Yeast homologs of human MCUR1 regulate mitochondrial proline metabolism

Mitochondria house evolutionarily conserved pathways of carbon and nitrogen metabolism that drive cellular energy production. Mitochondrial bioenergetics is regulated by calcium uptake through the mitochondrial calcium uniporter (MCU), a multi-protein complex whose assembly in the inner mitochondrial membrane is facilitated by the scaffold factor MCUR1. Intriguingly, many fungi that lack MCU contain MCUR1 homologs, suggesting alternate functions. Herein, we characterize Saccharomyces cerevisiae homologs Put6 and Put7 of MCUR1 as regulators of mitochondrial proline metabolism. Put6 and Put7 are tethered to the inner mitochondrial membrane in a large hetero-oligomeric complex, whose abundance is regulated by proline. Loss of this complex perturbs mitochondrial proline homeostasis and cellular redox balance. Yeast cells lacking either Put6 or Put7 exhibit a pronounced defect in proline utilization, which can be corrected by the heterologous expression of human MCUR1. Our work uncovers an unexpected role of MCUR1 homologs in mitochondrial proline metabolism.

1. The authors have extensively characterized the phenotype of the put6∆ and put7∆ mutants and have shown Put6 and Put7 proteins localize to the inner mitochondrial membrane, where they form a large hetero-oligomeric complex, whose abundance is regulated by proline. Whilst this provides strong evidence to implicate Put6 & Put7 in the regulation of proline metabolism, the authors do not provide enough evidence to support the mechanism linking Put6 & Put7 to ROS generation, or the claim that Put6 and Put7 are necessary to prevent redox imbalance. Further experiments are needed to support this claim.
2. The growth defect upon proline supplementation is clear but would benefit from additional experiments in SM + glucose background (to clearly show that the growth defect upon proline supplementation is independent of MRC function). The authors' claim that the growth defect is completely independent of defects in mitochondrial respiratory function is not fully supported by their experiments. Although they have demonstrated the physical independence of Put6/7 and 3 out of the 4 ETC complexes, this does not rule out functional interdependence. The claim that growth of Δput6 mutant strain is rescue in SM + galactose with proline as sole nitrogen source by the addition of arginine (Fig. 4b) seems contrast with the observations of VanderLuis et al (cited by the authors as reference 26), where the growth of an analogous Δput6 mutant strain in presence of the two amino acids is similarly impaired in comparison to the wild type reference.

