The EMBO Journal Peer Review Process File - EMBO-2015-91440
Manuscript EMBO-2015-91440
Phospholipid methylation controls Atg32mediated mitophagy and Atg8 recycling
Kaori Sakakibara, Akinori Eiyama, Sho W. Suzuki, Machiko Sakoh-Nakatogawa, Nobuaki
Okumura, Motohiro Tani, Ayako Hashimoto, Sachiyo Nagumo, Noriko Kondo-Okamoto, Chika
Kondo-Kakuta, Eri Asai, Hiromi Kirisako, Hitoshi Nakatogawa, Osamu Kuge, Toshifumi Takao,
Yoshinori Ohsumi and Koji Okamoto
Corresponding author: Koji Okamoto, Osaka University
Review timeline:
Submission date:
Editorial Decision:
Revision received:
Editorial Decision:
Revision received:
Accepted:
03 March 2015
14 April 2015
08 August 2015
31 August 2015
03 September 2015
04 September 2015
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.)
Editor: Andrea Leibfried
1st Editorial Decision
14 April 2015
Thank you for submitting your manuscript entitled 'Functional link between Atg32-mediated
mitophagy and phospholipid methylation'. I have now received reports of all referees, which are
enclosed below.
As you will see, both referees consider that your work is well-performed and potentially interesting.
Referees 1, however, thinks that the study does not offer at this stage the sufficient insight that s/he
would expect for publication in The EMBO Journal. The referee thinks that it remains unclear how
relevant your findings are in a more physiological context and where the Atg8-PMME conjugate
accumulates within the cell. This referee also raises concerns regarding the specific effect observed
for mitophagy versus other Atg8-dependent processes (see major issues mentioned in the report).
Referee number 2 is more supportive, but also thinks that the work could be further improved by
additional experiments and amendments.
Given the interest into the topic and the constructive comments provided by the referees, I can offer
to consider a revised version should you be able to substantiate your model along the lines suggested
by the referees. This might demand a lot of work, and I will be happy to extend the revision time
should that be useful. I am certain that adding more insight into the described process will make
your paper an outstanding one. Please let me know in case you want to discuss the necessary
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revisions further, and also if you foresee a problem in addressing all the comments. Please also note
our scooping protection; we will not reject your manuscript based on novelty issues in case similar
work is published elsewhere while yours is under consideration here. Thank you for the opportunity
to consider your work for publication. I look forward to your revision.
-----------------------------------------------REFEREE COMMENTS
Referee #1:
In their manuscript Sakakibara et al describe a possible link between the phospholipid biosynthesis
pathway and mitophagy. The authors identify the enzyme Opi3, a methylene-fatty-acylphospholipid synthase, as a crucial factor for mitophagy.
In their assay utilizing mito-DHFR-mCherry, opi3∆ cells reveal a drastic decrease in mitophagy
under mitophagy inducing conditions (growth for 72 hours in non-fermentable carbon source).
Deletion of Cho2, a phosphatidylethanolamine methyltransferase, acting downstream of Opi3, did
not affect mitochondrial degradation, excluding the possibility that mitophagy depends on PC
homeostasis. One-dimensional thin-layer chromatography in opi3∆ cells reveals a drastic increase in
the PMME, a product of Cho2 and subsequent substrate of Opi3 for conversion to PC. The
accumulation of PMME leads to the generation of Atg8-PMME conjugate. Atg8-PMME appears a
to be a poor substrate of Atg4 and is therefore not efficiently deconjugated.
Furthermore, loss of Opi3 leads to an accumulation of GSH, which has been previously linked to
Atg32 expression. Indeed the authors show a strong decrease in Atg32 expression in opi3∆ cells.
Taken together these data allow the conclusion that disruption of conversion of PMME to PC leads
to the impairment of mitophagy.
