Phospholipids alter activity and stability of mitochondrial membrane-bound ubiquitin ligase MARCH5

This study shows that lipids can act as regulators for the ubiquitination process and can control the stability and activity of a membrane-embedded E3 ubiquitin ligase.

1. The authors carefully perform titrations of various lipids. Have authors consider additional ways to distinguish the activity and stability of the MARCH5? Would mutants in the putative cardiolipin binding site or testing modified forms of cardiolipin (oxidized CL) provide additional support that the increased activity is indeed not a result of aggregation? Can higher-order species formation be reversed if a de-ubiquitinating enzyme is added?
2. The authors also mention MARCH6 regulation by cholesterol. What are the sequence differences between the different MARCH family members, and could this be informative in understanding lipid regulation?
3. Finally, the authors cite that there is an increased rate for auto-ubiqutination in the presence of cardiolipin. Could the authors comment on how this compares to typical rates for ubiquitination?
Overall, this is a well executed study. I recommend publication following minor revision.
Reviewer #2 (Comments to the Authors (Required)): In this manuscript by Merklinger et al., the authors showed that mitochondrial membrane-bound ubiquitin ligase MARCH5 is controlled by phospholipid species. Cardiolipin and phosphatidic acid bound to purified MARCH5 and regulated the stabilization and the ubiquitination pattern. Some of the observations of the study are interesting, however some issues need to be addressed.

Major
(1) Lipid binding assay using PVDF membrane was shown in Fig. 1B. Dose the detergent affect this result? Have the authors examined this experiment using a crude membrane fraction without detergent? CL signal was not clear. In Fig. C, is the degree of detergent replacement altered by the phospholipid species? Is this difference affect the result, Fig. 1D?
(2) Authors showed the potential CL and PA binding sites. Mutagenetic assay such as replacement of some residues by alanine will enhance this manuscript in Figs 1-4.
(3) In all assays, do fatty acid compositions of phospholipids affect each result? It is an important point.
(4) In Fig. 5 and its discussion, authors suggested CL as regulator of MARCH5 upon externalization to the OMN. However, this manuscript did not show the results supporting the author's suggestion.

Minors
Authors should provide better explanation for Fig. 4 in the results and the legend.

Responses to reviewers' comments are in blue.
Line numbering refers to the revised version of the manuscript.

Reviewer #1 (Comments to the Authors (Required)):
Merklinger and colleagues investigate the regulation of MARCH5 by lipids, and report altered activity and stability specific to cardiolipin. The authors compare auto-ubiquitination by detergent solubilized MARCH5 over a range of lipid conditions and detergent concentrations, and also find cardiolipin binding induces a decrease in thermal stability, while phosphatidic acid increases thermal stability. A few questions/suggestions to help improve the manuscript: We thank the reviewer for the appreciation of our manuscript.
1. The authors carefully perform titrations of various lipids. Have authors consider additional ways to distinguish the activity and stability of the MARCH5? Would mutants in the putative cardiolipin binding site or testing modified forms of cardiolipin (oxidized CL) provide additional support that the increased activity is indeed not a result of aggregation? Can higher-order species formation be reversed if a de-ubiquitinating enzyme is added?
We very much appreciate the comment and given the nature of our study, we did consider this particular issue. The binding of CL to MARCH5 and the induced destabilization might relate to higher conformational flexibility of MARCH5, which allows the auto-ubiquitination.
It is difficult to assess if the binding of CL to MARCH5 is causing auto-ubiquitination or the decrease in thermal stability and therefore probably the higher conformational flexibility induced by CL, which might result in aggregation. The binding of CL or the possible aggregation is always directly or indirectly related to the enhanced auto-ubiquitination of MARCH5. Therefore, mutational studies would only confirm or reject the hypothesis of specific CL binding sites. However, would not give insight into what causes the autoubiquitination, the specific CL interaction or the conformational flexibility induced by CL. Additionally, there are inevitable limitations to the biophysical method one want to perform when working with membrane proteins, which also relates to MARCH5.
We included a cross-linking experiment of MARCH5 in presence of CL and in absence of 2. The authors also mention MARCH6 regulation by cholesterol. What are the sequence differences between the different MARCH family members, and could this be informative in understanding lipid regulation?
The MARCH-family is defined and characterized by the cytosolic RING domain, via the arrangement of His and Cys residues, which coordinate the two zinc ions. The MARCH family contains a Cys and His on the fourth and fifth zinc coordinating residue (RING-CH), whereas the classic RING domain includes a His and a Cys (RING-HC), respectively (Dodd et al, 2004). The coordinating residues are highly conserved among the family. However, the MARCH family members highly vary in the number of transmembrane domains (some have no transmembrane domains) as well as their sequence. The transmembrane domain within the MARCH family is not conserved (Samji et al, 2014;Bauer et al, 2017). Therefore, we do not think that we can conclude anything in regards to lipid regulation by looking at the sequence of the different transmembrane domains.
3. Finally, the authors cite that there is an increased rate for auto-ubiqutination in the presence of cardiolipin. Could the authors comment on how this compares to typical rates for ubiquitination?
This is an interesting question. However, we cannot conclude anything about the rate of autoubiquitination at the stage of experimental data we have and we do not discuss about rate of auto-ubiquitination but enhancement of auto-ubiquitination when compared to the control. Overall, this is a well executed study. I recommend publication following minor revision.
We thank the referee for their thorough assessment of our work and their recommendation.

