A conserved ion channel function of STING mediates noncanonical autophagy and cell death

The cGAS/STING pathway triggers inflammation upon diverse cellular stresses such as infection, cellular damage, aging, and diseases. STING also triggers noncanonical autophagy, involving LC3 lipidation on STING vesicles through the V-ATPase-ATG16L1 axis, as well as induces cell death. Although the proton pump V-ATPase senses organelle deacidification in other contexts, it is unclear how STING activates V-ATPase for noncanonical autophagy. Here we report a conserved channel function of STING in proton efflux and vesicle deacidification. STING activation induces an electron-sparse pore in its transmembrane domain, which mediates proton flux in vitro and the deacidification of post-Golgi STING vesicles in cells. A chemical ligand of STING, C53, which binds to and blocks its channel, strongly inhibits STING-mediated proton flux in vitro. C53 fully blocks STING trafficking from the ER to the Golgi, but adding C53 after STING arrives at the Golgi allows for selective inhibition of STING-dependent vesicle deacidification, LC3 lipidation, and cell death, without affecting trafficking. The discovery of STING as a channel opens new opportunities for selective targeting of canonical and noncanonical STING functions.

1. Authors include further work on STING trafficking to distinguish their work from that previously published (PMID: 37535724).However, there is much work already published showing the transit of STING from ER, through the golgi and to the endolysosomal system, much of which is cited in this manuscript.So, it is less clear as to what extra this brings.The only extra information is that LC3 lipidation occurs on post golgi vesicles rather than the golgi itself.
2. Can authors block the movement of STING into post-golgi vesicles and, this increasing the dwell time at the golgi, and see if LC3 lipidation now occurs at the golgi membranes?Or is the lipidation really specific for the post golgi compartment.This was previously achieved by blocking GCC2 and different Rab GTPases (PMID: 36379959).
3. Can the authors provide any evidence that STING ion channel activation of LC3 lipidation on endolysosomal vesicles is in anyway related to the degradation of STING following activation?
Reviews from The EMBO Journal 4. The final section on ion channel activity dependent effects on cell death seem not to be linked with the previous work on LC3 lipidation.Is STING induced cell death affected by blocking LC3 lipidation? 5.The pH sensor used (Lyso-pHluorin), targets to late endosome/lysosomes via CD63.This would likely be separate from the Rab5 compartment where authors mainly locate STING.Is there any evidence that the robust Lyso-pHluorin signal overlaps with Rab5? 6.On Line 485 there is a mis-spelling of lipidation.
Referee #2: In the manuscript, the authors demonstrated that agonist-bound STING can act as a proton channel and mediates non-canonical lipidation of LC-3.They claimed that this function of STING is a primary cause of cell death, which is often accompanied with STING activation in several cell types.The biochemical part in the study was sound, and this reviewer thinks it is worth reporting despite the same content has recently been published in Science.In contrast, cell biological part was underdeveloped, and requires thorough experiments to make the conclusion clear.
(1) They used Lyso-pHluorin throughout the study.I see several problems with this imaging.* The images with Lyso-pHluorin are not consistent.For example, Fig. 4C (mSTING, 0 h) vs Fig. 4G (without stimulation).The former showed even distribution in cells, the latter showed the strong staining at periphery.
(4) The effect of C53 addition to LC3 membrane localization should be provided (related to Fig 3D).
(5) The cell death data is interesting but needs more validation.What type of cell death is occurring?In Fig. S5, they found one mutant that accelerates the increase of pH.Does this mutant increase the rate of cell death?
27th Sep 2023 1st Editorial Decision Dear Jay, Thank you for transferring your manuscript from The EMBO Journal to EMBO Reports.As discussed, I would like to invite you to revise your manuscript along the lines outlined in your revision plan and as discussed.We had penciled in a revision within 1 month, given the competitive situation.
