The mechanism of Atg15-mediated membrane disruption in autophagy

  1. Yoko Kagohashi
  2. Michiko Sasaki
  3. Alexander I. May
  4. Tomoko Kawamata
  5. Yoshinori Ohsumi

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Abstract

Autophagy is a lysosomal/vacuolar delivery system that degrades cytoplasmic material. During autophagy, autophagosomes deliver cellular components to the vacuole, resulting in the release of a cargo-containing autophagic body (AB) into the vacuole. AB membranes must be disrupted for degradation of cargo to occur. The lipase Atg15 and vacuolar proteases Pep4 and Prb1 are known to be necessary for this disruption and cargo degradation, but the mechanistic underpinnings remain unclear. In this study, we establish a system to detect lipase activity in the vacuole and show that Atg15 is the sole vacuolar phospholipase. Pep4 and Prb1 are required for the activation of Atg15 lipase function, which occurs following delivery of Atg15 to the vacuole by the MVB pathway. In vitro experiments reveal that Atg15 is a phospholipase B of broad substrate specificity that is likely implicated in the disruption of a range of membranes. Further, we use isolated ABs to demonstrate that Atg15 alone is able to disrupt AB membranes.

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  1. Version published to 10.1083/jcb.202306120
  2. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    1. General Statements [optional]

    This study represents a detailed analysis of the mechanistic bases of Atg15 function in autophagy, which relates back to our initial studies of autophagy published by our group 30 years ago in JCB (10.1083/jcb.119.2.301), where we reported autophagy by disrupting vacuolar protease function. We report that Atg15 is the sole vacuolar lipase in yeast, and that it exhibits broad activity on a range of lipids. Following submission of our study to Review Commons, we have received favorable feedback from all three reviewers. We plan to perform the revisions below within one month, following which we will submit a full revision to JCB with complete point-by-point responses to the reviewers. We are confident that this study will be of interest to the broad readership of JCB and trust that you will find it worthy of further consideration for publication.

    2. Description of the planned revisions

    Reviewers 1 and 3 suggested that we should confirm whether Atg15 is indeed the sole vacuolar lipase using lipids other than NBD-PE. While we have already shown that the kinase-dead S332A variant is non-function in vacuolar lysates, we will further address this comment by determining whether vacuolar lysates isolated from atg15Δ cells are able to process other lipid species. We will also collect replicates and quantify data for all figures to address comments made by the reviewers.

    We also received a comment from reviewer 2 asking us to determine the function and expression level of vector-borne ATG15 and ATG15-Flag expressed in the atg15Δ background strain. We will provide these data, along with a comparison with results from WT cells, in our revision.

    Reviewer 1 indicated that we need to address the localization of Atg15 in more detail. We plan to better explain the results of our initial analyses, as well as collecting more detailed data using super-resolution microscopy, and these data will be analyzed and discussed in further detail.

    Regarding the text, we will update the results, discussion and methods to make these easier to follow, as pointed out by reviewer 1.

    A complete point-by-point response to reviewer comments will be provided in the full revision.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    No revisions yet carried out.

    4. Description of analyses that authors prefer not to carry out

    Reviewer 3 suggested that when considering Atg15 lipase activity we provide information about the lipid makeup of autophagic body membranes. While we agree that this is an interesting suggestion, our pilot experiments have indicated to us that this analysis is complex and will generate a very large amount of additional data and technical details that would need to be supplemented, clearly exceeding the scope of this study. Further, while we are currently performing these analyses, it will take significant additional time to bring these experiments to a conclusion in line with the reviewer’s comment.

    On a related note, reviewer 3 suggested that we provide more detailed analyses of the degradation products arising from Atg15 lipase activity to provide some context into our finding that Atg15 acts on purified autophagic body membranes. We have collected initial lipidomic data for vacuolar extracts following the induction of autophagy (see attached figure), and have confirmed that we detect lysophospholipids in a manner that quantitatively depends on the amount of Atg15. However, as we feel that these data require further careful and time-consuming lipidomic analyses, as well as a nuanced discussion of results arising, we plan to publish these data in a separate, detailed paper and not in the present study.

