Autophagosome formation is initiated at phosphatidylinositol synthase-enriched ER subdomains

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1 Manuscript EMBO Autophagosome formation is initiated at phosphatidylinositol synthase-enriched ER subdomains Taki Nishimura, Norito Tamura, Nozomu Kono, Yuta Shimanaka, Hiroyuki Arai, Hayashi Yamamoto, Noboru Mizushima Corresponding author: Noboru Mizushima, Taki Nishimura, The University of Tokyo, Graduate School and Faculty of Medicine Review timeline: Submission date: 06 July 2016 Editorial Decision: 23 August 2016 Revision received: 19 February 2017 Editorial Decision: 10 March 2017 Revision received: 28 March 2017 Accepted: 11 April 2017 Editor: Andrea Leibfried Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 23 August 2016 Thank you for submitting your manuscript for consideration by the EMBO Journal. It has now been seen by three referees whose comments are shown below. As you will see, the referees have a mixed opinion on the suitability of your manuscript for publication here. I therefore initiated a consultation session to allow all referees to cross-comment on each other. The outcome of this - which is also reflected in the individual reports of the referees - is that the referees appreciate your approach and the identified role for PI and PIS. However, they also think that the membrane composition in the fractions and the changes induced by genetic or chemical manipulations needs much further insight and that alternative models for the sequences of events regarding FIP200 and Atg9 need to be considered throughout the manuscript. Furthermore, the imaging data, lipid profile analyses and the PIS localization data need additional insight. Should you be able to address these criticisms in full, we could consider a revised manuscript. Please note that I would need strong support from the referees on a revised version for publication here. I should remind you that it is EMBO Journal policy to allow a single round of revision only and that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses in this revised version. I do realize that addressing all the referees' criticisms will require a lot of additional time and effort and be technically challenging, and I can extend the revision time to 6 months should this be helpful. I would also understand if you wish to publish the manuscript elsewhere, in which case please let us know so we can withdraw it from our system. European Molecular Biology Organization 1

2 If you decide to thoroughly revise the manuscript for the EMBO Journal, please include a detailed point-by-point response to the referees' comments. Please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process, please visit our website: Thank you for the opportunity to consider your work for publication. I look forward to your revision REFEREE COMMENTS Referee #1: In this manuscript by Nishimura and colleagues, a biochemical assay was applied to characterize the autophagic precursor membrane, termed isolation membrane or phagophore, in terms of its relation to ER-membranes and its lipid composition. The whole study is based on floatation experiments in which membranes are separated according to their density. The authors investigated the distribution of ATG-proteins in gradient fractions based on western blots, focusing on subunits of the ULK-1 complex and ATG9. The authors used deletions to enrich autophagic precursor membranes, which are stalled at various stages (e.g. deletion of ATG3 to inhibit maturation). The authors found that FIP200 binds to ER-membranes independently of other members of the ULK-1 complex and that assembly of the complex occurs at ER-domains enriched in the lipid phosphatidylinositol. The association of autophagic precursor membranes with the ER was intensively characterized in the past using various techniques including electron tomography for visualization, co-localization studies and biochemical characterizations. It was known that so called omegasomes, which are PI3P-enriched membrane domains of the ER, serve as nucleation sites for autophagosomes and that ATG-proteins are recruited to those sites in a hierarchical manner. Regarding those previous data, the insights obtained from this study are rather limited. Moreover, the entire study appears preliminary and lacks many important controls. I therefore do not recommend publication of this manuscript in the EMBO J. Fractions from gradient centrifugations where analyzed in terms of distribution of ATG-proteins and it was claimed that phagophore membranes are enriched in certain fractions. One surprising observation is that upon starvation the profile of ATG-protein distribution in these fractions did not change. Moreover, no other experimental methods were used (e.g. ultrastructural analysis) to confirm that the distribution of ATG-proteins correlate with enrichment of autophagic membranes. Furthermore, many claims of the authors are not supported by data or are in odds with what was observed experimentally (e.g. " ATG proteins, except for membrane protein ATG9A and... LC3, mostly remained in the bottom cytosolic fraction", although a significant portion of FIP200 and ATG13 are found in floating fractions. Moreover, against the author's interpretation, a significant offset in the distribution of ATG9 or LC3 (peak in fraction 5) and FIP200 or ATG13 (peak in fraction 6 and 7) was observed. The authors concluded from KO experiments that autophagic precursors are enriched in floating fractions without characterizing these fractions carefully. All fractions contain an ensemble of various membranes and deletion of ATG-proteins might also affect the composition of membranes in these fractions and not only the presence or absence of various autophagic precursors. ULK-1 was found to be membrane associated even during vegetative growth previously (Chan et al, 2009), but the authors reported here that most of ULK-1 was cytosolic without commenting on this discrepancy. Moreover, the difference in distribution profiles of ULK-1 and ATG13 or FIP200 is in conflict with what has previously been published by the authors of this study (i.e. ULK-1, ATG13, and FIP200 form a stable complex independently of nutrient status of cells). Finally, the authors depleted PI since they observed a strong co-localization of FIP200 and PIsynthase. The authors observed strong co-localization of FIP200 and PIS in starved wortmannin treated cells, but did not show colocalization in starved cells w/o WM-treatment. The latter colocalization was only performed with ULK-1. Furthermore, the authors depleted PI to investigate the European Molecular Biology Organization 2

3 effect on protein localization by expressing inducible PISHQ-GFP-PIPLC. The major problem with depleting PI is that PIPs are also depleted which are important regulators of membrane identity and vesicular trafficking. Thus depleting PI might also affect organelles and transport processes and the observed effects on autophagy are thus a result of disturbed overall cellular morphology. Referee #2: This manuscript provides data that contributes to the increasing evidence that autophagosome formation occurs at sub-domains of the ER. Nishimura and colleagues have investigated the nature and composition of these ER subdomains with a powerful biochemical approach that combines subcellular fractionation, isolation of vesicles, and lipid analysis. Furthermore, they provide data on the lipid biosynthetic enzymes, which produce and hydrolyse PI suggesting this is metabolic function occurs at this ER subdomain. They identify membranes in both WT and AtgKO MEFs that contain subsets of Atg proteins. These membranes have distinct characteristic allowing them to be separated on gradients. By analysing focusing on the ULK complex member FIP200, Atg9, WIPI2 and ER components they develop a hypothesis that the ULK complex is recruited to an ER subdomain which provides a platform to form isolation membranes from Atg9-positive membranes. Overall the data is technically excellent, well controlled and address the aim of the study, and is an important contribution to the autophagy field and others interested in the role of lipids. Specific points: 1. The main issue is the final interpretation of the data in particular the conclusion that transfer of FIP200 to an Atg9-positive vesicle creates the isolation membrane. This conclusion is flawed as the authors only consider the data in yeast on Atg9, to inform them in the interpretation of the experiments, and the formulation of the final model. In particular, Atg9 in mammalian cells has been shown to dynamically associate with early membranes. Indeed in Fig. S2 the authors also show the WIPI2/FIP200 positive puncta have very low amounts of Atg9. The data and model should be reconciled with the mammalian data, in particular Orsi et al., 2012, and Lamb et al., Figure 1D. Is Atg14 part of the Beclin1 complex? Is Vps34 or Vps15 found in these fractions? 3. There are slight differences in the distribution of Flag-Atg9 and endogenous Atg9 in Fig. 1D and E. There is less flag-atg9 in the top fractions in the Atg3KO although this is proposed to be the expanding isolation membrane. 4. Page 6, end of 1st paragraph. Orsi et al also showed the ULK1 puncta form in Atg9-/- MEFs. 5. The isolation of Atg9 vesicles was done from gradient fractions obtained from gradients which are not included in the data. The authors should add subcellular fractionation analysis for the WT/Flag-Atg9 cells and Atg14KO/Flag Atg9 both under growing conditions. 6. Figure 2B-G the authors should clarify if the gradients were all prepared with lysates from growing cells. 7. In Figure 2E, the population of flag-atg9 found in the gradient shown in Fig. 1E is a very minor population (perhaps less than 10%) of the total flag-atg9 and is distributed differently from the endogenous shown in Fig. 1F, suggesting the tagged transfected Atg9 may be accumulating in the ER due to saturation of the export machinery. This would result in the large amounts of Atg9 seen in the middle of the gradient. The authors use the fraction 1 pool for Fig. 2E which is a crucial experiment for the model of recruitment of the FIP200 complex to the Atg9-positive compartment. Do these membranes in fraction 1 with Flag-Atg9 have Atg14? Can the authors demonstrate that the flag-atg9 exits the ER and traffics normally during fed and starved conditions? 8. In Figure 2H, derived from the data in Figure 1 and 2, the final membrane compartment diagrammed "Atg3 KO fr. 1" shows Atg9 vesicle with WIPI2 and FIP200. Given that this fraction contains Atg5 and WIPI2 (Figure 1D) can the authors exclude the possibility that FIP200 is bound via the Atg complex which is bound to WIPI2 (as shown by Dooley et al., 2014) and not directly to the vesicle. In the wortmannin experiment there are small amounts of both WIPI2 and Atg5 in fraction 1, and WIPI2 association with membranes has been shown to be wortmannininsensitive (Polson et al., 2010). 9. In Figure 3 the authors should unify the colors in panel A and B. In panel A blue indicates no role but in panel B blue is used for positive colocalization. 10. Figure 4E, the authors show that in the Atg14 KO that flag-pis can immunoprecipitate FIP200 from fraction 4-7 with Rab1 and Sec61B suggesting this is the ER subdomain. The authors model would suggest that this region is devoid of Atg9. Is there Atg9 in this immunoprecipitate? 11. Regarding point 10, and other experiments, the authors should consider the enrichment of the European Molecular Biology Organization 3