This is potentially interesting observation, however there are some major issues that must be
addressed to convincingly demonstrate the physiological relevance of the phenomena described in
this study: The entire study is based on an observation in Opi3 null strain, which according to the
authors' data leads to abnormal accumulation of a PE-derivative that cannot be processed by Atg4
thus inhibiting mitophagy; The authors should try to look for physiological or pathophysiological
conditions that lead to the accumulation of PMME; It is crucial to determine at which organelle
PMME and Atg8-PMME conjugate accumulate in the opi3 null strain; Finally, the authors do not
provide a clear explanation for the specific inhibition of mitophagy.
Several additional points need to be addressed:
Figure 1 B/H (e.g.): Utilizing mito-DHFR-mCherry in degradation assays, the authors show the
reduction in mitophagy in opi3∆ cells. Strikingly, the authors ignore several additional bands that
are formed under these conditions.
Fig S1D shows also a drastic increase in the level of lipidated Atg8 but no effect on pexophagy in
opi3∆ cells. This observation should be addressed in more detail. Furthermore, utilizing the GFPAtg8 degradation assay would allow the verification of Atg8 lipidation under regular autophagyinducing conditions (SD-N under fermentable conditions) and might additionally support the ALP
assay.
Figure 2A: to solidify authors conclude that the Cho2 function negatively affects mitophagy in the
absence of Opi3, the authors should try to over express it in a wild type strain?
The ratio of Atg8-PE to Atg8-PMME in the different strains and time points should be determined.
This is rather important since based on the data presented in figure 4B, most of Atg8 in the opi3 null
strain is "locked" in its lipidated form. In case the majority of Atg8 is conjugated to PMME, one
would expect inhibition of the other Atg8-dependent autophagic processes.
Figure 2E: why is the GSH level in the double knock out opi3∆cho2∆ smaller than in cho2∆ cells?
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The data presented in Figure S3A are very important and should therefore included in figure 3.
Additionally, testing the effect of BSO treatment would strengthen the hypothesis that the elevated
GSH level in opi3∆ cells suppresses Atg32 expression.
Figure S3 B-E: the authors mix up the description in the text and the names of the figures, 2 parts of
the figure are obviously missing.
Figure S4B: co-localization of the PAS and Atg32 should be examined under mitophagy-inducing
conditions in opi3∆ cells compared to wild type.
Figure7: Atg8 lipidation should be examined.
Minor comments:
Figure 3A: the authors don't provide the rationale for the use of a pep4∆prb1∆ strain.
The model is not very clear and not too informative. For example it indicates a cytosolic localization
of lipidated Atg8 which is very unlikely.
Several times the authors refer to the "expression level" of proteins (e.g. Por1 in Figure 1D/E,
Atg32-HA in Figure 3, Cho2 in the discussion), the description "protein level" is appropriate.
Referee #2:
EMBOJ-2015-91440
Sakakibara et al
Summary:
This comprehensive study describes a series of experiments that illuminates previously recognized,
but mechanistically not understood, aspects of the specific pathway for autophagic destruction of
mitochondria (mitophagy). The work focuses on the derangement of mitophagy by mutations in the
terminal steps of the de novo pathway for phosphatidylcholine (PC) biosynthesis. The authors
demonstrate defects in the terminal methylation steps in the conversion of phosphatidylethanolamine
(PE) to PC compromise mitophagy. The underlying mechanisms are complex and involve at least
two branches, but both focus on a known mediator of mitophagy; the Atg32 protein. What
recommends this MS is that the study makes interesting observations, in several different contexts,
regarding how phospholipid metabolism impinges on mitophagy, and describes the novel mislipidation of a core component of the autophagic pathway (Atg8) that disqualifies this component
from productive potentiation of mitophagy. Given the central role played by mitochondria in
eukaryotic cell physiology and mammalian disease, this reviewer believed the EMBO J readership
(and the cell biology community in general) will find the work of interest. The authors identify a
number of interesting points, these points are well supported experimentally, and the impact of the
work is supported by some surprising discoveries.