Minor comments
The title suggests that put6 and put7 mutants are auxotrophs, but they only have an effect when cells need to use proline as the sole nitrogen source (which is unlikely in physiological conditions) (figure 4a SM+(NH4)2SO4 vs SM+Proline). Authors should re-write the title to reflect the results. At line 81, the authors state that they performed a phylogenetic analysis across 15 selected species to demonstrate the presence of the MCUR1 protein (results are shown in Fig 1a). It would be appreciated if they justified the choice of these species. The authors may need to acknowledge that Put6 and Put7 have individual roles, further to their interaction in a complex, as suggested by: 1. whilst it's clear that Put6 and Put7 interact in a hetero-oligomeric complex, as they co-precipitate with strong statistical significance, the absence of common IP proteins suggests that these proteins individually interact with other proteins/complexes. 2. Presence of smaller complexes in fig 4 g for Put7 but not Put6 3. Effect of proline supplementation on gene expression increase expression of Put6 20 fold compared to just 1.5 fold for Put7 In figure 3 and 4, the authors claim that Put6 and Put7 are not required for MRC, but the experiments don't fully disprove this requirement: Authors should show how cells respond to growth in SM+Glucose+Proline vs SM+Galactose+Proline. Why did the authors do all supplementation experiments in SM+galactose instead of SM+glucose? (clarification in the text) How did the authors choose the 10 amino acids used for the supplementation experiments? (clarification required in the text). If there is no clear specific reason, then, the authors should perform experiments with the remaining proteinogenic aa, to check whether put6 and put7 affect the metabolism of other amino acids too). Why did the authors choose to supplement with arginine? Mechanistic link to Redox imbalance : As mentioned previously, the experiments conducted to demonstrate a link between Put6 & Put7 and redox imbalance do not offer sufficient proof. Firstly, a two-fold increase in MitoSox is not particularly striking, especially when accompanied by a simultaneous increase in NADPH/NADH levels. Additionally, previous work (https://onlinelibrary.wiley.com/doi/epdf/10.1002/yea.3154) has shown that owing to the antioxidant properties of proline, its intracellular accumulation can help S.cerevisiae cells survive ethanol stress. The increased levels of NADPH & NADH would be consistent with previous work suggesting an increased antioxidant capacity under proline accumulation. However, further experiments are needed to reconcile the increase in ROS observed by the authors with the same. Perhaps, measuring other indicators of the cellular redox state, such as glutathione mediated antioxidant response will provide further clarity on this paradox. Secondly, although the authors' claim that ROS or NADPH/NADH imbalance is leading to the observed cell cycle arrest, is very plausible, there is no clear mechanism that can be proposed from their work. In order to claim that the cell cycle arrest is caused by redox imbalance, the authors need to probe mechanisms that are known to directly affect cell cycle progression, for eg. the DNA damage response. Lastly, the fact that redox equivalents have altered concentrations could directly affect mitochondrial respiratory chain activity. This undermines the authors' claim about the independence of Put6 & Put7 and mitochondrial respiration.
Reviewer #3 (Remarks to the Author): Human mitochondrial calcium uniporter regulator 1 (MCUR1) is involved in the assembly of mitochondrial complexes in the inner membrane (IM) such as the Ca2+ uniporter channel complex and cytochrome c oxidase. Here, the authors conduct a comprehensive study of the MCUR1 yeast homolog Fmp32 and the related protein Ylr283. Previous studies of Fmp32 have indicated it is required for proline metabolism and that it has a conserved role in mitochondrial bioenergetics. The novel findings of this study are that deletion of Fmp32 and Ylr283 block the ability of yeast strains to use proline as a nitrogen source and that proline uptake and accumulation are significantly upregulated. Because of the strong link to proline metabolism, the authors propose to rename Fmp32 and Ylr283 as Put6 and Put7, respectively. Biochemical analysis shows that Put6 and Put7 interact with each other in the mitochondrial matrix and have increased protein levels in the presence of proline. Depletion of Put6/Put7 does not impair mitochondrial bioenergetics and the assembly of respiratory complexes. The authors suggest that the Put6 is negative regulator of proline import and metabolism. Strikingly, human MCUR1 is able to rescue the growth phenotypes of the Put6 and Put7 deletion strains in addition to restoring proline metabolic levels. The mechanism by which proline accumulates to high levels in the Put6/Put7 deletion strains is unclear, however, the results convincingly show that both proteins are critical for proline transport. The findings from the study significantly advance the understanding of human MCUR1 and its conserved function in yeast. The manuscript is of broad interest to investigators in mitochondria research and metabolism.
Some questions for the authors to consider.
1. How confident are the authors that HsMCUR1 has two transmembrane domains? In the alignment the other proteins just have one TM. A 2019 structure paper by Alvarez et al., (https://doi.org/10.1016Alvarez et al., (https://doi.org/10. /j.str.2018, which the authors should include and consider, has just one TM at the C-terminal end of HsMCUR1. The authors should consider updating the cartoon alignment in Figure 1b accordingly. This would also better support the ability of HsMCUR1 to rescue the Put6 and Put7 mutants.
2. MitoSOX is used to conclude that the Put6 and Put7 mutant have higher levels of ROS. The authors should complement this with another method such as aconitase activity assays to assess oxidative stress.
3. The band for MCUR1 is 28 kDa instead of 39 kDa for the full-length protein. What post-translational processing are the authors referring to? More explanation is needed to explain the much lower molecular weight band? 4. Could the authors provide more insight into why MCUR1 alone can complement the double knockout put6put7 strain but individually Put6 and Put7 are not able to complement the put6put7 double knockout (Figure 7)? 5. The growth profiles of the Put6 and Put7 deletion strains are comparable to wild-type and there is no disruption in the formation of mitochondrial respiratory complexes. Even so, have the authors confirmed that the mitochondrial membrane potential is similarly unperturbed? TMRM staining of mitochondria in the Put6 and Put7 deletion strains would help confirm this.
6. With the arrest of the put6 and put7 mutants at G1, did the authors examine whether there were any problems with mitochondrial dynamics that may contribute to the cell cycle delay. Are more fragmented mitochondria observed in the mutant strains? These data could provide additional support for the conclusion by the authors on Line 263-265 that "perturbation in the cellular and mitochondrial redox state may explain the growth arrest of the mutants in the proline-containing medium." 7. One line 146 and 147, the authors state "Tomar et al., did not observe any perturbation in any MRC complexes in MCUR1 deficient cells." Previous work by Tomar did show MCUR1 deletion impairs mitochondrial bioenergetics, whether loss of MCUR1 also disrupted mitochondrial respiratory complexes is not clear as it does not seem to have been directly tested. The authors should clarify the point they are trying to make in regards to the paper by Tomar et al.

