Inhibition of mitochondrial protein import and proteostasis by a pro-apoptotic lipid

Reviewer #2 (Public Review):

This study elucidates the toxic effects of the lipid aldehyde trans-2-hexadecenal (t-2-hex). The authors show convincingly that t-2-hex induces a strong transcriptional response, leads to proteotoxic stress and causes the accumulation of mitochondrial precursor proteins in the cytosol.

The data shown are of high quality and well-controlled. The genetic screen for mutants that are hyper-and hypo-sensitive to t-2-hex is elegant and interesting, even if the mechanistic insights from the screen are rather limited. Moreover, the authors show evidence that t-2-hex affects subunits of the TOM complex. However, they do not formally demonstrate that the lipidation of a TOM subunit is responsible for the toxic effect of t-2-hex. A t-2-hex-resistant TOM mutant was not identified. Nevertheless, this is an interesting and inspiring study of high quality. The connection of proteostasis, mitochondrial biogenesis and sphingolipid metabolism is exciting and will certainly lead to many follow-up studies.

Reviewer #3 (Public Review):

Summary: The authors investigate the effect of high concentrations of the lipid aldehyde trans-2-hexadecenal (t-2-hex) in a yeast deletion strain lacking the detoxification enzyme. Transcriptomic analyses as global read out reveal that a large range of cellular functions across all compartments are affected (transcriptomic changes affect 1/3 of all genes). The authors provide additional analyses, from which they built a model that mitochondrial protein import caused by modification of Tom40 is blocked.

Strengths:
Global analyses (transcriptomic and functional genomics approach) to obtain an overview of changes upon yeast treatment with high doses of t-2-hex.

Weaknesses:
The use of high concentrations of t-2-hex in combination with a deletion of the detoxifying enzyme Hfd1 limits the possibility to identify physiological relevant changes. From the hundreds of identified targets the authors focus on mitochondrial proteins, which are not clearly comprehensible from the data. The main claim of the manuscript that t-2-hex targets the TOM complex and inhibits mitochondrial protein import is not supported by experimental data as import was not experimentally investigated. The observed accumulation of precursor proteins could have many other reasons (e.g. dissipation of membrane potential, defects in mitochondrial presequence proteases, defects in cytosolic chaperones, modification of mitochondrial precursors by t-2-hex rendering them aggregation prone and thus non-import competent). However, none of these alternative explanations have been experimentally addressed or discussed in the manuscript.
Furthermore, many of the results have been reported before (interaction of Tom22 and Tom70 with Hfd1) or observed before (TOM40 as target of t-2-hex in human cells).

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Fita-Torró et al. study the toxic effects of the intermediary lipid degradation product trans-2-hexadecenal (t-2-hex) on yeast mitochondria and suggest a mechanism by which Hfd1 safeguards Tom40 from lipidation by t-2-hex and its consequences, such as mitochondrial protein import inhibition, cellular proteostasis deregulation, and stress-responses.

The authors aimed to dissect a mechanism for t-2-hex' apoptotic consequences in yeast and they suggest it is via lipidation of Tom40 but really under the tested conditions everything seems lipidated. Thus, it is unclear whether Tom40 is the crucial causal target. They also do not provide much biochemical experiments to investigate this phenomenon further functionally. Tom40 is one possible and perhaps, given the cellular consequences, a reasonable candidate but not validated beyond in vitro lipidation by exogenous t-2-hex.

In the revised version of our manuscript, we have now included extensive new experimentation, which shows that protein import at the TOM complex is a physiologically important target of the pro-apoptotic lipid t-2-hex and that enzymes such as the Hfd1 dehydrogenase sensitively regulate this inhibition. In vitro chemoproteomic experiments have now been performed at more physiological t-2hex concentrations of 10µM, which is lower than published data in human cell models. Consistently, several TOM and TIM subunits are enriched in these in vitro lipidation studies (new Fig. 8B). Tom40 lipidation alone is not sufficient to explain t2-hex toxicity, as a cysteine-free version of Tom40 does not confer tolerance to the apoptotic lipid (new Fig. 8D). Importantly however, the loss of function of nonessential accessory Tom subunits 70 or 20 confers t-2-hex tolerance (new Fig. 8D) indicating that pre-protein import at the TOM complex is a physiological target of t2-hex most likely dependent on lipidation of more Tom subunits than just the essential Tom40 pore. Moreover, we now show that mitochondrial protein import is inhibited by the lipid at low physiological doses of 10µM and that this inhibition is modulated by the gene dose of the t-2-hex degrading Hfd1 enzyme (new Fig. 5G).

