, there was a lack of STING-induced\nGolgin-97 degradation in BJAB cells. However, we don’t know if this is specific to BJAB\ncells or if there are cell type specific differences in STING-induced Golgi degradation.\nWe did not include this data in the manuscript since we plan to follow up on this finding\nin a new paper. Regarding TAX1BP1 and autoimmune disease, a previous study\nidentified mutations in TAX1BP1 in patients with systemic lupus erythematosus (SLE).\nThis paper further showed that TAX1BP1 expression was reduced in SLE patients\nwhich was associated with increased inflammation mediated by macrophages (Qian et\nal. Int. Immunol.; PMID: 37465957). We have cited and discussed this paper in the\nrevised manuscript.\nrekraM\nANRg-noC\nOK-1PB1XAT\n75\nh\n0\nh\n4\nh\nVinculin\n100\nh\n0\nh\n4\nh\n8\n150\nrekraM\ndiABZI\nBJAB\nMinor comments:\n1) TAX1BP1 is an abbreviation and should be spelled out in full when first introduced in\nthe text.\nDone\n2) In Supplementary Figure 4 and Figure 6, it would be more illustrative to include a\nmerged figure to show the colocalization of different markers.\nDone\nReviewer 2:\nMajor points:\n1. Particularly, overexpression of STING in some vital experiments raise significant\ndoubt about the interpretation of the results since the results may be based on a non-\nphysiological model system with artefacts affecting the results. The fluorescence\nmicroscopy imaging data need to be improved. The imaging data in this paper will also\nbe very much improved to obtain more secure conclusions if the authors used\ncorrelative light and electron microscopy to determine the fate of STING from the Golgi\nto the lysosome. Another improvement would be to use Halo-tagged STING to follow\nthe lysosomal turnover of STING in a quantitative manner to enable comparison with\nother pathways like ESCRT-dependent microautophagy (ref. 11) and STING-dependent\nmacroautophagy.\nWe thank the reviewer for these comments and agree that the imaging data required\nimprovement. To this end, we generated HeLa cells stably expressing STING-eGFP\nand have repeated all the imaging experiments. Our new data, which is consistent with\nour previous data, indicate an impairment in STING trafficking to recycling endosomes\nand lysosomes in TAX1BP1 knockout cells. Correlative light and electron microscopy is\nan excellent suggestion, but we did not perform the experiment because the new and\nimproved imaging data provided strong evidence of an impairment in STING trafficking\nfrom the Golgi to lysosomes in the absence of TAX1BP1. As recommended, we have\nperformed STING degradation assays using THP-1 cells stably expressing STING-\neGFP. Our new quantitative flow cytometry data (Fig. 4A-E) provides additional\nevidence that TAX1BP1 is involved in STING degradation.\n2. As a continuation of point 1: How important is this pathway described here relative to\nESCRT-dependent degradation of STING by microautophagy? Can the authors\ndetermine the fraction of STING that is degraded by the Golgi-fragmentation pathway\naided by TAX1BP1? What about KO of p62 and other SLRs to determine if they have a\nrole together with TAX1BP1?\nThese are excellent points raised by the reviewer. Our new data indicates that STING\ninteraction with the ESCRT-0 protein HGS is diminished in the absence of TAX1BP1\n(Fig. 4F-H). Furthermore, knockdown of TSG101, a core component of the ESCRT-0\ncomplex significantly inhibits STING degradation (Fig. 4C-E). However, knockdown of\nTSG101 together with TAX1BP1 knockout completely impairs STING degradation (Fig.\n4C-E). These data suggest that TAX1BP1 mediates STING degradation through both\nESCRT-dependent and independent mechanisms. Although STING+ vesicles that have\negressed from the Golgi are degraded by microautophagy, STING-induced Golgiphagy\nappears to be independent of macroautophagy and microautophagy. These data are\nshown in Supplementary Fig. 9. Since LC3 lipidation is unimpaired in TAX1BP1 KO\nmacrophages treated with diABZI, we suspect that STING degradation by Golgiphagy\noccurs through FIP200-independent noncanonical autophagy. We have also generated\np62/SQSTM1 knockout THP-1 cells and showed that both STING and Golgi\ndegradation are impaired (Fig. 8F). A previous study showed that p62 colocalizes with\nboth STING and HGS (Gentili et al. Nat. Comm. 2023; PMID: 36739287). Together, our\ndata suggests that both TAX1BP1 and p62 are involved in STING-induced Golgiphagy\nand ESCRT-mediated degradation of STING by microautophagy. Regarding other\nSLRs, a recent study from the Youle lab found that TNIP1 and other autophagy\nreceptors including p62, NBR1, NDP52, TAX1BP1 and OPTN all associate with STING-\ninduced ubiquitin and LC3+ vesicles (Bunker et al. 2025 BioRxiv). Future studies are\nneeded to assess other SLRs for potential roles in STING-induced Golgiphagy.\n3. Could it be that TAX1BP1 is primarily important here as an autophagy receptor for\ndegradation of fragmented Golgi and that STING is simply degraded as a bystander?\nDo the authors provoke Golgi fragmentation and thereby TAX1BP1-dependent\nGolgiphagy by overexpressing STING too much?\nOur new data indicate that TAX1BP1 directly interacts with STING and links STING to\nthe ESCRT machinery (Figs. 4, 5). This suggests that STING is not degraded as a\nbystander but is specifically targeted for degradation by TAX1BP1. TAX1BP1 appears\nto serve as an autophagy receptor for fragmented Golgi after STING activation but may\nalso promote STING degradation of STING vesicles that have egressed from the Golgi\nto traffic to lysosomes. New experiments were repeated with cells stably expressing\nSTING to avoid potential artifacts from STING transient overexpression. Golgi\nfragmentation is also observed by transmission electron microscopy using primary WT\nand Tax1bp1–/– macrophages (Fig. 9) again suggesting that STING-induced Golgi\nfragmentation is not caused by overexpression artifacts.\n4. KO of TAX1BP1 in human monocytic THP-1 cells led to increased resistance to\nHerpes Simplex Virus type 1 (HSV-1) infection and increased expression of mRNAs for\nIfn-b, Ifna4, Mx1, Isg15 and Irf7 (Fig. 2B, D-G). However, I find the increase in the IFN-b\nprotein level to be relatively small in Fig.2C with no change at 12h and a small increase\nat 24h. Can the authors comment on this?\nThe reviewer raises a good point about the observed upregulation in HSV-induced IFN-\nbeta production in TAX1BP1-KO THP-1 cells (Fig. 2). While there are statistically\nsignificant differences at some of the time points, we don’t know the reason why some\nof the changes are relatively small. We suspect it could be due to the influence of HSV-\n1-encoded antagonists of STING such as UL38.