Mitochondrial‐derived vesicles retain membrane potential and contain a functional ATP synthase

  1. Reut Hazan (Ben‐Menachem)
  2. Dvora Lintzer
  3. Tamar Ziv
  4. Koyeli Das
  5. Irit Rosenhek‐Goldian
  6. Ziv Porat
  7. Hila Ben Ami Pilo
  8. Sharon Karniely
  9. Ann Saada
  10. Neta Regev‐Rudzki
  11. Ophry Pines

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Abstract

Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle‐to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial‐derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP‐producing vesicles, which can potentially regenerate ATP‐deficient mitochondria and may participate in organelle‐to‐organelle communication.

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  1. Version published to 10.15252/embr.202256114
  2. Version published to 10.1101/2022.07.10.499450v2 on bioRxiv
  3. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    August 17, 2022

    RE: Review Commons Refereed Preprint #RC-2022-01442

    Dear Editor of the EMBO Journal,

    Please find our updated manuscript and response to the reviewers’ comments. We appreciate the effort that the reviewers have put into the evaluation of our manuscript.

    We are happy with the potential importance the reviewers realise in the study:

    Reviewer 1: The finding that ubiquitination occurs inside mitochondria would be an important conceptual advance, which would open new perspectives both for ubiquitination and mitochondrial biology

    Reviewer 2: This work would represent a significant/exceptional discovery if supported by compelling data.

    Reviewer 3: the results are interesting and very important, as mentioned in the major comments section…

    With regard to the major comments raised by the reviewers, you will find below our specific response point by point with explanations and suggested novel experiments (highlighted in yellow). In summary we suggest the following actions to fully support our model:

    • We will perform a-complementation with ubiquitin (lacking the GG motif) fused at its C-terminus to the short fragment of b-galactosidase (a). Blue colonies with ωm will indicate import.
    • As shown in Figure S2, now added to the manuscript, we show detection of ubiquitinated proteins and mono ubiquitin in extracts of mitochondria pre-treated with trypsin.
    • A bio-archives address of our other manuscript will be provided.
    • The use of a-complementation for protein localization was developed by us 15 years ago and since then has been used by us and other groups verifying its use as a screening tool. One point is clear, ωm or ωc do not leak into other subcellular compartments. Nevertheless, in the research of specific genes validation is important. Yes!!! ωm and ωc are exclusively located in mitochondria or the cytosol respectively.
    • We will highly purify mitochondria on gradients and treat them with protease.
    • We cannot be sure that we will be able to detect a protein with ubiquitin modifying activity which functions solely on certain proteins in mitochondria, so publication cannot rely on this.
    • Repeat mass spectrometry with careful editing will be undertaken as suggested by the reviewer.
    • We will attempt to perform protease protection assays in the presence of specific detergents.

    Before tackling the very tough revision, we would like to know if EMBO Journal would positively consider acceptance of our manuscript based on the review and planned revision.

    Prof. Ophry Pines Microbiology & Molecular Genetics Hebrew University of Jerusalem Jerusalem 91220 Israel

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary:

    In this manuscript, Zhang et al. investigate whether ubiquitination occurs inside mitochondria of the budding yeast S. cerevisiae. They first observe thanks to a sensitive complementation assay that several components of the yeast ubiquitination (and deubiquitination) machinery can localize inside mitochondria. To be able to specifically probe ubiquitin conjugates assembled inside mitochondria they fused HA-tagged ubiquitin to a mitochondrial targeting sequence. Using this construct, they demonstrate that ubiquitin conjugates can be assembled in mitochondria. A series of elegant experiments demonstrates that the pattern of ubiquitin conjugates depends on the mitochondrial localization and the activity of the ubiquitin conjugating enzyme Rad6. Altogether, these results convincingly demonstrate that ubiquitination can occur inside yeast mitochondria when ubiquitin is intentionally targeted inside this organelle. It however remains unclear whether mitochondrial ubiquitination occurs in endogenous conditions (without targeting ubiquitin into this compartment) and whether it affects mitochondrial functions.

    Response: Regarding the question whether mitochondrial ubiquitination occurs in endogenous conditions, we feel that this is obvious based on our results. We detect numerous ubiquitination related enzymes (E1, E2, E3, DUB) eclipsed in mitochondria but none of the proteasome subunits. As pointed out by the reviewer “these results convincingly demonstrate that ubiquitination can occur inside yeast mitochondria”. With that said, additional data will be incorporated into the manuscript as suggested by the reviewer and can be seen below.

    Major comments:

    1. The materials and methods section is lacking important information (western blot protocol, details of antibodies, strains, plasmids...). It is thus difficult to evaluate how several experiments were performed and how their design (e.g. the promoters chosen to express tagged proteins) could impact the interpretation of the results. This is a major issue that needs to be corrected. The main text should also explicitly indicate whether tagged proteins used in the alpha-complementation assay are overexpressed or not.

    Response: The materials and methods section will be updated accordingly.

    1. Despite the previous comment, the data presented in the manuscript convincingly demonstrate that multiple components of the ubiquitination machinery can localize within mitochondria and that ubiquitin conjugates can be assembled in mitochondria when ubiquitin is modified to be intentionally targeted into this compartment. However, little data is shown to support the hypothesis that ubiquitin conjugates can be assembled in mitochondria when ubiquitin is not fused to a mitochondrial targeting sequence. Thus, in my opinion, the evidences presented in the current manuscript are not sufficient to conclude that ubiquitin conjugates are assembled in mitochondria in endogenous conditions (as this is done implicitly). Additional evidences are needed to draw this conclusion (see some experimental suggestions hereafter). Without further evidences, the speculative aspects of the claim that "ubiquitination occurs in the mitochondrial matrix" should be discussed explicitly.

    Response: See the discussion above why we are confident that ubiquitination occurs in mitochondria. Our major problem with ubiquitin and the ubiquitination enzymes is that they are eclipsed in mitochondria. We propose as suggested by the reviewer (item 4 of his review) to perform a-complementation with ubiquitin fused at its C-terminus to the short fragment of b-galactosidase (a). Blue colonies with ωm will indicate import.

    1. The authors used a mass spectrometry approach to identify mitochondrial ubiquitination substrates. However, they have not yet succeeded in identifying a substrate whose modification is specifically regulated by a given component of the mitochondrial ubiquitination machinery. They have also not identified a phenotype or process impacted by mitochondrial ubiquitination. Thus, at this stage, the biological consequences of mitochondrial ubiquitination remain elusive.