Comments:
This detailed study is carefully done and is of high quality. The authors are to be congratulated for
submitting such a complete and well-supported body of work. This reviewer suggests the work can
be improved in one respect. The most interesting aspect is the evidence showing that Atg8-PMME is
defective in the dynamic recycling process because it is resistant to the deconjugating enzyme.
While the in vitro data to this point are compelling, it would be nice to buttress the in vivo
demonstration of such. Perhaps this could be done in a pulse-chase analysis using ethanolamine
tracer to pulse-label the newly lipidated Atg8-PE and Atg8-PMME and demonstrate persistence of
the latter in a chase with cold ethanolamine.
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1st Revision - authors' response
08 August 2015
I. Referee #1’s comments
(1) The authors should try to look for physiological or pathophysiological conditions that lead to the
accumulation of PMME.
This point is truly a fundamental and central problem in order to elucidate the biological relevance
on the link between mitophagy and phospholipid methylation. We thank the Referee #1 for asking
us to challenge it. We have tried two different experimental approaches. First, we attempted to
promote PMME synthesis in wild-type cells supplemented with monomethylethanolamine
(MMEA). Although PMME can be generated from MMEA via the Kennedy pathway, we found no
effect on mitophagy and Atg8 lipidation (see the attached Figure 1). Perhaps, the Kennedy pathway
is not enough active to significantly increase the PMME levels in wild-type yeast cells. Second, we
sought to overexpress Cho2 in wild-type cells. Notably, overexpression of Cho2 caused partial
mitophagy defects in wild-type cells, likely due to a moderate increase in PMME-conjugated Atg8.
We added the results and sentences into the supplemental figure and
text, respectively, as described below.
(Results in page 11, line 17): Finally, we investigated the impact of Cho2 overexpression on
mitophagy in wild-type cells. Although the protein levels of Cho2 under the strong, constitutive
GPD promoter were still significantly reduced during respiratory growth, mitophagy was partially
suppressed, likely due to moderate accumulation of PMME-conjugated Atg8 (Supplementary Fig
S7D). Thus, it seems possible that Cho2-mediated PE methylation can act as a physiological process
to affect degradation of mitochondria.
(2) It is crucial to determine at which organelle PMME and Atg8-PMME conjugate accumulate in
the opi3 null strain.
Except for a few examples such as PI(3)P visualized by a FYVE domain-containing marker, assays
for localization of phospholipids are still not feasible due to the lack of specific labeling methods.
Hence, we think that determination of subcellular PMME composition is beyond the scope of our
study. Given the fact that Atg8 is mostly conjugated to PMME in cells lacking Opi3, we performed
density gradient centrifugation analysis, and found that lipidated Atg8 (likely Atg8–PMME) in the
opi3-null mutant was enriched in the late endosome fraction. We added the results and a sentence
into the main figure and text, respectively, as described below.
(Results in page 9, line 1): Moreover, our subcellular fractionation assays revealed Atg8 coexisted
with the
cytosol/vacuole and late endosome in wild-type and opi3-null cells, respectively (Fig 4F),
suggesting that
Atg8-phospholipid conjugates (likely Atg8–PMME) aberrantly associate with the late endosomelike structure.
(3) The authors do not provide a clear explanation for the specific inhibition of mitophagy.
We apologize for confusions on this point. Loss of Opi3 leads to at least two prominent defects
related to mitophagy: strong suppression of Atg32 induction, and drastic activation of Atg8–PMME
generation. The former is probably “specific” to mitophagy. The latter is likely to be “general” to
autophagosome formation. The reasons why mitophagy is predominantly impaired in the absence of
Opi3 could be that strong Atg32 suppression and abnormal Atg8 lipidation synergistically impact
mitophagy, and that Atg8–PE (still present at least to some extent) is preferentially loaded to the
pre-autophagosomal structure (PAS) for the Cvt pathway, pexophagy, and nonselective autophagy,
but not to the PAS for mitophagy. Accordingly, we modified sentences in the text as described
below.