\n5. In Figure 3A and -B, the blots for pTBK1 (S172), pIRF3 (S396), pSTING (S366) as\nwell as pIKKalpha/beta (S176/180 and pIkBalpha (S32/36) need quantifications with\nstatistics.\nDone as requested. The pTBK1, pIRF3, pSTING and pIKK bands were quantified from\nthree independent experiments. We removed the pIkBa blots since our previous\nantibody was depleted and a new antibody that originated from a different lot no longer\nworked.\n6. Importantly, also the total levels of STING should be quantified in Fig. 3A and 3B\nfrom the blots shown. From just visual inspection there does not seem to be a profound\nturnover of STING, but this may of course be balanced by increased expression of\nSTING?\nThe experiments were repeated, and STING bands were quantified from three\nindependent experiments. See Fig. 3A-L.\n7. Lines 168-169: The authors write that “However, in Tax1bp1–/– BMDMs treated with\ndiABZI, STING degradation was largely impaired (Fig. 3C).” From the top blot in Figure\n3C this clearly seems an overstatement and the authors need to quantify the STING\nlevels from the experiments in Fig. 3C.\nThe STING bands were quantified by densitometry. We have also added new\nquantitative flow cytometry STING degradation assays in Fig. 4 in the manuscript that\nprovides additional support for TAX1BP1 in STING degradation.\n8. In Fig. 4A the band of IPed TAX1BP1 (IPed with a TAX1BP1 antibody) migrates\nbelow the 75 kDa MW marker while the band of TAX1BP1 IPed with STING (IPed with a\nSTING antibody) migrates clearly above the 75 kDa MW marker. How can the authors\nexplain these seemingly very different molecular weights of TAX1BP1 observed in the\ntwo IP experiments?\nWe have repeated the STING-TAX1BP1 co-IP assay and IPed TAX1BP1 in both STING\nand TAX1BP1 immunoprecipitates now migrates above the 75 kDa band as expected.\n9. Does TAX1BP1 bind directly to STING? Importantly, the authors need to show that\nSTING and TAX1BP1 can directly interact with each other. IP from cells and PLA\nsuggest that STING and TAX1BP1 may be in a complex but assays for direct\ninteractions are needed such as for example GST pulldowns of recombinant proteins\nand/or bimolecular fluorescence complementation assays.\nWe have performed a nanoBiT complementation assay to demonstrate a direct\ninteraction between TAX1BP1 and STING. The new data is shown in Fig. 5F, G.\n10. The imaging shown in Figure 4D needs quantifications of colocalizations and an\nestimate of how relevant the fluorescence signal distributions shown are as only single\ncells are shown in the top and bottom panels.\nQuantification of the colocalization is now provided in the new Fig. 5E.\n11. The resolution of the images in Fig 4D should be significantly improved. This is\nespecially important for the lower panel diABZI (2h). See for example the imaging\nshown in reference 11 (Kuchitsu et al. 2023).\nWe have repeated the immunofluorescence experiments (new Fig. 5D) and the\nresolution is now significantly improved.\n12. The quality and resolution of the imaging shown in Fig. 5A and D need to be\nimproved.\nWe have repeated the immunofluorescence experiments and the resolution is now\nsignificantly improved.\n13 (Related to point 1 and 3). Importantly, the overexpression of STING affects the\nexperiment a lot since Golgi looks completely fragmented in the TGN46 staining in Fig\n5A. The impression that STING is degraded as a bystander by TAX1BP1 mediated\nGolgiphagy caused by a non-physiological level of STING due to overexpression\nbecomes strong from looking at these data.\nThis is a valid concern. In the revised manuscript, we generated HeLa cells stably\nexpressing STING-eGFP and repeated the immunofluorescence experiments.\nImportantly, we have confirmed STING-induced Golgi fragmentation using Golgin-97\nstaining as well as using a newly generated Golgiphagy reporter (Fig. 8C, D;\nSupplementary Fig. 8D, E).\n14. (Related to 1, 3 and 13). In Fig.7C STING is highly overexpressed completely\ncompromising the imaging data making it impossible to trust the conclusions made.\nWe have repeated the immunofluorescence experiments with HeLa cells stably\nexpressing STING-eGFP and the resolution is now significantly improved.\n15. The imaging data in Supplementary Figure 7B are far from publication quality and\nthe experiments need quantification of the number of red only dots per cells and fraction\nof red to yellow dots.\nWe agree and have modified the Golgiphagy reporter with a minimal Golgi targeting\nmotif (FLWRIFCFRK) as previously reported (Kitta et al. EMBO J. 2024; PMID\n38822137). Instead of THP-1 cells, new experiments were performed in control and\nTAX1BP1-KO HeLa cells stably expressing the Golgiphagy reporter. The new images\nare significantly improved and shown in Fig. 8C, D and Supplementary Fig. 8D, E.\nQuantification of mCherry+GFP– puncta is provided as described in Kitta et al. EMBO J.\nWe also validated the Golgiphagy reporter and showed colocalization of red and green\nperinuclear fluorescence intensity with a Golgi marker (Supplementary Fig. 8E). The\ndata indicate that TAX1BP1 is required for STING-induced Golgiphagy and likely\nfunctions as a Golgiphagy receptor for STING-induced fragmented Golgi.\nMinor comments:\nLine 56 in the Introduction: The authors refer to the “ULK-Beclin kinase complex”. This\nneeds to be corrected since these are two complexes with the ULK1/2 kinase complex\nbeing distinct from the PI3K class III complex 1 containing Beclin1.\nDone\nReviewer 3:\nThe authors identified a novel mechanism by which TAX1BP1 regulates anti-viral\nresponse pathways through STING inhibition. This regulatory mechanism involves\nTAX1BP1 facilitating the degradation of STING via Golgi autophagy (Golgiphagy),\nthereby downregulating the cGAS-STING pathway and suggesting its role in modulating\ninflammatory responses. Although the negative regulation of the cGAS-STING pathway\nthrough the Golgi is highly intriguing, there are significant gaps that need to be\naddressed before accepting the authors’ claims. The primary concern is that the\nsuppression mechanism of the cGAS-STING pathway by TAX1BP1 appears to be\nhighly limited in humans or mice, making the interpretation of the data unclear. I strongly\nrecommend that the authors conduct a more detailed analysis in both humans and\nmice.