Dear Dr Koch,

Thank you for submitting your revised manuscript entitled "Post-translational insertion of small membrane proteins by the bacterial signal recognition particle" for consideration as a Research Article by PLOS Biology.

Your revision has now been evaluated by the PLOS Biology editorial staff, as well as by the original Academic Editor, and I am writing to let you know that we would like to send your submission out for external peer review.

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Gabriel Gasque, Ph.D.,

Senior Editor

PLOS Biology

Dear Dr Koch,

Thank you very much for submitting a revised version of your manuscript "Post-translational insertion of small membrane proteins by the bacterial signal recognition particle" for consideration as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors and by the original Academic Editor and reviewers 1, 2, and 4.

In light of the reviews (below), we are positive about your study and pleased to offer you the opportunity to address the lingering points raised by the reviewer. As you will see, reviewer 1 thinks that additional in vivo data could resolve standing ambiguities in your study. Together with the Academic Editor, we encourage you to seriously consider this request. Although we will not make addressing it experimentally a prerequisite for publication, adding these additional data seems relatively straightforward and would significantly strengthen the robustness and in vivo relevance of the work. Please also address the outstanding minor concerns of reviewers 1 and 2.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.

We expect to receive your revised manuscript within 2 months.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

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You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Gabriel Gasque, Ph.D.,

Senior Editor

PLOS Biology

*****************************************************

REVIEWS:

Reviewer #1: The authors have addressed most of my comments during the last round of review. In general, biochemical data are much stronger and provide strong evidence that YohP can be post-translationally targeted by SRP/FtsY. Additional reconstitutions using proteoliposomes further pointed to SecYEG or YidC as potential translocases for YohP, which is a nice addition. Overall the paper is much stronger. My major remaining concern is still the lack of in vivo data to show that YohP insertion is SRP/FtsY dependent and post-translational. The additional in vivo experiment showing the membrane localization of the YohP mRNA further introduces ambiguity to the interpretation of the targeting route in vivo. Since the authors already have an in vivo assay for the membrane localization of YohP by pulse chase, it seems within their reach to at least show that YohP insertion in vivo is SRP/FtsY dependent.

Additional minor comments:

The topology of YohP via proteinase K is opposite of the conclusion in the previous draft using a different method. Given that YohP is small, could its topology be influenced by the His6 tag? I note that the authors have already shown good evidence that YohP is membrane-localized / inserted, so the topology information is not necessary in this paper and may be left for a different characterization given the current ambiguity.

Since OmpA translocation is SecA and SecYEG-dependent, is it possible that the effect of IpomF on OmpA translocation could be due to inhibition of SecA, rather than SecYEG?

Reviewer #2: The authors have made a commendable effort to address the reviewers' comments.

1. However, many of their statements are still too definitive (for example in the abstract). The authors should simply state their observations for the constructs they examined and the assays they carried out. As just a few examples, I suggest the following changes (these are just examples, modifications need to be made throughout the manuscript):

--line 194: "…these data show that the vast majority of YohP-His is oriented in a Cin-Nout topology, …"

--line 223: "…does not efficiently occur spontaneously in our assay, suggesting that insertion requires…"

--line 267: "…strongly stimulated YohP-His insertion."

--lines 298-299: "In order to further test whether…"

--line 366: "…whereas our data indicate that the…"

--line 370: "To obtain further information about the insertion mode…"

Small proteins are tricky to study and as found by the authors, different assays can lead to completely different conclusions. In the previous draft, the authors concluded, based on AMS labeling, that YohP has a Nin-Cout topology. Now the authors conclude the protein has a Cin-Nout topology. Both assays have limitations. The authors should avoid stating that they have "proof".

2. Some non-standard phrasing that should be changed:

--line 203: "allows to determine"

--line 233: "for preventing possible saturation"

--line 283: "10-fold up-scaled"

--line 326: "were UV-exposed"

--line 329: "decorated with a-Ffh antibodies"

Reviewer #4: I find that the authors have done a formidable job in addressing the reviewers comments and now present a much stronger case for they study with more controls and clarification of issues that were confusing or not directly justified by the available data.

I therefore fully support its publication.

Dear Dr Koch,

Thank you for submitting your revised Research Article entitled "Post-translational insertion of small membrane proteins by the bacterial signal recognition particle" for publication in PLOS Biology. I have now discussed this version with the Academic Editor, and we're delighted to let you know that we're now editorially satisfied with your manuscript.

However before we can formally accept your paper and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication. Please also make sure to address the data and other policy-related requests noted at the end of this email.

*Copyediting*

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

*Published Peer Review History*

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https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

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*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

*Submitting Your Revision*

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include a cover letter, a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable), and a track-changes file indicating any changes that you have made to the manuscript.

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

Sincerely,

Gabriel Gasque, Ph.D.,

Senior Editor,

ggasque@plos.org,

PLOS Biology

=======================

DATA AVAILABILITY:

Please rename your "Quantification" file as S1 Data, making sure it has a legend and is mentioned in each figure legend.

Dear Dr Koch,

On behalf of my colleagues and the Academic Editor, Frederick M Hughson, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology.

The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. If there is a chance that you'll be unavailable during the copy editing/proof review period, please provide us with contact details of one of the other authors whom you nominate to handle these stages on your behalf. This will ensure that any requested corrections reach the production department in time for publication.

Early Version

The version of your manuscript submitted at the copyedit stage will be posted online ahead of the final proof version, unless you have already opted out of the process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

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 submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact me if I can provide any assistance during the production process.

Kind regards,

Alice Musson

Publishing Editor,

PLOS Biology

on behalf of

Gabriel Gasque,

Senior Editor

PLOS Biology