Strengths:

The effects of lipids and their metabolic intermediates on protein function are understudied thus the authors' research contributing to elucidating direct effects of a single lipid is appreciated. It is particularly unknown by which mechanism t-2hex causes cell death in yeast. The authors elegantly use modulation of the levels of enzyme Hfd1 that endogenously catabolizes t-2-hex as an approach to studying t2-hex stress. Understanding the cause and consequences of this stress is relevant for understanding fundamental regulation mechanisms, and also to human health since the human homolog of Hfd1, ALDH3A2, is mutated in Sjögren-Larsson Syndrome. The application of a variety of global transcriptomic, functional genomic, and chemoproteomic approaches to study t-2-hex stress targets in the yeast model is laudable.

Weaknesses:

- The extent of the contribution of Tom40 lipidation to the general t-2-hex stress phenotype is unclear. Is Tom40 lipidation alone enough to cause the phenotype? An alteration of the cysteine residue in question could help answer this key question.

Deletion of all four cysteine residues in Tom40 is not sufficient to confer resistance to t-2-hex stress. This result had been included in the original manuscript, but was somehow hidden in the Discussion. The revised manuscript now includes t-2hex tolerance assays for the Tom40 cysteine free mutant in new Figure 8. As a result, cysteine lipidation of Tom40 alone is not sufficient to confer t-2-hex toxicity. This implies most likely other lipidation targets within the TOM and TIM complexes, as indicated by our in vitro lipidation studies. We therefore included the non-essential adaptor proteins Tom70 and Tom20 of the TOM complex and tested the tolerance of the respective deletion mutants in t-2-hex tolerance assays. As shown in new Figure 8, the absence of Tom70 and Tom20 function significantly increases tolerance to t-2hex and the tom20 mutant accumulates less Aim17 pre-protein upon t-2-he stress, indicating that the TOM complex is a physiologically important target of the proapoptotic lipid, which acts most likely via lipidation of more subunits than the Tom40 import channel.

- It is unclear whether the exogenously applied amounts of t-2-hex (concentrations chosen between 25-200 uM) are physiologically relevant in yeast cells. For comparison, Chipuk et al. (2012) used at most 1 uM on mitochondria of human cells, while Jarugumilli et al. (2018) considered 25 uM a 'lower dose' on human cells. Since the authors saw responses below 10 uM (Fig. 3B) and at the lowest selected concentration of 25 uM (Fig. 8), why were no lower, likely more specific, concentrations applied for the global transcriptomic and chemoproteomic experiments? Key experiments have to be repeated with the lower concentrations.

We have now performed several experiments with lower t-2-hex concentrations. A new chemoproteomic study with 10µM t-2-hex-alkyne has been conducted and the new results added to the supplementary information, combining 10µM and 100µM in vitro lipidation studies (Suppl. Table 6). Many subunits of the TOM and TIM complexes consistently are enriched significantly in both chemoproteomic experiments. These new data are summarized in revised Figure 8. Additionally we have performed in vivo pre-protein assays with lower t-2-hex concentrations. As shown in new Figure 5, Aim17 mitochondrial import is already inhibited by t-2-hex doses as low as 10µM in a wild type strain, and that this inhibition is enhanced in a hfd1 mutant and alleviated in a Hfd1 overexpressor. It is important to note that a dose of 10µM of external t-2-hex addition is significantly lower than doses applied to human cell cultures such as in Jarugumilli et al. (2018). It proves that mitochondrial protein import is a sensitive and physiologically relevant t2-hex target in our yeast models and that t-2-hex detoxification by enzymes such as the Hfd1 dehydrogenase sensitively regulates this specific inhibition.