Reviewer #2 (Comments to the Authors (Required)):
In this manuscript by Merklinger et al., the authors showed that mitochondrial membranebound ubiquitin ligase MARCH5 is controlled by phospholipid species. Cardiolipin and phosphatidic acid bound to purified MARCH5 and regulated the stabilization and the ubiquitination pattern. Some of the observations of the study are interesting, however some issues need to be addressed.
We thank the reviewer for the appreciation of our study.

Major
(1) Lipid binding assay using PVDF membrane was shown in Fig. 1B. Dose the detergent affect this result? Have the authors examined this experiment using a crude membrane fraction without detergent? CL signal was not clear. In Fig. C, is the degree of detergent replacement altered by the phospholipid species? Is this difference affect the result, Fig. 1D?
We performed the lipid binding with two different detergent DDM and LMNG, which show the same results. Furthermore, we performed lipid-binding assays, including the lipids tested in the ubiquitination assay and stability measurements detecting with a MARCH5 antibody excluding possible interaction of GFP-His10 tag to interact with the lipids. We included an additional figure in the supplementary (Fig S1) and statement in line 107-108, and additional methods line 402-410.
We did not perform a lipid-binding assay using crude membrane fraction, because we would not expect MARCH5 binding to any of the lipids, since it is still incorporated in the natural lipid bilayer and would compete with the natural lipid carried over from the membrane.
The lipids were dissolved in the same detergent concentration to have same molar concentration in the lipid stock solution as well as the same molar ratio of protein to lipid in the experiment.
The detergent concentration does not have an effect on the thermal stability of MARCH5. This is shown by the lipid titration experiments, where the amount of detergent in the sample was 0.012% (w/v) LMNG (Table 2), which resulted in a melting temperature of 52.91 ± 0.65 °C. In contrast, the thermal stability measurement, where all the six different lipids were tested, the amount of detergent consisted of 0.005% (w/v) and resulted in a melting temperature of 52.86 ± 0.58 °C for the + control (Table1), which coincide with thermal stability measurement of the lipid titration experiments. This is also valid for the other lipids (see Table 1 and 2). Furthermore, there was no effect on the ubiquitination patters with variation in detergent concentration, showed by the ubiquitination assay of the lipid titration experiments, where the detergent concentration was 0.002% (w/v) ( Fig. 3 and Fig S5), whereas the ubiquitination assay including all the lipids 0.001% (w/v) LMNG was present ( Fig. 2)). Therefore, we can exclude that the detergent concentration has an influence on the thermal stability or stability.
Of note, the critical micelle concentration of the used detergent LMNG is 0.001% (1 CMC), which means at this concentration LMNG micelles are formed. Therefore, we always included detergent above this concentration. The detergent concentrations are stated in the respective material and method section.
(2) Authors showed the potential CL and PA binding sites. Mutagenetic assay such as replacement of some residues by alanine will enhance this manuscript in Figs 1-4.
We agree with the reviewer on this point; it would be interesting analyses to perform.
However, these would be quite extensive experiments as many additional mutants will have to be purified and verified for stability with CD to shown that they otherwise behave similarly to the wild type enzyme. Therefore, given the scope of the study, we decided early in the process that we will focus on the wild type enzyme, mainly since we amount of protein and lipids we require for each experiment is quite large. See also the response to point 1 from Reviewer 1.
(3) In all assays, do fatty acid compositions of phospholipids affect each result? It is an important point.
This is a very interesting question, but it would be beyond the scope of this paper to anlyse the impact of fatty acid composition in our assays. The experiments were preformed using natural lipids occurring in vertebrate tissue (stated in material and methods). Therefore, the specific fatty acid composition was not considered, rather the overall lipid class. However, MARCH5 might interact only with specific lipid species, but this was not the aim of this study. We wanted to give a broad overview about possible effect of different lipid classes towards activity and stability of MARCH5. Testing different fatty acid compositions would exceed the scope of this initial study considering the enormous possibility of fatty acid combinations.
Furthermore, using natural lipids in this study had the advantage to cover large range of fatty acid compositions of the lipids occurring in the cell. This allows us to exclude specific binding for example of the abundant lipids PE and PC. We included a statement in line 131 and 132 that we use natural lipids, which is a mix of different lipid species. Furthermore, we replaced the word lipid species by the word lipid class to prevent confusion.
We are currently looking into specific fatty acid compositions of the lipids interacting with MARCH5 for a further study to evaluate the fatty acid dependency, which might be solely responsible for the effect on MARCH5. We show some initial results here (see figure below), where we tested CL of E.coli and the CL with the fatty acid chains 18:1. In the thermal stability measurement, we can see different effects on MARCH5. E.coli CL seems not to have an effect on the thermal stability compared to the control. However, CL 18:1 shows minor stabilization. Initial ubiquitination assay reveals no significant change in presence of one of the CL species.
However, we do not know yet the cause of the difference, which we will investigate in the future, but CL is known to have specific fatty acid chains in different tissues, therefore might connect to different regulatory effects (Fajardo et al, 2017;Bradley et al, 2016). (4) In Fig. 5 and its discussion, authors suggested CL as regulator of MARCH5 upon externalization to the OMN. However, this manuscript did not show the results supporting the author's suggestion.
We thank the reviewer for bringing our attention to this. The model depicted in Fig 5