\nAs stated by the authors in the Introduction, TAX1BP1 has been previously shown to\ninhibit IFN induction through the RLRs pathway and some TLR pathways. Therefore,\nthe authors should investigate whether similar phenomena are observed in TAX1BP1\nKO THP-1 cells and BMDMs. At the very least, experiments using PolyIC stimulation of\nRLRs, TLR stimulation with agents like LPS, and RNA virus infection experiments\nshould be performed. Additionally, further induction of IFN through the cGAS-STING\npathway should be conducted using TAX1BP1 KO THP-1 cells and BMDMs. The\nauthors’ current experiments are limited to using G3-YSD, cGAMP, and HSV-1; hence,\nthey should assess IFN induction through the cGAS-STING pathway using ligands\nknown to activate this pathway, such as PolydAdT, ISD, and several DNA viruses.\nWe and others have already published that TAX1BP1 inhibits type I IFN induced by\nRLR and TLR signaling (see PMID: 27736772, 39193925, 17703191, 29358708,\n28898289). In fact, our recent study (PMID: 39193925) directly compared poly(I:C)\nstimulation versus virus infection for TAX1BP1 degradation and autophagy function. Our\nfocus in this manuscript is on TAX1BP1 regulation of the cGAS-STING pathway. We\nhave already used the cGAS agonist G3-YSD, cGAMP and HSV-1 infection in vitro. Our\nresults are in agreement and indicate that TAX1BP1 knockout macrophages exhibit\nincreased type I IFN induction in response to all these agonists. However, we have\nused an additional cGAS ligand, HT-DNA, and showed increased type I IFN responses\nin TAX1BP1-KO THP-1 cells (Supplementary Fig. 3). Furthermore, we have used an\nadditional DNA virus Ectromelia virus, a mouse poxvirus, for in vivo studies and showed\nthat myeloid-specific TAX1BP1 knockout mice are more resistant to Ectromelia infection\n(Fig. 10). In aggregate, we have used multiple cGAS and STING agonists in this study.\nUnderstanding the suppression mechanism of the cGAS-STING pathway by TAX1BP1\nin humans and mice is crucial and reveals an extremely interesting phenomenon.\nHowever, despite using TAX1BP1 KO THP-1 cells and BMDMs in some experiments,\nnot all experiments were conducted using these cells. This is insufficient as supporting\ndata for the authors' claims. The authors should conduct experiments using TAX1BP1\nKO THP-1 cells and BMDMs in all experimental setups, and discuss each result to\ncomprehensively examine the suppression mechanism of the cGAS-STING pathway by\nTAX1BP1 in both humans and mice.\nWe thank the reviewer for finding the TAX1BP1 suppression mechanism of cGAS-\nSTING to be extremely interesting. The reviewer raises a good point that not all\nexperiments were performed in TAX1BP1 knockout THP-1 cells and BMDMs. The\nreasons for this are practical. We initially attempted confocal/immunofluorescence\nexperiments in BMDMs and THP-1 cells but found that STING staining and resolution of\nthe Golgi were suboptimal in these cells. Many papers, including high profile studies in\nNature, Mol. Cell and others (see PMID: 36261523, 37086726, 33201170, 39578541,\n35649354) have used HeLa cells stably expressing STING-eGFP for studying STING\ntrafficking and localization owing to their relatively large cytoplasm and uniform\nmorphology.\nThe suppression mechanism of the cGAS-STING pathway is critical, as its breakdown\nis reported to be associated with autoimmune diseases like COPA syndrome and SAVI.\nPrevious studies have also reported that TAX1BP1 negatively regulates IFN induction,\nand the relationship between its malfunction and disease is an important issue.\nNevertheless, the authors have not provided sufficient results related to these matters,\nconducting only infection experiments with TAX1BP1flox/flox LysM-Cre mice. The\nauthors should analyze the relationship between diseases related to the cGAS-STING\npathway and TAX1BP1 by performing scRNA-seq on samples obtained from patients.\nThis approach could elucidate the biological functions of TAX1BP1 more effectively.\nAdditionally, a comprehensive analysis using samples from patients with\nneurodegenerative diseases caused by Golgi abnormalities should be conducted.\nWe appreciate the emphasis on disease raised by the reviewer. However, COPA\nsyndrome and SAVI are extremely rare diseases and we do not have access to these\npatient samples or mouse models. Similarly, we do not have access to samples from\npatients with neurodegenerative diseases caused by Golgi abnormalities. Although we\nagree that these would be very interesting avenues to pursue, we feel that these studies\nwill not directly address any questions about the mechanisms by which TAX1BP1\ninhibits STING and are also beyond the scope of this manuscript.\nThere are several errors and inconsistencies in the figure legends and the main text that\nthe authors should review and correct. For example, there is inconsistent terminology\nbetween \"Real-time qRT-PCR\" in L104 and \"qRT-PCR\" in L102, and an error in L786\nwhere \"yellow arrow\" should be \"yellow arrowhead.\" Additionally, in Fig. 3A, the\ncomparison targets are not shown on the same membrane, making the comparison\ninvalid and unacceptable. Furthermore, in the case of immunofluorescence staining,\nwhile some quantifications are provided, most figures show only a single cell in one field\nof view, and many lack quantification altogether. As a solution, the authors must provide\nquantitative data for all these experiments to address these issues comprehensively.\nWe thank the reviewer for pointing out these errors. We have fixed all the minor errors\nin terminology. The experiment in Fig. 3A has been repeated and all samples were\nloaded on the same gel. All immunofluorescence experiments in the manuscript were\nrepeated and quantification provided. We now show multiple cells in every field for\nimmunofluorescence experiments.\nReviewer 4:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer 5:\nIn this manuscript, the authors suggested that TAX1BP1 was involved in STING\ndegradation and the degradation was mediated by Golgiphagy. They exploited several\napproaches to address the in vivo significance, including generation of KO mice and\nvirus infections, and convincingly showed that TAX1BP1 was a negative regulator of\nSTING signalling. In contrast, the mechanistic insight of the study is weak as described\nbelow.\nThe authors might find the following specific comments useful to improve the study.