    __Response: __We have not identified a substrate whose modification is dependent on a given component of the mitochondrial ubiquitination machinery, even though we have tried. Again, the problem is low levels of these proteins eclipsed in mitochondria. Even when we do find a protein that is ubiquitinated (e.g. Aco1) its ubiquitination is not exclusively dependent on Rad6. Thus, different ubiquitin enzymes may have the same substrates.

    1. The authors have not directly investigated whether ubiquitin itself (without a mitochondrial targeting sequence) localizes in mitochondria. I encourage them to address this question since it would provide an important piece of evidence suggesting that mitochondrial ubiquitination can occur in endogenous conditions. This could be done using the alpha-complementation assay and the results could be presented within Figure 1. Ideally this experiment should be performed without overexpressing ubiquitin. Note that if the authors decide to use a C-terminally tagged form of ubiquitin for this experiment, the GG motif of ubiquitin should be mutated to avoid cleavage of the alpha tag by cellular DUBs. This form of ubiquitin will not be conjugatable, but this is not an issue for this experiment since its aim is to determine whether ubiquitin can be targeted to mitochondria, not to probe conjugates.

    Response: We will perform experiments as suggested by the reviewer including ubiquitin fused at its C-terminus to the short fragment of b-galactosidase (a), see item 2. We have previously made a PreSu9-Ubi lacking a GG motif but now will look at a different combination of this and other constructs.

    1. In the top panels of Figure 2 and S1, free ubiquitin is well detectable in the total and cytosolic fractions. It is however not clear to me whether it is also detectable in the concentrated mitochondrial fraction. If yes and if it would be resistant to trypsin digestion, it would provide additional evidence that endogenous ubiquitin can be targeted to the mitochondrial matrix (see previous comment).

    Response: See Item 6.

    1. The data shown in the top panel of Figure 2 and S1 also suggest that free ubiquitin is less concentrated in mitochondria than in the cytosol (since it is more difficult to detect in the concentrated mitochondrial fraction than in the cytosolic fraction, see previous comment). It is thus possible that the use of preSu9-HA-Ubi (or preFum1-HA-Ubi) lead to an artificially high intra-mitochondrial concentration of free ubiquitin. As the concentration of free ubiquitin is known to impact ubiquitination processes, I encourage the authors to compare the relative levels of free ubiquitin present in the mitochondrial fraction prepared from WT and preSu9-HA-Ubi (or preFum1-HA-Ubi) expressing cells. If free ubiquitin is detectable in mitochondrial fractions and resistant to trypsin (see previous comment), this could be done by repeating the experiment shown in Figure 3B and probing the blot with an antibody that recognizes free ubiquitin.

    Response to 5 and 6: Detection of ubiquitin in mitochondria is extremely difficult even when mitochondria are 15-fold concentrated versus the cytosol and when HA-Ubi is overexpressed. Thus, ubiquitin is eclipsed in mitochondria. Nevertheless, as shown in the Figure below which was not part of the submitted manuscript yet was performed in parallel to experiments done early on, shows detection of very weak bands of free ubiquitin in extracts of mitochondria pre-treated with trypsin.

    __Endogenous ubiquitination pattern in mitochondria of ____Δ____rad6 ____cells is restored to normal by Rad6-____α____. __WT or Δrad6 cells containing a Rad6-α construct or an empty plasmid were subjected to subcellular fractionation. Mitochondrial fractions with or without trypsin treatment, were probed for ubiquitin by WB. Aco1 is a matrix mitochondrial protein, and Tom70 is a mitochondrial outer membrane protein (MOM) facing the cytosol.

    1. I strongly encourage the authors to provide more data indicating that "ubiquitination occurs in mitochondria" by performing experiments that do not rely on the use of the preSu9-HA-Ubi or other forms of ubiquitin that are intentionally targeted to mitochondria. For instance, they could analyse the pattern of HA-Ubi conjugates of trypsin digested mitochondrial fractions prepared from wt, rad6-delta, and rad6-delta complemented with preSu9-Rad6-alpha-SL17. Note that if trypsin digested mitochondrial fractions are too contaminated by ubiquitinated proteins present outside mitochondria to perform this experiment, the authors may use the unspecific DUB Usp2 as an alternative protease to strip ubiquitinated proteins from the mitochondria periphery.

    Response: Concentrated mitochondrial extracts from WT and Δrad6 cells untreated or treated with trypsin were probed with anti-ubiquitin antibodies (Figure above). A very weak band corresponding to free ubiquitin can be detected in extracts of mitochondria treated with trypsin but these are very weak and are on the limit of detection.

    Minor comments:

    1. Overall, the manuscript is well organized and easy to follow. The text is clearly written; the figures are well annotated.

    2. The authors should provide full images of all the blots with anti-ubiquitin and anti-HA antibodies so that one can see the bands corresponding to free ubiquitin (or free HA-Ubi). For instance, in Figure 3B, it is not possible to see the presence (or absence) of the band corresponding to free HA-Ubi because the very bottom of the image is cut.

    3. The authors should indicate whether the MTS of Su9 (and Fum1) are expected to be cleaved after import of preSu9-HA-Ubi (and preFum1-HA-Ubi) in mitochondria. They should also label on the corresponding immunoblots the presence (or absence) of the band corresponding to the free preSu9-HA-Ubi (and preFum1-HA-Ubi) (or HA-Ubi if the MTS is expected to be cleaved from these constructs).

    4. In Figure 3B, the ubiquitin conjugates produced with preSu9-HA-Ubi and preFum1-HA-Ubi have different migration patterns. I think this should be explicitly mentioned and discussed. Could it be due to the presence of lysine residues in the Su9 or Fum1 MTS that could lead to the assembly of artificial ubiquitin chains?

    5. The authors indicate that "endogenous Rad6 [...] is expressed at very low levels and can hardly be detected in the mitochondrial fraction by WB (Figure S5)". I did not manage to observe the band corresponding to endogenous Rad6 in the mitochondrial fraction in the pdf. The authors should provide a more contrasted or better quality image.

    CROSS-CONSULTATION COMMENTS I agree with reviewer 2 that proper validation of the complementation assay is crucial for this manuscript. I was myself wondering whether it uses endogenously tagged proteins or whether it is based on an overexpression system. I imagine this information will be detailed in the manuscript in preparation mentioned by the authors. I am therefore wondering whether it would be possible to ask the authors to provide the draft of this manuscript (or at least the validation part).

    Response: A bio-archives address of our other manuscript will be provided upon resubmission. See other issues referred to the response Reviewer 2.

    I agree with most comments of reviewer 3. Regarding the hypothesis that preSu9-HA-Ubi could form aggregates on the cytosolic surface of the mitochondria, I think that the results presented on Figure 7B rather argue against it (since they indicate that Rad6 localized inside mitochondria can restore the pattern of ubiquitin conjugates). That's why (in my opinion) the major question the author now need to adress is whether intra-mitochondrial ubiquitination occurs in endogenous conditions (ie without forcing ubiquitin into this compartment and without E2 or E3 overexpression).