- The amount of t-2-hex applied is especially important to consider in light of over 1300 proteins lipidated to an extent equal to or greater than Tom40 (Supp. Table 6). This chemoproteomic experiment (Fig. 8B, Supp. Table 6) is also weakened by the inclusion of only 2 replicates, thus precluding assessment of statistical significance. The selection of targets in Fig. 8B as "among the best hits" is neither immediately comprehensible nor further explained and represents at best cherrypicking. Further evidence based on statistical significance or validation by other means should be provided.

We performed the chemoproteomic screens as described by Jarugumilli et al. (2018) with 2 replicates of mock treated versus 2 replicates of t-2-hex-alkyne treated cell extracts. A new chemoproteomic study with 10µM t-2-hex-alkyne has been conducted and the new results added to the supplementary information combining 10µM and 100µM in vitro lipidation studies (Suppl. Table 6). Differential enrichment analysis of the proteomic data was performed with the amica software (Didusch et al., 2022). Proteins were ranked according to their log2 fold induction comparing lipid- and mock-treated samples with a threshold of ≥1.5, and the adjusted p-value was calculated. Several TOM and TIM subunits were consistently identified as differentially enriched proteins, which is summarized in new Figure 8B.

- The authors unfortunately also underuse the possible contribution of mass spectrometry technology to in addition determine the extent and localization of lipidation on a global scale (especially relevant since Cohen et al. (2020) suggest site-specific mechanisms).

We agree that site-specific modifications of t-2-hex will be most likely important in the inhibition or other type of regulation of specific target proteins. Our collective data show that in the case of the inhibition of mitochondrial protein import, several lipidation events on TOM and TIM are involved. Dissection of individual cysteine lipidations on those subunits will be interesting, but we feel that this is out of the scope of the present work.

- The general novelty of studying t-2-hex stress is lowered in light of existing literature in humans (see e. g. Chipuk et al., 2012; Cohen et al., 2020; Jarugumilli et al., 2018), and in yeast by the same authors (Manzanares-Estreder et al., 2017) and as the authors comment themselves, a significant part of the manuscript may represent rather a confirmation of the already described consequences of t-2-hex stress

We do not agree and we have not commented that our present study is a mere confirmation of t-2-hex stress previously applied in yeast and human models. In humans, t-2-hex has been identified as an efficient pro-apoptotic lipid, which causes mitochondrial dysfunction via direct lipidation of Bax, however the studies of Jarugumilli et al. (2018) revealed that many other direct t-2-hex targets exist, which remained uninvestigated to date. This work continues our previous studies (Manzanares-Estreder et al., 2017), where we show that t-2-hex is a universal proapoptotic lipid applicable in yeast models and contributes important novel findings, such as the massive transcriptional response resembling proteostatic defects caused by t-2-hex, mitochondrial protein import as a physiologically important and direct target of t-2-hex, the function of detoxifying enzymes such as Hfd1 in modulating lipid-mediated inhibition of mitochondrial protein import and general proteostasis. Additionally, we provide transcriptomic, chemoproteomic and functional genomic data to the scientific community, which will be a rich source for future studies on yet undiscovered pro-apoptotic mechanisms employed by t-2-hex.

Reviewer #2 (Public Review):

This study elucidates the toxic effects of the lipid aldehyde trans-2-hexadecenal (t-2-hex). The authors show convincingly that t-2-hex induces a strong transcriptional response, leads to proteotoxic stress, and causes the accumulation of mitochondrial precursor proteins in the cytosol.