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You are able to opt out of this by letting the editorial office know (emboreports@embo.org).If you do opt out, the Review Process File link will point to the following statement: "No Review Process File is available with this article, as the authors have chosen not to make the review process public in this case." We would also welcome the submission of cover suggestions, or motifs to be used by our Graphics Illustrator in designing a cover. 1 We thank the reviewers and the editor for their supportive and insightful comments which have further improved the quality of this study.The additional experiments performed during the revision further strengthened our conclusions that STING de-acidifies endosome-like vesicles for LC3 lipidation, a new function that depends on the channel function of STING which also triggers cell death.
Here is a quick summary of the new data added to the revised manuscript: (1) Confirmed that STING-induced Lyso-pHluorin puncta are positive for RAB5 (Fig 4K).Although Lyso-pHluorin is based on a late endosome marker CD63, the fraction of the probe de-quenched by STING is not on preexisting late endosome/lysosomes (Fig EV4F, G).
(3) We have further characterized Lyso-pHluorin puncta formation in live cell imaging, which confirmed that the vesicles de-acidified by STING are endosome-like compartments, with more colocalization with early and recycling endosome markers RAB5 and RAB11, and less with late endosome/lysosome markers RAB7, Editor's comments: Please address all referee concerns and provide all controls required.It will be important to verify the compartment in which STING resides (early vs late endosomes) and whether LC3 lipidation occurs specifically in post-Golgi vesicles, to verify the Lyso-pHluorin probe and to strengthen the functional data on cell death.Taguchi's group (Kuchitsu, Mukai et al., 2023), we observed a strong colocalization between STING and RAB11.We further quantified the colocalization of STING-induced Lyso-pHluorin puncta with different endosome/lysosome markers (Fig EV4F,quantified in Fig 4L).This confirmed that these Lyso-pHluorin puncta mostly overlapped with both the early and recycling endosome markers.We concluded that STING vesicles that are de-acidified represent endosome-like post-Golgi vesicles.

Whether LC3 lipidation occurs specifically in post-Golgi vesicles
Our data indicate that the endogenous LC3 puncta induced by STING has little overlap with the Golgi complex.However, we noticed that, when EGFP-LC3B was overexpressed, a low level of EGFP-LC3B signal was detected on the Golgi 30 min after STING activation -when most STING was still on the Golgi (Fig R1).Thus, it seems the Golgi membrane is a minor site of LC3 lipidation downstream of STING, which can be detected when LC3 is overexpressed.It is possible that blocking STING at the Golgi can cause more LC3 lipidation on the Golgi, but this does not seem to change our conclusion that the endogenous LC3 puncta mostly occurred in post-Golgi vesicles.This probe was verified using a proton ionophore monensin in our original submission (currently Fig EV4E) as well as by other lysosomal pH neutralization methods in other studies (Rost, Schneider et al., 2015a, Tan & Finkel, 2022).It has now been further validated with more experiments in the revised manuscript (Fig EV4A, E,  G).

Strengthen the functional data on cell death.
We have added more cell death tests as suggested by the reviewers.However, we believe the reviewers might agree that although cell death has been found to depend on the channel of STING, the exact mechanism by which the channel function kills the cell is beyond the scope of this study.c.We also tested whether ferroptosis (Wu, Liu et al., 2022) is a major mechanism for STING-dependent cell death using two different ferroptosis inhibitors.However, ferroptosis appeared to play a minor role (Fig EV7B ).
Referee #1: In the paper by Xun et al, authors report an ion channel activity for STING, which is required for its activation of non-canonical autophagy and cell death.STING activation is known to initiate innate immune signalling through TBK1, and had previously been reported to induce non-canonical LC3 lipidation; although the mechanism of activation of LC3 lipidation was not known.Here authors report that following STING activation and trafficking from ER to the golgi, an ion channel property of STING is induced in post golgi -endosome like vesicles that results in their deacidification.This neutralisation of pH triggers non-canonical autophagy via the ATG16L1-V-ATPase axis.