    Response: See response to the other reviewers

    Reviewer #1 (Significance (Required)):

    The finding that ubiquitination occurs inside mitochondria would be an important conceptual advance, which would open new perspectives both for ubiquitination and mitochondrial biology research. However, the significance of the current manuscript is limited because the presented evidences heavily rely on the use of artificial conditions (ubiquitin tagged with a mitochondrial-targeting sequence) that may trigger irrelevant ubiquitination events. The significance would be much higher if the authors would provide further evidences indicating that intra-mitochondrial ubiquitination occurs in endogenous conditions and/or if they had identified a mitochondrial process specifically impacted by mitochondrial ubiquitination.

    Expertise of the reviewer: Ubiquitination, Yeast biology, protein-protein interactions. No specific expertise in mitochondrial biology

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

    In the manuscript by Yu et al., the authors test the concept that certain proteins are unevenly distributed within distinct cell compartments. Due to this localization discrepancy, protein detection in some subcellular compartments can be "eclipsed" by a predominant subset of specific protein localizing in another cell compartment their actual distribution. Therefore, tiny amounts of physiologically relevant proteins could be biologically relevant. Still, their function in some locations can be overlooked (or eclipsed) because of the high expression level of the same protein in another subcellular compartment(s). Although, this concept is not particularly novel. For example, it is already known that many different proteins can localize to distinct cellular locations (e.g., permanent mitochondrial and peroxisomal localization of many proteins or transient localization of particular proteins to separate cell compartments). The authors apply a yeast system and an α-complementation assay to test further the role of such eclipsed proteins in mitochondrial biology. Specifically, they focus on the ubiquitin (Ub, or as abbreviated incorrectly in this manuscript; Ubi) conjugation pathway, components of which have never been convincingly shown to localize inside the mitochondria. This work proposes that certain ubiquitination events can occur inside yeast mitochondria. This work would represent a significant/exceptional discovery if supported by compelling data. However, the major problem with this work is that the conclusions are based on the ectopic expression of distinct proteins. This approach is not failproof in precise protein expression/delivery to the specific subcellular locations and is likely to result in a non-specific localization. Thus, the problem of eclipsed proteins is addressed by the methodology that may lead to the artificial generation of eclipsed overexpressed proteins. A more effective approach would be if the authors found a way to study this issue with endogenous proteins. The need for overexpression of mitochondria-targeted ubiquitin makes it challenging to reconcile the physiological role of these fundings. In addition, some critical technical issues and omissions further reduce the potential impact of this work (see Specific comments below). For example, strong evidence of mitochondria fraction purity and additional evidence that all the essential constructs used in this work are not misdirected to a different compartment are needed.

    Response: “Although, this concept is not particularly novel” is a very disappointing remark by the reviewer!! While dual targeting of proteins has been known for many, many years, how widespread the phenomenon was unknown and thought to be negligible. We are leaders for the last 30 years in the field of dual targeting and distribution and in particular distribution of single translation products. We coined the terms “echoforms” and “eclipsed distribution” and developed methods to detect and screen for dual targeting. The concept of eclipsed distribution and in particular eclipsed targeting to mitochondria is very new, and is leading to a novel perception of the mitochondrial proteome (see MS submission). While the reviewer appears to be an expert on ubiquitination, we are experts on dual targeting.

    • Ub was abbreviated incorrectly in this manuscript, Ubi. __Response: __This will be corrected.

    Other comments will be referred to in the response to Specific comments.

    Specific comments

    1. The authors should demonstrate beyond doubt that the ω components of their assay (ω-C, which supposedly stays in the cytosol-ONLY and the ω-M component, which seemingly remains in the mitochondria-ONLY) are in the compartment that the authors claim. These two proteins are transfected into yeast cells and overexpressed. Therefore it is possible that they leak to other, not intended, subcellular compartments. The authors assume that ω-M and ω-C are exclusively located either in the mitochondria or the cytosol. However, this should be shown as validation of the assay. The indicated reference from 2005 (Ref.13) and others are irrelevant since assays have variations and are often researcher/lab dependent. This validation is very important since a misallocation of the overexpressed ω-M or ω-C, leaking into other subcellular compartments, may cause misdetection of the α-constructs.

    Response: The use of a-complementation for protein localization was developed by us 15 years ago and since then has been used by us and other groups verifying its use as a screening tool. One point is clear, ωm or ωc do not leak into other subcellular compartments. Nevertheless, in the research of specific genes validation is important. Yes!!! ωm and ωc are exclusively located in mitochondria or the cytosol respectively.

    It is not surprising that Ub conjugates are detected in mitochondrial fractions. It could be due to ubiquitination of the OMM (coming from the cytosol) or perhaps since the subcellular fractions were not pure mitochondria free from contamination (the likely culprit could be the ER). The mitochondrial fractions in this work were obtained by 10,000 g separation between cytosolic and mitochondrial crude fractions. Indeed, these 10,000 g crude fractions are highly impure with membranes from other compartments (i.e., microsomes, lysosomes, and so on). Therefore, more sophisticated purification methods should be used. In addition, the authors should also test these fractions for non-mitochondrial proteins from other membrane organelles.

    __Response: __ We agree with the reviewer and therefore will take the following approaches:

    1. i) We will treat isolated mitochondria with protease in order to remove adhering proteins and digest OMM proteins…… see attached figure.
    2. ii) We will highly purify mitochondria on gradients and this will be straight forward since we are now employing such methods in other projects in the lab. iii) Matrix protein enrichment (by mass spec) is associated with IP for preSu9-HA-Ub conjugates which is three-fold higher than for HA-Ub. In any case the fact that we identify conjugates of proteins not known to be mitochondrial, strongly supports our thesis.

    Figure 2. Coomassie blue staining does not show any signal in the "M" fraction. It can be interpreted that the authors do not get any mitochondria there, and therefore the lack of Ub signal is due to the absence of the protein in the samples. Using the same amount of protein from each fraction would probably reduce the necessity of 15x enrichment.

    Response: The Coomassie blue staining does show a signal in the "M" fraction which is weak yet when a 15x enrichment is run, the protein level by Coomassie blue staining is similar to the cytosolic fraction.

