ATG2A interacts with RAB1A and ARFGAP1 positive membranes during autophagosome biogenesis

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of 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.

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.

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.

Comments on the previous 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 previous reviews

(1) Interpretation of LC3-II accumulation and phenocopying

Therefore, LC3-II accumulation alone is insufficient to support phenocopying in my view.

We agree with this assessment. Upon reconsideration, we concluded that LC3-II accumulation alone does not justify the use of the term "phenocopying." We have therefore removed this language from the manuscript.

(2) Strength of conclusions regarding autophagosome biogenesis

As presented, the findings support a correlative relationship rather than a defined role in autophagosome biogenesis.

We agree that our original wording overstated the strength of the conclusions. To better reflect the data, we revised the text to state that our findings "expound upon" rather than "elucidate" the role of these membranes in autophagosome biogenesis.

(3) Title wording

The title states that ATG2A ‘engages’ Rab1A- and ARFGAP1-positive membranes during autophagosome formation... A more descriptive term, such as ‘associates,’ would more accurately reflect the data.

We appreciate this suggestion. We revised the title to avoid implying a causal dependency. The title now states that ATG2A interacts with Rab1A- and ARFGAP1-positive membranes, emphasizing the membrane association observed in our study rather than a direct interaction with the proteins themselves.

(4) ARFGAP1 knockdown phenotype

The authors claim: ‘siRNA against ARFGAP1 had very little effect’ but the quantification and blots show actually no effect.

We agree that the original wording was imprecise. The sentence has been revised to state:

“siRNA against ARFGAP1 had no effect on flux.”

This more accurately reflects both the data and our original interpretation.

(5) Interpretation of ARFGAP knockdown experiments

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.

We agree with this caution and have revised the text accordingly. The manuscript now states:

“Knockdown of ARFGAP2, ARFGAP3 and ARFGAP1-3 marginally increased autophagic flux (Fig. S2B,C), suggesting either no role or a minor role as negative regulators of autophagy.”

This wording more appropriately reflects the limitations of the experiment.

(6) Discussion of ERGIC/ERES remodeling literature

It would strengthen the manuscript to discuss previous studies reporting ERES and ERGIC remodeling and formation of ERES-ERGIC contact sites (PMID: 34561617; PMID: 28754694).

We appreciate the suggestion. These studies were already cited and discussed in the original submission, and therefore no additional changes were required.

(7) Figure readability

The font size in Figure 1A and Supplementary Figure S1G is too small for comfortable reading.

We agree and have enlarged the labels in both figures to improve readability.

(8) Clarification of starvation conditions in figure legends

In Figures 2A-C and Figure 4, it is unclear how the cells were treated. Were they starved in EBSS?

We have updated the corresponding figure legends to explicitly state the starvation conditions used in these experiments.

(9) Interpretation of ARFGAP1 knockdown and LC3 lipidation

In Figure 2A, ARFGAP1 knockdown appears to reduce LC3 lipidation without affecting Halo-LC3 cleavage.

We do not observe a reproducible reduction in LC3 lipidation following ARFGAP1 knockdown and therefore do not believe this conclusion is supported by the data. No changes were made in response to this comment.

(10) Clarification of protein-protein interaction statement

The phrase ‘but protein-protein interactions appear to be limited to RAB1’ would benefit from clarification.

We agree and have adopted the suggested wording. The manuscript now states:

“but stable protein-protein interactions appear to be limited to RAB1.”

We thank the reviewers again for their constructive feedback and for helping us improve the clarity and accuracy of the manuscript.