Reviewer1:
General: In the manuscript entitles "Put6 and Put7 are essential for mitochondrial proline metabolism", the authors convincingly show that the yeast orthologues of the human calcium carrier regulator (MCUR1) are required for proline metabolism. This is particularly interesting since S. cerevisiae does not possess a mitochondrial calcium uniporter but has retained MCUR1 orthologues. Moreover, the work presented clarifies an existing discrepancy relative to the role of Put6 and Put7 in OXPHOS biogenesis. The study is well designed, includes a thorough phenotypic analysis and provide novel information on the role of yeast Put6 and Put7 in proline utilization. In my opinion, the manuscript is suitable for publication in Nature Communications.

Response:
We thank the reviewer for finding our manuscript "suitable for publication in Nature Communications."

Major Comments:
1.I do however have concerns regarding the lack of insights into the molecular mechanism governing Put6 and Put7 function that should be addressed. Human MCUR1 represses calcium uptake by binding to the calcium uniporter MCU and regulating its function. In the case of Put6 and Put7, the authors did not identify any potential interacting partners by immunoprecipitation and MS analysis. However, it is not specified under which conditions were grown the cells used for mitochondria isolation. Growth in presence or absence of proline could be key to identify interactors which binding to Put6 and Put7 depends on proline metabolism. Weak regulatory interactions could be lost during the immunoprecipitation process. Have the authors considered cross-linking or proximity-dependent labelling approaches?