    Figure 3. It is puzzling why the HA-UBQ presence is so strong in the crude mitochondrial fraction, but the preSu9-HA-Ub signal (mito-matrix) is comparatively weak. These data suggest that the crude mito-fraction could be highly contaminated with OTHER membranes. On the other hand, the preSu9-HA-UBQ signal is no more than 1-5% of the total mitochondrial signal. The high enrichment of the HA-Ubi in both cytosols and the mitochondria could indicate the OMM ubiquitination or (again) contamination by other compartments. The constructs with MTS are detected in the mitochondria. However, the localization of tagged MTS-Ubi in a non-targeted compartment (e.g., cytosol) should be excluded by additional exposure times. Because the manuscript talks about eclipsed proteins, this is important.

    Response: The HA-Ub is strong in the mitochondrial fraction, in the absence of trypsin, but is very weak in the presence of the protease indicating that most of the ubiquitinated proteins are externally attached to mitochondria. In contrast, PreSu9-HA-Ub is imported into the mitochondrial matrix and is protected from trypsin. This manuscript refers to “eclipsed in mitochondria” (not the cytosol) and this is true for ubiquitination enzymes as well as for ubiquitin.

    Figure 3C-E. These data indeed suggest that the Ub-conjugates could be formed inside the mitochondria. However, the above-discussed possibility that other than mitochondria compartments co-sediment in the 10,000g fractions makes the data interpretation highly challenging.

    __Response: __We will highly purify mitochondria on gradients and this will be straight forward since we are now employing such methods in other projects in the lab.

    Figure 4. Unsurprisingly, mitochondrial targeting of Ub leads to detecting some co-immunoprecipitating mitochondrial proteins. However, these data do not support the notion that Ub conjugation machinery acts inside the mitochondria and that the target proteins are indeed conjugated with Ub (the interaction with Ub is not equal to being conjugated). At the minimum, the authors should provide a validation that some of the detected mitochondrial matrix proteins are indeed ubiquitinated. To this end, purified mitochondria could be used for the candidate protein IP under denaturing conditions and then blotted for the candidate protein and Ub.

    __Response: __As shown in Table S2 and figure S7, forms of Ilv5, a mitochondrial protein, are ubiquitinated in WT and Drad6 cells. These modified forms of Ilv5 can be eluted from mitochondrial extracts of WT and Drad6 cells. However, the ubiquitination of ilv5 is not dependent or effected by the Drad6 mutation. We cannot be sure that we will be able to detect a protein with ubiquitin modifying activity which functions solely on certain proteins in mitochondria.

    Figure 5. The knock-out of the E2 Rad6 causes a change in the mitochondria ubiquitination pattern. This is an interesting observation, but again it does not prove that the change in the mitochondrial ubiquitination is due to the activity of Rad6 inside of the mitochondria, as opposed to ubiquitination of the OMM proteins or contaminating fractions. One also wonders why overexpression of mitochondria-targeted Ub would be necessary to detect the ubiquitination if this process was physiologically relevant, especially given that detecting endogenous Ub is not challenging. Furthermore, the apparent increase in ubiquitination in E2 mutant cells (Fig. 5) should also be addressed in more detail. Finally, data from one WB is shown, and quantification of several independent experiments should also be provided.

    __Response: __We show in the MS that RAD6 is exclusively targeted to mitochondria (Su9MTS) while unimported molecules are degraded (SL17; degron). This hybrid Rad6 can restore the WT ubiquitin pattern, while a rad6 active site mutant cannot.

    Figure 6. Can the authors provide Western blot data showing the expression of Rad6? Furthermore, quantifying these rescue experiments is necessary to make this conclusion more solid.

    Response: Even though we did not succeed in making good Rad6 antisera, we can clearly detect Rad6-a fusion proteins (Figure 7B).

    Figure 7. The authors found that preSu9-Rad6-α have problems being imported into the mitochondria matrix; therefore, they rebuild it as a preSu9-Rad6-α-SL17 protein. SL17 is a degron that targets the cytosolic protein (not imported into the mitochondria) to the proteasome and degraded (Figs. 7A-B-C). These issues could be a red flag for the rest of the manuscript, suggesting that other constructs (that were not critically evaluated for their localization in this work) could leak to different cellular compartments.

    Response: The wording used by the reviewer is particularly disturbing since current understanding in cell biology of eukaryotic cells does not accept “leaking” of proteins to different cellular compartments. One wouldn’t want DNAses, RNAses, Proteases etc leaking from one compartment to another. The localization of proteins to different cellular compartments involves very precise signals on the proteins, and specific cellular components, such as translocases, are required to target proteins to their exact destination. This is true for Rad6; it contains an MTS like sequence which when removed blocks import of the protein into mitochondria. Rad6 according to our analysis is an eclipsed dual targeted protein, so it no surprise that it is in two compartments and the trick with the SL17 degron solves the problem.

    The manuscript needs to be carefully edited, some references are in the not correct format, and there are issues with figure labels.

    Response: Careful editing will be undertaken as suggested by the reviewer.

    CROSS-CONSULTATION COMMENTS I agree with a great summary by reviewer 1. This discovery should be validated by top-quality data.

    __Reviewer #2 (Significance (Required)): __

    In the manuscript by Yu et al., the authors test the concept that certain proteins are unevenly distributed within distinct cell compartments. Due to this localization discrepancy, protein detection in some subcellular compartments can be "eclipsed" by a predominant subset of specific protein localizing in another cell compartment their actual distribution. Therefore, tiny amounts of physiologically relevant proteins could be biologically relevant. Still, their function in some locations can be overlooked (or eclipsed) because of the high expression level of the same protein in another subcellular compartment(s). Although, this concept is not particularly novel. For example, it is already known that many different proteins can localize to distinct cellular locations (e.g., permanent mitochondrial and peroxisomal localization of many proteins or transient localization of particular proteins to separate cell compartments). The authors apply a yeast system and an α-complementation assay to test further the role of such eclipsed proteins in mitochondrial biology. Specifically, they focus on the ubiquitin (Ub, or as abbreviated incorrectly in this manuscript; Ubi) conjugation pathway, components of which have never been convincingly shown to localize inside the mitochondria. This work proposes that certain ubiquitination events can occur inside yeast mitochondria. This work would represent a significant/exceptional discovery if supported by compelling data. However, the major problem with this work is that the conclusions are based on the ectopic expression of distinct proteins. This approach is not failproof in precise protein expression/delivery to the specific subcellular locations and is likely to result in a non-specific localization. Thus, the problem of eclipsed proteins is addressed by the methodology that may lead to the artificial generation of eclipsed overexpressed proteins. A more effective approach would be if the authors found a way to study this issue with endogenous proteins. The need for overexpression of mitochondria-targeted ubiquitin makes it challenging to reconcile the physiological role of these fundings. In addition, some critical technical issues and omissions further reduce the potential impact of this work (see Specific comments above). For example, strong evidence of mitochondria fraction purity and additional evidence that all the essential constructs used in this work are not misdirected to a different compartment are needed.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary: In this study, the authors detected a set of components of a ubiquitination system in the mitochondrial matrix in budding yeast using the subcellular compartment-dependent α-complementation assay. The authors detected the conjugates of mitochondrial targeting signal sequence-directed HA-Ub (preSu9-HA-Ub) in the mitochondrial matrix. The immunoprecipitates of the preSu9-HA-Ubi conjugates were highly enriched for the mitochondrial matrix proteins. Subsequently, the authors focused on the Rad6 E2 ubiquitin conjugating enzyme in the mitochondrial matrix and evaluated its inactivation-altered ubiquitination pattern in the organelle. The authors conclude that ubiquitination occurs in the mitochondrial matrix because of the eclipsed targeted components of the ubiquitination machinery.