(Discussion in page 13, line 4): Assuming that Atg8–PE is produced with a decreased level but
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functional for autophagy-related processes in respiring cells lacking Opi3, it seems probable that
PMME-conjugated Atg8 is not competent for generation of isolation membranes, thereby being
excluded from autophagy flux. Consistent with this idea, transport of Ape1 (Cvt cargo) and
autophagic bodies to the vacuole is partially impaired in the absence of Opi3 under mitophagyinducing conditions (Supplementary Fig S1A, and data not shown).
(4) Figure 1 B/H (e.g.): Utilizing mito-DHFR-mCherry in degradation assays, the authors show the
reduction in mitophagy in opi3Δ cells. Strikingly, the authors ignore several additional bands that
are formed under these conditions.
Most of the additional bands are also found in cells lacking Atg32 (Fig 1B), indicating that they are
not generated via mitophagy. There is a band at the middle of the blot in wild-type and atg32 Δ, but
not in opi3 Δ. We do not understand what this band means, but do see no need to consider it for
quantification of mitophagy.
(5) Fig S1D shows also a drastic increase in the level of lipidated Atg8 but no effect on pexophagy
in opi3 Δ cells. This observation should be addressed in more detail. Furthermore, utilizing the
GFP-Atg8 degradation assay would allow the verification of Atg8 lipidation under regular
autophagy-inducing conditions (SD-N under fermentable conditions) and might additionally support
the ALP assay.
Pexophagy was induced by a shift from oleic acid medium to nitrogen starvation medium. We think
that, under this condition, Atg8–PE is still generated and able to promote pexophagy in the opi3-null
mutant. Similarly, autophagy occurs in starving cells lacking Opi3. We added a sentence into the
text as described below.
(Discussion in page 12, line 26): We speculate that Atg8 is conjugated to both PE and PMME in
starving cells lacking Opi3, and that Atg8–PE can promote pexophagy and bulk autophagy even in
the presence of Atg8–PMME.
We performed GFP–Atg8 processing assays under starved conditions, and found normal autophagy
flux in the absence of Opi3. We thus added the results and a sentence into the supplemental figure
and text, respectively, as described below.
(Results in page 5, line 26): We also monitored vacuolar protease-dependent processing of GFP–
Atg8, and found wild-type-like autophagy flux in the opi3-null mutant under starved conditions
(Supplementary Fig S1G and H).
(6) Figure 2A: to solidify authors conclude that the Cho2 function negatively affects mitophagy in
the absence of Opi3, the authors should try to over express it in a wild type strain?
We addressed this point in (1).
(7) The ratio of Atg8-PE to Atg8-PMME in the different strains and time points should be
determined. This is rather important since based on the data presented in figure 4B, most of Atg8 in
the opi3 null strain is "locked" in its lipidated form. In case the majority of Atg8 is conjugated to
PMME, one would expect inhibition of the other Atg8-dependent autophagic processes.
It is highly challenging to distinguish between Atg8–PE and Atg8–PMME by any of means
available at the moment. Mass spectrometry for phospholipids could be the only way, however, it is
very difficult to be quantitative anyway. Even in the opi3-null cells under respiratory (mitophagyinducing) conditions, there is a considerable amount of PE (Fig 2D) that can be conjugated to Atg8.
This PE-conjugated form may be partially competent for autophagosome formation. How
mitophagy is strongly suppressed in the absence of Opi3 is an important question that requires
further studies in the future. Please also see our response in (3).
(8) Figure 2E: why is the GSH level in the double knock out opi3Δ cho2Δ smaller than in cho2Δ
cells?
It is not entirely clear why it is. We speculate that the double knock out strain might contain higher
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levels of cellular stress compared to opi3Δ and cho2Δ cells, which causes a decrease in the GSH
levels.
(9) The data presented in Figure S3A are very important and should therefore included in figure 3.
Additionally, testing theeffect of BSO treatment would strengthen the hypothesis that the elevated
GSH level in opi3Δ cells suppresses Atg32 expression.
