Author response:
We thank the editors and reviewers for their thoughtful and constructive evaluation of our manuscript. We are pleased that the reviewers found the study valuable and the evidence supporting a role for Yme1 in MDC formation solid. As described below, we plan to modify the manuscript to clarify the lipid model, better explain the relationship between Ups-family proteins and MICOS, distinguish MDC formation from Atg32-dependent mitophagy, clarify metabolic conditions, add statistical analyses where missing, and strengthen Yme1 validation with immunoblotting.
eLife Assessment
This valuable study demonstrates that the inner membrane protease YME1 contributes to the formation of mitochondrial-derived compartments in yeast through the modulation of both the lipid transporter UPS2 and the MICOS complex. The evidence supporting this model is solid, although this manuscript could be improved by providing additional evidence supporting the independent roles for UPS2 and MICOS regulation in this process. This work will be of interest to cell biologists, biochemists, and geneticists interested in understanding the molecular basis of mitochondrial regulation and function.
We appreciate this positive assessment and agree that the roles of Ups-family lipid transport and MICOS in MDC regulation could be expanded further. This will be an important topic for future studies, especially with regard to how MICOS contributes to MDC formation. In the current revision, we will add new genetic data focused on PA-linked lipid metabolism through the yeast Pah1/Lipin pathway, which we think will help strengthen and clarify the lipid arm of the model. Our current interpretation is that Yme1-regulated Ups-family lipid transport and MICOS may both influence a shared mitochondrial membrane state that permits MDC formation. This interpretation is consistent with our genetic data and with known connections between Ups proteins, MICOS, and mitochondrial membrane organization.
Reviewer #1 (Public review):
Summary:
In this manuscript, Balasubramaniam and colleagues continue this group's efforts to understand mitochondrial-derived compartments (MDCs) that bud off from yeast mitochondria in response to metabolic stress. In a previous genetic screen, they identified Ups lipid transfer proteins and the AAA-protease Yme1 as components that modulate MDC formation. In this study, the authors link these observations by showing that Yme1 modulates levels of Ups1, Ups2, as well as MICOS complex members in the mitochondrial proteome. Using genetic approaches, they then show that Yme1's role on MDCs is dependent on its catalytic activity (via an inactive mutant) and that YME1 shows genetic interactions with UPS1/2 and MIC10/MIC60. The overall model is that Yme1 activity responds to metabolic cues and acts via proteolysis of these two distinct mitochondrial machineries to regulate MDC biogenesis.
Strengths:
The strengths of the study are its integration of mitochondrial proteomics with strong genetic approaches, as well as synergy with the authors' previous studies on the role of lipids in MD genesis. The work is overall well carried-out and experiments are thoughtfully discussed.
Weaknesses:
The major weaknesses are a lack of mechanistic resolution surrounding the model, e.g., proposed or tested mechanisms by which Yme1 activity is regulated by metabolic cues, or how Ups1/2 activity and the MICOS contribute to MDC generation. The authors acknowledge these as open questions, but addressing them would still enhance the significance of the study.
We thank the reviewer for the positive assessment, and we agree that the upstream regulation of this response remains an important open question. Yme1-dependent MDC regulation could involve changes in Yme1 activity, substrate accessibility, or broader changes in mitochondrial lipid and protein organization. Fully resolving how metabolic state gates this response will require future work, likely outside the scope of the current study.
We also agree that the manuscript would benefit from a more developed discussion of how lipid changes could contribute to MDC formation. Our prior work showed that reduced mitochondrial PE promotes MDC formation, whereas cardiolipin is required for MDC biogenesis (Xiao et al., 2024). We proposed that reduced PE changes the membrane environment of mitochondrial outer membrane proteins, potentially affecting their stability, abundance, insertion, or lateral organization within the membrane. Such changes could increase the pool of proteins available for sorting into MDCs or make the outer membrane more permissive for domain formation. In the revision, we will connect this model more directly to Yme1-dependent regulation of Ups-family lipid transport.
We will also expand the model to incorporate PA-linked metabolism. We did not initially focus heavily on Ups1 because complete loss of UPS1, or loss of downstream cardiolipin synthesis through CRD1, blocks MDC formation because cardiolipin is required. Thus, complete disruption of Ups1-dependent lipid transport may obscure the effects of more moderate changes in PA flux. To address this, we will include additional lipid measurements and new genetic data targeting PA metabolism through the yeast Pah1/Lipin pathway. Because Pah1 converts PA to DAG, this provides a way to alter PA-linked metabolism without simply eliminating cardiolipin synthesis. Our new data suggest that PA accumulation or altered PA-linked lipid flux may also promote MDC formation. Together, these findings support a broader model in which reduced PE and increased PA alter both the organization of OMM proteins and the physical properties of the membrane, including curvature and domain formation, thereby creating a membrane state that is more permissive for MDC biogenesis.
