Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function

Reviewer #1 (Public Review):

Summary:
In this study, Masroor Ahmad Paddar and his/her colleagues explore the noncanonical roles of ATG5 and membrane atg8ylation in regulating retromer assembly and function. They begin by examining the interactomes of ATG5 and expand the scope of these effects to include homeostatic responses to membrane stress and damage.

Strengths:
This study provides novel insights into the noncanonical function of ATG8ylation in endosomal cargo sorting process.

Weaknesses:
The direct mechanism by which ATG8ylation regulates the retromer remains unsolved.

Reviewer #2 (Public Review):

Summary: Padder et al. demonstrate that ATG5 mediates lysosomal repair via the recruitment of the retromer components during LLOMe-induced lysosomal damage and that mAtg8-ylation contributes to retromer-dependent cargo sorting of GLUT1. Although previous studies have suggested that during glucose withdrawal, classical autophagy contributes to retromer-dependent GLUT1 surface trafficking via interactions between LC3A and TBC1D5, the experiments here demonstrate that during basal conditions or lysosomal damage, ATGs that are not involved in mATG8ylation, such as FIP200, are not functionally required for retromer-dependent sorting of GLUT1. Overall, these studies suggest a unique role for ATG5 in the control of retromer function, and that conjugation of ATG8 to single membranes (CASM) is a partial contributor to these phenotypes.

Strengths:

(1) Overall, these studies suggest a unique non-autophagic role for ATG5 in the control of retromer function. They also demonstrate that conjugation of ATG8 to single membranes (CASM) is a partial contributor to these phenotypes. Overall, these data point to a new role for ATG5 and CASM-dependent mATG8ylation in lysosomal membrane repair and trafficking.

(2) Although the studies are overall supportive of the proposed model that the retromer is controlled by CASM-dependent mATG8-ylaytion, it is noteworthy that previous studies of GLUT1 trafficking during glucose withdrawal (Roy et al. Mol Cell, PMID: 28602638) were predominantly conducted in cells lacking ATG5 or ATG7, which would not be able to discriminate between a CASM-dependent vs. canonical autophagy-dependent pathway in the control of GLUT1 sorting. Is the lack of GLUT1 mis-sorting to lysosomes observed in FIP200 and ATG13KO cells also observed during glucose withdrawal? Notably, deficiencies in glycolysis and glucose-dependent growth have been reported in FIP200 deficient fibroblasts (Wei et al. G&D, PMID: 21764854) so there may be differences in regulation dependent on the stress imposed on a cell.

Weaknesses:

(1) Additional controls are needed to clarify the role of CASM in the control of retromer function. Because the manuscript proposes both CASM-dependent and independent pathways in the ATG5 mediated regulation of the retromer, it is important to provide robust evidence that CASM is required for retromer-dependent GLUT1 sorting to the plasma membrane vs. lysosome. The experiments with monsensin in Fig. 7C-E are consistent with but not unequivocally corroborative of a role for CASM. Based on the results shown with ATG16KO in Fig 4A-D, rescue experiments of these 16KO cells with WT vs. C-terminal WD40 mutant versions of ATG16 will specifically assess the requirement for CASM and potentially provide more rigorous support for the conclusions drawn.

(2) Also, the role of TBC1D5 should be further clarified. In Fig S7, are there any changes in the interactions between TBC1D5 and VPS35 in response to LLOMe or other agents utilized to induce CASM? Does TBC1D5 loss-of-function modulate the numbers of GLUT1 and Gal3 puncta observed in ATG5 deficient cells in response to LLOMe?

(3) Finally, the studies here are motivated by experiments in Fig. S1 (as well as other studies from the Deretic and Stallings labs) suggesting unique autophagy-independent functions for ATG5 in myeloid cells and neutrophils in susceptibility to Mycobacterium tuberculosis infection. However, it is curious that no attempt is made to relate the mechanistic data regarding the retromer or GLUT1 receptor mis-sorting back to the infectious models. Do myeloid cells or neutrophils lacking ATG5 have deficiencies in glucose uptake or GLUT1 cell surface levels?

Reviewer #3 (Public Review):

In this manuscript, Padder et al. used APEX2 proximity labeling to find an interaction between ATG5 and the core components of the Retromer complex, VPS26, VPS29, and VPS35. Further studies revealed that ATG5 KO inhibited the trafficking of GLUT1 to the plasma membrane. They also found that other autophagy genes involved in membrane atg8ylation affected GLUT1 sorting. However, knocking out other essential autophagy genes such as ATG13 and FIP200 did not affect GLUT1 sorting. These findings suggest that ATG5 participates in the function of the Retromer in a noncanonical autophagy manner. Overall, the methods and techniques employed by the authors largely support their conclusions. These findings are intriguing and significant, enriching our understanding of the non-autophagic functions of autophagy proteins and the sorting of GLUT1. Nevertheless, there are several issues that the authors need to address to further clarify their conclusions.

