Atco, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase

Leticia Veloso Ribeiro Franco; Chen-Hsien Su; Julia Burnett; Lorisa Simas Teixeira; Alexander Tzagoloff

doi:10.1371/journal.pone.0233177

Peer Review History

Original SubmissionFebruary 18, 2020
17 Mar 2020 Decision Letter - Yidong Bai, Editor PONE-D-20-04750 ATCO, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase PLOS ONE Dear Dr., Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We would appreciate receiving your revised manuscript by May 01 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Yidong Bai Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 3. Thank you for stating the following in your Competing Interests section: "NO" Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript by the Tzagoloff’s group builds on their previously reported observation that in yeast mitochondria, newly-synthesized Atp9, a mitochondrion encoded ATPase subunit, forms high molecular weight complexes with Cox6, a nucleus encoded subunit of cytochrome c oxidase. These complexes, now called ATCO, are proposed to coordinate the assembly of the two oxphos complexes. The experiments presented clearly demonstrate that the ATCO complexes are the source of Atp9 for Atp9 ring formation, are well executed and grant publication. However, the fact that in the absence of Cox6, Atp9 assembly proceeds normally, makes the model of co-assembly regulation contra intuitive. Unfortunately, the authors did not explore here whether ATCO is a precursor of COX and the sole source of Cox6 for COX assembly. Is has been previously reported by several groups that defects in mitochondrial ATPase biogenesis lead to a COX assembly defect, but not vice versa. Could this be related to the unavailability of Cox6 for COX assembly? The authors should at least discuss these possibilities. Minor points: 1- There are a few typos/grammar issues to be fixed. For example - Line 89: “nuclear encoded” should be “nucleus-encoded”. - Line 169: “does not translated Cox1” should be “does not translate Cox1” Reviewer #2: The authors previously reported the existence of high molecular weight complexes (referred to as ATCO) containing the mtDNA-encoded Cox6 and Atp9 proteins. The present study shows that newly synthesized Atp9 associates with ATCO before being incorporated into ATP synthase as an oligomeric ring. The use of cysteine atp9 mutants provides solid evidence for Atp9-Atp9 interactions in ATCO similar to those in assembled ATP synthase, arguing against non-specific aggregation of Atp9 and Cox6. Based on these findings, the authors propose that ATCO may regulate the relative amounts of COX and ATP synthase in being a source of Cox6 and Atp9 to be incorporated synchronously into their cognate complexes. This is an important and well executed study that helps to better understand the mechanisms involved in formation of the mitochondrial energy-transduction system. Minor points. - A comment on ‘the stand-alone Atp9-ring would be appreciated. Does its detection indicate that the Atp9-ring forms from ATCO independently of any other ATP synthase component, which is suggested by the model. How then the ring is produced in stoichiometric amounts with the other ATP synthase modules? Could the authors evoke a possible mechanism? -As nicely discussed, COX is much more sensitive to glucose repression than ATP synthase is, which provides evidence that Cox3 is not essential for formation of the Atp9-ring and its incorporation into ATP synthase. The converse is seemingly not true. Indeed, yeast ATP synthase defective mutants show a decreased content in COX, except those with FO-mediated proton leaks. It has been argued recently (Su et al, Hum Mol Genet. 2019 Nov 15;28(22):3792-3804, and references therein) that the proton-translocation activity of ATP synthase modulates the rate of COX biogenesis through the mitochondrial membrane electrical potential. Although this is not a requirement, this reviewer would appreciate if the authors could comment these reported findings in the light of the present study. -In Fig. 6B, RKY28 should be replaced by RKY26. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: di Rago Jean-Paul [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0233177.r001
Revision 1
28 Apr 2020 Author Response Dear Editor, Thank you for the prompt review of our manuscript “ATCO, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase ”. We also thank the two reviewers for their thoughtful comments and suggestions, which we have tried to incorporate into the revised manuscript. The changes made are summarized below. Reviewer #1: This manuscript by the Tzagoloff’s group builds on their previously reported observation that in yeast mitochondria, newly-synthesized Atp9, a mitochondrion encoded ATPase subunit, forms high molecular weight complexes with Cox6, a nucleus encoded subunit of cytochrome c oxidase. These complexes, now called ATCO, are proposed to coordinate the assembly of the two oxphos complexes. The experiments presented clearly demonstrate that the ATCO complexes are the source of Atp9 for Atp9 ring formation, are well executed and grant publication. However, the fact that in the absence of Cox6, Atp9 assembly proceeds normally, makes the model of co-assembly regulation contra intuitive. Unfortunately, the authors did not explore here whether ATCO is a precursor of COX and the sole source of Cox6 for COX assembly. Is has been previously reported by several groups that defects in mitochondrial ATPase biogenesis lead to a COX assembly defect, but not vice versa. Could this be related to the unavailability of Cox6 for COX assembly? The authors should at least discuss these possibilities. The question of whether Cox6 of ATCO is a precursor for COX biogenesis is being actively studied in our lab but has yet to be confirmed or refuted. At present we have evidence that the pleiotropic deficiency of cytochrome oxidase in mitochondria of ATP synthase mutants is related to decreased translation of all three mitochondrially encoded subunits of this respiratory complex. We do not know if Cox6 is involved in this translational regulation. This will require overcoming some sticky technical problems that we are working on now. The discussion of the effect of ATP synthase mutations on cytochrome oxidase has been expanded. Our recent experiments