We repeated the qPCR analysis for ATG32 in the absence or presence of BSO, and found strong
correlations between the mRNA and protein levels. We added the results and a sentence into the
main figure and text, respectively, as described below.
(Results in page 7, line 18): A reduction in the Atg32 content and its partial recovery by BSO
treatment were also detected at the transcriptional level in cells lacking Opi3 (Fig 3D).
(10) Figure S3 B-E: the authors mix up the description in the text and the names of the figures, 2
parts of the figure are obviously missing.
We corrected the misdescriptions and added the missing data into the supplemental figure.
(11) Figure S4B: co-localization of the PAS and Atg32 should be examined under mitophagyinducing conditions in opi3Δ cells compared to wild type.
We observed wild-type and opi3-null cells expressing Atg32–GFP and the PAS marker Atg11– or
Atg17–mCherry under a fluorescence microscope, and found colocalization of the mitophagy
receptor with the PAS in both cells. We added the results and a sentence into the supplemental
figure and text, respectively, as described below.
(Results in page 8, line 29): The PAS involved in mitophagy appears unaffected, as Atg32–GFP
colocalized with the PAS markers Atg11– and Atg17–mCherry even in the absence of Opi3
(Supplementary Fig S4B).
(12) Figure7: Atg8 lipidation should be examined.
Choline treatment also caused a partial reduction of Atg8 lipidation in cells overexpressing Atg32.
We added the results and a sentence into the supplemental figure and text, respectively, as described
below.
(Results in page 11, line 11): We also confirmed partially abrogated Atg8 lipidation (probably
PMME-conjugation) in opi3-null cells supplemented with choline (Supplementary Fig S7A and B).
(13) Figure 3A: the authors don't provide the rationale for the use of a pep4Δ prb1Δ strain.
Since Atg32 is a mitochondrial protein, it is also degraded in the vacuole via mitophagy. Thus, to
assess changes in the Atg32 protein levels, it is necessary to inactivate vacuolar proteases. We added
sentences into the text as described below.
(Results in page 7, line 9): Thus, we monitored the expression profiles of chromosomally HAtagged Atg32 in vacuolar protease-deficient cells under respiratory conditions. In this variant,
vacuole-dependent degradation of Atg32 is mostly blocked, thereby allowing us to examine the
protein levels irrespective of mitophagy activity.
(14) The model is not very clear and not too informative. For example it indicates a cytosolic
localization of lipidated Atg8 which is very unlikely.
We modified the model and added it into the main figure.
(15) Several times the authors refer to the "expression level" of proteins (e.g. Por1 in Figure 1D/E,
Atg32-HA in Figure 3, Cho2 in the discussion), the description "protein level" is appropriate.
According to the referee #1’s point, we corrected the descriptions in the text and figure legends.
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II. Referee #2’s comment
(1) The most interesting aspect is the evidence showing that Atg8-PMME is defective in the dynamic
recycling process because it is resistant to the deconjugating enzyme. While the in vitro data to this
point are compelling, it would be nice to buttress the in vivo demonstration of such. Perhaps this
could be done in a pulse-chase analysis using ethanolamine tracer to pulse-label the newly lipidated
Atg8-PE and Atg8-PMME and demonstrate persistence of the latter in a chase with cold
ethanolamine.
We realized that the Referee #2 raised an important criticism, and performed an Atg4 processing
assay in vivo. Unfortunately, as described in our response to the Referee #1’s point (1), a pulsechase analysis using ethanolamine is not appropriate for our purpose since the Kennedy pathway
does not efficiently generate PE in wild-type yeast cells. Alternatively, we constructed a strain
expressing Atg8G116 (a variant that does not need Atg4 for activation and following lipidation) and
Atg4 under the control of the inducible GAL promoter (GALP-ATG4). Using this strain, we can first
accumulate lipidated Atg8 in the presence of dextrose, since GALP-ATG4 is mostly suppressed,
thereby preventing Atg8 delipidation. After 24 h respiratory growth to facilitate Atg8–PMME
generation in opi3-null cells, we can then induce GALP-ATG4 to promote Atg8 delipidation by
galactose supplementation. The results clearly indicated that
lipidated Atg8 (mostly Atg8–PMME) cannot be efficiently processed by Atg4 in vivo. We thus
added the results and sentences into the main figures and text, respectively, as described below.