    With regard to reviewer 1’s comment about the vulnerability of Atg15 to the presence of detergent, we agree that this is an important point, but we can not eliminate the possibility that a very small amount of Atg15 exhibits lipase activity that is detected by this assay. The key message of our study is that Atg15 is activated by proteases in the vacuole to function as a broad-activity lipase; we feel that a detailed investigation of the active fragment of Atg15 is secondary to this finding and would unfortunately be very difficult to determine using currently available techniques, especially when considering the expression of Atg15. While it would be very nice to have these data, we therefore cannot provide these data in the full revision.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript by Kagohashi and colleagues provide evidence of the enzymatic activity of ATG15 as a phospholipase B with a broad substrate specificity and dissect the mechanisms by which this protein is activated in the vacuole to promote the hydrolysis of autophagic bodies. The manuscript is very clear and well written, the scientific and experimental concepts are clearly presented and easy to follow. This is excellent biochemistry with very well-thought concepts and well-designed experiments. I am convinced by the authors' results and conclusions which, for the most part, are justified by appropriate experiments and conclusive data.

    Main points:

    • The authors claim that they show that ATG15 is the sole vacuolar phospholipase (see abstract). This conclusion is based on the in vitro analysis of the hydrolysis of NBD-PE (a fluorescent, thus not physiological, phospholipid). Fig. 1 indeed show the absence of NBB-PE hydrolysis in cells lacking ATG15 or expressing a catalytic dead version of the protein, which supports the authors' conclusion. But (1) this does not reflect the hydrolysis of physiological phospholipids in the vacuole, (2) this is only based on the analysis of one phospholipid (PE), (3) this is not consistent with results presented in Fig.5 C-F, in which, in the absence of ATG15 or when the catalytic dead version of the protein is expressed, there is clearly still some hydrolysis of PC, PG and PI. Concerning Fig. 4, the authors state that 'the commercially available NBD-PC and NBD-PG had been somewhat decomposed' and it is unclear to me if the lanes marked by an oblique line correspond to the lysate of atg15D cells or to NBD-PL alone. If this corresponds to the NBD-PL alone, I suggest that the authors perform the experiment presented in Fig.1 (at least Fig. 1C), with additional NBD-PL to actually test for residual phospholipases activity in the absence of ATG15.

    I also suggest that the authors perform lipid analyses of purified vacuoles including engulfed organelles (autophagic bodies, MVBs etc.) to detect changes when put in contact with vacuolar lysates from WT, ATG15D cells and ATG15S332A, which will be more physiological that the use of NBD-PL, and could therefore support their conclusion.

    • The use of NBD-PL is very powerful and support the authors conclusions, but the paper lacks from physiological results as stated above. For instance, concerning the efficiency and substrate specificity of ATG15: what is the actual lipid composition of autophagic bodies ? How efficient is ATG15 in regard to the lipids that mainly compose the membrane of autophagic bodies ? Can the authors quantitatively compare the activity of ATG15 from one phospholipid to the other ? Here the experiments are performed on lipids in solution, what about hydrolysis activity on lipids in membranes ? As mentioned in my comment above, I suggest that the authors perform tests with purified autophagy bodies, or, at least, on reconstituted vesicles with a composition similar to what is found in autophagic bodies to assess the activity of ATG15 in physiological conditions.
    • The results and data presented by the authors are clear and seem unequivocal for the main parts but none of the results are quantified and there is no statistical analyses provided. How many times were the experiments repeated and how consistent are the results ? The authors must provide quantitative information and stats for all the figures.