    Major comments: The authors argued that the proteins that were modified with preSu9-HA-Ubi, which was forced to be imported into the mitochondria, are present in the mitochondrial matrix, because these species are resistant to trypsin digestion. However, it was possible that they formed severe aggregates on the cytosolic surface of the mitochondria, and hence, were resistant to the proteinase. In other words, a small amount of proteins that were not imported into the mitochondria could be deposited on the cytosolic surface of the mitochondria, where they were modified with preSu9-HA-Ubi by cytosolic Rad6. To confirm if the preSu9-HA-Ubi-modified proteins were really present in the mitochondrial matrix, they should perform the protease protection assay in the presence of an appropriate detergent (Figure 3D). In addition, subcellular fractionation of the organelle by density gradient centrifugation, indirect immunofluorescence microscopic analysis of the preSu9-HA-Ubi conjugates, and/or experiments on the in vitro import of preSu9-HA-Ubi and Rad6 into the mitochondria would strongly support the authors conclusion. Other experiments that might support the authors conclusion would be to test whether the band pattern for the preSu9-HA-Ubi conjugates changes when the mitochondrial import is impaired.

    Response: We will attempt to perform 1) Protease protection assay in the presence of a detergent (Figure 3D). 2) Subcellular fractionation of the organelle by density gradient centrifugation. 3) In vitro import of Rad6 into the mitochondria.

    Minor comments: In Figure 3B, the molecular weight distributions of the preSu9-HA-Ubi conjugates and those of the preFum-HA-Ubi conjugates are different. Is there any reason for this difference?

    In Figure 3E, the position of "-" (MG132) for lane 1 is not correct.

    In Figure 6A: The band pattern for preSu9-HA-Ubi (lane 13) in the rad6-delta cells expressing Ubc8-alpha is different from that of the wild-type cells expressing Ubc8-alpha (lane 12) as well as that obtained from the rad6-delta cells harboring empty plasmids (lane 9). Is there any explanation for this observation?

    In Figure 7B and S6: The level of preSu9-Rad6-alpha-SL17 in the rad6-delta cells is always lower than that in the wild-type cells (compare lanes 13 and 10 in Figure 7B, and lanes 13 and 12 in Figure S6). Is there any explanation for this observation? The protease protection assay (with detergent control) is needed to fully confirm that preSu9-Rad6-alpha-SL17 is present in the mitochondria.

    In Figure S7, the authors presented the matrix proteins, Ilv5 and Aco1, detected in the preSu9-HA-Ubi IPed samples and described this observation in the main text. However, the authors also showed the blots for Idh1 and Fum1, which were also pulled down with preSu9-HA-Ubi from the WT cells more than from the rad6-delta cells. Is this correct? If so, please elucidate this observation in the main text.

    Figure 8D and 8E are not cited in the main text. Although there are no explanations for these figures in the main text, it looks like Rad6-deltaN11-alpha resides in the mitochondrial fraction. However, the alpha-complementation assay suggests that it resides in the cytosol. Please explain this discrepancy.

    First page of the discussion section, item 6): E2 Rad6, but not E3 Rad6?

    Figure S7: HA-Ub (cytosolic form) control is needed in addition to the empty vector control.

    Figure S7, left panel: There is an unnecessary line break in "Hsp60" and "Ilv5."

    Figure S7, right panel: There is an unnecessary line break in "Hsp60."

    CROSS-CONSULTATION COMMENTS I agree with comments of reviewer 1 and 2. -Validation of the complementation assay. -I also think that it is important to address whether intra-mitochondrial ubiquitination can be observed with endogenous level of ubiquitin. If even a small amount of preSu9-HA-Ub is mistargeted to the cytosol, proteins at the cytosolic side of mitochondrial outer membrane could be ubiquitinated and detected in the mitochondrial fraction. -Preparation of mitochondria with more sophisticated purification methods (i.e. high resolution density gradient) would be needed to separate mitochondria from ER and other organelles. -More information is needed in the materials and methods section.

    Reviewer #3 (Significance (Required)): Significance Although the results are interesting and very important, as mentioned in the major comments section, additional experiments are needed to support their model. However, researchers working on the mitochondrial biology and ubiquitin systems might be interested in and influenced by the reported findings.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this study, the authors detected a set of components of a ubiquitination system in the mitochondrial matrix in budding yeast using the subcellular compartment-dependent α-complementation assay. The authors detected the conjugates of mitochondrial targeting signal sequence-directed HA-Ub (preSu9-HA-Ub) in the mitochondrial matrix. The immunoprecipitates of the preSu9-HA-Ubi conjugates were highly enriched for the mitochondrial matrix proteins. Subsequently, the authors focused on the Rad6 E2 ubiquitin conjugating enzyme in the mitochondrial matrix and evaluated its inactivation-altered ubiquitination pattern in the organelle. The authors conclude that ubiquitination occurs in the mitochondrial matrix because of the eclipsed targeted components of the ubiquitination machinery.

    Major comments:

    The authors argued that the proteins that were modified with preSu9-HA-Ubi, which was forced to be imported into the mitochondria, are present in the mitochondrial matrix, because these species are resistant to trypsin digestion. However, it was possible that they formed severe aggregates on the cytosolic surface of the mitochondria, and hence, were resistant to the proteinase. In other words, a small amount of proteins that were not imported into the mitochondria could be deposited on the cytosolic surface of the mitochondria, where they were modified with preSu9-HA-Ubi by cytosolic Rad6. To confirm if the preSu9-HA-Ubi-modified proteins were really present in the mitochondrial matrix, they should perform the protease protection assay in the presence of an appropriate detergent (Figure 3D). In addition, subcellular fractionation of the organelle by density gradient centrifugation, indirect immunofluorescence microscopic analysis of the preSu9-HA-Ubi conjugates, and/or experiments on the in vitro import of preSu9-HA-Ubi and Rad6 into the mitochondria would strongly support the authors conclusion. Other experiments that might support the authors conclusion would be to test whether the band pattern for the preSu9-HA-Ubi conjugates changes when the mitochondrial import is impaired.

