Dear Dr Li,

Thank you for submitting the revision of your manuscript entitled "A conserved ubiquitin- and ESCRT-dependent pathway to regulate human lysosomal membrane proteins" for consideration as a Research Article by PLOS Biology in the Review Commons program.

Your manuscript and the reviews have now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission back to the original reviewers.

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Kind regards,

Ines

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Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

Dear Dr Li,

Thank you for submitting your revised Research Article entitled "A conserved ubiquitin- and ESCRT-dependent pathway to regulate human lysosomal membrane proteins" for publication in PLOS Biology. I have now obtained advice from two of the original reviewers from Review Commons and have discussed their comments with the Academic Editor. 

Based on the reviews (attached below), we will probably accept this manuscript for publication, provided you satisfactorily address the remaining points raised by Reviewer 3. Please also make sure to address all the data and policy-related requests included below. In addition, we would like you to consider a suggestion to improve the title to:

"A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation"

As you address these items, please take this last chance to review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the cover letter that accompanies your revised manuscript.

We expect to receive your revised manuscript within two weeks.

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https://journals.plos.org/plosbiology/s/supporting-information  

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Please do not hesitate to contact me should you have any questions.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD,

Senior Editor,

ialvarez-garcia@plos.org,

PLOS Biology

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DATA POLICY: IMPORTANT - PLEASE READ

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797 

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

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Figs. 1B; Fig. 2D; Fig. 3D, E; Fig. 4B, C, E, G; Fig. 5B, C, E, G, H; Fig. 6F; Fig. 7B, D, F, H; Fig. 8B; Fig. S2B; Fig. S3B, D, G and Fig. S4B, D

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BLOT AND GEL REPORTING REQUIREMENTS:

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements 

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Reviewers' comments

Rev. 2:

The authors have taken the effort to address all my previous comments. I do not have any further concerns. Nice work!

Rev. 3:

The authors have revealed details concerning how two lysosome membrane proteins, RNF152 and LAPTM4A, are internalized into lysosomes for degradation in a ubiquitin and ESCRT dependent manner. Although it remains uncertain to what extent these examples can be generalized to other lysosome membrane proteins, these are interesting new findings. Nonetheless, given that the authors have only characterized 2 proteins that follow this degradation pathway, caution is required with respect to generalization. The authors were responsive to reviewer comments. My only remaining concerns are minor and should not require additional experiments.

1. Data presented in the response to reviewers should be included in the manuscript. If the reviewers need this information to assess the results, then other readers will appreciate them as well. That said, the preliminary screening data need not be added.

2. It remains unclear whether the RNF152 degradation observed with the over-expressed protein reflects the normal degradation pathway for the endogenous protein. Either way, it could be a useful tool. However, the authors should be more up front with this limitation if they cannot show that the endogenous protein is degraded in a similar manner.

3. The authors state: "Without treatment,many LSD patients will develop severe neurodegeneration symptoms (Marques and Saftig, 2019)." This sentence in the introduction is problematic as it implies that there are treatments for the neurological aspects of lysosome storage diseases. Unfortunately, treatments such as enzyme replacement therapy are only effective for the non-neuronal symptoms.

4. Page 27: Gene transfer with replication incompetent lentivirus is referred to as transduction rather than infection.

Dear Dr Li,

On behalf of my colleagues and the Academic Editor, Ana Garcia-Saez, I am pleased to say that we can in principle offer to publish your Research Article entitled "A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation" in PLOS Biology, provided you address any remaining formatting and reporting issues. These will be detailed in an email that will follow this letter and that you will usually receive within 2-3 business days, during which time no action is required from you. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have made the required changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

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We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Ines

--

Ines Alvarez-Garcia, PhD 

Senior Editor 

PLOS Biology

ialvarez-garcia@plos.org