The data shown are of high quality and well controlled. The genetic screen for mutants that are hyper-and hypo-sensitive to t-2-hex is elegant and interesting, even if the mechanistic insights from the screen are rather limited. The last part of the study is less convincing. The authors show evidence that t-2-hex affects subunits of the TOM complex. However, they do not formally demonstrate that the lipidation of a TOM subunit is responsible for the toxic effect of t-2-hex. A t-2-hexresistant TOM mutant was not identified. Moreover, it is not clear whether the concentrations of t-2-hex in this study are physiological. This is, however, a critical aspect. The literature is full of studies claiming the toxic effects of compounds such as H2O2; even if such studies are technically sound, they are misleading if nonphysiological concentrations of such compounds were used.

Nevertheless, this is an interesting study of high quality. A few specific aspects should be addressed.

We have now performed t-2-hex toxicity assays using several mutants in Tom subunits, the cysteine free mutant of the essential Tom40 core channel and deletion mutants in the accessory subunits Tom70 and Tom20 (new Figure 8). As a result, cysteine lipidation of Tom40 alone is not sufficient to confer t-2-hex toxicity. This implies most likely other lipidation targets within the TOM and TIM complexes, as indicated by our in vitro lipidation studies. Indeed, as shown in new Figure 8, the absence of Tom70 and Tom20 function significantly increases tolerance to t-2-hex indicating that the TOM complex is a physiologically important target of the proapoptotic lipid, which acts most likely via lipidation of more subunits than the Tom40 import channel.

We have now performed several experiments with lower t-2-hex concentrations. A new chemoproteomic study with 10µM t-2-hex-alkyne has been conducted and the new results added to the supplementary information combining 10µM and 100µM in vitro lipidation studies (Suppl. Table 6). Many subunits of the TOM and TIM complexes consistently are enriched significantly in both chemoproteomic experiments. These new data are summarized in revised Figure 8.

Additionally we have performed in vivo pre-protein assays with lower t-2-hex concentrations. As shown in new Figure 5, Aim17 mitochondrial import is already inhibited by t-2-hex doses as low as 10µM in a wild type strain, and that this inhibition is enhanced in a hfd1 mutant and alleviated in a Hfd1 overexpressor. It is important to note that a dose of 10µM of external t-2-hex addition is significantly lower than doses applied to human cell cultures such as in Jarugumilli et al. (2018). It proves that mitochondrial protein import is a sensitive and physiologically relevant t2-hex target in our yeast models and that t-2-hex detoxification by enzymes such as the Hfd1 dehydrogenase sensitively regulates this specific inhibition.

Reviewer #3 (Public Review):

Summary: The authors investigate the effect of the lipid aldehyde trans-2hexadecenal (t-2-hex) in yeast using multiple omic analyses that show that a large range of cellular functions across all compartments are affected, e.g. transcriptomic changes affect 1/3 of all genes. The authors provide additional analyses, from which they built a model that mitochondrial protein import caused by modification of Tom40 is blocked.

Strengths: Global analyses (transcriptomic and functional genomics approach) to obtain an unbiased overview of changes upon t-2-hex treatment.

Weaknesses: It is not clear why the authors decided to focus on mitochondria, as only 30 genes assigned to the GO term "mitochondria" are increasing, and also the follow-up analyses using SATAY is not showing a predominance for mitochondrial proteins (only 4 genes are identified as hits). The provided additional experimental data do not support the main claims as neither protein import is investigated nor is there experimental evidence that lipidation of Tom40 occurs in vivo and impacts on protein translocation.

30 mitochondrial gene functions are very strongly (>10 fold) up-regulated by t-2-hex. However, when genes up-regulated (>2 log2FC) or down-regulated (<-2 log2FC) by t-2-hex were selected and subjected to GO category enrichment analysis, we found that “Mitochondrial organization” was the most numerous GO group activated by t-2-hex, while it was “Ribosomal subunit biogenesis” for t-2-hex repression (new data in Suppl. Tables 1 and 2).