4 proteins they study during the isolation of vesicles from the gradient fractions. Their model would be better supported if they could show an enrichment in the ER domain or isolation membrane over other membranes. This does not have to be experimentally addressed, just considered in the analysis. 12. The pathway and lipid analysis in Figures 3-5 is very well done, it provides interesting and significant new insights. 13. Discussion, on page 9, the authors do not conclusively show the ULK1 complex translocates to Atg9 vesicles to elongate the membrane and they should consider the data in mammalian cells not just yeast. Referee #3: Nishimura et al EMBO Journal This MS employs a powerful array of genetic, biochemical and imaging methods to tackle a central outstanding question in the autophagy field. That is, on which membranes is autophagy initiated and the autophagy initiation complex located? This is a controversial issue as a number of papers have appeared in high-profile journals claiming to have identified 'the site', but it seems almost every organelle in the cell is claimed to be the site at which autophagic membrane biogenesis occurs. Clearly, the issue is not resolved. In this MS, the authors collect and characterize biochemical fractions that satisfy several validation criteria for sites where autophagosome biogenesis is initiated. They biochemically resolve an ER-subdomain to which ULK complex is recruited and an ATG9Acontaining isolation membranes. They then use a variety of approaches to come to a pathway where the ULK complex is recruited in a PI3K and ATG9A-independent manner to a PIS-enriched ERsubdomain where autophagosome is nucleated. ATG9A-vesicles are then brought to this site to further expand the growing autophagosome. Finally, the ULK domain redistributes to foster autophagosome elongation. This MS makes unique inroads into the question by presenting a comprehensive analysis whose data hang together. Although one could quibble with issues of whether knockouts are accompanied by compensation mechanisms, or otherwise perturb the system so that key components are mislocalized, etc, it is the opinion of this referee that the work needs to appear on the scientific stage to contribute to debate on the subject. It has much to contribute. The following comments are suggested to the authors for strengthening this work. Comments: (a) The imaging experiments that demonstrate colocalization of FIP200 with PIS in an ER subdomain would benefit from either super-resolution imaging or immune-em. The confocal images shown, while of high quality, are not of the resolution where one can confidently assess the nature of the subdomain. (b) Balla and coworkers reported that an active Sar1 cycle is required for PIS to be recruited into the ER sub-domains they describe. Is this also true for formation of the ULK-containing ER subdomain? If so, perhaps this is one reason why ER exit sites are suggested (apparently erroneously) to be sites of autophagosome formation, and discussion to that effect would be warranted. (c) Balla and coworkers also reported that an active PIS could be recruited into the ER sub-domains they describe, but that a catalytic-dead PIS cannot. Is this also true for PIS localization to, or formation of, the ULK-containing ER subdomain? The catalytic dead PIS is used to target PI-PLC to the ER, and the data are interpreted as reflecting depletion of PI in that subdomain. But, unless I missed it, there was no demonstration that the CD-PIS targets to the ULK subdomain. This point needs to be addressed explicitly. (d) The authors report quantification of PI and a host of other lipid profiles under the activated ER- PI-PLC conditions. They do not look at PI3P, PI4P or PI4,5P2, however. In particular, how much is PI3P reduced under these conditions? Are other phosphoinositides similarly affected. European Molecular Biology Organization 4

5 (e) Does interference with PC or PS synthesis or stability in the ULK-subdomain have any influence on its stability, or is the effect truly specific for PI? Do PLD1 KO MEFs form the ULK subdomain? (f) Throughout the legends, the authors state that the experiments shown were successfully repeated 2- or 3-times. What does this mean? Two biological replicates is insufficient. Three would be a minimum. 1st Revision - authors' response 19 February 2017 We have made the following major modifications in response to Reviewers' comments: l As we have added a significant amount of data, we have included one new Figure (Fig 6) and divided the original Fig 2 and Fig 5 into two figures Figs 2 and 3, and Figs 7 and 8, respectively. Also, we have moved Fig S1H and Fig S2 to Fig EV2C and EV1H, respectively. Now, we have 8 Figures and 5 Expanded Views. l We have included additional data of membrane flotation analysis (Figs 1D, and EV2A and B) (Referee #2, Comment #2 and 5). l We have evaluated the enrichement of the ER and isolation membrane in the ATG9Aprecipitation experiments (Figs 2B-D and 3A-C) (Referee #2, Comment #11). l We have examined whether ATG9A is coprecipitated with PIS-enriched membrane (Fig 5E) (Referee #2, Comment #10). l We have analyzed the association of FIP200 with PIS-positive structures under starvation condition in more detailed by super-resolution structured illumination microscopy (Fig 6) (Referee #1, Comment #9) (Referee #3, Comment #a). l We have shown that PISHQ-GFP-PIPLC overexpression could reduce the total cellular PI3P level and added a relevant discussion (Fig 8F) (Referee #1, Comment #10) (Referee #3, Comment #d). l We have discussed the possibility that ATG9A vesicles may have roles other than becoming a seed membrane for autophagosome biogenesis. This point has been reflected in our model figure, in which free ATG9 vesicles are now illustrated (Fig 8H) (Referee #2, Comment #1, 13). Response to the comments of Editor As you will see, the referees have a mixed opinion on the suitability of your manuscript for publication here. I therefore initiated a consultation session to allow all referees to crosscomment on each other. The outcome of this - which is also reflected in the individual reports of the referees - is that the referees appreciate your approach and the identified role for PI and PIS. However, they also think that the membrane composition in the fractions and the changes induced by genetic or chemical manipulations needs much further insight and that alternative models for the sequences of events regarding FIP200 and Atg9 need to be considered throughout the manuscript. We have included additional data of the membrane fractions in Figures 1D and EV2A and B and new immunoprecipitation analysis in Figure 5E. We have also calculated co-immunoprecipitation efficiency in Figs 2B-D and 3A-C. Regarding the model how FIP200 and ATG9A function, we have added a discussion to clarify a potential discrepancy between the present and previous studies (Orsi et al, 2012; Lamb et al, 2016) (page 10, line 335). The previous studies reported by Dr. Tooze's lab suggest that ATG9A is mostly detected near the isolation membrane/phagophore, but is not contained in the isolation membrane or autophagosomal membranes by fluorescence microscopy, electron microscopy and membrane sedimentation assays. On the other hand, our membrane flotation and subsequent immunoprecipitation assay suggest that at least a part of ATG9A can be incorporated into the isolation membrane. This may be caused by a difference in the sensitivity of the purification methods. However, we think that these two possibilities are not mutually exclusive; our data do not necessarily rule out the possibility that most ATG9A vesicles only transiently localize to autophagosome formation sites. This point has been reflected in our model figure (free ATG9A vesicles are illustrated in Fig 8H). We have also expanded our discussion. European Molecular Biology Organization 5

6 Furthermore, the imaging data, lipid profile analyses and the PIS localization data need additional insight. We have added detailed data on the colocalization between FIP200 and PIS-positive structures using a super-resolution structured illumination microscope (SR-SIM) (Fig 6). We have also shown that the expression of PISHQ-GFP-PIPLC decreased the cellular PI3P level using an RFP-2xFYVE domain reporter (Fig 8F). References Lamb CA, Nuhlen S, Judith D, Frith D, Snijders AP, Behrends C, Tooze SA (2016) TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic. EMBO J 35: Orsi A, Razi M, Dooley HC, Robinson D, Weston AE, Collinson LM, Tooze SA (2012) Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol Biol Cell 23: Response to the comments of Reviewer #1 In this manuscript by Nishimura and colleagues, a biochemical assay was applied to characterize the autophagic precursor membrane, termed isolation membrane or phagophore, in terms of its relation to ER-membranes and its lipid composition. The whole study is based on floatation experiments in which membranes are separated according to their density. The authors investigated the distribution of ATG-proteins in gradient fractions based on western blots, focusing on subunits of the ULK-1 complex and ATG9. The authors used deletions to enrich autophagic precursor membranes, which are stalled at various stages (e.g. deletion of ATG3 to inhibit maturation). The authors found that FIP200 binds to ER-membranes independently of other members of the ULK-1 complex and that assembly of the complex occurs at ER-domains enriched in the lipid phosphatidylinositol. 1) The association of autophagic precursor membranes with the ER was intensively characterized in the past using various techniques including electron tomography for visualization, co-localization studies and biochemical characterizations. It was known that so called omegasomes, which are PI3P-enriched membrane domains of the ER, serve as nucleation sites for autophagosomes and that ATG-proteins are recruited to those sites in a hierarchical manner. Regarding those previous data, the insights obtained from this study are rather limited. Although it is known that early autophagic structures are formed on the omegasome, a PI3Penriched domain of the ER, in mammalian cells, how the ER membrane contributes to autophagy initiation remain largely unknown. In this study, we have identified the PIS-enriched ER domain as a site for the initiation of autophagosome formation (Figs 4-7). We have also shown that phosphatidylinositol on the PIS-enriched membrane is required for autophagosome formation (Fig 8) and the PIS-enriched domain could contain other phospholipid synthesis enzymes such as PSS1 and CEPT1 (Fig 4). Furthermore, we propose that the ULK complex is recruited to these PISenriched ER-related membranes and, at a later step, to ATG9A-containing isolation membranes (Figs 2 and 3). We believe that these are novel and valuable findings rather than simple confirmation of previous data. 2) Moreover, the entire study appears preliminary and lacks many important controls. I therefore do not recommend publication of this manuscript in the EMBO J. We have added data and appropriate controls to make our conclusion is more convincing. If important controls are still lacking, we can include them upon specific suggestions. 3) Fractions from gradient centrifugations where analyzed in terms of distribution of ATGproteins and it was claimed that phagophore membranes are enriched in certain fractions. One surprising observation is that upon starvation the profile of ATG-protein distribution in these fractions did not change. European Molecular Biology Organization 6

7 To address this issue, we have quantified the intensity of ATG proteins in each fraction in Figs 1A and EV1A and calculated their distribution (Figure 1 for Reviewers). The distribution of at least FIP200, ATG13, ATG101, and WIPI2 was shifted to lighter density fractions (Fr. 1, 2 and Fr. 5-7), in which the autophagosomal marker LC3-II was enriched under starvation conditions. These results suggest that intermediate autophagic membranes are floated into light density fractions under starvation conditions. However, these starvation-induced changes are modest compared to those observed in ATG-depleted cells because autophagic flux is maintained in wild-type cells. In the revised version, we have described that the distribution of at least FIP200, ATG13, ATG101, and WIPI2 was changed in response to starvation (p4, line 110). European Molecular Biology Organization 7

8 Figure 1 for Reviewers. Distribution of ATG proteins in Figs 1A and EV1A. The intensities of the bands were quantified using Image J software. Data are expressed as the percentage of total counts. European Molecular Biology Organization 8

9 4) Moreover, no other experimental methods were used (e.g. ultrastructural analysis) to confirm that the distribution of ATG-proteins correlate with enrichment of autophagic membranes. We thank the reviewer for this comment. However, we speculate that early autophagic membranes show vesicular structures without known morphological features. It would be difficult to identity structures before the isolation membrane. More extensive investigation would be required, which we believe would be a separate complete work. 5) Furthermore, many claims of the authors are not supported by data or are in odds with what was observed experimentally (e.g. " ATG proteins, except for membrane protein ATG9A and... LC3, mostly remained in the bottom cytosolic fraction", although a significant portion of FIP200 and ATG13 are found in floating fractions. We thank this reviewer for pointing this out. According to the reviewer s comment, we have modified these sentences to carefully explain the results. For example, in p4, line 108 Consistent with a previous report (Chan et al, 2009), small proportions of the ULK complex components were found in floated membrane fractions under growing conditions. 6) Moreover, against the author's interpretation, a significant offset in the distribution of ATG9 or LC3 (peak in fraction 5) and FIP200 or ATG13 (peak in fraction 6 and 7) was observed. The authors concluded from KO experiments that autophagic precursors are enriched in floating fractions without characterizing these fractions carefully. All fractions contain an ensemble of various membranes and deletion of ATG-proteins might also affect the composition of membranes in these fractions and not only the presence or absence of various autophagic precursors. We thank the reviewer for this comment. We agree that the peak distributions of ATG proteins are not completely identical under starvation conditions, which could reflect that all stages of autophagic structures co-exist in WT cells (Fig EV1A). A large portion of LC3 localizes to autophagosomes and autolysosomes, rather than early autophagic structures in WT cells. On the other hand, ATG9A localizes to not only autophagic structures but also other vesicular/tubular structures. To overcome this problem, we used ATG KO cells to synchronize the stage of autophagosome formation and found that early autophagic markers were consistently floated into light and middle density fractions (Fig 1C and D). We confirmed that the distribution to those floated fractions was dependent on the autophagic machinery by seeing the effect of acute inhibition of PI 3-kinase by wortmannin treatment (Fig 1E and F). To further overcome the potentially mixed nature of these fractions, we immune-purified autophagic membranes from those fractions using magnetic beads (Figs 2 and 3) and found that FIP200 was co-precipitated with the ER membrane and ATG9A-enriched membrane. It is interesting to test whether the composition or density of autophagic precursor membranes is changed by deletion of ATG genes, but it requires a lot of experiments and we believe that it is beyond the scope of this manuscript. 7) ULK-1 was found to be membrane associated even during vegetative growth previously (Chan et al, 2009), but the authors reported here that most of ULK-1 was cytosolic without commenting on this discrepancy. As we respond to Comment #5, we have modified the sentence and added a phrase explaining that a part of ULK1 was found in floated membrane fractions and cited the paper by Chan et al. (page 4, line 109). 8) Moreover, the difference in distribution profiles of ULK-1 and ATG13 or FIP200 is in conflict with what has previously been published by the authors of this study (i.e. ULK-1, ATG13, and FIP200 form a stable complex independently of nutrient status of cells). We previously reported that ULK1, ATG13, FIP200, and ATG101 form a stable complex in the cytosol (Hosokawa et al, 2009a; Hosokawa et al, 2009b). By contrast, we characterize the ULK1 complex in the membrane fraction in this study and found that behaviors of ATG13 and FIP200 are different from that of ULK1 as this reviewer points out. There are two possibilities. First, FIP200 and ATG13 could associate with the membrane more tightly than ULK1 and, therefore, ULK1 is European Molecular Biology Organization 9