    Minor comments:

    • In Figure 3B, the molecular weight distributions of the preSu9-HA-Ubi conjugates and those of the preFum-HA-Ubi conjugates are different. Is there any reason for this difference?

    • In Figure 3E, the position of "-" (MG132) for lane 1 is not correct.

    • In Figure 6A: The band pattern for preSu9-HA-Ubi (lane 13) in the rad6-delta cells expressing Ubc8-alpha is different from that of the wild-type cells expressing Ubc8-alpha (lane 12) as well as that obtained from the rad6-delta cells harboring empty plasmids (lane 9). Is there any explanation for this observation?

    • In Figure 7B and S6: The level of preSu9-Rad6-alpha-SL17 in the rad6-delta cells is always lower than that in the wild-type cells (compare lanes 13 and 10 in Figure 7B, and lanes 13 and 12 in Figure S6). Is there any explanation for this observation? The protease protection assay (with detergent control) is needed to fully confirm that preSu9-Rad6-alpha-SL17 is present in the mitochondria.

    • In Figure S7, the authors presented the matrix proteins, Ilv5 and Aco1, detected in the preSu9-HA-Ubi IPed samples and described this observation in the main text. However, the authors also showed the blots for Idh1 and Fum1, which were also pulled down with preSu9-HA-Ubi from the WT cells more than from the rad6-delta cells. Is this correct? If so, please elucidate this observation in the main text.

    • Figure 8D and 8E are not cited in the main text. Although there are no explanations for these figures in the main text, it looks like Rad6-deltaN11-alpha resides in the mitochondrial fraction. However, the alpha-complementation assay suggests that it resides in the cytosol. Please explain this discrepancy.

    • First page of the discussion section, item 6): E2 Rad6, but not E3 Rad6?

    • Figure S7: HA-Ub (cytosolic form) control is needed in addition to the empty vector control.

    • Figure S7, left panel: There is an unnecessary line break in "Hsp60" and "Ilv5."

    • Figure S7, right panel: There is an unnecessary line break in "Hsp60."

    CROSS-CONSULTATION COMMENTS

    I agree with comments of reviewer 1 and 2.

    • Validation of the complementation assay.
    • I also think that it is important to address whether intra-mitochondrial ubiquitination can be observed with endogenous level of ubiquitin. If even a small amount of preSu9-HA-Ub is mistargeted to the cytosol, proteins at the cytosolic side of mitochondrial outer membrane could be ubiquitinated and detected in the mitochondrial fraction.
    • Preparation of mitochondria with more sophisticated purification methods (i.e. high resolution density gradient) would be needed to separate mitochondria from ER and other organelles.
    • More information is needed in the materials and methods section.

    Significance

    Significance

    Although the results are interesting and very important, as mentioned in the major comments section, additional experiments are needed to support their model. However, researchers working on the mitochondrial biology and ubiquitin systems might be interested in and influenced by the reported findings.

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    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript by Yu et al., the authors test the concept that certain proteins are unevenly distributed within distinct cell compartments. Due to this localization discrepancy, protein detection in some subcellular compartments can be "eclipsed" by a predominant subset of specific protein localizing in another cell compartment their actual distribution. Therefore, tiny amounts of physiologically relevant proteins could be biologically relevant. Still, their function in some locations can be overlooked (or eclipsed) because of the high expression level of the same protein in another subcellular compartment(s). Although, this concept is not particularly novel. For example, it is already known that many different proteins can localize to distinct cellular locations (e.g., permanent mitochondrial and peroxisomal localization of many proteins or transient localization of particular proteins to separate cell compartments). The authors apply a yeast system and an α-complementation assay to test further the role of such eclipsed proteins in mitochondrial biology. Specifically, they focus on the ubiquitin (Ub, or as abbreviated incorrectly in this manuscript; Ubi) conjugation pathway, components of which have never been convincingly shown to localize inside the mitochondria. This work proposes that certain ubiquitination events can occur inside yeast mitochondria. This work would represent a significant/exceptional discovery if supported by compelling data. However, the major problem with this work is that the conclusions are based on the ectopic expression of distinct proteins. This approach is not failproof in precise protein expression/delivery to the specific subcellular locations and is likely to result in a non-specific localization. Thus, the problem of eclipsed proteins is addressed by the methodology that may lead to the artificial generation of eclipsed overexpressed proteins. A more effective approach would be if the authors found a way to study this issue with endogenous proteins. The need for overexpression of mitochondria-targeted ubiquitin makes it challenging to reconcile the physiological role of these fundings. In addition, some critical technical issues and omissions further reduce the potential impact of this work (see Specific comments below). For example, strong evidence of mitochondria fraction purity and additional evidence that all the essential constructs used in this work are not misdirected to a different compartment are needed.