\nCritiques:\n(1) Many studies excluded the contribution of macroautophagy to STING degradation\n(For example, Nature (2019) 567, 262-266, Extended Fig.6). Furthermore, recent\npapers suggested that microautophagy, which does not require Atg genes and thus\nisolation membrane, is the critical process that mediates STING degradation.\nWe thank the reviewer for raising this point and agree that macroautophagy does not\nplay a role in STING degradation. To address this point, we generated THP-1 cells\nlacking the core autophagy gene Atg3 which is required for LC3 lipidation. ATG3\nknockout cell lines were treated with diABZI to examine STING and Golgi degradation.\nIndeed, our data agrees with earlier studies that macroautophagy is dispensable for\nSTING degradation (Supplementary Fig. 9B). Therefore, we have removed all\nstatements in the manuscript that macroautophagy is involved in STING degradation.\nInterestingly, STING-induced Golgi degradation occurs in the absence of ATG3 and\nFIP200 (Supplementary Fig. 9D), as well as TSG101 knockdown (Supplementary Fig.\n9E), suggesting that STING-induced Golgiphagy occurs independently of\nmacroautophagy and microautophagy. We suspect it is a FIP200-independent\nnoncanonical autophagy pathway.\nThe data in Figure 3C, in which the blot of LC3 was shown, did not address which\npathway was involved in STING degradation. The authors should be cautious about that\nthe emergence of LC3-II does not necessarily indicate that macroautophagy is in\nprocess. In particular in the case of STING activation, CASM (conjugation of Atg8 to\nsingle membrane) occurs at the TGN/endosomes, which is not relevant to STING\ndegradation.\nTherefore, the authors should test if knockout or knockdown of the Atg genes impacts\nSTING degradation in BMDMs.\nThese are excellent points raised by the reviewer. We agree that STING-induced LC3\nlipidation is associated with its ion channel function and CASM. We experienced\ntechnical issues attempting to knock out ATG proteins in BMDMs and therefore we\ngenerated the knockouts in THP-1 cells. In the absence of ATG3 or FIP200\n(Supplementary Fig. 9), STING degradation is not impaired suggesting that\nmicroautophagy is the main pathway leading to STING degradation. To address the role\nof microautophagy we knocked down the ESCRT-0 protein TSG101 and examined\nSTING degradation using a quantitative flow cytometry assay. The results in Fig. 4C-E\nindicate that microautophagy is indeed the main pathway leading to STING degradation.\n(2) The degradation of STING did not appear to be significantly affected by TAX1BP1-\nKO (Figure 3). The STING becomes ubiquitinated and thus upshifted (or smeared) in\nSDS-PAGE, which sometimes makes interpretation of the results difficult. This reviewer\nsuggests to use fluorescence protein-tagged STING to validate the degradation rate of\nSTING by FACS (For example, Nat Cell Biol (2023) 25, 453-466).\nWe thank the reviewer for this excellent suggestion. We generated THP-1 cells stably\nexpressing STING-eGFP and examined diABZI-induced STING degradation by flow\ncytometry. TAX1BP1 knockout macrophages have a significant impairment in STING\ndegradation (Fig. 4A, B).\n(3) In related to the critique (2), WB needs quantitation throughout the manuscript.\nDone as requested\n(4) In the Nat Cell Biol paper, STING was shown to move to recycling endosomes after\nits exit from the Golgi/TGN, where STING becomes ubiquitinated. Therefore, the\nauthors should investigate carefully the site of STING arrest in TAX1BP1 KO (the Golgi\nvs recycling endosomes). Also, the authors should investigate the ubiquitinated status\nof STING in TAX1BP1 KO.\nWe performed immunofluorescence experiments to examine diABZI-induced STING\ntrafficking from the trans-Golgi (Golgin-97) to recycling endosomes (Transferrin\nreceptor) and lysosomes (LAMP2). Our data indicate that STING trafficking to recycling\nendosomes and lysosomes is impaired (although not completely) with most of STING\nlocalized at the trans-Golgi in the absence of TAX1BP1 (Fig. 6). Our TEM data suggests\nthat this could be due to impaired Golgiphagy with an accumulation of fragmented Golgi\nin TAX1BP1 knockout macrophages (Fig. 9). We attempted a STING ubiquitination\nassay with endogenous ubiquitin several times but were unable to get this assay to\nwork. We hope the reviewer appreciates the technical challenges associated with this\nassay since there are very few studies (only one to our knowledge; PMID: 37139896)\nshowing endogenous STING ubiquitination in macrophages.\n(5) The STING-stimulation-dependent degradation of Golgin97 is impressive. The\nauthors should examine if the degradation of Golgin97 requires the function of Atg\ngenes. It is possible that STING degradation proceeds with microautophagy,\nwhereas Golgin97 degradation does with macroautophagy, both processes requiring TAX1BP1.\nThe authors would like to check if the degradation of Golgin 97 requires the function of\nESCRT.\nWe thank the reviewer for this excellent suggestion. Interestingly, STING-induced Golgi\ndegradation appears to be independent of ATG3, FIP200 and TSG101 (Supplementary\nFig. 9) suggesting that macroautophagy and microautophagy are not involved in\nSTING-induced Golgiphagy. We suspect that a noncanonical autophagy pathway\nmediates STING-induced Golgiphagy.\n(6) If TAX1BP1 is involved in microautophagic degradation of STING, the authors\nshould provide the clear mechanistic insights.\nOur data indeed suggests that TAX1BP1 is involved in STING degradation by\nmicroautophagy. Given that p62/SQSTM1 was previously shown to colocalize with\nSTING and the ESCRT-0 protein HGS (Gentili et al. Nat. Comm. 2023; PMID:\n36739287), we hypothesized that TAX1BP1, STING and HRS were in the same\ncomplex and TAX1BP1 and p62 linked ubiquitinated STING with the ESCRT machinery.\nIndeed, our new data shows that diABZI-induced STING-HGS colocalization was\npartially impaired in TAX1BP1 knockout macrophages (Fig. 4F, G). Furthermore,\ndiABZI-induced STING-HGS interaction was also diminished in TAX1BP1 knockout\nmacrophages as shown by a co-IP assay (Fig. 4H). These data indicate that STING is\ndegraded by selective microautophagy dependent on p62/SQSTM1 and TAX1BP1.\nWe again thank the reviewers for their constructive comments which have helped\nus to further improve the manuscript which we hope is now acceptable for\npublication in Nature Communications. The following is a point-by-point response\nto the reviewers’ comments:\nReviewer #1 (Remarks to the Author):\nThe authors have responded to main concerns raised and this revised manuscript is\nnow acceptable for publication.\nWe thank the reviewer for the positive comments.