(1) The authors confirmed the interaction between Atg5 and the Retromer complex through Co-IP experiments. Is the interaction between Atg5 and the Retromer direct? If it is direct, which Retromer complex protein regulates the interaction with Atg5? Additionally, does ATG5 K130R mutant enhance its interaction with the Retromer?

(2) To more directly elucidate how ATG5 regulates Retromer function by interacting with the Retromer and participates in the trafficking of GLUT1 to the plasma membrane, the authors should identify which region or crucial amino acid residues of ATG5 regulate its interaction with the Retromer. Additionally, they should test whether mutations in ATG5 that disrupt its interaction with the Retromer affect Retromer function (such as participating in the trafficking of GLUT1 to the plasma membrane) and whether they affect Atg8ylation. They also need to assess whether these mutations influence canonical autophagy and lysosomal sensitivity to damage.

Author response:

Reviewer #1 (Public Review):

Summary:

In this study, Masroor Ahmad Paddar and his/her colleagues explore the noncanonical roles of ATG5 and membrane ATG8ylation in regulating retromer assembly and function. They begin by examining the interactomes of ATG5 and expand the scope of these effects to include homeostatic responses to membrane stress and damage.

Strengths:

This study provides novel insights into the noncanonical function of ATG8ylation in endosomal cargo sorting process.

Weaknesses:

The direct mechanism by which ATG8ylation regulates the retromer remains unsolved.

We agree with the reviewer. We do however show how at least one aspect of ATG8ylation contributes to the proper retromer function, which occurs via lysosomal membrane maintenance and repair. Understanding the more direct effects on retromer will require a separate study. We will emphasize this in the revised manuscript and point out the limitations of the present work.

Reviewer #2 (Public Review):

Summary:

Padder et al. demonstrate that ATG5 mediates lysosomal repair via the recruitment of the retromer components during LLOMe-induced lysosomal damage and that mAtg8-ylation contributes to retromer-dependent cargo sorting of GLUT1. Although previous studies have suggested that during glucose withdrawal, classical autophagy contributes to retromer-dependent GLUT1 surface trafficking via interactions between LC3A and TBC1D5, the experiments here demonstrate that during basal conditions or lysosomal damage, ATGs that are not involved in mATG8ylation, such as FIP200, are not functionally required for retromer-dependent sorting of GLUT1. Overall, these studies suggest a unique role for ATG5 in the control of retromer function, and that conjugation of ATG8 to single membranes (CASM) is a partial contributor to these phenotypes.

Strengths:

(1) Overall, these studies suggest a unique non-autophagic role for ATG5 in the control of retromer function. They also demonstrate that conjugation of ATG8 to single membranes (CASM) is a partial contributor to these phenotypes. Overall, these data point to a new role for ATG5 and CASM-dependent mATG8ylation in lysosomal membrane repair and trafficking.

(2) Although the studies are overall supportive of the proposed model that the retromer is controlled by CASM-dependent mATG8-ylaytion, it is noteworthy that previous studies of GLUT1 trafficking during glucose withdrawal (Roy et al. Mol Cell, PMID: 28602638) were predominantly conducted in cells lacking ATG5 or ATG7, which would not be able to discriminate between a CASM-dependent vs. canonical autophagy-dependent pathway in the control of GLUT1 sorting. Is the lack of GLUT1 mis-sorting to lysosomes observed in FIP200 and ATG13KO cells also observed during glucose withdrawal? Notably, deficiencies in glycolysis and glucose-dependent growth have been reported in FIP200 deficient fibroblasts (Wei et al. G&D, PMID: 21764854) so there may be differences in regulation dependent on the stress imposed on a cell.

We thank the reviewer on the overall assessment of the strengths of the study.

We have discussed in the manuscript the elegant study by Roy et al., PMID 28602683. To accommodate reviewer’s comment, we will additionally emphasize in the text that our study is focused on basal conditions and conditions that perturb endolysosomal compartments. We agree with the reviewer that under metabolic stress conditions (such as glucose limitation) more complex pathways may be engaged and will acknowledge that in the discussion.

Weaknesses:

(1) Additional controls are needed to clarify the role of CASM in the control of retromer function. Because the manuscript proposes both CASM-dependent and independent pathways in the ATG5 mediated regulation of the retromer, it is important to provide robust evidence that CASM is required for retromer-dependent GLUT1 sorting to the plasma membrane vs. lysosome. The experiments with monsensin in Fig. 7C-E are consistent with but not unequivocally corroborative of a role for CASM.

We fully agree with the reviewer. In fact, our data with bafilomycin A1 treatment causing GLUT1 miss-sorting (manuscript line 317) show that it is the perturbance of lysosomes and not CASM per se that leads to mis-sorting of GLUT1 (Fig. 7D,E). Note that it has been shown (PMIDs: 28296541, 25484071 and 37796195) that although bafilomycin A1 deacidifies lysosomes it does not induce but instead inhibits CASM. This is because bafilomycin A1 cases dissociation of V1 and V0 sectors of V-ATPase, unlike other CASM-inducing agents which promote V1 V0 association. Complementing this, our data with ATG2AB DKO and ESCRT VPS37A KO (Fig. 8A-F) indicate that the repair of lysosomes is important to keep the retromer machinery functional (as illustrated in Fig. 8G). This may be one of the effector mechanisms downstream of membrane atg8ylation in general and hence also downstream of CASM. We will revise Fig. 7 title to read “Lysosomal damage causes GLUT1 mis-sorting” and will explain these relationships in the text.