show that growth of an atp9 null mutant under conditions that produce only 50% �-/0 cells result in a 10 fold reduction of Cox6 (unpublished). The explanation for this could that the absence of Atp9 leads to a decreased membrane potential that prevents Cox6 transport (see response last question). Alternatively, transport of Cox6 is normal but in absence of Atp9, it is rapidly proteolyzed. In both cases Cox6 would be unavailable for COX biogenesis. Reviewer #2: The authors previously reported the existence of high molecular weight complexes (referred to as ATCO) containing the mtDNA-encoded Cox6 and Atp9 proteins. The present study shows that newly synthesized Atp9 associates with ATCO before being incorporated into ATP synthase as an oligomeric ring. The use of cysteine atp9 mutants provides solid evidence for Atp9-Atp9 interactions in ATCO similar to those in assembled ATP synthase, arguing against non-specific aggregation of Atp9 and Cox6. Based on these findings, the authors propose that ATCO may regulate the relative amounts of COX and ATP synthase in being a source of Cox6 and Atp9 to be incorporated synchronously into their cognate complexes. This is an important and well executed study that helps to better understand the mechanisms involved in formation of the mitochondrial energy-transduction system. - A comment on ‘the stand-alone Atp9-ring would be appreciated. Does its detection indicate that the Atp9-ring forms from ATCO independently of any other ATP synthase component, which is suggested by the model. How then the ring is produced in stoichiometric amounts with the other ATP synthase modules? Could the authors evoke a possible mechanism? The ATP9 ring by itself has also been reported to be present in an atp6 mutant, a mutant with an unprocessed N-terminal presequence on Atp6 and an atp10 mutant that contains only 5% of the normal concentration of ATP synthase (unpublished). Although these observations indicated that Atp9 can oligomerize into the ring independent of the assembly of the rest of the complex, they do not exclude the possibility that At9 translation and/or ring formation is regulated as the amount of ring formed in these studies was not measured. Mitochondria from cells that have been incubated for two hour growth in the presence of chloramphenicol have a significantly higher concentration of ATCO. The incubation in the presence of chloramphenicol inhibits mitochondrial translation of Atp9 but not of Cox6, which would be expected to increase the mitochondrial pool of this subunit and hence also increase the amount of ATCO when Atp9 translation is restored in isolated mitochondria. This may indicated that Atp9 translation and ATCO formation may be regulated by Cox6. This is also supported by previous evidence that cells undergoing derepression from glucose assemble significantly less Atp9 ring in a cox6 but not other oxidase mutants (Su et al [15], Fig. 6). This is now mentioned in the discussion. -As nicely discussed, COX is much more sensitive to glucose repression than ATP synthase is, which provides evidence that Cox3 is not essential for formation of the Atp9-ring and its incorporation into ATP synthase. The converse is seemingly not true. Indeed, yeast ATP synthase defective mutants show a decreased content in COX, except those with FO-mediated proton leaks. It has been argued recently (Su et al, Hum Mol Genet. 2019 Nov 15;28(22):3792-3804, and references therein) that the proton-translocation activity of ATP synthase modulates the rate of COX biogenesis through the mitochondrial membrane electrical potential. Although this is not a requirement, this reviewer would appreciate if the authors could comment these reported findings in the light of the present study. Su et al (2019) have shown that overexpression of the oxodicarboxylate carrier OCD1 an atp6 mutant increases the concentrations of ATP synthase and COX a factor 2.5 and 4, respectively. These authors interpreted their results to indicate the higher abundance of COX enhances substrate-level ATP synthesis as a result of increased TCA cycle flux, thereby restoring the membrane potential of mitochondria in the atp6 mutant. Since most of the ATP in the ODC1/atp6 mutant is made by substrate level phosphorylation during conversion of α-ketoglutarate to succinate Su et al (2019) proposed that COX biogenesis is modulated by the membrane potential rather than by ATP from oxidative phosphorylation. The proposed function of ATCO in establishing the stoichiometry ATP synthase relative to cytochrome oxidase is predicated on the absence in normal cells of monomeric Atp9 and Cox6. This is supported by our results in the case Atp9. Although we do not have similar direct evidence for Cox6, the observed increase synthesis of ATCO by isolated mitochondria from cells that have been grown in the presence of chlroramphenicol, support the idea that synthesis of Atp9 and more to the point also of ATCO depends on the supply of Cox6. This point is now brought out in the discussion. Like other proteins that are transported by the TIM23 translocase of mitochondria, uptake of Cox6 depends on a membrane potential. The restoration of a membrane potential in the atp6 mutant overexpressing ODC1 strain would be expected to improve transport of newly synthesized Cox6 from the cytoplasm to the matrix, which in turn could account for the increase of COX observed by Su et al (2019). The typos and other minor points picked up the reviewers have been corrected. We hope that with these improvements the paper will be deemed acceptable for publication. Sincerely yours, Alex Tzagoloff Attachments Attachment Submitted filename: response to reviewers.docx https://doi.org/10.1371/journal.pone.0233177.r002
30 Apr 2020 Decision Letter - Yidong Bai, Editor ATCO, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase PONE-D-20-04750R1 Dear Dr. Tzagoloff, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Yidong Bai Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0233177.r003
Formally Accepted
4 May 2020 Acceptance Letter - Yidong Bai, Editor PONE-D-20-04750R1 Atco, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase Dear Dr. Tzagoloff: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Yidong Bai Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0233177.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .