ATG2A engages RAB1A and ARFGAP1 positive membranes during autophagosome biogenesis

Reviewer #1 (Public review):

Summary:

D. Fuller et al. set out to study the molecular partners that cooperate with ATG2A, a lipid transfer protein essential for phagophore elongation, during the process of autophagy. Through a series of experiments combining microscopy and biochemistry, the authors identify ARFGAP1 and Rab1A as components of early autophagic membranes, which accumulate at the periphery of aberrant pre-autophagosomal structures induced by loss of ATG2. While ARFGAP1 has no apparent function in autophagy, the authors show that RAB1A is implicated in autophagy, although the precise mechanisms are not explored in the manuscript.

Strengths:

The work presented by Fuller et al. provides new insights into the composition of early autophagic membranes. The authors provide a series of MS experiments identifying proteins in close proximity to ATG2A, which is a valuable dataset for the field. Furthermore, they show for the first time the interaction between ATG2A and RAB1A both in fed and starved conditions, which extends the characterisation of the pre-autophagosomal structures observed in ATG2 DKO cells.

Weaknesses / Specific comments:

(1) The authors claim that Rab1A/B knockdown phenocopies the LC3-II accumulation observed in ATG2 DKO cells. While LC3-II accumulation is consistent with this interpretation, depletion of many autophagy-related proteins can give rise to a similar phenotype, even when they function at distinct stages of the autophagic cascade. Therefore, LC3-II accumulation alone is insufficient to support phenocopying in my vew. Immunofluorescence analyses demonstrating comparable cellular phenotypes-such as membrane accumulation of pre-autophagosomal structures-following Rab1 knockdown should be provided. Moreover, p62 does not accumulate upon Rab1 depletion, suggesting that loss of Rab1 does not fully phenocopy ATG2 deficiency. Consequently, it remains unclear whether Rab1A depletion truly phenocopies ATG2A depletion with respect to autophagy progression or the accumulation of pre-autophagosomal structures.

(2) Interpretation of the significance of the data

(2.1) The significance statement asserts that "this study elucidates the role of early secretory membranes in autophagosome biogenesis." While the data convincingly demonstrate an association between the RAB1A GTPase and ATG2A, the study does not provide mechanistic insight into how this interaction functionally contributes to autophagy. As presented, the findings support a correlative relationship rather than a defined role in autophagosome biogenesis.

(2.2) The title states that ATG2A "engages" Rab1A- and ARFGAP1-positive membranes during autophagosome formation. However, both Rab1A and ARFGAP1 are shown to localize to pre-autophagosomal structures independently of ATG2A. In the absence of evidence demonstrating a functional or causal dependency, the term "engages" appears overstated. A more descriptive term, such as "associates," would more accurately reflect the data.

(2.3) In the Discussion, the authors state that previous studies have reported increased LC3-II levels following knockdown of Rab1 proteins (refs. 38 and 49). However, it is unclear where this observation is documented in the cited references.

(3) Some concerns remain in specific figures, as outlined below:
• Quantification is missing in Fig S2D.
• The authors claim: "siRNA against ARFGAP1 had very little effect" but the quantification and blots show actually no effect.
• Conclusions drawn from KD experiments in Fig. S2 should be interpreted with caution, as knockdown efficiency is very low, particularly for ARFGAP1/3 in the triple knockdown.
• In New Fig. 4, the representative blot is not representative of the results showed in the quantification as previously noted.

Reviewer #2 (Public review):

The mechanisms governing autophagic membrane expansion remain incompletely understood. ATG2 is known to function as a lipid transfer protein critical for this process; however, how ATG2 is coordinated with the broader autophagic machinery and endomembrane systems has remained elusive. In this study, the authors employ an elegant proximity labeling approach and identify two ER-Golgi intermediate compartment (ERGIC)-localized proteins-Rab1 and ARFGAP1-as novel regulators of ATG2 during autophagic membrane expansion.

Their findings support a model in which autophagosome formation occurs within a specialized subdomain of the ER that is enriched in both ER exit sites (ERES) and ERGIC, providing valuable mechanistic insight. The overall study is well executed and offers an important contribution to our understanding of autophagy. I support its publication in eLife and offer the following minor comments for clarification and improvement.

Specific Comments

(1) Integration with Prior Literature
The data convincingly implicate the ERES-ERGIC interface in autophagosome biogenesis. It would strengthen the manuscript to discuss previous studies reporting ERES and ERGIC remodeling and formation of ERERS-ERGIC contact sites (PMID: 34561617; PMID: 28754694) in the context of the current findings.

(2) Figure Labeling
The font size in Figure 1A and Supplementary Figure S1G is too small for comfortable reading. Please consider enlarging the labels to improve clarity.

(3) Experimental Conditions
In Figures 2A-C and Figure 4, it is unclear how the cells were treated. Were they starved in EBSS? Please include this information in the corresponding figure legends.

(4) LC3 Lipidation vs. Cleavage
In Figure 2A, ARFGAP1 knockdown appears to reduce LC3 lipidation without affecting Halo-LC3 cleavage. Clarifying this observation would help readers better understand the functional specificity of ARFGAP1 in the pathway.

(5) Use of HT-mGFP in Figure 2C
Please clarify why the assay in Figure 2C was performed in the presence of HT-mGFP. Explaining the rationale would aid interpretation of the results.

(6) FIB-SEM Imaging
For the FIB-SEM images in Figures 3 and S3, directly labeling the cellular structures in the images would greatly facilitate interpretation for the reader.

(7) Supplementary Figures
Many of the supplemental figures are high quality and contain key data. If space permits, I suggest moving these into the main figures. In particular, the FLASH-PAINT experiment could be presented as part of Figure 1.

(8) Text Revision for Clarity
In line 242, the phrase "but protein-protein interactions appear to be limited to RAB1" would benefit from clarification. A more precise formulation could be: "but stable protein-protein interactions appear to be limited to RAB1."

(9) COPII Inhibition Strategy
The authors used the dominant-active SAR1(H79G) mutant to inhibit COPII function. While this is effective in in vitro budding assays, the GDP-locked mutant SAR1(T39N) has been shown to be more effective in blocking COPII-mediated trafficking in cells. Including SAR1(T39N) in the analysis would provide stronger support for the conclusions.

Reviewer #3 (Public review):

The manuscript by Fuller et al describes a crosstalk between ARTG2A with components of the early secretory pathway, namely RAB1A and ARFGAP1. They show that ATG2A is recruited to membranes positive for RAB1A, which they also show to interact with ATG2A. In agreement with earlier findings by other groups, silencing RAB1A negatively affects autophagy. While ARFGAP1 was also found on ATG2A positive membranes, silencing ARFGAP1 had no impact autophagy. Notably, these ARFGAP1 positive membranes are not Golgi membranes.

The findings are interesting and the data are in general of good quality. I think the story is good enough to be published in eLife and I have the following questions, which the authors may attend to:

(1) Are the membranes to which ATG2A is recruited a form of ERGIC?

(2) Figure 3A/B: Is it possible to show a better example? The difference is barely detectable by eye. Since Immunoblotting is not really a quantitative method, I think that such a weak effect is prone to be wrong. Is there another tool/assay to validate this result?

(3) Is the curvature-sensitive region of ARFGAP1 required for its co-localization with ATG2A?

(4) What does Rab1A do? What is its effector? Or does the GTPase itself remodel the membrane?

(5) What about Arf1? It appears that this role of ARFGAP1 is unrelated to Arf1 and COPI? Thus, one would predict that Arf1 does not localize to these structures and does not affect ATG2A function

(6) Does ARFGAP1 promote fission of the membrane from its donor compartment?

(7) What are ARFGAP1 and Rab1A recruited to? What is the lipid composition, or protein that recruits these two players to regulate autophagy?

Comments on the latest version:

The revisions carried out by the authors are fine. The new data on ArfGAP1 and about the indirectness of the ATG2A and Rab1A interaction improve both clarity and strength of the manuscript. I have no further comments.