\nReviewer #2 (Remarks to the Author):\nThe authors have answered all my comments and criticisms and improved the\nmanuscript substantially. There is still more work to be done to sort out the mechanistic\ndetails of the microautophagy and Golgiphagy pathways implicated here. However,\ngiven all that has been done in this paper now, I regard that as future studies which are\nbeyond the scope of this study.\nWe thank the reviewer for the positive comments.\nReviewer #3 (Remarks to the Author):\nThe authors make an interesting and internally consistent claim that TAX1BP1\nsuppresses the cGAS–STING pathway during DNA virus responses using myeloid cells.\nHowever, there are insufficient data to fully support this claim, and we strongly\nrecommend more detailed analyses as follows;\n“We and others have already published that TAX1BP1 inhibits type I IFN induced by\nRLR and TLR signaling (see PMID: 27736772, 39193925, 17703191, 29358708,\n28898289). In fact, our recent study (PMID: 39193925) directly compared poly(I:C)\nstimulation versus virus infection for TAX1BP1 degradation and autophagy function. Our\nfocus in this manuscript is on TAX1BP1 regulation of the cGAS-STING pathway. We\nhave already used the cGAS agonist G3-YSD, cGAMP and HSV-1 infection in vitro. Our\nresults are in agreement and indicate that TAX1BP1 knockout macrophages exhibit\nincreased type I IFN induction in response to all these agonists. However, we have used\nan additional cGAS ligand, HT-DNA, and showed increased type I IFN responses in\nTAX1BP1-KO THP-1 cells (Supplementary Fig. 3). Furthermore, we have used an\nadditional DNA virus Ectromelia virus, a mouse poxvirus, for in vivo studies and showed\nthat myeloid-specific TAX1BP1 knockout mice are more resistant to Ectromelia infection\n(Fig. 10). In aggregate, we have used multiple cGAS and STING agonists in this study.”\nWe partially agree with the authors’ conclusions. Nonetheless, because the authors’\nexperiments are performed consistently in myeloid cells while many of the cited studies\nrely primarily on MEFs and other non-myeloid cells, they should determine whether the\nTAX1BP1-dependent suppression of the cGAS–STING pathway is a myeloid-cell-\nspecific phenomenon. To this end, the authors should assess IFN expression and\nproduction after RLR and TLR pathway activation in myeloid TAX1BP1-deficient cells\nsuch as BMDMs and THP-1.\nWe have performed experiments to examine RLR and TLR activation in TAX1BP1-\nknockout (KO) THP-1 cells. It should be noted that we and others have already\npublished that TAX1BP1 restricts RLR and TLR pathways (PMID: 27736772; 39193925;\n17703191; 29358708; 28898289). As expected, loss of TAX1BP1 potentiates RLR and\nTLR4 activation in THP-1 cells (see data below). Control and TAX1BP1-KO THP-1 cells\nwere infected with VSV at an MOI of 1 and qRT-PCR was performed to examine the\ninduction of Ifn and Il6 mRNAs. There was more Ifn and Il6 at 8h of VSV infection in\nTAX1BP1-KO THP-1 cells (see below). We also treated control and TAX1BP1-KO THP-\n1 cells with LPS (500 ng) and examined Ifn and Il6 mRNAs by qRT-PCR. There was\nsignificantly more Ifn and Il6 in LPS-treated TAX1BP1-KO THP-1 cells. This data was\nnot included in the revised manuscript because the cGAS-STING pathway is the focus\nof this study and we intend to use this data in a future manuscript.\n“We thank the reviewer for finding the TAX1BP1 suppression mechanism of\ncGASSTING to be extremely interesting. The reviewer raises a good point that not all\nexperiments were performed in TAX1BP1 knockout THP-1 cells and BMDMs. The\nreasons for this are practical. We initially attempted confocal/immunofluorescence\nexperiments in BMDMs and THP-1 cells but found that STING staining and resolution of\nthe Golgi were suboptimal in these cells. Many papers, including high profile studies in\nNature, Mol. Cell and others (see PMID: 36261523, 37086726, 33201170, 39578541,\n35649354) have used HeLa cells stably expressing STING-eGFP for studying STING\ntrafficking and localization owing to their relatively large cytoplasm and uniform\nmorphology.”\nOther reviewers have likewise suggested performing confocal/immunofluorescence\nexperiments using BMDMs, THP-1, or other myeloid cells. The authors respond that\n“STING staining and resolution of the Golgi were suboptimal in these cells,” but\nconfocal/immunofluorescence analyses of STING and the Golgi have been performed in\nnumerous published studies. Therefore, the authors should carry out these\nconfocal/immunofluorescence experiments in the indicated myeloid cell types.\nReviewer 1 expressed a similar concern about using non-myeloid cells for imaging\nstudies but nevertheless was satisfied with our imaging experiments in the revised\npaper using HeLa control and TAX1BP1-KO STING-eGFP cells. To confirm that the\nresults obtained in myeloid cells are congruent with those obtained in HeLa cells, we\nexamined diABZI-induced STING trafficking to the trans-Golgi in THP-1 cells stably\nexpressing STING-eGFP and control gRNA (control THP-1 STING-eGFP) and\nTAX1BP1-KO STING-eGFP THP-1 cells. Similar to the results obtained in HeLa cells,\nthere was significantly more diABZI-induced STING localized to the trans-Golgi in\nTAX1BP1-KO STING-eGFP THP-1 cells compared to control THP-1 STING-eGFP cells\n(see data below). Therefore, we conclude that the mechanisms of STING trafficking and\nregulation by TAX1BP1 are conserved in HeLa cells and myeloid cells.\n“We appreciate the emphasis on disease raised by the reviewer. However, COPA\nsyndrome and SAVI are extremely rare diseases and we do not have access to these\npatient samples or mouse models. Similarly, we do not have access to samples from\npatients with neurodegenerative diseases caused by Golgi abnormalities. Although we\nagree that these would be very interesting avenues to pursue, we feel that these studies\nwill not directly address any questions about the mechanisms by which TAX1BP1\ninhibits STING and are also beyond the scope of this manuscript.”\nThe authors also state that “COPA syndrome and SAVI are extremely rare diseases and\nwe do not have access to these patient samples or mouse models.” However, multiple\npatient-derived datasets are publicly available in repositories such as NCBI. The\nauthors should therefore discuss the relationship between TAX1BP1 and these\ndisorders—or with other IFN-related or Golgi-disruption-associated diseases—using\nthese publicly available datasets.