In preparation of this manuscript, many of the key findings were published by another group (PMID: 37535724), including the ion channel property of STING, ion channel dependent deacidification of STING vesicles and subsequent activation of non-canonical autophagy.Blockade of STING ion channel activity with C53 was shown to inhibit activation of non-canonical autophagy.
In this manuscript, authors highlight further insights into the post-golgi trafficking of STING and how this impacts activation of LC3 lipidation.Specifically, they distinguish between the effects of C53 in blocking STING trafficking versus ion channel activity.This is a well-executed set of experiments with clear conclusions.There is clearly value in solidifying these interesting findings regarding novel activities of STING.However, there are obviously questions on novelty.
We thank this reviewer for the supportive comments.
Comments 1. Authors include further work on STING trafficking to distinguish their work from that previously published (PMID: 37535724).However, there is much work already published showing the transit of STING from ER, through the golgi and to the endolysosomal system, much of which is cited in this manuscript.So, it is less clear as to what extra this brings.The only extra information is that LC3 lipidation occurs on post golgi vesicles rather than the golgi itself.
We thank the reviewer for bringing this important point.We aimed to clarify in Fig. 2 that STING traffics through the Golgi to form post-Golgi vesicle clusters instead of staying in the Golgi body for signaling purpose.We have now added discussions about this to the revised manuscript.
The literature is controversial regarding the signaling compartments of STING (ERGIC, Golgi, or post-Golgi vesicles).2. Can authors block the movement of STING into post-golgi vesicles and, this increasing the dwell time at the golgi, and see if LC3 lipidation now occurs at the golgi membranes?Or is the lipidation really specific for the post golgi compartment.This was previously achieved by blocking GCC2 and different Rab GTPases (PMID: 36379959).
We appreciate these discussions and suggestions.The LC3 lipidation down stream of STING did not seem to be specific to post-Golgi membranes, because we noticed that when we overexpressed EGFP-LC3B, the earliest, weak EGFP-LC3B puncta colocalized with the Golgi, but stronger puncta developed afterwards outside the Golgi (Fig R1 on Page 1 of this document).It is thus likely that a relatively small fraction of LC3 lipidation happens on the Golgi.However, endogenous LC3 puncta never showed obvious colocalization with Golgi markers, no matter STING is endogenous or ectopic (Fig EV3A, C).It is possible that upon STING retention on the Golgi, LC3 lipidation might now occur at the Golgi membrane.However, this does not seem to change our conclusion that most LC3 puncta were detected on post-Golgi, endosome-like STING vesicles.We have now added discussions about this in the revised manuscript.
3. Can the authors provide any evidence that STING ion channel activation of LC3 lipidation on endolysosomal vesicles is in anyway related to the degradation of STING following activation?
We have previously shown that ATG5-KO cells do not have major defects in STING degradation (Gui, Yang et al., 2019).Thus, we do not expect a major impact of LC3 lipidation on STING degradation.Instead, recent data from multiple other groups have shown that STING vesicle clusters are directly delivered to lysosomes through endosomal sorting complex required for transport (ESCRT)-mediated micro-autophagy (Balka, Venkatraman et al., 2023, Gentili, Liu et al., 2023, Kuchitsu et al., 2023).
4. The final section on ion channel activity dependent effects on cell death seem not to be linked with the previous work on LC3 lipidation.Is STING induced cell death affected by blocking LC3 lipidation?
STING has several non-canonical functions including LC3 lipidation and cell death.We asked whether these functions relied on the new channel function of STING.Our data showed that both LC3 lipidation and cell death were fully dependent on the channel of STING.However, LC3 lipidation is not required for cell death.We tested two different methods to block STING-mediated LC3 lipidation, neither of which suppressed STING-dependent cell death.
(1) Overexpression of a bacterial effector protein SopF, which is known to block V-ATPase-mediated ATG16L1 recruitment downstream of STING (Fischer, Wang et al., 2020) Thus, STING-dependent cell death is not mediated through LC3 lipidation, but both cell death and LC3 lipidation required the channel of STING.