Minors
Authors should provide better explanation for Fig. 4 in the results and the legend.
We thank the reviewer for highlighting this. We have included the following explanation in the paragraph "MARCH5 stability is affected by the lipid phospholipid environment" in line 202-205 and lines 210, 214, 216 and 220 marked in red. Furthermore, we added an explanatory sentence in the caption in line 720 also marked in red. Thank you for submitting your revised manuscript entitled "Phospholipids alter activity and stability of mitochondrial membranebound ubiquitin ligase MARCH5". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
Along with points mentioned below, please tend to the following: -please upload your main and supplementary figures as single files -please add Keywords and Category for your manuscript in our system -please note that the titles in the system and manuscript file must match -please add callouts for Figures S1A-B; S3A-B; S4A-C and S5A, B, D-F to your main manuscript text; -there is a splice between the second and third set of blots in figure 2B, third panel. Also there are a few flaws in the blots in Figure 3B ( e.g. white stripes). Same for S5B. Please provide source data files for figure 2B, 3B and S5B.
If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
LSA now encourages authors to provide a 30-60 second video where the study is briefly explained. We will use these videos on social media to promote the published paper and the presenting author (for examples, see https://twitter.com/LSAjournal/timelines/1437405065917124608). Corresponding or first-authors are welcome to submit the video. Please submit only one video per manuscript. The video can be emailed to contact@life-science-alliance.org To upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plex You will be guided to complete the submission of your revised manuscript and to fill in all necessary information. Please get in touch in case you do not know or remember your login name.
To avoid unnecessary delays in the acceptance and publication of your paper, please read the following information carefully.
A. FINAL FILES: These items are required for acceptance.
--An editable version of the final text (.DOC or .DOCX) is needed for copyediting (no PDFs).
--High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max. 200 characters including spaces). This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title. It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person. Author names should not be mentioned.

B. MANUSCRIPT ORGANIZATION AND FORMATTING:
Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files.
**Submission of a paper that does not conform to Life Science Alliance guidelines will delay the acceptance of your manuscript.** **It is Life Science Alliance policy that if requested, original data images must be made available to the editors. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original data images prior to final submission.** **The license to publish form must be signed before your manuscript can be sent to production. A link to the electronic license to publish form will be sent to the corresponding author only. Please take a moment to check your funder requirements.** **Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript. If you do want to opt out of having the reviewer reports and your point-by-point responses displayed, please let us know immediately.** Thank you for your attention to these final processing requirements. Please revise and format the manuscript and upload materials within 7 days.
Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. The authors have addressed all comments from this Reviewer.
It is a nice piece of work. I recommend publication without delay.
Reviewer #2 (Comments to the Authors (Required)): In this manuscript by Merklinger et al., the authors showed that phospholipid species affect mitochondrial membrane-bound ubiquitin ligase MARCH5. Purified MARCH5 bound to cardiolipin and phosphatidic acid. Additionally, phospholipids regulated the stabilization and the ubiquitination pattern. This manuscript suggest that phospholipids can regulate the dynamics and turnover of mitochondria.
The authors have satisfyingly answered all my questions.