10 released from the membranes during sample preparation and flotation experiments. Second, the stoichiometry of the complex subunits is different between the cytosolic and membrane complexes. We have added our interpretation in the revised manuscript (p11, line 368). 9) Finally, the authors depleted PI since they observed a strong co-localization of FIP200 and PI-synthase. The authors observed strong co-localization of FIP200 and PIS in starved wortmannin treated cells, but did not show colocalization in starved cells w/o WM-treatment. The latter co-localization was only performed with ULK-1. We thank the reviewer for this helpful comment. According to the reviewer's suggestion, we investigated the co-localization of FIP200 and PIS-GFP in starved cells without wortmannin treatment (new data in Fig 6A and C). Approximately 20% of FIP200 puncta almost perfectly colocalized with PIS-GFP and more than 50% of FIP200 puncta partially associated with PIS-GFPpositive structures. We have tried live-imaging of GFP-FIP200, but exogenous FIP200 had a tendency to form aggregated structures, which showed a localization pattern different from that of endogenous FIP200. Therefore, we used Venus-ULK1 for live-imaging analysis. 10) Futhermore, the authors depleted PI to investigate the effect on protein localization by expressing inducible PISHQ-GFP-PIPLC. The major problem with depleting PI is that PIPs are also depleted which are important regulators of membrane identity and vesicular trafficking. Thus depleting PI might also affect organelles and transport processes and the observed effects on autophagy are thus a result of disturbed overall cellular morphology. We thank this reviewer for this critical comment. According to the suggestion, we have tested whether the expression of PISHQ-GFP-PIPLC also affects intracellular localization of RFP- 2xFYVE, a PI3P probe that primarily labels endosomes. As a result, RFP-2xFYVE punctate structures were abolished after induction of PISHQ-GFP-PIPLC (new data in Fig 8F). In line with this, previous reports showed that an ER-anchored PIPLC interferes with homeostasis of other PIPs, such as PI4P and PI4,5P 2 (Kim et al, 2011; Chang & Liou, 2015). These results suggest that forced expression of PIPLC at the ER membrane affects the total cellular PIPs levels and that the PI derived from ER membrane is critical not only for autophagy but also other cellular events. Thus, our previous conclusion that PI produced at the PIS domain is important for autophagy is still valid. However, we agree that it is difficult to evaluate the importance of the PI pool in the ER membrane in autophagy and other cellular events separately. Given this situation, we have added sentences to state that we could not rule out a possibility that PI depletion affects autophagosome formation through indirect effects in Discussion (page 12, line 398). References Chan EY, Longatti A, McKnight NC, Tooze SA (2009) Kinase-inactivated ULK proteins inhibit autophagy via their conserved C-terminal domains using an Atg13-independent mechanism. Mol Cell Biol 29: Chang CL, Liou J (2015) Phosphatidylinositol 4,5-Bisphosphate Homeostasis Regulated by Nir2 and Nir3 Proteins at Endoplasmic Reticulum-Plasma Membrane Junctions. J Biol Chem 290: Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, Iemura S, Natsume T, Takehana K, Yamada N, Guan JL, Oshiro N, Mizushima N (2009a) Nutrient-dependent mtorc1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell 20: Hosokawa N, Sasaki T, Iemura S, Natsume T, Hara T, Mizushima N (2009b) Atg101, a novel mammalian autophagy protein interacting with Atg13. Autophagy 5: Kim YJ, Guzman-Hernandez ML, Balla T (2011) A highly dynamic ER-derived phosphatidylinositol-synthesizing organelle supplies phosphoinositides to cellular membranes. Dev Cell 21: Lamb CA, Nuhlen S, Judith D, Frith D, Snijders AP, Behrends C, Tooze SA (2016) TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic. EMBO J 35: European Molecular Biology Organization 10

11 Orsi A, Razi M, Dooley HC, Robinson D, Weston AE, Collinson LM, Tooze SA (2012) Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol Biol Cell 23: Response to the comments of Reviewer #2 This manuscript provides data that contributes to the increasing evidence that autophagosome formation occurs at sub-domains of the ER. Nishimura and colleagues have investigated the nature and composition of these ER subdomains with a powerful biochemical approach that combines subcellular fractionation, isolation of vesicles, and lipid analysis. Furthermore, they provide data on the lipid biosynthetic enzymes, which produce and hydrolyse PI suggesting this is metabolic function occurs at this ER subdomain. They identify membranes in both WT and AtgKO MEFs that contain subsets of Atg proteins. These membranes have distinct characteristic allowing them to be separated on gradients. By analysing focusing on the ULK complex member FIP200, Atg9, WIPI2 and ER components they develop a hypothesis that the ULK complex is recruited to an ER subdomain which provides a platform to form isolation membranes from Atg9-positive membranes. Overall the data is technically excellent, well controlled and address the aim of the study, and is an important contribution to the autophagy field and others interested in the role of lipids. We thank this reviewer for the positive assessment of our manuscript. Specific points: 1. The main issue is the final interpretation of the data in particular the conclusion that transfer of FIP200 to an Atg9-positive vesicle creates the isolation membrane. This conclusion is flawed as the authors only consider the data in yeast on Atg9, to inform them in the interpretation of the experiments, and the formulation of the final model. In particular, Atg9 in mammalian cells has been shown to dynamically associate with early membranes. Indeed in Fig. S2 the authors also show the WIPI2/FIP200 positive puncta have very low amounts of Atg9. The data and model should be reconciled with the mammalian data, in particular Orsi et al., 2012, and Lamb et al., We have added a discussion to clarify a potential discrepancy between the present and previous studies (Orsi et al, 2012; Lamb et al, 2016). The previous studies reported by Dr. Tooze's lab showed that ATG9A dynamically associates with the isolation membrane/phagophore, but is not contained in the isolation membrane or autophagosomal membranes by fluorescence microscopy, electron microscopy, and membrane sedimentation assays. On the other hand, our membrane flotation and subsequent immunoprecipitation assay suggest that at least a part of ATG9A can be incorporated into the isolation membrane. However, these two possibilities are not mutually exclusive; our data do not necessarily rule out the possibility that most ATG9A vesicles only transiently localize to autophagosome formation sites. We have added this discussion, cited these two related papers (page 6, line 168) (page 10, line 335) and modified our model in Fig 8H. 2. Figure 1D. Is Atg14 part of the Beclin1 complex? Is Vps34 or Vps15 found in these fractions? We thank this reviewer for this suggestion. During our experiments using ATG14 KO cells, we realized that the strongest band that reacts to our ATG14 antibody is a non-specific band and a weaker band at a lower position represents true ATG14 (indicated by an arrow in Fig 1D). This band was detected in both the top and middle fractions together with VPS34 and BECLIN1 in ATG3 KO cells. We have replaced the previous blots with the new one (new data in Fig 1D). We thank this reviewer for letting us realize the mistake. 3. There are slight differences in the distribution of Flag-Atg9 and endogenous Atg9 in Fig. 1D and E. There is less flag-atg9 in the top fractions in the Atg3KO although this is proposed to be the expanding isolation membrane. European Molecular Biology Organization 11

12 Because of overexpression, FLAG-ATG9A is mostly present in the perinuclear region (likely the Golgi and/or recycling endosomes), whereas endogenous ATG9A is mainly present on small vesicles throughout the cytoplasm in ATG3 KO MEFs (Figure 2 for Reviewers). This could cause a difference in their distribution in the flotation analysis. The ATG9A population in the middle fractions is increased rather than that in the top fractions is decreased. Figure 2 for Reviewers. Overexpressed FLAG-ATG9A accumulates in the perinuclear region. ATG3 KO MEFs or ATG3 KO MEFs stably expressing FLAG-ATG9A were cultured in regular DMEM. The cells were fixed and stained with anti-atg9a and anti-flag antibodies. Yellow arrows indicate the perinuclear localization of overexpressed FLAG-ATG9A. Scale bar, 10 µm. 4. Page 6, end of 1st paragraph. Orsi et al also showed the ULK1 puncta form in Atg9-/- MEFs. We have cited this previous observation (page 6, line 186). 5. The isolation of Atg9 vesicles was done from gradient fractions obtained from gradients which are not included in the data. The authors should add subcellular fractionation analysis for the WT/Flag-Atg9 cells and Atg14KO/Flag Atg9 both under growing conditions. We thank the reviewer for this suggestion. We have added these results of subcellular fractionation analysis, which were used for subsequent isolation experiments (new data in Fig EV2A and B). 6. Figure 2B-G the authors should clarify if the gradients were all prepared with lysates from growing cells. These were from growing cells. We have added this information in the legends to Fig 2 (page 20, line 676) and Fig 3 (page 21, line 695). 7. In Figure 2E, the population of flag-atg9 found in the gradient shown in Fig. 1E is a very minor population (perhaps less than 10%) of the total flag-atg9 and is distributed differently from the endogenous shown in Fig. 1F, suggesting the tagged transfected Atg9 may be accumulating in the ER due to saturation of the export machinery. This would result in the European Molecular Biology Organization 12

13 large amounts of Atg9 seen in the middle of the gradient. The authors use the fraction 1 pool for Fig. 2E which is a crucial experiment for the model of recruitment of the FIP200 complex to the Atg9-positive compartment. Do these membranes in fraction 1 with Flag-Atg9 have Atg14? Can the authors demonstrate that the flag-atg9 exits the ER and traffics normally during fed and starved conditions? We thank the reviewer for this comment. To address this issue, we have analyzed intracellular localization of FLAG-ATG9A. As shown above (see comment #3), FLAG-ATG9A is localized to the perinuclear region (likely the Golgi and/or recycling endosomes) and vesicular structures under growing condition. However, an ER-like reticular pattern was not clearly observed, indicating that FLAG-ATG9A is not accumulating in the ER. In addition, we have investigated FLAG-ATG9A trafficking to the autophagosome formation site. Wortmannin treatment under starvation condition induces accumulation of FLAG-ATG9A with FIP200 (Figure 3 for Reviewers). This result suggests that FLAG-ATG9A is normally transported to the autophagosome formation site in response to autophagy induction. Figure 3 for Reviewers. Inhibition of PI 3-kinase causes accumulation of ATG9A at FIP200- positive autophagosome formation site. ATG3 KO MEFs stably expressing FLAG-ATG9A were cultured in starvation medium in the presence of wortmannin for 1 h. The cells were fixed and stained with anti-fip200 and anti-flag antibodies. Scale bar, 10 µm. 8. In Figure 2H, derived from the data in Figure 1 and 2, the final membrane compartment diagrammed "Atg3 KO fr. 1" shows Atg9 vesicle with WIPI2 and FIP200. Given that this fraction contains Atg5 and WIPI2 (Figure 1D) can the authors exclude the possibility that FIP200 is bound via the Atg complex which is bound to WIPI2 (as shown by Dooley et al., 2014) and not directly to the vesicle. In the wortmannin experiment there are small amounts of both WIPI2 and Atg5 in fraction 1, and WIPI2 association with membranes has been shown to be wortmannin-insensitive (Polson et al., 2010). We thank this reviewer for raising this possibility. We agree that we cannot exclude the possibility that a small portion of FIP200 is recruited to the membranes via its interaction with ATG12 ATG5 ATG16L1 and the wortmannin-insensitive population of WIPI2. We have added this discussion in the revised manuscript (p11, line 350). 9. In Figure 3 the authors should unify the colors in panel A and B. In panel A blue indicates no role but in panel B blue is used for positive colocalization. We thank the reviewer for the helpful comment. We have changed the colors in the graph in Fig 4B to be consistent with Fig 4A. European Molecular Biology Organization 13

14 10. Figure 4E, the authors show that in the Atg14 KO that flag-pis can immunoprecipitate FIP200 from fraction 4-7 with Rab1 and Sec61B suggesting this is the ER subdomain. The authors model would suggest that this region is devoid of Atg9. Is there Atg9 in this immunoprecipitate? We thank the reviewer for this critical comment. We investigated whether ATG9A was coprecipitated with PIS-enriched membranes purified from Fractions 4-7 of ATG14 KO MEFs. Consistent with our model (Fig 3D), FIP200 and SEC61B, but not ATG9A, were clearly coprecipitated with PIS-containing membranes (new data in Fig 5E). These results support our conclusion that FIP200 is recruited to PIS-enriched ER membranes, which are devoid of ATG9A at the initiation stage of autophagy. 11. Regarding point 10, and other experiments, the authors should consider the enrichment of the proteins they study during the isolation of vesicles from the gradient fractions. Their model would be better supported if they could show an enrichment in the ER domain or isolation membrane over other membranes. This does not have to be experimentally addressed, just considered in the analysis. We thank the reviewer for this suggestion. We have measured the intensities of the bands both in the input and IP fractions and estimated enrichment of the ER and isolation membrane following immunoprecipitation. The quantification data show the enrichment of FIP200 in the FLAG-ATG9A precipitates from ATG3 KO homogenates (Fig 3A) and the enrichment of the ER marker SEC61B in the FLAG-ATG9A precipitates from middle fractions of ATG3 KO homogenates, but not of WT and ATG14 homogenates (Fig 2B and C, and Fig 3B and C). These results indicate that the ER membrane and isolation membrane were efficiently enriched by immunoprecipitation with FLAG- ATG9A in ATG3 KO homogenates, which support the model shown in Fig 3D. 12. The pathway and lipid analysis in Figures 3-5 is very well done, it provides interesting and significant new insights. We would like to appreciate this positive comment. 13. Discussion, on page 9, the authors do not conclusively show the ULK1 complex translocates to Atg9 vesicles to elongate the membrane and they should consider the data in mammalian cells not just yeast. As we have responded to Comment #1, we have added more discussion regarding the potential discrepancy with the previous studies. This point has also been reflected in our revised model (Fig. 8H). References Lamb CA, Nuhlen S, Judith D, Frith D, Snijders AP, Behrends C, Tooze SA (2016) TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic. EMBO J 35: Orsi A, Razi M, Dooley HC, Robinson D, Weston AE, Collinson LM, Tooze SA (2012) Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol Biol Cell 23: Response to the comments of Reviewer #3 This MS employs a powerful array of genetic, biochemical and imaging methods to tackle a central outstanding question in the autophagy field. That is, on which membranes is autophagy initiated and the autophagy initiation complex located? This is a controversial issue as a number of papers have appeared in high-profile journals claiming to have identified 'the site', but it seems almost every organelle in the cell is claimed to be the site at which autophagic membrane biogenesis occurs. Clearly, the issue is not resolved. In this MS, the authors collect and characterize biochemical fractions that satisfy several validation criteria European Molecular Biology Organization 14

15 for sites where autophagosome biogenesis is initiated. They biochemically resolve an ERsubdomain to which ULK complex is recruited and an ATG9A-containing isolation membranes. They then use a variety of approaches to come to a pathway where the ULK complex is recruited in a PI3K and ATG9A-independent manner to a PIS-enriched ERsubdomain where autophagosome is nucleated. ATG9A-vesicles are then brought to this site to further expand the growing autophagosome. Finally, the ULK domain redistributes to foster autophagosome elongation. This MS makes unique inroads into the question by presenting a comprehensive analysis whose data hang together. Although one could quibble with issues of whether knockouts are accompanied by compensation mechanisms, or otherwise perturb the system so that key components are mis-localized, etc, it is the opinion of this referee that the work needs to appear on the scientific stage to contribute to debate on the subject. It has much to contribute. The following comments are suggested to the authors for strengthening this work. We thank this reviewer for the positive assessment of our manuscript. Comments: (a) The imaging experiments that demonstrate colocalization of FIP200 with PIS in an ER subdomain would benefit from either super-resolution imaging or immune-em. The confocal images shown, while of high quality, are not of the resolution where one can confidently assess the nature of the subdomain. We thank the reviewer for the helpful comment. According to the reviewer's suggestion, we have analyzed colocalization of FIP200 with PIS-GFP using a super-resolution structured illumination microscope. In starved cells, approximately 20% of FIP200 puncta almost perfectly colocalized with PIS-GFP and more than 50% of FIP200 puncta partially associated with PIS-GFP-positive structures (new data in Fig 6A and C). These results indicate that FIP200 colocalizes with PIS-GFP even under wortmannin-untreated condition. By contrast, most of FIP200 well colocalized with PIS-GFP puncta in wortmannin-treated starved cells (new data in Fig 6B and C), which is consistent with our previous observation by conventional confocal microscopy (Fig 5A and B). Collectively, these results support our conclusion that FIP200 is recruited to the PIS-enriched ER subdomains at the initiation stage of autophagy. b) Balla and coworkers reported that an active Sar1 cycle is required for PIS to be recruited into the ER sub-domains they describe. Is this also true for formation of the ULK-containing ER subdomain? If so, perhaps this is one reason why ER exit sites are suggested (apparently erroneously) to be sites of autophagosome formation, and discussion to that effect would be warranted. As the reviewer pointed out, Sar1 activity is required for both the generation of PIS puncta (Kim et al, 2011) and autophagosome formation (Zoppino et al, 2010; Ge et al, 2013). During preparation of this manuscript, it was reported that the drug H89, which blocks recruitment of Sar1 to the ER membrane, reduces the number of ATG13 puncta under starvation conditions (Karanasios et al, 2016), indicating that inhibition of Sar1 cycle affects the formation of ULK puncta. Although it is possible that inhibition of Sar1-depdendent formation of the PIS domain impairs autophagy, this is merely a correlation, not a causal relationship. Therefore, we would prefer not to specifically mention this point in this manuscript. (c) Balla and coworkers also reported that an active PIS could be recruited into the ER subdomains they describe, but that a catalytic-dead PIS cannot. Is this also true for PIS localization to, or formation of, the ULK-containing ER subdomain? The catalytic dead PIS is used to target PI-PLC to the ER, and the data are interpreted as reflecting depletion of PI in that subdomain. But, unless I missed it, there was no demonstration that the CD-PIS targets to the ULK subdomain. This point needs to be addressed explicitly. We prepared two different catalytic-dead PIS mutants, H105Q and H105Y, and analyzed their localization. Consistent with the previous report, the PIS H105Y mutant did not clearly target to the ER subdomain. In contrast, the PIS H105Q mutant still showed punctate structures and localized to European Molecular Biology Organization 15

16 ER subdomain, though the number of PIS puncta was reduced (Figure 4 for Reviewers). Therefore, we used the H105Q mutant for targeting PI-PLC to the PIS-enriched ER subdomains. As we have already shown, PISH105Q-GFP-PIPLC can indeed colocalize with FIP200 (Fig 8C). Figure 4 for Reviewers. PIS H105Q-GFP, but not PIS H105Y-GFP, forms punctate structures. WT MEFs stably expressing PIS-GFP, PIS H105Q-GFP or PIS H105Y-GFP were cultured in regular DMEM. The cells were fixed and analyzed by confocal microscopy. Scale bar, 10 µm. (d) The authors report quantification of PI and a host of other lipid profiles under the activated ER-PI-PLC conditions. They do not look at PI3P, PI4P or PI4,5P2, however. In particular, how much is PI3P reduced under these conditions? Are other phosphoinositides similarly affected. We thank this reviewer for this critical comment. According to the suggestion, we have tested whether the expression of PISHQ-GFP-PIPLC also affects intracellular localization of RFP- 2xFYVE, a PI3P probe that primarily labels endosomes. As a result, RFP-2xFYVE punctate structures were abolished after induction of PISHQ-GFP-PIPLC (new data in Fig 8F). In line with this, previous reports showed that an ER-anchored PIPLC interferes with homeostasis of other PIPs, such as PI4P and PI4,5P 2 (Kim et al, 2011; Chang & Liou, 2015). These results suggest that forced expression of PIPLC at the ER membrane affects the total cellular PIPs levels and that the PI derived from ER membrane is critical not only for autophagy but also other cellular events. Thus, our previous conclusion that PI produced at the PIS domain is important for autophagy is still valid. However, we agree that it is difficult to evaluate the importance of the PI pool in the ER membrane in autophagy and other cellular events separately. Given this situation, we have added sentences to state that we could not rule out a possibility that PI depletion affects autophagosome formation through indirect effects in Discussion (page 12, line 398). (e) Does interference with PC or PS synthesis or stability in the ULK-subdomain have any influence on its stability, or is the effect truly specific for PI? Do PLD1 KO MEFs form the ULK subdomain? Regarding the involvement of other lipid synthesis enzymes or lipases that accumulate at the autophagosome formation sites, some were already reported and some were tested by our hands. It was reported that CPT1 positively regulates autophagosome formation under oleic acid-treated conditions (Dupont et al, 2014). The role for PLD1 is rather controversial; autophagic flux is reduced in PLD1 KO MEFs (Dall'Armi et al, 2010); Pharmacological inhibition by PLD inhibitors (VU , Cay10594) interferes with autophagic flux and clearance of autophagic substrates (Bae et al, 2014; Holland et al, 2016); By contrast, autophagic flux was increased in sipld1-treated HEK293 or HeLa cells via mtor pathway inhibition (Jang et al, 2014). Finally, we have examined the effect of double knockdown of PSS1 and PSS2 and found no significant inhibitory effect on European Molecular Biology Organization 16

17 autophagic flux and FIP200 puncta formation (Figure 5 for Reviewers). However, compared to the PI synthesis pathway, the pathways for PC, PS, and PA synthesis have salvage pathways and are closely connected with each other, which makes it difficult to assess their individual contribution to autophagy. In addition, so far there is no proper method to measure levels of individual phospholipids in autophagic membranes. Therefore, we do not include these results in this manuscript and keep them for a future study. We have discussed these previous findings by citing these papers (page 12, line 386). Figure 5 for Reviewers. Double knockdown of PSS1 and PSS2 shows no effect on autophagy. (A-C) HeLa cells were transfected with the indicated sir (Invitrogen) twice. (A) Total R was extracted using Isogen and reverse-transcribed using ReverTraAce. PCR reactions were performed European Molecular Biology Organization 17