(Results in page 10, line 13): To further prove that Atg4 does not efficiently catalyze delipidation of
PMME-conjugated Atg8 in living cells, we put the ATG4 gene under the control of the GAL
promoter in wild-type and opi3-null cells expressing His–FLAG–Atg8G116. In these cells, lipidated
Atg8 can initially be accumulated in the presence of dextrose where ATG4 expression is mostly
suppressed (at the Gly-0h time point). Cells are then shifted to a glycerol medium supplemented
with galactose that strongly induces ATG4 expression (at the Gly-24h time point). We found that
wild-type and opi3-null cells contained a similar amount of Atg4 at the Gly-48h time point.
Although the protein levels of lipidated His–FLAG–Atg8G116 were reduced by 28% compared with
those at the Gly-24h time point in wild-type cells, the lipidated protein was even increased to 127%
in opi3-null cells (Fig 5F).
Finally, to strengthen our argument that PMME could also be an unwanted target for lipidation of
their Atg8 homologs, we tried PMME-conjugation of GATE-16, a human Atg8 homolog, in vitro.
Although the recombinant human Atg7 was not fully active (due to unknown reasons), we
succeeded to generate both PE- and PMME-conjugated GATE-16. Notably, GATE-16–PE was
efficiently delipidated by the recombinant human Atg4, but GATE-16–PMME was not. We added
the results and a sentence into the supplemental figure and text, respectively, as described below.
(Discussion in page 14, line 12): Indeed, GATE-16–PMME can be formed but inefficiently
deconjugated by Atg4 in vitro (Supplementary Fig S5A), raising the possibility that autophagosome
formation might be compromised by PMME conjugation of this Atg8 homolog in mammals.
We, once again, thank the referees for their valuable comments. We think that our revised
manuscript with the new data has been significantly improved, and hope that the paper will be
acceptable for publication in The EMBO Journal.
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Figure 1 attached to the Cover letter
Wild-type cells expressing mito-DHFR–mCherry (depicted by arrowhead) were cultured in glycerol
medium (Gly) in the absence or presence of MMEA, harvested at the indicated time points, and
subjected to western blotting. Generation of free mCherry (depicted by arrow) indicated transport
of mitochondria to the vacuole.
2nd Editorial Decision
31 August 2015
Thank you for submitting your revised manuscript for our consideration. It has now been seen once
more by the original referees (see comments below), and I am happy to inform you that they are
both satisfied with the revisions and therefore have no further objections towards publication in The
EMBO Journal.
Before sending you a formal letter of acceptance, some editorial points need to be taken care of at
this stage:
1. please upload a modified manuscript text file as a word doc and include an author contribution
statement as well as a conflict of interest statement. Please include in the Material and Methods
sections the following references or alter your text slightly to avoid (self-)plagiarism (see also
attached screenshots for the relevant sections):
Strains and growth conditions: Okamoto et al., Dev Cell, 2009
Fluorescence microscopy: Eiyama et al., FEBS letters, 2013
Phospholipid composition assay: Motohiro Tani and Osamu Kuge, Mol Microbiology, 2012
2. your figures seem all to have a very good resolution, however, the typesetters would need a single
page for each figure (and individual figure files). I hope you'll be able to move the individual panels
around to adjust your figures to this requirement. If not, we can also break the largest figures up into
two figures
3. we have now the 'expanded view' in the HTML version of the manuscript to allow better visibility
of supplementary figures. At the moment, we can accommodate 5 expanded view figures. Therefore,
I would like to suggest to move the quantitation panels from figures S2 into the main figure 2 and to
use supplementary figures S1, S3, S4, S5 and a merged S6-7 as expanded view figures. For this, we
would need again one single page per figure as an individual file, i.e. you'd need to squeeze some of
your panels a bit. It would also be great to have the figure legends of these expanded view figures
included in the main text word document (see also our guide to authors).