Reviewer #2 (Public review):
In this manuscript, the authors report a novel regulation of the outer mitochondrial membrane remodeling domains called mitochondria-derived compartments, MDCs. The team has previously established the main principles behind this recently identified quality control pathway, but the mechanisms that control MDCs formation remain incompletely understood. Using the baker's yeast model, the authors identify the conserved mitochondrial protease Yme1 as a crucial factor that regulates MDC formation. Mechanistically, Yme1's proteolytic function controls the levels of Ups1 and Ups2 lipid transfer proteins and the components of the membrane organizing complex called MICOS, thus providing a plausible model as to how Yme1-dependent proteolysis permits MDC formation through the removal of lipid and MICOS-dependent constraints. Finally, the authors show that this Yme1-mediated activity is also defined by metabolic conditions. In principle, this study is interesting and novel, and holds potential to provide new insights into the regulation of the MDC pathway that emerged as a new fundamental mitochondrial quality control mechanism. However, the following points should be carefully addressed.
Major points:
(1) Yme1 has been previously shown to regulate mitochondria-specific autophagy through Atg32 processing. Given the high similarity of the MDC pathway to piecemeal autophagy and the fact that both pathways share some of the core components, the authors should address the involvement of Atg32 in their model. It would also be important to include a brief discussion addressing the differences between piecemeal autophagy and the MDC pathway.
We agree that this is an important point. The reason we did not focus on Atg32 in the current manuscript is that we previously investigated the relationship between MDC formation and Atg32-dependent mitophagy and found that Atg32 is dispensable for MDC formation (Hughes et al., 2016). Based on that result, we do not anticipate that Atg32 is required for the Yme1-dependent MDC phenotypes described here. This is also consistent with the different growth conditions associated with these pathways: Atg32-dependent mitophagy is stimulated under respiratory or post-diauxic conditions, whereas MDCs do not form under the respiratory conditions that stimulate Atg32-dependent mitophagy (Hughes et al., 2016; Raghuram and Hughes, 2024).
We will clarify this distinction in the revised manuscript. In addition, to be thorough, we plan to generate and test the Atg32-GFP variant previously shown to block Yme1-dependent Atg32 processing and mitophagy (Wang et al., 2013). This will allow us to test directly whether preventing Yme1-dependent Atg32 cleavage affects MDC formation. If successful and interpretable, we will include these data in the revised manuscript.
(2) The Rpt3 (P215L) expression experiment is interesting, but appears to be somewhat superficial due to the unclear mechanism by which the mitochondrial network morphology is restored in these cells. Could this result be replicated in the dnm1∆ mgm1∆ double deletion mutant, which is a well-established model for mitochondrial network restoration?
We agree that the Rpt3(P215L) experiment is best viewed as a morphology control. The purpose was to test whether abnormal mitochondrial morphology alone explains the MDC defect in yme1Δ cells. Because Rpt3(P215L) improved mitochondrial morphology but did not restore MDC formation, we interpret this as evidence that morphology alone is not sufficient.
We attempted to generate the requested dnm1Δ mgm1Δ yme1Δ triple-mutant combination, but that strain combination has not been viable in our hands. However, we do have dnm1Δ data showing that altering mitochondrial structure can rescue some morphological features but does not restore MDC formation in yme1Δ cells. We will include these data where appropriate and clarify that this experiment is intended as a morphology control.
(3) Figure 3E. The changes in PE levels appear to be minor. While statistically significant, the observed differences may not be physiologically relevant. More in-depth lipidomic analysis data should be presented to substantiate the authors' argument and better address the questions at hand. Related to that, could PE or PA supplementation stimulate MDC formation?
We agree that additional lipid data would strengthen this part of the manuscript. We initially streamlined the lipid section because we had previously examined the lipid requirements for MDC formation in detail, showing that reduced mitochondrial PE can promote MDC formation, whereas cardiolipin is required (Xiao et al., 2024). However, the current study would benefit from a broader analysis of the lipid changes associated with Yme1-dependent regulation.
In the revision, we will expand the lipid data to include additional lipid species and incorporate these results into the model. We will also add new genetic data targeting PA metabolism through the yeast Pah1/Lipin pathway. Together, these data suggest that PA accumulation or altered PA-linked lipid flux may also contribute to MDC formation. This supports a broader lipid-balance or lipid-shunting model in which reduced PE, increased PA, or altered lipid distribution between mitochondrial membranes could influence OMM remodeling through effects on membrane curvature, OMM protein organization, or mitochondrial membrane contacts.
We agree that direct PE or PA supplementation would be a valuable experiment. We have attempted lipid supplementation but have not been able to deliver these lipids effectively to yeast cells in a way that produces interpretable results. We are therefore focusing on lipid profiling and genetic approaches that alter lipid metabolism inside the cell.
(4) The connection between rapamycin treatment and Yme1-regulated MDC formation is unclear and puzzling and needs to be explained better.
We agree that this connection is not fully clear. In this manuscript, rapamycin is used primarily as a robust MDC-inducing condition. Our data do not define the full pathway connecting TORC1 inhibition to Yme1-dependent mitochondrial remodeling.
In the revision, we will either clarify this point or reduce the emphasis on rapamycin as a mechanistic entry point. Our current interpretation is that rapamycin creates a metabolic/mitochondrial state in which Yme1-dependent remodeling of lipid and membrane-organization pathways becomes important for MDC formation. Whether this involves direct regulation of Yme1, altered substrate availability, altered membrane composition, or a combination of these remains open.