18 with STBR Premix Ex Taq. The ΔΔCT method was employed to determine relative gene level differences with GAPDH qpcr products used as a control (n=3). (B) Autophagic flux assay. The cells were cultured in regular DMEM or starvation medium in the presence or absence of 100 nm bafilomycin A 1 (BafA 1 ) for 2 h. (C) The cells were cultured in starvation medium for 1 h, fixed and stained with anti-lc3 and anti-fip200 antibodies. Scale bar, 10 µm. (f) Throughout the legends, the authors state that the experiments shown were successfully repeated 2- or 3-times. What does this mean? Two biological replicates is insufficient. Three would be a minimum. We have obtained data with more than triplicates for statistical analysis. In some of the qualitative analyses, we have confirmed the results by repeating the same experiments. In these cases, we have done the experiments twice. We believe that not all experiments should be performed more than twice if the results are clear and reproducible. We have expanded the Data Analysis section (page 18, line 607). References Bae EJ, Lee HJ, Jang YH, Michael S, Masliah E, Min DS, Lee SJ (2014) Phospholipase D1 regulates autophagic flux and clearance of alpha-synuclein aggregates. Cell Death Differ 21: Chang CL, Liou J (2015) Phosphatidylinositol 4,5-Bisphosphate Homeostasis Regulated by Nir2 and Nir3 Proteins at Endoplasmic Reticulum-Plasma Membrane Junctions. J Biol Chem 290: Dall'Armi C, Hurtado-Lorenzo A, Tian H, Morel E, Nezu A, Chan RB, Yu WH, Robinson KS, Yeku O, Small SA, Duff K, Frohman MA, Wenk MR, Yamamoto A, Di Paolo G (2010) The phospholipase D1 pathway modulates macroautophagy. Nat Commun 1: 142 Dupont N, Chauhan S, Arko-Mensah J, Castillo EF, Masedunskas A, Weigert R, Robenek H, Proikas-Cezanne T, Deretic V (2014) Neutral lipid stores and lipase PNPLA5 contribute to autophagosome biogenesis. Curr Biol 24: Ge L, Melville D, Zhang M, Schekman R (2013) The ER-Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis. Elife 2: e00947 Holland P, Knaevelsrud H, Soreng K, Mathai BJ, Lystad AH, Pankiv S, Bjorndal GT, Schultz SW, Lobert VH, Chan RB, Zhou B, Liestol K, Carlsson SR, Melia TJ, Di Paolo G, Simonsen A (2016) HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels. Nat Commun 7: Jang YH, Choi KY, Min DS (2014) Phospholipase D-mediated autophagic regulation is a potential target for cancer therapy. Cell Death Differ 21: Karanasios E, Walker SA, Okkenhaug H, Manifava M, Hummel E, Zimmermann H, Ahmed Q, Domart MC, Collinson L, Ktistakis NT (2016) Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles. Nat Commun 7: Kim YJ, Guzman-Hernandez ML, Balla T (2011) A highly dynamic ER-derived phosphatidylinositol-synthesizing organelle supplies phosphoinositides to cellular membranes. Dev Cell 21: Lamb CA, Nuhlen S, Judith D, Frith D, Snijders AP, Behrends C, Tooze SA (2016) TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic. EMBO J 35: Orsi A, Razi M, Dooley HC, Robinson D, Weston AE, Collinson LM, Tooze SA (2012) Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol Biol Cell 23: European Molecular Biology Organization 18

19 Zoppino FC, Militello RD, Slavin I, Alvarez C, Colombo MI (2010) Autophagosome formation depends on the small GTPase Rab1 and functional ER exit sites. Traffic 11: nd Editorial Decision 10 March 2017 Thank you for submitting your manuscript for consideration by the EMBO Journal. It has now been seen by the three original referees again whose comments are shown below. As you will see, referee #1 still raises issues regarding the fractionation experiments. I asked the other referees to comment on these remaining issues (see additional comments of referee #2 below), and the consensus was to ask you to address them. I would thus like to invite you to submit a revised version of the manuscript, addressing the remaining issues REFEREE COMMENTS Referee #1: The authors of the manuscript made a strong attempt to revise their manuscript by including a substantial amount of new data. This attempt is appreciated. From the points raised below, it will become apparent that the part concerning separation of early autophagic precursors and isolation membranes by floatation is still not convincing. Many important control experiments are missing and statements of the authors are still in odds with the presented data. Thus, publication of this study is still not recommended by this reviewer. 1) The authors identified a shift in the distribution of FIP200, ATG13, ATG101, and ULK1 to middle density fractions in ATG14KO MEFs. The authors concluded that in these fractions autophagic precursor membranes are enriched. However, there is also a strong enrichment of FIP200, ATG101, ULK1, and ATG9A in light density fractions. A similar enrichment is seen in starved cells. Thus, the conclusion of the authors that middle density fractions contain autophagic precursor membranes is in conflict with their results. 2) The treatment with wortmannin markedly decreases the amount of ULK-1 kinase complex components in the top floating fractions. However, this drug is inhibiting many different cellular functions including endocytosis, cell-signaling cascades, D repair ect. This might explain why there is an even stronger decrease of top-floating proteins in WM-treated cells compared to ATG14 KO cells. Since no control experiment of starved wt-cells treated with wortmannin is presented, conclusions drawn from these experiments are limited. The combination of ATG3KO and wortmannin treatment under non-starved conditions is also not convincing without presenting adequate control experiments. 3) The only control experiment using starved cells is presented in Fig. EV1 with Atg9A being overexpressed. The most obvious control experiment which would show the unperturbed distribution of ATG-proteins in fed and starved cells is still missing. Taking points 1 to 3 into account, it appears that light AND middle density fractions contain mixtures of autophagic precursor membranes and elongating phagophors, arguing that the enrichment of a pure population of autophagic membranes in certain fractions is not possible. 4) The authors state that "FIP200 was not detected in these FLAG-ATG9A immunoprecipitates from the middle-density fractions of WT..." cells. However, the corresponding Fig. 2A shows a much weaker input band of FIP200 compared to WT-MEFs (without FLAG-tagged ATG9). This presents a strong limitation of the experiment and it is therefore not possible to compare wt with ATG14 KO experiment, in which the input of FIP200 is much stronger. The presented data are thus not convincing and did not support the conclusion drawn by the authors. Along these lines, Fig. 2D shows a FIP200 signal in the control and in the GFP-SEC61B sample. Although this could represent an unspecific band, it could also be related to posttranslational European Molecular Biology Organization 19

20 modifications in FIP200. The documented difference in IP-efficiencies is therefore unclear to this reviewer. 5) The authors stated that "Although FIP200 and Atg9A were not co-purified in the middle density fraction of Atg14KO homogenate, FIP200 and WIPI2, another isolation membrane protein, copurified with FLAG-Atg9A from the top fraction of Atg3 KO homogenates". From Fig. 1 it is apparent that FIP200 is also enriched in the top fraction of ATG14KO cells. Here, the authors only compare top (ATG3 KO) and middle (ATG14 KO) fractions without showing the corresponding top and middle fractions. Without the full set of data, it is not possible to draw conclusions. Moreover, there is still no complementary technique that independently confirms the conclusion that different types of autophagic membranes are enriched in specific fractions and the presented biochemical approach presented here still suffers from the mentioned limitations. Referee #2: The authors have appropriately revised the manuscript to address my comments and revision requests. Referee #3: The authors have positively addressed the bulk of the reviewers' many comments/criticisms and the revised version is now a stronger MS. one of the points of this MS that this referee likes is that the revised MS is not shy about laying out what remains unresolved. This is important. While the fact that PI-depletion in the ER has the expected pleiotropic depleting effects on PI3P (and likely other PIP) levels, and that this makes the effects less direct, the MS still has its share of strengths that will draw high interest. One point that requires some modification is that the authors overstate the case being made for actual transport of lipids through contact sites (lines ). There is no clear evidence to demonstrate this as other models are actually gaining more traction. Please soften that statement. Referee #2's comments on referee #1: I don't agree entirely with point 1. The authors define the middle and top fraction as two different types of early autophagosomes membranes (autophagosome precursors and isolation membranes). This is pretty clear in the text. One could argue that this is all semantics and essentially the membranes are the same and therefore are all autophagosome precursors. I agree that these are not purified fractions as the referee points out but I didn't think the authors claimed this. Regarding the other points- they are all valid and they can be addressed. It might be the lab has this data and this would be a good solution? Figure 2B is not good in particular. In fact the IP's in Figure 2B-D are not well controlled (no beads only control) but I missed this the first time around. The final point is correct-there is no independent complementary approach but the referee doesn't suggest one. Its easy to say this. The only ones I could think of would be an ontogenic approach, or some similar approach. Honestly my opinion of the fractionation data was moderated by the nice lipid analysis. These are very good experiments. 2nd Revision - authors' response 28 March 2017 We have made the following major modifications in response to Editor's and Reviewers' comments: European Molecular Biology Organization 20