Response:
The reviewer raises an important point. In the original submission, immunoprecipitation (IP) and mass spectrometry (MS) analyses were performed using mitochondria isolated from cells that were grown in synthetic media where ammonium sulfate was used as the sole nitrogen source. We now clearly describe these growth conditions in the Methods Section of the revised manuscript.
Our choice of media was guided by the fact that Put6 and Put7 mutants grow poorly in the media-containing proline as the sole nitrogen source. However, we understand and agree with reviewer's suggestion that proline supplementation may uncover novel interacting partners of Put6 and Put7. Therefore, we have now performed IP in proline conditioned growth media. Although the cell growth and mitochondrial yield was very low under this condition, we utilized 36 liters of growth media to obtain sufficient mitochondria for the IP/MS experiment. IP/MS analysis of the proline-cultured cells again identified Put6 and Put7 as the two interacting partners of the complex and did not uncover any additional common interacting proteins (See Figure below). These data are consistent with our original findings and further strengthens the existence of a higher order heteroligomeric complex of Put6 and Put7 in the mitochondria. The size of the Put6/Put7-containing higher order complex in the ammonium sulfate-grown cells Volcano plot showing the enrichment of Put6 and Put7 interacting proteins from yeast cells grown in proline-containing media. Immunoprecipitation was performed using mitochondria isolated from WT+EV, put6Δ + Put6-V5 and put7Δ + Put7-V5 yeast cells grown in proline containing media followed by MS analysis. The abundance of Put6-V5 and Put7-V5 interacting proteins were normalized using immunoprecipitate obtained from WT cells transformed with empty vector. forms translucent gel like structure at room temperature over 24 hour time period in a Ca 2+ -dependent manner." However, in our case the IP was carried out at much lower temperature (4°C) for short duration only. Therefore, we believe that Ca 2+ in buffer is unlikely to have an effect on the interactions under in vivo conditions.  Response: As suggested by reviewer, we have now overexpressed Put6 and Put7 using multicopy plasmids (pRS42N and pRS42H) and found that overexpression of PUT6 and PUT7 leads to a minor growth defect when grown on proline as the sole nitrogen source. This overexpression also led to a significant increase in mitochondrial Supplementary Fig.4 (a) Ten-fold serial dilutions of indicated yeast strains harboring empty vector (EV) or tagged versions of Put6/Put7 were seeded on synthetic media plates containing proline as the sole nitrogen source and incubated at 30°C. Pictures were taken after 3 days of seeding. ROS in both Put6 and Put7 overexpressing cells that are cultured in proline-containing media (below and in supplementary Fig. 6i &j). These data suggest that optimal levels of Put6 and Put7 are necessary for maintaining mitochondrial ROS homeostasis and are consistent with our proposed role of these proteins in cellular proline metabolism.  (i) Ten-fold serial dilutions of indicated yeast strains harboring empty vector (EV) or Put6 and Put7 cloned in 41N, 41H (single copy) or 42N, 42H (multi-copy) vectors, were seeded on synthetic media plates containing proline as the sole nitrogen source and incubated at 30°C. Pictures were taken after 3 days of seeding. (j) Mitochondrial ROS measured using mitoSOX fluorescence in yeast cells grown in proline as the sole nitrogen source. Data were normalized to unstained control and are expressed as mean ± SD (n = 3) relative to WT.

Reviewer2:
MCUR1 has been implicated in MCU-dependent mitochondrial Ca 2+ homeostasis and MRC complex IV biogenesis in a variety of eukaryotes.
Nevertheless, why many fungi lacking MCU still contain MCUR1 homologs still remained unknown. In the current study, the authors identified two previously uncharacterized mitochondrial genes, discovered their involvement in the regulation of mitochondrial proline metabolism and consequently, renamed them Put6 and Put7. This discovery, together with the rescue of put6∆ and put7∆ by MCUR1 expression, identifies a role for MCUR1 in proline metabolism, beyond calcium-dependent mitochondrial homeostasis, that might be conserved in higher eukaryotes.

Response:
We thank Reviewer 2 for their thoughtful comments and for recognizing the importance of the work.

Major Comments:
1. The authors have extensively characterized the phenotype of the put6∆ and put7∆ mutants and have shown Put6 and Put7 proteins localize to the inner mitochondrial membrane, where they form a large heterooligomeric complex, whose abundance is regulated by proline. Whilst this provides strong evidence to implicate Put6 & Put7 in the regulation of proline metabolism, the authors do not provide enough evidence to support the mechanism linking Put6 & Put7 to ROS generation, or the claim that Put6 and Put7 are necessary to prevent redox imbalance. Further experiments are needed to support this claim.