5. The pH sensor used (Lyso-pHluorin), targets to late endosome/lysosomes via CD63.This would likely be separate from the Rab5 compartment where authors mainly locate STING.Is there any evidence that the robust Lyso-pHluorin signal overlaps with Rab5?
We also initially thought they were late endosome/lysosomes, but these Lyso-pHluorin puncta had little overlap with late endosome/lysosomes.The Lyso-pHluorin puncta colocalized well with both STING and RAB5 (Fig 4K, three channel colocalization).We have now cloned mCherry-tagged RAB5, RAB7, RAB11, CD63, and LAPTM4B, and compared their colocalization with STING-induced Lyso-pHluorin puncta through live cell imaging.Lyso-pHluorin puncta showed the highest overlap with RAB5 and RAB11, with less association with late endosome/lysosome markers such as RAB7, CD63, and LAPTM4B (Fig EV4F,quantified in Fig 4L).Even when mCherry is directly fused to the C-terminal end of CD63 in the Lyso-pHluorin construct (Lyso-pHluorin-mCherry), the STING-induced pHluorin puncta still showed on vesicles separated from the pre-existing mCherry-positive late endosome/lysosomes (Fig EV4G).Thus, STING-induced pHluorin puncta come from a new pool of Lyso-pHluorin that is localized on STING vesicles which are different from the pre-existing late endosome/lysosomes.These STING vesicles developed endosome-like properties as they showed overlap with RAB5 and RAB11.
6. On Line 485 there is a mis-spelling of lipidation.
Corrected.Thanks!Referee #2: In the manuscript, the authors demonstrated that agonist-bound STING can act as a proton channel and mediates non-canonical lipidation of LC-3.They claimed that this function of STING is a primary cause of cell death, which is often accompanied with STING activation in several cell types.The biochemical part in the study was sound, and this reviewer thinks it is worth reporting despite the same content has recently been published in Science.In contrast, cell biological part was underdeveloped, and requires thorough experiments to make the conclusion clear.
(1) They used Lyso-pHluorin throughout the study.I see several problems with this imaging.
* The images with Lyso-pHluorin are not consistent.For example, Fig. 4C (mSTING, 0 h) vs Fig. 4G (without stimulation).The former showed even distribution in cells, the latter showed the strong staining at periphery.
The images in Fig. 4G were collected with confocal, whereas Fig. 4A/C/E were from wide-field, non-confocal microscope which detects more background signal from the plasma membrane.This has now been clarified in the figure legends.
* This probe does not have the internal fluorescence control, making it hard to exclude the possibility that the increase in the intensity may simply reflects the amount of the probe.The experiments should be performed with ratio-type pH probe.With the ratio-type pH probe, the actual pH value can also be measured accurately.
This is an important comment that we addressed through several points: (1) The observed Lyso-pHluorin puncta were not triggered by increased expression of the probe.The live cell imaging data in Fig. 4G/H showing a specific increase in the Lyso-pHluorin puncta instead of the whole signal in the cell provided a strict control that the new puncta were not due to increased expression levels of the probe during STING activation.
(2) There is a possibility that the probe level on STING vesicles might slowly increase over time, but such increase should not be visible without pH neutralization of these vesicles.First, these puncta did not occur if the STING channel is blocked (Fig 6E/F).Second, after the formation of these Lyso-pHluorin puncta, they can be re-quenched upon blocking of the STING channel by C53 (Fig 7A).Thus, the increased Lyso-pHluorin puncta were not simply due to a change of the probe levels on STING vesicles.
(3) We generated a potential ratio-type pH probe by fusing mCherry to the C-terminal end of CD63 in the Lyso-pHluorin construct (Lyso-pHluorin-mCherry).The pHluorin sequence is inserted in the small luminal loop of CD63.We performed live cell imaging to check the relative signals of pHluorin and mCherry before and after STING activation.STING-induced pHluorin puncta overlapped with very weak, new mCherry signal outside of the pre-existing mCherry vesicles on late endosome/lysosomes (Fig EV4G, left and middle).As a positive control for de-acidification-induced pHluorin puncta in this experiment, we detected robust pHluorin puncta that overlaps with pre-existing mCherry vesicles upon the addition of a proton ionophore monensin (Fig EV4G , right).