    Significance

    Although it has been long known that ATG15 is required for the degradation of autophagic bodies, how this protein which transits to the vacuole through the MVB pathway can be activated in the vacuole to specifically target autophagic bodies and/or its cargo, remained completely unknown. The results presented here, pending their confirmation with additional experiments, thus fill an important gap in knowledge to understand the last crucial steps of the autophagy pathway which had remained largely elusive across organisms. Autophagy is critical for the physiology and development of all eucaryotes with major implication in human diseases. This manuscript will thus be of interest not only for the autophagy community but also for a broader general scientific community with potential applications in medical sciences. The results presented here also provide crucial elements to understand lipid hydrolysis in the vacuole and how this is finely regulated to ensure proper disruption of autophagic bodies, and thus, to the support the finality of autophagy degradation, while maintaining the integrity of the vacuolar membrane. In that context, this paper will influence all cell biologists by providing knowledge of the function, activities and homeostasis of the vacuole. This work raises the question of how ATG15 is specifically addressed to the membrane of autophagic bodies in the vacuole which will be the subject for future exciting research.

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    Referee #2

    Evidence, reproducibility and clarity

    Atg15, a membrane-bound phospholipase targeted to the vacuole via the MVB pathway, has been studied in recent years. It is synthesized as an inactive proenzyme activated by vacuolar enzymes and is responsible for the degradation of the autophagic bodies' membranes. While its physiological function is well characterized, the biochemical details of its activation and overall activity remain largely unknown.

    In the present study, Kagohashi et al conducted a comprehensive investigation to understand the mechanisms involved in the disruption of autophagic bodies (ABs) membranes. They focused on the activities of Atg15 and Pep4/Prb1 and employed primarily in vitro methods to elucidate the functional mechanism of Atg15. Purified Atg15 and ABs were used in the experiments. According to the proposed model, Pep4/Prb1 processes and activates Atg15 during its localization to the AB membrane, and this activation is necessary for Atg15's lipase activity. To support this model, the authors performed in vitro assays using purified proteins, vacuole and ABs purification, genetics, mutations, lipase activity assays, and morphological examination of autophagic bodies using a super-resolution fluorescence microscope. Their findings demonstrated that the activity of Pep4/Prb1 is required for Atg15's lipase activity, and Atg15 functions as a vacuolar lipase. The importance of the lipase motif for Atg15's activity was confirmed through the use of hydrolase mutants and purified Atg15 from both wild-type and mutant samples. Overall, the manuscript provides a comprehensive and solid analysis of Atg15 that will most likely interest the cell biology community. The experiments are well-controlled, and the conclusions are based on solid experimental data.

    There are only a couple of minor issues that deserve the authors' attention:

    Figure 1c shows a lower level of NBD-LPE in cells expressing ATG15 from a plasmid compared to the wild type, indicating that the plasmid did not fully restore the lipase activity. Additionally, the exact details for Atg15 expression should be explicitly described.

    To ensure transparency and reproducibility, the authors should provide information about the specific expression vector(s) used for the plasmids in the study.

    Significance

    As indicated above, the study provides elaborate and solid characterization of an important enzyme that has been previously mainly functionally characterized.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The rupture of single membrane-bound autophagic bodies is essential to release and catabolize contents of autophagosomes deposited in the vacuole. The phospholipase Atg15 has been thought to play an important role in this process. This study establishes methods to analyze phospholipase activity in isolated Saccharomyces cerevisiae vacuoles and elucidates the mechanisms that activate Atg15. Using an elegant cell-free assay the authors demonstrate that vacuolar extracts can cleave phosphatidyl ethanolamine in an Atg15 and Pep4/Prb-dependent manner. Atg15 is cleaved in the presence of Pep4/Prb, likely causing the release of Atg15 cytosolic domain in the vacuole. An Atg15 construct lacking the transmembrane anchor retains its lipase activity and when artificially targeted to vacuole using CPY tag localizes to autophagic bodies. The authors also establish the minimum construct of Atg15 that is sufficient to execute lipase function. The authors then isolate Atg15 from vacuolar extracts using a FLAG tag-based pulldown and show that the FLAG eluate is sufficient to cleave a range of phospholipids. Finally, using a protease-protection assay the authors show that Atg15 isolated using FLAG resin can cause disruption of isolated autophagic bodies.