Response:
Previous studies have shown that increased mitochondrial proline oxidation is accompanied with increased mitochondrial ROS generation (Donald et al., 2001;PMID: 11280728). This is likely due overflow of electrons to the electron transport chain. (Hancock et al., 2016;PMID: 26660760). We recognize that we only used MitoSOXbased readout for ROS level measurements. Therefore, to further support our findings, we have now performed two additional experiments to demonstrate that mitochondrial ROS increases in put6∆ and put7∆ mutants when grown in media with proline as the sole nitrogen source.

(i) Oxidative inactivation of mitochondrial aconitase:
Since mitochondrial aconitase is sensitive to increased ROS levels, we measured aconitase activity in mitochondria isolated from WT, put6∆ and put7∆ yeast cells grown in ammonium sulfate or proline as the sole nitrogen source. As shown below and in Fig. 6g & h of the revised manuscript, aconitase activity is drastically reduced in put6∆ and put7∆ yeast cells only when proline is used as the sole nitrogen source.
(ii) Oxidative stress induced mitochondrial fragmentation: Mitochondria undergo fragmentation during oxidative stress (Willems et al., 2015 PMID: 26166745). Therefore, we also checked the mitochondrial morphology of WT and put6∆ and put7∆ mutants using Mitotracker TM Red after growing cells in ammonium sulfate or proline as the sole Fig. 6g & h: Aconitase activity in isolated mitochondria from WT, put6Δ, put7Δ yeast cells grown on synthetic media with ammonium sulfate (g) or proline (i) as sole nitrogen source. Data were normalized to citrate synthase activity and are expressed as mean ± SD (n = 3) relative to WT. Fig. 6i: Indicated yeast strains grown in ammonium sulfate or proline as sole nitrogen source were stained using Mitotracker TM Red CMXROS and visualized using confocal microscopy. Scale bar represent 2.44 microns. nitrogen source. As shown here and in Fig. 6i of the revised manuscript, the mutants displayed mitochondrial fragmentation when grown in proline.
2. The growth defect upon proline supplementation is clear but would benefit from additional experiments in SM + glucose background (to clearly show that the growth defect upon proline supplementation is independent of MRC function). The authors' claim that the growth defect is completely independent of defects in mitochondrial respiratory function is not fully supported by their experiments.

Response:
We have now performed the suggested experiment and found that growth defects of put6∆ and put7∆ mutants in proline persist in glucose-containing synthetic medium, suggesting that growth defect in proline is independent of mitochondrial respiratory function (see figure below and Fig. 4b & c in the revised manuscript).
3. Although they have demonstrated the physical independence of Put6/7 and 3 out of the ETC complexes, this does not rule out functional interdependence.

Response:
The reviewer's assertion is correct that physical independence of Put6/7 complex from the ETC complexes does not rule out functional interdependence. We did consider this possibility and had shown that activities of the ETC complexes and ATP synthase are intact in Put6 mutant (Fig. 3e of the original manuscript). To further exclude the involvement of Put6/Put7 complex in ETC function, we measured cellular oxygen Ten-fold serially diluted yeast cells were seeded on synthetic media (SM) plates with glucose (b) or galactose (c) as carbon source and ammonium sulfate or proline as the sole nitrogen source. Proline oxidase, Put1, which is required for growth in proline-containing media, was used as a control. Pictures were taken after 3 days of seeding. consumption under basal conditions, upon 2 µm antimycin treatment to inhibit mitochondrial respiration, and upon 5 µm CCCP treatment to observe maximal respiration under uncoupled conditions. As seen in the accompanying figure and Fig. 3F of the revised manuscript, our data revealed that mitochondrial, non-mitochondrial, and maximal respiration in put6Δ cells is equivalent to the WT cells. These results clearly show that the loss of Put6 do not impair ETC function.