Thus, STING-induced Lyso-pHluorin puncta likely represent a fraction of the probe localized to STING vesicles.The fluorescence signal of this fraction of Lyso-pHluorin requires the channel of STING to de-acidify these vesicles.
* The probe should be validated anyway with bafilomycin A or chloroquine.The positive control is missing.
Yes, we have now fully validated the probe with chloroquine (CQ, membrane-permeable weak base), monensin (proton ionophore), and nigericin (proton ionophore).In our lab submission, we showed that Lyso-pHluorin puncta were immediately induced upon the addition of monensin (current * The subcellular compartment in which the pH increases should be determined live. We have now cloned mCherry-tagged RAB5, RAB7, RAB11, CD63, and LAPTM4B, and compared their colocalization with STING-induced Lyso-pHluorin puncta through live cell imaging.Lyso-pHluorin puncta showed the highest overlap with RAB5 and RAB11, with less association with late endosome/lysosome markers such as RAB7, CD63, and LAPTM4B (Fig EV4F,quantified in Fig 4L).Even when mCherry is directly fused to the Cterminal end of CD63 in the Lyso-pHluorin construct (Lyso-pHluorin-mCherry), the STING-induced pHluorin puncta still showed on vesicles separated from the pre-existing mCherry-positive late endosome/lysosomes (Fig EV4G).Thus, STING-induced pHluorin puncta come from a different pool of Lyso-pHluorin that is localized on STING vesicles which are different from the pre-existing late endosome/lysosomes.These STING vesicles developed endosome-like properties as they showed overlap with RAB5 and RAB11.We believe these vesicles are not bona fide early or recycling endosomes, as they appear to be clusters of STING trafficking vesicles that recruit these RABs.Thus, we described them as endosome-like post-Golgi STING vesicles.
(2) The recent study showed that the post-Golgi traffic of STING to lysosomes involves recycling endosomes, rather than early endosomes [Nat. Cell Biol. 25, 453 (2023)].The authors should test Rab11 and/or tranferrin receptor in Figure 3 and determine the site of LC3 lipidation "together with STING".
Thanks for this important suggestion.We have examined STING/LC3 co-staining with EGFP-RAB11, which revealing extensive overlap between STING with RAB11, with a relatively less colocalization between LC3 and RAB11 (Fig 3I/J/K).As described above, we have also tried live cell imaging testing the colocalization between STING-induced Lyso-pHluorin puncta with mCherry-RAB11 as well as other organelle markers (Fig EV4F,quantified in Fig 4L).The Lyso-pHluorin puncta appeared to associate with both RAB5 and RAB11.STING vesicles might use these RAB proteins for their trafficking.Future studies are needed to fully define the properties of these vesicles, but we used "endosome-like vesicles" at this point to describe the vesicles de-acidified by STING.
We have now added quantification of these panels.(5) The cell death data is interesting but needs more validation.What type of cell death is occurring?In Fig. S5, they found one mutant that accelerates the increase of pH.Does this mutant increase the rate of cell death?
We have now compared the impact of STING-WT and -L54E on cell death.We expressed similar amounts of the two proteins in U2OS cells.L54E consistently triggered more cell death compared with STING-WT (Fig 7F/G), consistent with a role for proton release in STING-dependent cell death.