    Specific comments

    1. The authors should demonstrate beyond doubt that the ω components of their assay (ω-C, which supposedly stays in the cytosol-ONLY and the ω-M component, which seemingly remains in the mitochondria-ONLY) are in the compartment that the authors claim. These two proteins are transfected into yeast cells and overexpressed. Therefore it is possible that they leak to other, not intended, subcellular compartments. The authors assume that ω-M and ω-C are exclusively located either in the mitochondria or the cytosol. However, this should be shown as validation of the assay. The indicated reference from 2005 (Ref.13) and others are irrelevant since assays have variations and are often researcher/lab dependent. This validation is very important since a misallocation of the overexpressed ω-M or ω-C, leaking into other subcellular compartments, may cause misdetection of the α-constructs.
    2. It is not surprising that Ub conjugates are detected in mitochondrial fractions. It could be due to ubiquitination of the OMM (coming from the cytosol) or perhaps since the subcellular fractions were not pure mitochondria free from contamination (the likely culprit could be the ER). The mitochondrial fractions in this work were obtained by 10,000 g separation between cytosolic and mitochondrial crude fractions. Indeed, these 10,000 g crude fractions are highly impure with membranes from other compartments (i.e., microsomes, lysosomes, and so on). Therefore, more sophisticated purification methods should be used. In addition, the authors should also test these fractions for non-mitochondrial proteins from other membrane organelles.
    3. Figure 2. Coomassie blue staining does not show any signal in the "M" fraction. It can be interpreted that the authors do not get any mitochondria there, and therefore the lack of Ub signal is due to the absence of the protein in the samples. Using the same amount of protein from each fraction would probably reduce the necessity of 15x enrichment.
    4. Figure 3. It is puzzling why the HA-UBQ presence is so strong in the crude mitochondrial fraction, but the preSu9-HA-Ub signal (mito-matrix) is comparatively weak. These data suggest that the crude mito-fraction could be highly contaminated with OTHER membranes. On the other hand, the preSu9-HA-UBQ signal is no more than 1-5% of the total mitochondrial signal. The high enrichment of the HA-Ubi in both cytosols and the mitochondria could indicate the OMM ubiquitination or (again) contamination by other compartments. The constructs with MTS are detected in the mitochondria. However, the localization of tagged MTS-Ubi in a non-targeted compartment (e.g., cytosol) should be excluded by additional exposure times. Because the manuscript talks about eclipsed proteins, this is important.
    5. Figure 3C-E. These data indeed suggest that the Ub-conjugates could be formed inside the mitochondria. However, the above-discussed possibility that other than mitochondria compartments co-sediment in the 10,000g fractions makes the data interpretation highly challenging.
    6. Figure 4. Unsurprisingly, mitochondrial targeting of Ub leads to detecting some co-immunoprecipitating mitochondrial proteins. However, these data do not support the notion that Ub conjugation machinery acts inside the mitochondria and that the target proteins are indeed conjugated with Ub (the interaction with Ub is not equal to being conjugated). At the minimum, the authors should provide a validation that some of the detected mitochondrial matrix proteins are indeed ubiquitinated. To this end, purified mitochondria could be used for the candidate protein IP under denaturing conditions and then blotted for the candidate protein and Ub.
    7. Figure 5. The knock-out of the E2 Rad6 causes a change in the mitochondria ubiquitination pattern. This is an interesting observation, but again it does not prove that the change in the mitochondrial ubiquitination is due to the activity of Rad6 inside of the mitochondria, as opposed to ubiquitination of the OMM proteins or contaminating fractions. One also wonders why overexpression of mitochondria-targeted Ub would be necessary to detect the ubiquitination if this process was physiologically relevant, especially given that detecting endogenous Ub is not challenging. Furthermore, the apparent increase in ubiquitination in E2 mutant cells (Fig. 5) should also be addressed in more detail. Finally, data from one WB is shown, and quantification of several independent experiments should also be provided.
    8. Figure 6. Can the authors provide Western blot data showing the expression of Rad6? Furthermore, quantifying these rescue experiments is necessary to make this conclusion more solid.
    9. Figure 7. The authors found that preSu9-Rad6-α have problems being imported into the mitochondria matrix; therefore, they rebuild it as a preSu9-Rad6-α-SL17 protein. SL17 is a degron that targets the cytosolic protein (not imported into the mitochondria) to the proteasome and degraded (Figs. 7A-B-C). These issues could be a red flag for the rest of the manuscript, suggesting that other constructs (that were not critically evaluated for their localization in this work) could leak to different cellular compartments.
    10. The manuscript needs to be carefully edited, some references are in the not correct format, and there are issues with figure labels.

    CROSS-CONSULTATION COMMENTS

    I agree with a great summary by reviewer 1. This discovery should be validated by top-quality data.

    Significance

    In the manuscript by Yu et al., the authors test the concept that certain proteins are unevenly distributed within distinct cell compartments. Due to this localization discrepancy, protein detection in some subcellular compartments can be "eclipsed" by a predominant subset of specific protein localizing in another cell compartment their actual distribution. Therefore, tiny amounts of physiologically relevant proteins could be biologically relevant. Still, their function in some locations can be overlooked (or eclipsed) because of the high expression level of the same protein in another subcellular compartment(s). Although, this concept is not particularly novel. For example, it is already known that many different proteins can localize to distinct cellular locations (e.g., permanent mitochondrial and peroxisomal localization of many proteins or transient localization of particular proteins to separate cell compartments). The authors apply a yeast system and an α-complementation assay to test further the role of such eclipsed proteins in mitochondrial biology. Specifically, they focus on the ubiquitin (Ub, or as abbreviated incorrectly in this manuscript; Ubi) conjugation pathway, components of which have never been convincingly shown to localize inside the mitochondria. This work proposes that certain ubiquitination events can occur inside yeast mitochondria. This work would represent a significant/exceptional discovery if supported by compelling data. However, the major problem with this work is that the conclusions are based on the ectopic expression of distinct proteins. This approach is not failproof in precise protein expression/delivery to the specific subcellular locations and is likely to result in a non-specific localization. Thus, the problem of eclipsed proteins is addressed by the methodology that may lead to the artificial generation of eclipsed overexpressed proteins. A more effective approach would be if the authors found a way to study this issue with endogenous proteins. The need for overexpression of mitochondria-targeted ubiquitin makes it challenging to reconcile the physiological role of these fundings. In addition, some critical technical issues and omissions further reduce the potential impact of this work (see Specific comments above). For example, strong evidence of mitochondria fraction purity and additional evidence that all the essential constructs used in this work are not misdirected to a different compartment are needed.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Zhang et al. investigate whether ubiquitination occurs inside mitochondria of the budding yeast S. cerevisiae. They first observe thanks to a sensitive complementation assay that several components of the yeast ubiquitination (and deubiquitination) machinery can localize inside mitochondria. To be able to specifically probe ubiquitin conjugates assembled inside mitochondria they fused HA-tagged ubiquitin to a mitochondrial targeting sequence. Using this construct, they demonstrate that ubiquitin conjugates can be assembled in mitochondria. A series of elegant experiments demonstrates that the pattern of ubiquitin conjugates depends on the mitochondrial localization and the activity of the ubiquitin conjugating enzyme Rad6. Altogether, these results convincingly demonstrate that ubiquitination can occur inside yeast mitochondria when ubiquitin is intentionally targeted inside this organelle. It however remains unclear whether mitochondrial ubiquitination occurs in endogenous conditions (without targeting ubiquitin into this compartment) and whether it affects mitochondrial functions.

    Major comments:

    1. The materials and methods section is lacking important information (western blot protocol, details of antibodies, strains, plasmids...). It is thus difficult to evaluate how several experiments were performed and how their design (e.g. the promoters chosen to express tagged proteins) could impact the interpretation of the results. This is a major issue that needs to be corrected. The main text should also explicitly indicate whether tagged proteins used in the alpha-complementation assay are overexpressed or not.