Based on the results shown with ATG16KO in Fig 4A-D, rescue experiments of these 16KO cells with WT vs. C-terminal WD40 mutant versions of ATG16 will specifically assess the requirement for CASM and potentially provide more rigorous support for the conclusions drawn.

We will carry out the experiment proposed by the reviewer for the planned revision.

(2) Also, the role of TBC1D5 should be further clarified. In Fig S7, are there any changes in the interactions between TBC1D5 and VPS35 in response to LLOMe or other agents utilized to induce CASM?

We thank the reviewer for pointing this out. We do have data with VPS35 in co-IPs shown in Fig. S7. There is no change in the amounts of VPS35 or TBC1D5 in GFP-LC3A co-IPs. We will include a graph with quantification in the revised manuscript and emphasize this point.

Does TBC1D5 loss-of-function modulate the numbers of GLUT1 and Gal3 puncta observed in ATG5 deficient cells in response to LLOMe?

We agree that TBC1D5 is an interesting aspect. However, because TBC1D5 does not change its interactions in the experiments in our study, we consider this topic (i.e. whether TBC1D5 phenocopies VPS35 and ATG5 KOs in its effects on Gal3) to be beyond the scope of the present work. We underscore that LLOMe (lysosomal damage) mis-sorts GLUT1 even without any genetic intervention (e.g., in WT cells in the absence of ATG5 KO; Fig. 7). Thus, in our opinion the effects of TBC1D5 inactivation may be a moot point.

(3) Finally, the studies here are motivated by experiments in Fig. S1 (as well as other studies from the Deretic and Stallings labs) suggesting unique autophagy-independent functions for ATG5 in myeloid cells and neutrophils in susceptibility to Mycobacterium tuberculosis infection. However, it is curious that no attempt is made to relate the mechanistic data regarding the retromer or GLUT1 receptor mis-sorting back to the infectious models. Do myeloid cells or neutrophils lacking ATG5 have deficiencies in glucose uptake or GLUT1 cell surface levels?

Reviewer’s point is well taken. Glucose uptake, its metabolism, and diabetes underly resurgence in TB in certain populations and are important factors in a range of other diseases. This was alluded to in our discussion (lines 461-469). However, these are complex topics for future studies. We will expand this section of the discussion.

Reviewer #3 (Public Review):

In this manuscript, Padder et al. used APEX2 proximity labeling to find an interaction between ATG5 and the core components of the Retromer complex, VPS26, VPS29, and VPS35. Further studies revealed that ATG5 KO inhibited the trafficking of GLUT1 to the plasma membrane. They also found that other autophagy genes involved in membrane atg8ylation affected GLUT1 sorting. However, knocking out other essential autophagy genes such as ATG13 and FIP200 did not affect GLUT1 sorting. These findings suggest that ATG5 participates in the function of the Retromer in a noncanonical autophagy manner. Overall, the methods and techniques employed by the authors largely support their conclusions. These findings are intriguing and significant, enriching our understanding of the non-autophagic functions of autophagy proteins and the sorting of GLUT1. Nevertheless, there are several issues that the authors need to address to further clarify their conclusions.

(1) The authors confirmed the interaction between Atg5 and the Retromer complex through Co-IP experiments. Is the interaction between Atg5 and the Retromer direct? If it is direct, which Retromer complex protein regulates the interaction with Atg5? Additionally, does ATG5 K130R mutant enhance its interaction with the Retromer?

AlphaFold modeling in the initial submission of our study to eLife (absent from the current version) suggested the possibility of a direct interaction between ATG5 and VPS35 with ATG12—ATG5 complex facing outwards, in which case K130R would not matter. However, mutational experiments in putative contact residues did not alter association in co-IPs. So either ATG5 interacts with other retromer subunits or more likely is in a larger protein complex containing retromer. It will take a separate study to dissect associations and find direct interaction partners. We can provide our data on the currently available modeling and mutational analyses in a full point-for-point rebuttal but believe that since they are inconclusive, they should not be included in the study.

(2) To more directly elucidate how ATG5 regulates Retromer function by interacting with the Retromer and participates in the trafficking of GLUT1 to the plasma membrane, the authors should identify which region or crucial amino acid residues of ATG5 regulate its interaction with the Retromer. Additionally, they should test whether mutations in ATG5 that disrupt its interaction with the Retromer affect Retromer function (such as participating in the trafficking of GLUT1 to the plasma membrane) and whether they affect Atg8ylation. They also need to assess whether these mutations influence canonical autophagy and lysosomal sensitivity to damage.

Please see the response to point 1.

We thank the editors and reviewers for their assessment, constructive criticisms and recommendations.