4.
The claim that growth of put6Δ mutant strain is rescue in SM + galactose with proline as sole nitrogen source by the addition of arginine (Fig. 4b)  There is high likelihood of artifacts in such high-throughput studies. With that in mind, we carefully performed single gene studies on experimentally validated yeast mutants to show that the growth defect of Put6 and Put7 is observed only in proline-containing media out of all the 20 amino acids tested (Fig. 4a). In our case, we measured growth of Fig. 3f: BY4741 WT and put6Δ cells were grown in YPGE media, counted and oxygen consumption was measured at 30°C using O2K FluoRespirometer. Mitochondrial respiration refers to antimycin sensitive respiration, non-mitochondrial respiration refers to antimycin resistant respiration, and maximal respiratory rates were calculated after addition of 5 µM CCCP. Data are expressed as mean ± SD (n = 3). cells in 7 mL media with a starting OD 600nm of 0.1 and maximal OD 600nm of ~5.0 over 30 hours. Thus, our low-throughput growth assay accounted for ~5-6 doublings. In contrast, VanderSlius et al., performed a 96-well plate based high-throughput growth assay measuring only two doublings from 0.1 to 0.5 over the period of 30-40 hours. Thus, we attribute differences in results from the two studies to differences in the experimental setup. Additionally, we constructed Put6 in different genetic background (CEN.PK) and further show that put6Δ mutants display a growth defect in proline that can be rescue by arginine supplementation. Together, our data show that Put6 is specifically required for proline utilization and this phenotype is observed in different prototrophic yeast strains.
Minor comments: 1. The title suggests that Put6 and Put7 mutants are auxotroph, but they only have an effect when cells need to use proline as the sole nitrogen source (which is unlikely in physiological conditions) (figure 4a SM+(NH4)2SO4 vs SM+Proline). Authors should re-write the title to reflect the results.

Response:
We have reworded our title to: "Put6 and Put7 are novel regulators of mitochondrial proline metabolism".
2. At line 81, the authors state that they performed a phylogenetic analysis across 15 selected species to demonstrate the presence of the MCUR1 protein (results are shown in Fig 1a). It would be appreciated if they justified the choice of these species.
Growth measurement of WT and put6Δ yeast cells in CEN.PK genetic background in prolinecontaining media. 4 mM arginine was after 18 hours of growth in put6Δ yeast cells (indicated by an arrow).   Proline oxidase, Put1, which is required for growth in proline-containing media, was used as a control. Pictures were taken after 3 days of seeding.

Response
experiments with the remaining proteinogenic aa, to check whether put6 and put7 affect the metabolism of other amino acids too).

Response:
We have now performed the supplementation experiments with the remaining 10 amino acids (see figure below and figure 4a in the revised manuscript) and found that put6∆ and put7∆ mutants show growth defect only when proline was used as sole nitrogen source. Consistent with the previous study (Ljungdahl et. al., 2012 PMID: 22419079), we found that histidine, lysine, and cysteine cannot be used by yeast as sole nitrogen source (see figure below and figure 4a in the revised manuscript). Fig. 4 (a) Prototrophic WT, put6Δ, and put7Δ yeast cells were cultured at 30°C in synthetic liquid media containing the indicated amino acids as the sole nitrogen source. Growth was monitored by measuring absorbance at 600nm at the indicated time intervals. Ammonium sulfate was used as the most favoured nitrogen source.
6. Why did the authors choose to supplement with arginine?

Response:
We used glutamate and arginine because they are favorable nitrogen sources and are downstream products of proline oxidation. Perhaps, measuring other indicators of the cellular redox state, such as glutathione mediated antioxidant response will provide further clarity on this paradox.
Secondly, although the authors' claim that ROS or NADPH/NADH imbalance is leading to the observed cell cycle arrest, is very plausible, there is no clear mechanism that can be proposed from their work. In order to claim that the cell cycle arrest is caused by redox imbalance, the authors need to probe mechanisms that are known to directly affect cell cycle progression, for eg. the DNA damage response.