\nWe have accessed publicly available datasets for single-cell RNA sequencing (scRNA-\nseq) from SAVI patients. We examined TAX1BP1, p62/SQSTM1 and NBR1 expression\nin SAVI patients to determine if these key negative regulators of STING activation are\ndownregulated in SAVI patients. However, as seen in the volcano plot below, there are\nno statistically significant differences in TAX1BP1, p62/SQSTM1 or NBR1 expression in\nSAVI patients compared to controls.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #5 (Remarks to the Author):\nThe authors mostly addressed to the original critiques and the quality of the study has\nbeen much improved. The elucidation of the site of STING accumulation in TAX1BP KO\ncells is neat.\nMajor critiques:\n(1) Some papers including (PMID: 36918692) showed that HGS was dispensable for\nSTING degradation in mouse embryonic fibroblasts. Therefore, the requirement of HGS\n(or HRS) for STING degradation has to be validated in their experimental settings.\nThere may be cell type-specific differences in HGS regulation of STING degradation. A\nprevious study performed in human fibroblasts (PMID: 36739287) showed that STING\ndegradation is indeed dependent on HGS. More relevant to our study, another group\nknocked out HGS using CRISPR-Cas9 in iBMDMs, and this resulted in enhanced\nSTING, TBK1 and IRF3 phosphorylation and STING degradation was impaired (PMID:\n37139896). Nevertheless, we attempted this experiment several times but HGS\nknockdown led to significant cell death that prevented us from obtaining reliable western\nblot results.\n(2) Virtually, all of the ESCRT-mediated processes require VPS4a/b function. The\nauthors should test whether VPS4a/b KD (in addition to TSG101 KD performed in the\nrevision), affects Golgiphagy.\nWe thank the reviewer for this suggestion. We knocked down VPS4a and VPS4b\nseparately in THP-1 cells and neither had any effect on the degradation of Golgin-97.\nWe then combined the siRNAs for VPS4a and VPS4b and again there was no effect on\nGolgin-97 degradation. This data is presented in Supplementary Fig. 9G. Together,\nwith our TSG101 knockdown results, we conclude that microautophagy is not involved in\nSTING-induced Golgi degradation.\nMinor critique:\n(3) It has been well established that TBK1 is not required for STING degradation.\nTherefore, texts regarding the interaction between TAX1BP and TBK1 needs to be\ncarefully revised.\nWe thank the reviewer for raising this point. We have toned down our conclusions from\nthe rescue experiment with the TAX1BP1 A114Q mutant that is impaired in binding to\nboth FIP200 and TBK1. This mutant is impaired in the inhibition of STING induction of\nIFN and IL-6 (Supplementary Figure 7); however, we don’t know if TAX1BP1\ninteraction with FIP200 or TBK1 mediates suppresses STING induction of type I\ninterferon and cytokines. Since STING-induced Golgiphagy still occurs in the absence\nof FIP200, our findings suggest that TAX1BP1 interacts with TBK1 to promote a\nnoncanonical form of autophagy that leads to Golgi degradation.\n(4) The authors would like to examine if p62 rescue into p62-KO cells restores the\nSTING degradation.\nWe have rescued p62 expression in p62-KO THP-1 cells using a lentiviral vector and\nexamined STING and Golgin-97 degradation. As expected, restoration of p62 rescued\nboth STING and Golgin-97 degradation. This data is presented in Supplementary Fig.\n9A.\nWe again thank the reviewers for their constructive comments which have helped\nus to further improve the manuscript which we hope is now acceptable for\npublication in Nature Communications. The following is a point-by-point response\nto the reviewers’ comments:\nReviewer #1 (Remarks to the Author):\nThe authors have responded to main concerns raised and this revised manuscript is\nnow acceptable for publication.\nWe thank the reviewer for the positive comments.\nReviewer #2 (Remarks to the Author):\nThe authors have answered all my comments and criticisms and improved the\nmanuscript substantially. There is still more work to be done to sort out the mechanistic\ndetails of the microautophagy and Golgiphagy pathways implicated here. However,\ngiven all that has been done in this paper now, I regard that as future studies which are\nbeyond the scope of this study.\nWe thank the reviewer for the positive comments.\nReviewer #3 (Remarks to the Author):\nThe authors make an interesting and internally consistent claim that TAX1BP1\nsuppresses the cGAS–STING pathway during DNA virus responses using myeloid cells.\nHowever, there are insufficient data to fully support this claim, and we strongly\nrecommend more detailed analyses as follows;\n“We and others have already published that TAX1BP1 inhibits type I IFN induced by\nRLR and TLR signaling (see PMID: 27736772, 39193925, 17703191, 29358708,\n28898289). In fact, our recent study (PMID: 39193925) directly compared poly(I:C)\nstimulation versus virus infection for TAX1BP1 degradation and autophagy function. Our\nfocus in this manuscript is on TAX1BP1 regulation of the cGAS-STING pathway. We\nhave already used the cGAS agonist G3-YSD, cGAMP and HSV-1 infection in vitro. Our\nresults are in agreement and indicate that TAX1BP1 knockout macrophages exhibit\nincreased type I IFN induction in response to all these agonists. However, we have used\nan additional cGAS ligand, HT-DNA, and showed increased type I IFN responses in\nTAX1BP1-KO THP-1 cells (Supplementary Fig. 3). Furthermore, we have used an\nadditional DNA virus Ectromelia virus, a mouse poxvirus, for in vivo studies and showed\nthat myeloid-specific TAX1BP1 knockout mice are more resistant to Ectromelia infection\n(Fig. 10). In aggregate, we have used multiple cGAS and STING agonists in this study.”\nWe partially agree with the authors’ conclusions. Nonetheless, because the authors’\nexperiments are performed consistently in myeloid cells while many of the cited studies\nrely primarily on MEFs and other non-myeloid cells, they should determine whether the\nTAX1BP1-dependent suppression of the cGAS–STING pathway is a myeloid-cell-\nspecific phenomenon. To this end, the authors should assess IFN expression and\nproduction after RLR and TLR pathway activation in myeloid TAX1BP1-deficient cells\nsuch as BMDMs and THP-1.