In the revised version of our manuscript, we have now included extensive new experimentation, which shows that protein import at the TOM complex is a physiologically important target of the pro-apoptotic lipid t-2-hex and that enzymes such as the Hfd1 dehydrogenase sensitively regulate this inhibition. In vitro chemoproteomic experiments have now been performed at more physiological t-2hex concentrations of 10µM, which is lower than published data in human cell models. Consistently, several TOM and TIM subunits are enriched in these in vitro lipidation studies (new Fig. 8B). Tom40 lipidation alone is not sufficient to explain t2-hex toxicity, as a cysteine-free version of Tom40 does not confer tolerance to the apoptotic lipid (new Fig. 8D). Importantly however, the loss of function of nonessential accessory Tom subunits 70 or 20 confers t-2-hex tolerance (new Fig. 8D) indicating that pre-protein import at the TOM complex is a physiological target of t2-hex most likely dependent on lipidation of more Tom subunits than just the essential Tom40 pore. Moreover, we now show that mitochondrial protein import is inhibited by the lipid at low physiological doses of 10µM and that this inhibition is modulated by the gene dose of the t-2-hex degrading Hfd1 enzyme (new Fig. 5G).

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Private recommendations for the authors

- On the existing data from Supp. Table 6, the authors may include a global assessment of whether or not the protein included a cysteine (the likely site for lipidation).

Although free cysteines in target proteins are the most frequent sites of modification by LDEs such as t-2-hex, other amino acids such as lysines or histidines can be lipidated by these lipid derivatives. Therefore we would like to exclude this information from our chemoproteomic data.

- What determines whether a gene is labeled in Fig. 6B other than fold change? Why is MAC1 with the highest FC not shown?

We analyzed the potential anti-apoptotic SATAY hits with a log2 < -0.75 according to expected detoxification pathways (heat shock response, pleiotropic drug response), to their function in the ER (the intracellular site where t-2-hex is generated) or in mitochondria (the major t-2-hex target identified so far). This is now better described in the text. As for the potential pro-apoptotic SATAY hits, we analyzed gene functions with a log2 > 1.5 and marked the predominant groups “Cytosolic ribosome and translation” and “Amino acid metabolism”. In any case, the interested reader has all SATAY data available in supplemental tables 4 and 5 to find alternative gene functions with a potential role in cellular adaptation to t-2-hex.

- Supplementary Table numbering should be double-checked.

Ok, numbering has been double-checked.

Reviewer #2 (Recommendations For The Authors):

Major points

(1) Identification of the t-2-hex target. Neither Tom70, Tom20 nor the cysteine in Tom40 is essential. If one of these components is critical for the t-2-hex-mediated toxicity, mutants should be t-2-hex-resistant. This is a straight-forward, simple, and critical experiment.

We have now performed t-2-hex toxicity assays in the cysteine free Tom40 mutant, and tom20 and tom70 deletion mutants. As shown in new Figure 8, cysteine lipidation of Tom40 alone is not sufficient to confer t-2-hex toxicity. However, the absence of Tom70 and Tom20 function significantly increases tolerance to t-2-hex indicating that the TOM complex is a physiologically important target of the proapoptotic lipid, which acts most likely via lipidation of more subunits than the Tom40 import channel.

(2) The authors claim that t-2-hex blocks the TOM complex. Since in vitro import assays with yeast mitochondria are a well established and simple technique, the authors should isolate mitochondria from their cells and perform import experiments. It is expected that those mitochondria show reduced import rates, however, swelling of these mitochondria to mitoplasts should suppress the import defect.

We agree that our study does not investigate a direct effect of t-2-hex on the import capacity of purified mitochondria. However, we determine the in vivo accumulation of several mitochondrial precursor proteins, which is widely used to assay for the efficiency of mitochondrial protein import, for example the recent hallmark paper discovering the mitoCPR protein import surveillance pathway exclusively uses epitope-tagged mitochondrial precursors to determine the regulation of mitochondrial protein import (Weidberg and Amon, Science 2018 360(6385)). Additionally, our new results that mutants in accessory TOM subunits 20 and 70 are hyperresistant to t-2-hex (Figure 8D) and that deletion of TOM20 decreases the t-2-hex induced pre-protein accumulation (Suppl. Figure 1) identify the TOM complex and hence protein import at the outer mitochondrial membrane as a physiologically important t-2-hex target.