Response:
The reviewer raises a critical point. Elevated NAD(P)H levels and proline accumulation in put6∆ and put7∆ mutants appear paradoxical to the increased mitochondrial ROS levels we observed. Therefore, to validate our findings, we performed additional experiments including aconitase activity assay and show that both put6∆ and put7∆ mutants indeed have higher mitochondrial ROS when grown in proline as the sole nitrogen source (See our response to this reviewer's major Comment 1). The elevated mitochondrial ROS we observed is consistent with rapid mitochondrial uptake of proline and its oxidation via ETC. Indeed, a previous study has shown that increased mitochondrial proline oxidation by Put1 is accompanied with increased mitochondrial ROS generation (Donald et al., 2001;PMID: 11280728). The study by Takagi et al., 2016, which is referred by this reviewer, used put1∆ cells. The loss of Put1 prevents transfer of electrons from proline to the mitochondrial ETC and thus would not cause an increase in ROS. However, in our experimental set up Put1 is intact, thus with its excess substrate available, an elevation in ROS is expected.
To test whether redox imbalance and increased ROS is the cause of cell cycle arrest, we performed glutathione supplementation experiment. We found that glutathione is not able to rescue the growth defect of put6∆ and put7∆ cells (Fig. 6j in the revised manuscript and below). We also tested other antioxidants including ascorbic acid and Nacetyl cysteine, both of which failed to rescue put6∆ and put7∆ growth defect in prolinecontaining medium. Together, these results suggest that the oxidative stress is not the primary cause of cell cycle arrest.
However, we can rescue this growth defect upon supplementation with alternate nitrogen source (arginine or glutamate) (Fig. 6k & l in revised manuscript and above), suggesting that the primary cause of cell cycle arrest in put6 and put7 mutants is nitrogen starvation likely due to the inability to utilize nitrogen from proline. Our cell cycle data showing that put6∆ and put7∆ mutants grown in proline are mostly stuck in G0/G1 phase ( Fig. 6m

Response:
In the revised manuscript, we clearly show that Put6 and Put7 are dispensable for mitochondrial respiratory chain formation or function (Fig. 3). The alteration in redox equivalents are seen only when proline is used as sole nitrogen source. Therefore, Put6/Put7 are not expected to impact mitochondrial respiratory chain function in complex media where variety of nitrogen sources is available, the conditions that were used in Fig. 3. Our data shows that Put6 and Put7 do not impact mitochondrial carbon-based energy metabolism but they impact nitrogen metabolism, specifically pertaining to proline utilization.

Reviewer 3:
Human mitochondrial calcium uniporter regulator 1 (MCUR1) is involved in the assembly of mitochondrial complexes in the inner membrane (IM) such as the Ca2+ uniporter channel complex and cytochrome c oxidase. The authors should consider updating the cartoon alignment in Figure 1b accordingly. This would also better support the ability of HsMCUR1 to rescue the Put6 and Put7 mutants.  Because expression of each protein is dependent on the presence of other, expressing

Response
Put6 or Put7 individually in put6Δput7Δ double knockout is not able to rescue proline phenotypes (Fig. 7a & c). However, MCUR1 expression is independent of the presence of either Put6 and Put7 (Fig. 7b), therefore MCUR1 alone is able to complement put6Δput7Δ double knockout (Fig, 7a & c).

Response:
As suggested by the reviewer, we checked the mitochondrial morphology of WT and mutants grown in ammonium sulfate or proline as the sole nitrogen source using Mitotracker TM Red. The mutants displayed mitochondrial fragmentation when grown in proline (below and Fig. 6i in the revised manuscript). This data is consistent with ROS induced perturbation in mitochondrial morphology as predicted by the reviewer. 8. Figure 1, replace "mitochondrial" with "mitochondrial fraction"

Response:
We have now replaced mitochondrial with mitochondrial fraction in Figure 1 legend. Fig. 6i: Indicated yeast strains grown in ammonium sulfate or proline as sole nitrogen source were stained using Mitotracker TM Red CMXROS and visualized using confocal microscopy. Scale bar represent 2.44 microns.