    Major comments:

    1. Throughout the manuscript, TLC data and Ape1 maturation data are not quantified. The authors should include data on replicates and quantitation for all TLC and Ape1 processing data.
    2. The conclusion that Atg15 is the sole source of phospholipase activity is based on cleavage of NBD-PE alone. It is not clear why specifically PE was chosen to test lipase activity of Atg15. It is possible that Atg15 has a higher preference for PE as has been shown previously (Ramya and Rajsekaran 2016). Have the authors tested to see if other phospholipids can be cleaved by vacuolar lysates derived from Atg15 knockout cells? This should be investigated further before concluding that Atg15 is the sole source of all lipase activity in vacuolar extracts.
    3. Atg15 overexpressed and purified from Saccharomyces cerevisiae is shown to be sufficient to catalyze the cleavage of PE (among other phospholipids). How do the authors reconcile this finding with their observations on the requirement of Pep4 and Prb? This information should be included in the discussion.
    4. Regarding Figure 3 and movie EV3, especially the lower panel, the overlap of cherry-Atg8 (autophagic bodies) and CPY(1-50)-Atg15(DN35)-mNG is not very clear. There appear to be several CPY(1-50)-Atg15(DN35)-mNG rings that do not surround Atg8.
    • a. Are these images from a single stack or represent the entire volume of the cell? This result could be better represented as a line profile and through a correlation analysis.
    • b. The finding that CPY(1-50)-Atg15(DN35) binds autophagic bodies is interesting, but it should be demonstrated with native/wild type protein. This can be achieved by expressing lipase deficient Atg15-mNG in rapamycin-treated cells, which should have intact accumulated autophagic bodies.
    • c. Atg15-mNG also localizes to a ring-like structure outside the vacuole. The authors should comment on the potential impact of this finding.
    1. The rationale for using detergent solubilized and FLAG-eluted Atg15 to test lipase activity with other phospholipids (LPC, PI, PC and PG) is not clear. Detergent solubilized and FLAG-eluted Atg15 is degraded (Figure4C). Does this mean that degraded forms of Atg15 exhibit broader lipase activity? The authors should test for breakdown of other phospholipids with whole vacuolar extracts or vacuolar pellet fraction that has intact membrane bound Atg15. If only degraded forms of Atg15 show broad phospholipid lipase activity, then this will be informative about regulation of Atg15 function.
    2. Figure6B: ProteinaseK is a broad-spectrum protease. It is unclear why it would specifically cleave GST-GFP and prApe1 to produce single bands (and not a smear) corresponding to free-GFP and dApe1. This result can be explained better.

    Minor comments:

    1. Fig1E legend states, "Each vacuolar lysates were added at a volume ratio of 1:5:25". It's not clear what this means or what this ratio is for. In general figure legends need to be more descriptive on how the experiment was performed.
    2. It's not clear what processed Atg15 (pcrAtg15) refers to in Figure4C. Is it indicating the smear around the 75kDa band? This should be explained clearly in the figure legend and the results section.

    Significance

    The phospholipase Atg15 is known to play a crucial role in the degradation of autophagic bodies within the vacuole. However, the regulatory mechanisms that prevent detrimental lipase activity of Atg15 have remained unclear. This study shows that proteolytic processing and membrane binding could activate Atg15, thereby providing important insights into the mechanism of Atg15 regulation.
    Using isolated autophagic bodies and vacuolar extract, the results here show direct disruption of autophagic bodies by Atg15. The cell-free assay to assess lipase activity can be further utilized to analyze vacuolar function. These finding will be of interest to a audience interested in various forms of autophagy and vacuolar degradation.

  6. Version published to 10.1101/2023.06.26.546516v1 on bioRxiv
  7. Version published to 10.1101/2023.06.26.546516v2 on bioRxiv