STING-dependent cell death likely involves multiple mechanisms including ferroptosis (Tang, Zundell et al., 2016, Wu, Chen et al., 2019, Wu et al., 2022, Xu, Chen et al., 2023).We found that two different ferroptosis inhibitors partially suppressed STING-dependent cell death (Fig EV7B).However, they were not able to fully block cell death, suggesting the presence of additional cell death mechanisms activated by STING.We further investigated whether this is a type of autophagic cell death as the channel also mediates non-canonical LC3 lipidation.Deletion of ATG7 or overexpression of a bacterial effector protein SopF strongly suppressed STING-dependent LC3 lipidation (Fig 7H/I, EV7C/D).However, neither of them blocked STING-dependent cell death (Fig 7J , EV7E).Thus, although the channel of STING mediates both LC3 lipidation and cell death, the two downstream processes appear to be independent of each other.We plan to further pursue the mechanistic studies regarding STING-dependent cell death as a future direction.

6th Dec 2023 1st Revision -Editorial Decision
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We look forward to seeing a final version of your manuscript as soon as possible.Authors have addressed all my previous questions and comments.The expanded discussion and extra experimental data further strengthen and clarify the manuscript.I believe the current manuscript to be suitable for publication in EMBO Reports.

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(<https://www.embopress.org/page/journal/14693178/authorguide#authorshipguidelines>) 6) We replaced Supplementary Information with Expanded View (EV) Figures and Tables that are collapsible/expandable online.A maximum of 5 EV Figures can be typeset.EV Figures should be cited as 'Figure EV1, Figure EV2" etc... in the text and their respective legends should be included in the main text after the legends of regular figures.
CD63, and LAPTM4B (Fig EV4F, quantified in Fig 4L).(4)More experiments testing STING-dependent cell death: the hyperactive channel mutant of STING (L54E) triggers more cell death than wild-type STING (Fig 7F, G) See below for our point-by-point responses to all comments.

1.
Verify the compartment in which STING resides (early vs late endosomes) STING colocalization with the early endosome marker RAB5 and late endosome/lysosome markers CD63/LAMP1 were quantified in our original manuscript (currently Fig 3F/G/H, EV3F-L), which indicated more colocalization with RAB5.We have now also quantified the colocalization of STING with the recycling endosome marker RAB11 (Fig 4I/J/K), as suggested by reviewer 2. Consistent with the recent work from Tomohiko
a. New experiments using LC3-lipidation-deficient cells ruled out the requirement of LC3 lipidation in STINGdependent cell death.We used two different ways to block STING-dependent LC3 lipidation: (1) expression of a bacterial effector protein SopF (Fig 7H/I/J) which modifies V-ATPase to block ATG16L1 recruitment; (2) ATG7 depletion by CRISPR KO (Fig EV7C/D/E).b.The impact of the L54E mutation on STING-dependent cell death has been tested, which showed more cell death downstream of this mutant compared with the wild-type STING expressed in the same level (Fig 7F/G).
Several reasons might have contributed to such controversy: (1) the post-Golgi STING vesicles form clusters around the microtubule organization center (MTOC) where the Golgi body is also located and considered as part of the MTOC (Fig. R2, right; Fig EV7F in the revised manuscript).The partial overlap between the Golgi and the post-Golgi STING vesicle clusters likely led to some mis-interpretation that STING stays on the Golgi for signaling purpose; (2) Some trans-Golgi markers traffics out of the Golgi (e.g., TGN46) upon STING activation, which makes the tracking dynamics of STING more complicated; (3) lower resolution confocal images precluded the differentiation between the Golgi and post-Golgi vesicle clusters; (4) STING first traffics through the Golgi to the perinuclear vesicle clusters, after which it again slowly traffics out of this region for lysosomal degradation(Fig R2, right).Both steps happen around the MTOC area, which could cause misunderstanding of this process.(5) Wrong online cartoon models of the relative localizations of key subcellular organelles (Fig.R2, left).

Fig R2 .
Fig R2.Schematic illustration of STING trafficking to the area around the perinuclear microtubule organization center (MTOC).Left: wrong model of the relative localizations of key organelles involved in STING trafficking.Right: the correct model.

( 4 )
The effect of C53 addition to LC3 membrane localization should be provided (related to Fig 3D).Yes, consistent with the western result, C53 fully blocks LC3 puncta formation in immunofluorescence.The new IF images have been added to Fig 6I/J.
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