4. as you might know, we encourage our authors to provide original source data, particularly
uncropped/-processed electrophoretic blots for the main figures of your manuscript. If you would
like to add source data to your beautiful blots, we would welcome one PDF-file per figure for this
information. These will be linked online as supplementary "Source Data" files.
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I am therefore formally returning the manuscript to you for a final round of minor revision, only to
allow you to easily modify/replace these files. Once we should have received them, we should then
be able to swiftly proceed with formal acceptance and production of the manuscript!
-----------------------------------------------Referee #1:
The authors addressed most of my comments and the revised manuscript meets the scientific merit
of EMBO J.
Referee #2:
In my initial review, I found particularly interesting the evidence showing that Atg8-PMME is
defective in the dynamic recycling process because it is resistant to the deconjugating enzyme. My
sole recommendation for improving an already strong and interesting MS was to strengthen the in
vitro data regarding the inability of Atg8-PMME to be recycled with an in vivo pulse-chase
experiment. The authors have responded strongly and creatively to this suggestion. Moreover, in this
referee's opinion, the authors also responded strongly to Reviewer 1's criticisms. Requests for
additional work would now extend beyond any reasonable scope of this quality study.
2nd Revision - authors' response
03 September 2015
Thank you very much for handling our manuscript and inviting us to submit a final version for
formal acceptance. We have carefully read all editorial comments made by you. Please see the
following point-by-point responses to the comments:
Editorial comments
(1) Please upload a modified manuscript text file as a word doc and include an author contribution
statement as well as a conflict of interest statement. Please include in the Material and Methods
sections the following references or alter your text slightly to avoid (self-)plagiarism (see also
attached screenshots for the relevant sections): Strains and growth conditions: Okamoto et al., Dev
Cell, 2009 Fluorescence microscopy: Eiyama et al., FEBS letters, 2013 Phospholipid composition
assay: Motohiro Tani and Osamu Kuge, Mol Microbiology, 2012.
We added an author contribution statement and a conflict of interest statement into the modified
manuscript. We also included the references listed above into the Materials and Methods section.
All changes have been indicated in blue.
(2) Your figures seem all to have a very good resolution, however, the typesetters would need a
single page for each figure (and individual figure files). I hope you'll be able to move the individual
panels around to adjust your figures to this requirement. If not, we can also break the largest figures
up into two figures.
We reduced the magnification of all figures, and placed them into single panels. Please see if those
EPS files look OK.
(3) We have now the 'expanded view' in the HTML version of the manuscript to allow better
visibility of supplementary figures. At the moment, we can accommodate 5 expanded view figures.
Therefore, I would like to suggest to move the quantitation panels from figures S2 into the main
figure 2 and to use supplementary figures S1, S3, S4, S5 and a merged S6-7 as expanded view
figures. For this, we would need again one single page per figure as an individual file, i.e. you'd
need to squeeze some of your panels a bit. It would also be great to have the figure legends of these
expanded view figures included in the main text word document (see also our guide to authors).
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We moved the quantitation panels from Figure S2A and B to Figure 2E (combined), and the Atg32
expression blot from Figure S6 to Figure 6C. Then, Figures S1, S3, S4, S5, and S7 were converted
to Figures EV1, EV2, EV3, EV4, and EV5, respectively. We reduced the magnification of all
figures, and placed them into single panels. Please see if those EPS files look OK.
4) As you might know, we encourage our authors to provide original source data, particularly
uncropped/-processed electrophoretic blots for the main figures of your manuscript. If you would
like to add source data to your beautiful blots, we would welcome one PDF-file per figure for this
information. These will be linked online as supplementary "Source Data" files.
At this moment, we do not take the opportunity to provide the original source data. We, once again,
thank you for your support on our study. We look forward to formal acceptance for publication in
The EMBO Journal.
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