\nWe have performed experiments to examine RLR and TLR activation in TAX1BP1-\nknockout (KO) THP-1 cells. It should be noted that we and others have already\npublished that TAX1BP1 restricts RLR and TLR pathways (PMID: 27736772; 39193925;\n17703191; 29358708; 28898289). As expected, loss of TAX1BP1 potentiates RLR and\nTLR4 activation in THP-1 cells (see data below). Control and TAX1BP1-KO THP-1 cells\nwere infected with VSV at an MOI of 1 and qRT-PCR was performed to examine the\ninduction of Ifn and Il6 mRNAs. There was more Ifn and Il6 at 8h of VSV infection in\nTAX1BP1-KO THP-1 cells (see below). We also treated control and TAX1BP1-KO THP-\n1 cells with LPS (500 ng) and examined Ifn and Il6 mRNAs by qRT-PCR. There was\nsignificantly more Ifn and Il6 in LPS-treated TAX1BP1-KO THP-1 cells. This data was\nnot included in the revised manuscript because the cGAS-STING pathway is the focus\nof this study and we intend to use this data in a future manuscript.\n“We thank the reviewer for finding the TAX1BP1 suppression mechanism of\ncGASSTING to be extremely interesting. The reviewer raises a good point that not all\nexperiments were performed in TAX1BP1 knockout THP-1 cells and BMDMs. The\nreasons for this are practical. We initially attempted confocal/immunofluorescence\nexperiments in BMDMs and THP-1 cells but found that STING staining and resolution of\nthe Golgi were suboptimal in these cells. Many papers, including high profile studies in\nNature, Mol. Cell and others (see PMID: 36261523, 37086726, 33201170, 39578541,\n35649354) have used HeLa cells stably expressing STING-eGFP for studying STING\ntrafficking and localization owing to their relatively large cytoplasm and uniform\nmorphology.”\nOther reviewers have likewise suggested performing confocal/immunofluorescence\nexperiments using BMDMs, THP-1, or other myeloid cells. The authors respond that\n“STING staining and resolution of the Golgi were suboptimal in these cells,” but\nconfocal/immunofluorescence analyses of STING and the Golgi have been performed in\nnumerous published studies. Therefore, the authors should carry out these\nconfocal/immunofluorescence experiments in the indicated myeloid cell types.\nReviewer 1 expressed a similar concern about using non-myeloid cells for imaging\nstudies but nevertheless was satisfied with our imaging experiments in the revised\npaper using HeLa control and TAX1BP1-KO STING-eGFP cells. To confirm that the\nresults obtained in myeloid cells are congruent with those obtained in HeLa cells, we\nexamined diABZI-induced STING trafficking to the trans-Golgi in THP-1 cells stably\nexpressing STING-eGFP and control gRNA (control THP-1 STING-eGFP) and\nTAX1BP1-KO STING-eGFP THP-1 cells. Similar to the results obtained in HeLa cells,\nthere was significantly more diABZI-induced STING localized to the trans-Golgi in\nTAX1BP1-KO STING-eGFP THP-1 cells compared to control THP-1 STING-eGFP cells\n(see data below). Therefore, we conclude that the mechanisms of STING trafficking and\nregulation by TAX1BP1 are conserved in HeLa cells and myeloid cells.\n“We appreciate the emphasis on disease raised by the reviewer. However, COPA\nsyndrome and SAVI are extremely rare diseases and we do not have access to these\npatient samples or mouse models. Similarly, we do not have access to samples from\npatients with neurodegenerative diseases caused by Golgi abnormalities. Although we\nagree that these would be very interesting avenues to pursue, we feel that these studies\nwill not directly address any questions about the mechanisms by which TAX1BP1\ninhibits STING and are also beyond the scope of this manuscript.”\nThe authors also state that “COPA syndrome and SAVI are extremely rare diseases and\nwe do not have access to these patient samples or mouse models.” However, multiple\npatient-derived datasets are publicly available in repositories such as NCBI. The\nauthors should therefore discuss the relationship between TAX1BP1 and these\ndisorders—or with other IFN-related or Golgi-disruption-associated diseases—using\nthese publicly available datasets.\nWe have accessed publicly available datasets for single-cell RNA sequencing (scRNA-\nseq) from SAVI patients. We examined TAX1BP1, p62/SQSTM1 and NBR1 expression\nin SAVI patients to determine if these key negative regulators of STING activation are\ndownregulated in SAVI patients. However, as seen in the volcano plot below, there are\nno statistically significant differences in TAX1BP1, p62/SQSTM1 or NBR1 expression in\nSAVI patients compared to controls.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #5 (Remarks to the Author):\nThe authors mostly addressed to the original critiques and the quality of the study has\nbeen much improved. The elucidation of the site of STING accumulation in TAX1BP KO\ncells is neat.\nMajor critiques:\n(1) Some papers including (PMID: 36918692) showed that HGS was dispensable for\nSTING degradation in mouse embryonic fibroblasts. Therefore, the requirement of HGS\n(or HRS) for STING degradation has to be validated in their experimental settings.\nThere may be cell type-specific differences in HGS regulation of STING degradation. A\nprevious study performed in human fibroblasts (PMID: 36739287) showed that STING\ndegradation is indeed dependent on HGS. More relevant to our study, another group\nknocked out HGS using CRISPR-Cas9 in iBMDMs, and this resulted in enhanced\nSTING, TBK1 and IRF3 phosphorylation and STING degradation was impaired (PMID:\n37139896). Nevertheless, we attempted this experiment several times but HGS\nknockdown led to significant cell death that prevented us from obtaining reliable western\nblot results.\n(2) Virtually, all of the ESCRT-mediated processes require VPS4a/b function. The\nauthors should test whether VPS4a/b KD (in addition to TSG101 KD performed in the\nrevision), affects Golgiphagy.\nWe thank the reviewer for this suggestion. We knocked down VPS4a and VPS4b\nseparately in THP-1 cells and neither had any effect on the degradation of Golgin-97.\nWe then combined the siRNAs for VPS4a and VPS4b and again there was no effect on\nGolgin-97 degradation. This data is presented in Supplementary Fig. 9G. Together,\nwith our TSG101 knockdown results, we conclude that microautophagy is not involved in\nSTING-induced Golgi degradation.\nMinor critique:\n(3) It has been well established that TBK1 is not required for STING degradation.\nTherefore, texts regarding the interaction between TAX1BP and TBK1 needs to be\ncarefully revised.\nWe thank the reviewer for raising this point. We have toned down our conclusions from\nthe rescue experiment with the TAX1BP1 A114Q mutant that is impaired in binding to\nboth FIP200 and TBK1. This mutant is impaired in the inhibition of STING induction of\nIFN and IL-6 (Supplementary Figure 7); however, we don’t know if TAX1BP1\ninteraction with FIP200 or TBK1 mediates suppresses STING induction of type I\ninterferon and cytokines. Since STING-induced Golgiphagy still occurs in the absence\nof FIP200, our findings suggest that TAX1BP1 interacts with TBK1 to promote a\nnoncanonical form of autophagy that leads to Golgi degradation.