(3) The first part of the study is very strong. The last figure is also of good quality, however, it is not clear whether the effects on TOM subunits are really causal for the observed t-2-hex effect on gene expression. The authors might cure this by improved data or by avoiding bold statements such as: 'Hfd1 associates with the Tom70 subunit of the TOM complex and t-2-hex covalently lipidates the central Tom40 channel, which altogether indicates that transport of mitochondrial precursor proteins through the outer mitochondrial membrane is directly inhibited by the pro-apoptotic lipid and thus represents a hotspot for pro- and anti-apoptotic signaling.' (Abstract).

We now show that several TOM and TIM subunits are lipidated in vitro by physiological low t-2-hex concentrations, that loss of function of accessory subunits Tom20 or Tom70 rescues t-2-hex toxicity (new Figure 8) and that the gene dose of Hfd1 determines the degree of mitoprotein import block (new Figure 5). These data identify the TOM complex as a physiologically important target of the pro-apoptotic lipid. The Abstract has been modified accordingly.

(4) If the t-2-hex levels are in a physiological range, one would expect that overexpression of Hfd1 prevents the t-2-hex-induced import arrest.

We have now confirmed that overexpression of Hfd1 indeed prevents inhibition of mitochondrial protein import by t-2-hex. As shown in new Figure 5, Aim17 mitochondrial import is already inhibited by t-2-hex doses as low as 10µM in a wild type strain, and that this inhibition is enhanced in a hfd1 mutant and alleviated in a Hfd1 overexpressor.

(5) The authors claim that Fmp52 is a t-2-hex-detoxifying enzyme, but do not show evidence. They should rewrite this sentence and be more cautious, or they should show that increased Fmp52 levels indeed deplete t-2-hex from mitochondria.

We show that loss of Fmp52 function leads to a strong t-2-hex sensitivity. Fmp52 belongs to the NAD-binding short-chain dehydrogenase/reductase (SDR) family and localizes to highly purified mitochondrial outer membranes (Zahedi et al, 2006). These are the indications that suggest that Fmp52 participates in the enzymatic detoxification of t-2-hex in addition to Hfd1. The Results section has been modified accordingly.

Minor points:

(6) Aim17 was recently identified as a characteristic constituent of cytosolic protein aggregates named MitoStores (Krämer et al., 2023, EMBO J). The authors might test whether the cytosolic Aim17 protein colocalizes with the Hsp104-GFP granules that accumulate upon t-2-hex exposure as shown in Fig. 4A.

We agree that determining the fate of unimported mitochondrial precursors upon t-2-hex stress would be interesting. We have made some attempts to co-visualize Aim17-dsRed and Hsp104-GFP upon t-2-hex treatment, but we still have some technical issues. While we clearly see that Aim17 accumulates in cytoplasmic foci upon prolonged t-2-hex exposure, we are not able to determine colocalization with Hsp104, in great part because t-2-hex causes mitochondrial fragmentation, which leads to the appearance of Aim17-stained foci in the cytosol independently of protein aggregates. While so far we are not able to localize Aim17 unambiguously in Hsp104 containing aggregates (mitoStores) upon lipid stress, we would like to move the manuscript farther without those experiments.

(7) In Fig. 1A, the figures of the different lines are difficult to distinguish. Lines of one color with different intensities would be better suited.

We have been working before with dose-response profiles generated by the destabilized luciferase system and found that the color-coded representation of the plots is the most effective way to represent the data, see for example Fita-Torró et al. Mol Ecol. 2023 32(13):3557-3574, Pascual-Ahuir et al. BBA 2019 1862(4):457-471, Rienzo et al., Mol Cell Biol. 2015 35(21):3669-83, and several other publications. Therefore we want to keep the format of the Figure.

(8) A title page should be added to each of the supplemental data files with short descriptions of the information that is provided in the columns of the tables. Response: Explanatory title pages have been now added to the supplemental data files.