21 l We have added a long-exposed blot of FIP200 in immunoprecipitation analysis (Figure 2B) (Reviewer #1, Comment #4) (Reviewer #2, Additional comment). l We have softened the statement about the lipid transport model in the discussion part (page 12, line 403, ) (Reviewer #3, Comment). l We have improved the resolution of the movies (Movies EV1-3) (Editor s comment). l We have removed unneeded legends from the main text and figure files (Editor s comment). Response to the comments of Editor As you will see, referee #1 still raises issues regarding the fractionation experiments. I asked the other referees to comment on these remaining issues (see additional comments of referee #2 below), and the consensus was to ask you to address them. I would thus like to invite you to submit a revised version of the manuscript, addressing the remaining issues. We appreciate your decision after consultation with Reviewers. As outlined below, we have addressed all the remaining issues, including those from referee #1. We have added a long-exposed blot of FIP200 in immunoprecipitation analysis (Fig 2B) and softened the statement about the lipid transport model (page 12, line 403, ). In addition, we show additional data of membrane flotation analysis in this letter, but as these new results are redundant, we did not add them to the main figures and Expanded Views. European Molecular Biology Organization 21

22 Response to the comments of Reviewer #1 The authors of the manuscript made a strong attempt to revise their manuscript by including a substantial amount of new data. This attempt is appreciated. From the points raised below, it will become apparent that the part concerning separation of early autophagic precursors and isolation membranes by floatation is still not convincing. Many important control experiments are missing and statements of the authors are still in odds with the presented data. Thus, publication of this study is still not recommended by this reviewer. 1) The authors identified a shift in the distribution of FIP200, ATG13, ATG101, and ULK1 to middle density fractions in ATG14KO MEFs. The authors concluded that in these fractions autophagic precursor membranes are enriched. However, there is also a strong enrichment of FIP200, ATG101, ULK1, and ATG9A in light density fractions. A similar enrichment is seen in starved cells. Thus, the conclusion of the authors that middle density fractions contain autophagic precursor membranes is in conflict with their results. This comment is not very clear for us, which we think is also reflected by the supportive comments from Reviewer #2. In WT cells, FIP200, ATG13, ATG101, and WIPI2 were floated into lightdensity (top) fractions under starvation condition. As autophagic flux is maintained in WT cells, autophagic structures at several different stages are distributed into different fractions. Therefore, autophagic markers are floated broadly and do not show strong enrichment in specific fractions. In contrast, autophagic structures at specific stages accumulate in ATG KO cells because autophagosome formation is blocked at specific stages. It is clear that FIP200, ATG13, ATG101, and ULK1 accumulate more in middle-density fractions than in light-density fraction in ATG14 KO cells. Our main purpose of these experiments is to analyze autophagic membranes of these major populations. We understand that, as pointed out by this Reviewer, a part of autophagic markers are floated into the light-density (top) fractions of ATG14 KO cells (Fig 1C), suggesting that these fractions may also contain a smaller amount of autophagic precursor membranes. However, we think that the most straightforward approach is to investigate the major population rather than the minor population. In fact, we show that the top fraction rather contains isolation membranes, which are at a more advanced stage. We believe that the comments from Reviewer #2 to this issue should also clarify this point. 2) The treatment with wortmannin markedly decreases the amount of ULK-1 kinase complex components in the top floating fractions. However, this drug is inhibiting many different cellular functions including endocytosis, cell-signaling cascades, D repair ect. This might explain why there is an even stronger decrease of top-floating proteins in WM-treated cells compared to ATG14 KO cells. Since no control experiment of starved wt-cells treated with wortmannin is presented, conclusions drawn from these experiments are limited. The combination of ATG3KO and wortmannin treatment under non-starved conditions is also not convincing without presenting adequate control experiments. As suggested by the reviewer, we have examined the effect of wortmannin under starvation conditions in WT MEF cells, but did not observe a clear change in the amount of ULK1 kinase complex components in the top fractions compared to non-treated cells (Figure 1A for Reviewers). It suggests that the flotation of a small population of these proteins is PI3P-independent and perhaps represent a kind of background signals. We do not think that there is a significant difference in the top fraction between wortmannin-treated cells and ATG14 KO cells. We understand that any drugs have potential side effects. This reviewer suggests including wild-type controls, but we think that even it cannot validate the specificity of wortmannin. What we would like to do here is to test whether the light-density membranes accumulating in the top fractions of ATG3 KO cells indeed represent isolation membranes by seeing the effect of wortmannin, which is already known to inhibit the formation of isolation membranes. To further support our conclusion, we have performed a flotation analysis using ATG5 KO cells, in which isolation membranes are known to accumulate. Consistent with the results using ATG3 KO cells, the ULK1 complex components and WIPI2 were floated into the top fractions in ATG5 KO cells (Figure 1B for Reviewers, **). We think that all these results are sufficient to conclude that isolation membranes are enriched in the top fractions. As these data are rather redundant and the space is limited, we would like not to include these data in our manuscript. European Molecular Biology Organization 22

23 Figure 1 for Reviewers. Flotation of ATG proteins derived from starvation/wortmannin-treated WT and non-treated ATG5 KO cell homogenates. (A) WT MEFs were cultured in starvation medium in the presence of 200 nm wortmannin (Starv. + WM) for 2 h and subjected to Optiprep flotation analysis. (B) ATG5 KO MEFs were cultured in regular DMEM and subjected to Optiprep flotation analysis. Asterisks indicate the flotation of Atg proteins. 3) The only control experiment using starved cells is presented in Fig. EV1 with Atg9A being overexpressed. The most obvious control experiment which would show the unperturbed distribution of ATG-proteins in fed and starved cells is still missing. We have already shown that overexpression of FLAG-ATG9A does not affect the distribution of ATG proteins in the flotation assay (please compare Fig 1A with EV2A, Fig 1C with EV2B, and Fig 1D with 1E). In addition, we performed the flotation and immunoprecipitation assays under growing conditions (only the exceptions are Fig 1F and Fig EV1A). We think that the experiments suggested by the reviewer would not be essential control experiments. Taking points 1 to 3 into account, it appears that light AND middle density fractions contain mixtures of autophagic precursor membranes and elongating phagophors, arguing that the enrichment of a pure population of autophagic membranes in certain fractions is not possible. As pointed out by Reviewer #2, we do not claim that a complete pure population is enriched in each fraction. We analyzed major populations accumulating in ATG KO cells. Even if there are some small contaminations, we believe that our conclusion is still valid. 4) The authors state that "FIP200 was not detected in these FLAG-ATG9A immunoprecipitates from the middle-density fractions of WT..." cells. However, the corresponding Fig. 2A shows a much weaker input band of FIP200 compared to WT-MEFs (without FLAG-tagged ATG9). This presents a strong limitation of the experiment and it is therefore not possible to compare wt with ATG14 KO experiment, in which the input of FIP200 is much stronger. The presented data are thus not convincing and did not support the conclusion drawn by the authors. Along these lines, Fig. 2D shows a FIP200 signal in the control and in the GFP-SEC61B sample. Although this could represent an unspecific band, it could also be related to posttranslational modifications in FIP200. The documented difference in IP-efficiencies is therefore unclear to this reviewer. As we used the same volume of each input fraction for immunoblot analysis, the amount of input fraction reflects flotation of each protein. In Fig 2, a reduction in the FIP200 signals in WT MEF samples is due to a reduction in the floated population as shown in Fig 1A. Therefore, it is impossible (and would be inappropriate) to load the same amount of FIP200 protein in all the European Molecular Biology Organization 23