Author response:

The following is the authors’ response to the original reviews.

We thank the reviewers and editors for their thoughtful comments, which substantially improved the quality and clarity of our manuscript. We have attempted to address each major concern with either new experiments or significant textual revisions.

Reviewer 1 noted that “this research is conducted exclusively in HEK293 cells… including at least one additional cell line would significantly strengthen the main findings.” To directly address this concern, we repeated our RAB1A/B double-knockdown experiments in H4 neuroglioma cells, which endogenously express a tandem fluorescent-tagged LC3B reporter. Using flow cytometry to quantify autophagic flux, we confirmed that RAB1 depletion in H4 cells recapitulates the flux defects observed in HEK293 cells, thereby validating the generality of our findings across distinct lineages.

To validate the robustness of the ATG2 DKO phenotype and the localization of ARFGAP1-positive membranes, we acquired an ATG2 double knockout HeLa cell line. We confirmed the presence of the characteristic large ATG2-deficient PAS compartment in HeLa cells, and the recruitment of ARFGAP1 membranes, but note that ARFGAP1 displays a solid distribution through the compartment in these cells, in contrast to the more peripheral enrichment observed in HEK293 cells. These data are now included and discussed in the revised manuscript.

Multiple reviewers asked for greater clarity around the interaction between ATG2A and RAB1A. Although our original data showed that these proteins co-immunoprecipitate in cells, we had not established whether their association was direct. In response, we attempted in vitro co-immunoprecipitations from purified components. As we could not detect interactions in this simplified system, we now speculate that the ATG2A–RAB1A interaction is indirect. This clarification is now incorporated into the results section.

Multiple reviewers also raised questions regarding the nature of the membranes recruiting ARFGAP1 and the potential relationship to Arf1 and Golgi trafficking. In particular, Reviewer 3 asked: “(5) What about Arf1? … one would predict that Arf1 does not localize to these structures and does not affect ATG2A function.” To examine whether ARFGAP1 recruitment depends on Golgi integrity or Arf1-regulated trafficking, we perturbed the Golgi using three mechanistically distinct methods: Brefeldin A, mitotic entry, and SidM expression, each of which dissolves Golgi architecture. In each condition, ARFGAP1 localization to the enlarged PAS compartment in ATG2 DKO cells was unchanged. These results indicate that ARFGAP1 recruitment is independent of Golgi structure and provide indirect support for the notion that Arf1 does not participate in this process. Reviewer 3 also asked: “Is the curvature-sensitive region of ARFGAP1 required for its co-localization with ATG2A?” To address this, we generated ARFGAP1 mutants lacking either GAP catalytic activity or the ALPS curvature-sensing domain. When expressed in ATG2 DKO cells, all mutants retained full recruitment to the PAS compartment. Thus, neither GAP activity nor ALPS-mediated curvature sensing is required for ARFGAP1 localization in this context.

Response to Reviewer 3 -“(2) Figure 3A/B: … is there another tool/assay to validate this result?”—we quantified autophagic flux following SAR1B(H79G) overexpression using the flow-cytometry tandem-fluorescent LC3 assay. These experiments confirmed that SAR1B(H79G) causes only a modest reduction in autophagic flux, consistent with partial inhibition of COPII, thereby supporting our original interpretation.

We also took steps to improve the integration of our findings with prior literature. Reviewer 2 requested that we strengthen the manuscript by incorporating studies on ERES–ERGIC remodeling (“It would strengthen the manuscript to discuss previous studies…”). We now cite and discuss the studies corresponding to PMIDs 34561617 and 28754694, aligning our observations with mechanistic models of early secretory pathway remodeling. More broadly, Reviewer 1 commented that our discussion “overlooks some important aspects,” and Reviewer 3 asked, “Are the membranes to which ATG2A is recruited a form of ERGIC?” In response, we substantially rewrote the discussion, expanding our integration of existing literature and explicitly addressing models in which ATG2A acts at an ERGIC-derived membrane.