Reviewer #3 (Recommendations For The Authors):

Figure 5A: The authors aim to assess protein import, however, their experimental set-up is not suited and does not allow conclusions about protein translocation into mitochondria. The authors monitor protein steady state levels, which does not reflect import capacity. For this e.g. pulse-chase experiments coupled to coIP or in organello import assays with radiolabeled substrate proteins would be required. In addition, the authors lack a non-treated control to show that no precursor accumulates in the absence of CCCP and t-2-hex. At the moment, the conclusion of blocked import cannot be made, as there are many other explanations for the observed steady state levels, e.g. the TAP tag interfered with the import competence of the precursor or t-2-hex could impact on MPP function (in particular as Figure 8B shows that also intra-mitochondrial proteins undergo modification by t-2-hex).

We agree that our study does not investigate a direct effect of t-2-hex on the import capacity of purified mitochondria. However, we determine the in vivo accumulation of several mitochondrial precursor proteins, which is widely used to assay for the efficiency of mitochondrial protein import, for example the recent hallmark paper discovering the mitoCPR protein import surveillance pathway exclusively uses epitope-tagged mitochondrial precursors to determine the regulation of mitochondrial protein import (Weidberg and Amon, Science 2018 360(6385)). Figure 5 contains several non-treated control experiments, which show that no (or less in the case of Ilv6) precursors of Tap-tagged Aim17, Cox5a, Ilv6, or Sdh4 accumulate in the absence of CCCP or t-2-hex. This is shown in Figure 5A for untreated cells or in Figure 5B and new Figure 5G for solvent (DMSO) treated cells. This demonstrates that the Tap-tag does not interfere with the import of the respective precursors. Additionally, our new results that mutants in accessory TOM subunits 20 and 70 are hyperresistant to t-2-hex (Figure 8D) identify the TOM complex and hence protein import at the outer mitochondrial membrane as a physiologically important t-2-hex target.

Figure 8: The conclusion that Tom40 is directly lipidated comes from an in vitro assay, with the conclusion that Tom40 is the main target, because it is the only Tom protein with a cysteine (Tom70 as not being part of the Tom core is excluded, however, lack of Tom70 function would also have detrimental consequences for mitochondrial protein import). However, there is no experiment showing a modification of Tom40 and a consequence for protein import. The proposed model is therefore very far-fetched and several aspects are speculation but not supported by experimental data. To propose such a model, the author needs to show experimental evidence, e.g. by generating a yeast strain in which the cysteine i Tom40 are replaced by e.g. Serine residues, and then assess if protein import (e.g. pulse-chase assays) are not affected anymore upon addition of t-2-hex.

Deletion of all four cysteine residues in Tom40 is not sufficient to confer resistance to t-2-hex stress. This result had been included in the original manuscript, but was somehow hidden in the Discussion. The revised manuscript now includes t-2hex tolerance assays for the Tom40 cysteine free mutant in new Figure 8D. As a result, cysteine lipidation of Tom40 alone is not sufficient to confer t-2-hex toxicity. This implies most likely other lipidation targets within the TOM and TIM complexes, as indicated by our in vitro lipidation studies. We therefore included the non-essential adaptor proteins Tom70 and Tom20 of the TOM complex and tested the tolerance of the respective deletion mutants in t-2-hex tolerance assays. As shown in new Figure 8D, the absence of Tom70 and Tom20 function significantly increases tolerance to t2-hex indicating that the TOM complex is a physiologically important target of the pro-apoptotic lipid, which acts most likely via lipidation of more subunits than the Tom40 import channel.

Figure 8A: The pulldown experiments lack positive (other Tom subunits) and negative controls and were performed with (large) tags on all proteins, which can easily result in false positive interactions. The conclusion that Hfd1 interacts with Tom70 and Tom22 cannot be made. Also, the conclusion if an interaction is robust or not cannot be made as the pull-down lacks control fractions, it is also not clear how much of the eluate was loaded. Finally, Hfd1-HA was not expressed from its endogenous promoter, likely resulting in over-expression, which again strongly hampers conclusions about bona fide interaction partners.