\n(4) The authors would like to examine if p62 rescue into p62-KO cells restores the\nSTING degradation.\nWe have rescued p62 expression in p62-KO THP-1 cells using a lentiviral vector and\nexamined STING and Golgin-97 degradation. As expected, restoration of p62 rescued\nboth STING and Golgin-97 degradation. This data is presented in Supplementary Fig.\n9A."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have responded to main concerns raised and this revised manuscript is now acceptable for publication.", "reviewer_2": "The authors have answered all my comments and criticisms and improved the manuscript substantially. There is still more work to be done to sort out the mechanistic details of the microautophagy and Golgipahgy pathways implicated here.\nHowever, given all that has been done in this paper now, I regard that as future studies which are beyond the scope of this study.", "reviewer_3": "The authors make an interesting and internally consistent claim that TAX1BP1 suppresses the cGAS–STING pathway during DNA virus responses using myeloid cells. However, there are insufficient data to fully support this claim, and we strongly recommend more detailed analyses as follows;\n“We and others have already published that TAX1BP1 inhibits type I IFN induced by RLR and TLR signaling (see PMID: 27736772, 39193925, 17703191, 29358708, 28898289). In fact, our recent study (PMID: 39193925) directly compared poly(I:C) stimulation versus virus infection for TAX1BP1 degradation and autophagy function. Our focus in this manuscript is on TAX1BP1 regulation of the cGAS-STING pathway. We have already used the cGAS agonist G3-YSD, cGAMP and HSV-1 infection in vitro. Our results are in agreement and indicate that TAX1BP1 knockout macrophages exhibit increased type I IFN induction in response to all these agonists. However, we have used an additional cGAS ligand, HT-DNA, and showed increased type I IFN responses in TAX1BP1-KO THP-1 cells (Supplementary Fig. 3). Furthermore, we have used an additional DNA virus Ectromelia virus, a mouse poxvirus, for in vivo studies and showed that myeloid-specific TAX1BP1 knockout mice are more resistant to Ectromelia infection (Fig. 10). In aggregate, we have used multiple cGAS and STING agonists in this study.”\nWe partially agree with the authors’ conclusions. Nonetheless, because the authors’ experiments are performed consistently in myeloid cells while many of the cited studies rely primarily on MEFs and other non-myeloid cells, they should determine whether the TAX1BP1-dependent suppression of the cGAS–STING pathway is a myeloid-cell-specific phenomenon. To this end, the authors should assess IFN expression and production after RLR and TLR pathway activation in myeloid TAX1BP1-deficient cells such as BMDMs and THP-1.\n“We thank the reviewer for finding the TAX1BP1 suppression mechanism of cGASSTING to be extremely interesting. The reviewer raises a good point that not all experiments were performed in TAX1BP1 knockout THP-1 cells and BMDMs. The reasons for this are practical. We initially attempted confocal/immunofluorescence experiments in BMDMs and THP-1 cells but found that STING staining and resolution of the Golgi were suboptimal in these cells. Many papers, including high profile studies in Nature, Mol. Cell and others (see PMID: 36261523, 37086726, 33201170, 39578541, 35649354) have used HeLa cells stably expressing STING-eGFP for studying STING trafficking and localization owing to their relatively large cytoplasm and uniform morphology.”\nOther reviewers have likewise suggested performing confocal/immunofluorescence experiments using BMDMs, THP-1, or other myeloid cells. The authors respond that “STING staining and resolution of the Golgi were suboptimal in these cells,” but confocal/immunofluorescence analyses of STING and the Golgi have been performed in numerous published studies. Therefore, the authors should carry out these confocal/immunofluorescence experiments in the indicated myeloid cell types.\n“We appreciate the emphasis on disease raised by the reviewer. However, COPA syndrome and SAVI are extremely rare diseases and we do not have access to these patient samples or mouse models. Similarly, we do not have access to samples from patients with neurodegenerative diseases caused by Golgi abnormalities. Although we agree that these would be very interesting avenues to pursue, we feel that these studies will not directly address any questions about the mechanisms by which TAX1BP1 inhibits STING and are also beyond the scope of this manuscript.”\nThe authors also state that “COPA syndrome and SAVI are extremely rare diseases and we do not have access to these patient samples or mouse models.” However, multiple patient-derived datasets are publicly available in repositories such as NCBI. The authors should therefore discuss the relationship between TAX1BP1 and these disorders—or with other IFN-related or Golgi-disruption-associated diseases—using these publicly available datasets.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "The authors mostly addressed to the original critiques and the quality of the study has been much improved. The elucidation of the site of STING accumulation in TAX1BP KO cells is neat.\nMajor critiques:\n(1) Some papers including (PMID: 36918692) showed that HGS was dispensable for STING degradation in mouse embryonic fibroblasts. Therefore, the requirement of HGS (or HRS) for STING degradation has to be validated in their experimental settings.\n(2) Virtually, all of the ESCRT-mediated processes require VPS4a/b function. The authors should test whether VPS4a/b KD (in addition to TSG101 KD performed in the revision), affects Golgiphagy.\nMinor critique:\n(3) It has been well established that TBK1 is not required for STING degradation. Therefore, texts regarding the interaction between TAX1BP and TBK1 needs to be carefully revised.\n(4) The authors would like to examine if p62 rescue into p62-KO cells restores the STING degradation."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have responded to all of my comments, and I find the revised manuscript acceptable for publication in Nature Communications.", "reviewer_5": "The authors have adequately responded to my concerns."}, "author_response": ""}]

Decision letter