    2. Despite the previous comment, the data presented in the manuscript convincingly demonstrate that multiple components of the ubiquitination machinery can localize within mitochondria and that ubiquitin conjugates can be assembled in mitochondria when ubiquitin is modified to be intentionally targeted into this compartment. However, little data is shown to support the hypothesis that ubiquitin conjugates can be assembled in mitochondria when ubiquitin is not fused to a mitochondrial targeting sequence. Thus, in my opinion, the evidences presented in the current manuscript are not sufficient to conclude that ubiquitin conjugates are assembled in mitochondria in endogenous conditions (as this is done implicitly). Additional evidences are needed to draw this conclusion (see some experimental suggestions hereafter). Without further evidences, the speculative aspects of the claim that "ubiquitination occurs in the mitochondrial matrix" should be discussed explicitly.

    3. The authors used a mass spectrometry approach to identify mitochondrial ubiquitination substrates. However, they have not yet succeeded in identifying a substrate whose modification is specifically regulated by a given component of the mitochondrial ubiquitination machinery. They have also not identified a phenotype or process impacted by mitochondrial ubiquitination. Thus, at this stage, the biological consequences of mitochondrial ubiquitination remain elusive.

    4. The authors have not directly investigated whether ubiquitin itself (without a mitochondrial targeting sequence) localizes in mitochondria. I encourage them to address this question since it would provide an important piece of evidence suggesting that mitochondrial ubiquitination can occur in endogenous conditions. This could be done using the alpha-complementation assay and the results could be presented within Figure 1. Ideally this experiment should be performed without overexpressing ubiquitin. Note that if the authors decide to use a C-terminally tagged form of ubiquitin for this experiment, the GG motif of ubiquitin should be mutated to avoid cleavage of the alpha tag by cellular DUBs. This form of ubiquitin will not be conjugatable, but this is not an issue for this experiment since its aim is to determine whether ubiquitin can be targeted to mitochondria, not to probe conjugates.

    5. In the top panels of Figure 2 and S1, free ubiquitin is well detectable in the total and cytosolic fractions. It is however not clear to me whether it is also detectable in the concentrated mitochondrial fraction. If yes and if it would be resistant to trypsin digestion, it would provide an additional evidence that endogenous ubiquitin can be targeted to the mitochondrial matrix (see previous comment).

    6. The data shown in the top panel of Figure 2 and S1 also suggest that free ubiquitin is less concentrated in mitochondria than in the cytosol (since it is more difficult to detect in the concentrated mitochondrial fraction than in the cytosolic fraction, see previous comment). It is thus possible that the use of preSu9-HA-Ubi (or preFum1-HA-Ubi) lead to an artificially high intra-mitochondrial concentration of free ubiquitin. As the concentration of free ubiquitin is known to impact ubiquitination processes, I encourage the authors to compare the relative levels of free ubiquitin present in the mitochondrial fraction prepared from wt and preSu9-HA-Ubi (or preFum1-HA-Ubi) expressing cells. If free ubiquitin is detectable in mitochondrial fractions and resistant to trypsin (see previous comment), this could be done by repeating the experiment shown in Figure 3B and probing the blot with an antibody that recognizes free ubiquitin.

    7. I strongly encourage the authors to provide more data indicating that "ubiquitination occurs in mitochondria" by performing experiments that do not rely on the use of the preSu9-HA-Ubi or other forms of ubiquitin that are intentionally targeted to mitochondria. For instance, they could analyse the pattern of HA-Ubi conjugates of trypsin digested mitochondrial fractions prepared from wt, rad6-delta, and rad6-delta complemented with preSu9-Rad6-alpha-SL17. Note that if trypsin digested mitochondrial fractions are too contaminated by ubiquitinated proteins present outside mitochondria to perform this experiment, the authors may use the unspecific DUB Usp2 as an alternative protease to strip ubiquitinated proteins from the mitochondria periphery.

    Minor comments:

    1. Overall, the manuscript is well organized and easy to follow. The text is clearly written; the figures are well annotated.

    2. The authors should provide full images of all the blots with anti-ubiquitin and anti-HA antibodies so that one can see the bands corresponding to free ubiquitin (or free HA-Ubi). For instance, in Figure 3B, it is not possible to see the presence (or absence) of the band corresponding to free HA-Ubi because the very bottom of the image is cut.

    3. The authors should indicate whether the MTS of Su9 (and Fum1) are expected to be cleaved after import of preSu9-HA-Ubi (and preFum1-HA-Ubi) in mitochondria. They should also label on the corresponding immunoblots the presence (or absence) of the band corresponding to the free preSu9-HA-Ubi (and preFum1-HA-Ubi) (or HA-Ubi if the MTS is expected to be cleaved from these constructs).

    4. In Figure 3B, the ubiquitin conjugates produced with preSu9-HA-Ubi and preFum1-HA-Ubi have different migration patterns. I think this should be explicitly mentioned and discussed. Could it be due to the presence of lysine residues in the Su9 or Fum1 MTS that could lead to the assembly of artificial ubiquitin chains?

    5. The authors indicate that "endogenous Rad6 [...] is expressed at very low levels and can hardly be detected in the mitochondrial fraction by WB (Figure S5)". I did not manage to observe the band corresponding to endogenous Rad6 in the mitochondrial fraction in the pdf. The authors should provide a more contrasted or better quality image.

    CROSS-CONSULTATION COMMENTS

    • I agree with reviewer 2 that proper validation of the complementation assay is crucial for this manuscript. I was myself wondering whether it uses endogenously tagged proteins or whether it is based on an overexpression system. I imagine this information will be detailed in the manuscript in preparation mentioned by the authors. I am therefore wondering whether it would be possible to ask the authors to provide the draft of this manuscript (or at least the validation part).

    • I agree with most comments of reviewer 3. Regarding the hypothesis that preSu9-HA-Ubi could form aggregates on the cytosolic surface of the mitochondria, I think that the results presented on Figure 7B rather argue against it (since they indicate that Rad6 localized inside mitochondria can restore the pattern of ubiquitin conjugates). That's why (in my opinion) the major question the author now need to adress is whether intra-mitochondrial ubiquitination occurs in endogenous conditions (ie without forcing ubiquitin into this compartment and without E2 or E3 overexpression).

    Significance

    The finding that ubiquitination occurs inside mitochondria would be an important conceptual advance, which would open new perspectives both for ubiquitination and mitochondrial biology research. However, the significance of the current manuscript is limited because the presented evidences heavily rely on the use of artificial conditions (ubiquitin tagged with a mitochondrial-targeting sequence) that may trigger irrelevant ubiquitination events. The significance would be much higher if the authors would provide further evidences indicating that intra-mitochondrial ubiquitination occurs in endogenous conditions and/or if they had identified a mitochondrial process specifically impacted by mitochondrial ubiquitination.

    Expertise of the reviewer: Ubiquitination, Yeast biology, protein-protein interactions. No specific expertise in mitochondrial biology

  7. Version published to 10.1101/2022.07.10.499450v1 on bioRxiv