We agree that our pulldown studies are done in an artificial context, such as Hfd1 overexpression needed for sufficient protein level for detection or use of Tapfusion proteins. However, the conclusion that Tom70 is a potential interactor of Hfd1 can be made based on the following observations: Hfd1-HA is preferentially pulled down from total protein extracts containing Tom70-Tap, but not from extracts containing no Tap-protein and significantly less from extracts containing Tom22-Tap, another TOM associated subunit. The pulldown assay has been repeated now several times and the efficiency of Hfd1 pulldown has been quantified and statistically analyzed with respect to the quantity of purified Tom protein, which is shown in modified Figure 8A.

Figure 4A and C: Depletion of proteasomal activity results in larger aggregates in Figure 4A. However, the addition of t-2-hex blocks proteasomal activity (Figure 4C). How can proteasome inhibition result in bigger aggregates if the proteasomal activity is lost upon t-2-hex addition?

The negative effect of t-2-hex on proteasomal activity is most likely an indirect effect caused by protein aggregation (Bence et al., Science 2001 292-1552) and occurs in wild type and rpn4 mutant cells with reduced proteasomal activity (Fig. 4C). t-2-hex causes cytosolic protein aggregation in wild type cells, which is aggravated (more and larger protein aggregates) in rpn4 mutants because of their lower levels of active proteasome (Fig. 4A). The observed protein aggregates will further diminish proteasomal activity, which is confirmed in Fig. 4C.

Figure 1B: The authors use a reporter to determine HFD1 expression that consists of the promoter region of HFD1 fused to luciferase. These fusion constructs have been shown to often not reflect the bona fide expression levels of genes (Yoneda et al., J Cell Sci 2004). qPCR analysis of transcript levels should be included to support the induction of HFD1.

We agree that the live cell luciferase reporters used here are not suitable for the determination of absolute mRNA levels. However, the aim of these reporter experiments is to quantify the inducibility of different genes (HFD1, GRE2) dependent on increasing stress doses. These dose response profiles cannot be obtained by qPCR analysis, while the destabilized reporters are an excellent tool for this, which have been used to accurately describe numerous dynamic stress responses (for example: Dolz-Edo et al. 2013 MCB 33:2228-40, Rienzo et al. 2015 MCB 35:3669-83, PascualAhuir et al. 2019 BBA 862:457-471). Additionally, the induction of HFD1 mRNA levels by salt (NaCl) and oxidative (menadione) stress determined by qPCR has been published before (Manzanares-Estreder et al. 2017 Oxid Med Cell Longevity 2017:2708345).

The authors conclude from Figure 1 that entry into apoptotic cell death is modulated by efficient t-2-hex detoxification. However, this is based on growth curves and no analysis of apoptotic cell death is performed. The data show that the addition of hexadecenal results in a growth arrest, that is overcome likely upon degradation of t-2-hex (depending on the amount of Hfd1).

We agree that our experiments measure growth inhibition and not specifically apoptotic cell death. The text has been changed accordingly.

Figure 4A: Microscopy images show between 1-2 yeast cells. Either more cells need to be shown or quantifications of the aggregates are required. In addition, it is not clear if the control received the same DMSO concentration as the treated cells and also the time point for the control is not specified.

We have now quantified the number of aggregates across cell populations in new Figure 4A in DMSO, t-2-hex and t-2-hex-H2 treated wt and rpn4 mutants. These data show specific aggregate induction by t-2-hex and not by DMSO or the saturated t-2-hex-H2 control, which is aggravated in rpn4 mutants and avoided by CHX pretreatment.

Figure 5: Western blots in figure 5A, B, D, E and F lack a loading control. Without this, conclusions about increases in protein abundance cannot be made. Response: We have now included additional panels with the loading controls for the Western blots in new figure 5, except figure 5B, where the appearance or not of the pre-protein can be compared to the amount of mature protein in the same blot.

Figure 2B: Complex II assembly factors SDH5,6,9 are described here as ETC complexes. As the proteins are not part of the mature complex II, the heading should be modified into ETC complexes and ETC assembly.

Figure 2B has been revised and the classification of ETC proteins changed accordingly.