(5) The MICOS complex is clearly involved in the regulation of MDC, but the manuscript misses the mark on providing compelling evidence and a clear explanation as to how MICOS contributes to said regulation.
We agree that the mechanism by which MICOS regulates MDC formation remains an important open question and will be a major focus of future work. Our current data show that MICOS perturbation can partially restore MDC formation in yme1Δ cells, supporting a role for MICOS in this pathway. This analysis was motivated in part by the incomplete genetic suppression achieved through the lipid pathway alone, which suggested that additional Yme1-regulated factors contribute to MDC formation.
MICOS therefore represents a strong candidate for this additional regulatory input. However, defining whether MICOS acts through lipid distribution, OMM-IMM organization, membrane architecture, or another mechanism will require a deeper investigation than is possible within the scope of the current study. We will clarify this point in the revised manuscript and present the current findings as the beginning of a broader investigation into how MICOS contributes to MDC biogenesis.
Minor points:
(1) The authors should discuss potential reasons for the dramatically different rates of MDC formation in the S288C and W303 background cells. Does this have anything to do with generally more robust mitochondrial functions in the latter cells?
We agree this is worth discussing. One likely explanation is that the difference reflects broader differences in mitochondrial activity and metabolic state between these strain backgrounds. We and others have shown that W303 cells have more robust respiratory mitochondrial function than BY/S288C-derived cells, and in our hands W303 also shows lower MDC formation. This fits our broader model that MDCs are favored in glucose-grown or metabolically perturbed cells and do not form under respiratory conditions (Raghuram and Hughes, 2024). We do not yet know the genetic basis for this difference, so we will present this as an interesting future direction.
(2) Proper statistical analyses should be provided for all the graphs presented.
We will add statistical analyses where missing.
(3) The authors should include Yme1 immunoblots to confirm the identity of strains being studied and validate the presence or overexpression of Yme1 and its catalytic mutant in their experiments.
We agree that direct validation of Yme1 protein levels will strengthen the manuscript. Our quantitative mitochondrial proteomics already confirms strong depletion of Yme1 in yme1Δ cells, and we will also include quantitative proteomics showing increased Yme1 abundance in the overexpression strain. In addition, we have now obtained a Yme1 antibody from a colleague and will include immunoblots validating Yme1 loss, re-expression, catalytic mutant expression, and overexpression where appropriate.
Reviewer #3 (Public review):
Summary:
Since describing MDCs over a decade ago, the lab of the corresponding author, Hughes, has been at the forefront of further characterizing these structures. Here, they follow up on recent work (PMID: 38497895), where a screen identified Yme1 as a potential regulator of MDCs. After confirming that Yme1-ko prevents MDCs that are usually induced via various established treatments (Rapamycin, cycloheximide, Concanavalin A), the authors confirmed that the proteolytic activity of Yme1 is required. Next, using proteomics, they identified how loss of Yme1 impacts the mitochondrial proteome with and without Rapamycin treatment to induce MDCs. From this result and based on insight from other published data implicating lipids, the focused initially on the lipid transfer protein Usp2, a known target of Yme1. Here, they showed that loss of Usp2 could partially rescue MDC formation in Yme1-ko cells. To look for other Yme1 targets that might also be involved in MDC formation, next, they investigated the MICOS complex, which was also notable in their proteomics data. They then showed that inhibiting MICOS also partially restored MDC formation in Yme1-ko cells. They then tested the combined effects of Usp2 and MDC inhibition on MDCs, which was limited by the fact that the combination of full MICOS disruption, Usp2-KO, and Yme1-KO was not viable. To circumvent this limitation, they investigated the knockout of individual MICOS subunits in combination with Usp2 and/or Yme1. Finally, they showed that growth conditions also mediate MDC formation in the context of Yme1 overexpression. In rich media, Yme1 overexpression induces MDCs on its own. However, this induction is lost upon amino acid starvation, suggesting that there are still other as-yet-unidentified factors regulating the formation of MDCs.
Strengths:
The authors use unbiased approaches and genetic models to begin unraveling a novel regulatory role of Yme1 in the formation of MDCs.
Weaknesses:
(1) The authors find both Ups1 and Ups2 in their screens, but only focus on Ups2 in this paper. It would be good to know why they did not also investigate Ups1, and its other protease Atp23, which could potentially act similarly to Yme1, or even rescue the loss of Yme1.
We agree that Ups1 and Atp23 are important to consider. We initially focused on Ups2 because its deletion partially restores MDC formation in yme1Δ cells and because of its connection to mitochondrial PE synthesis, which we had previously shown to regulate MDC formation (Xiao et al., 2024). Ups1 is more difficult to assess genetically because complete loss of UPS1, or of downstream cardiolipin synthesis through CRD1, blocks MDC formation due to the requirement for cardiolipin. Thus, an ups1Δ phenotype cannot readily reveal whether a more moderate reduction in Ups1 activity, and the resulting accumulation or redistribution of PA, might promote MDC formation.
In the revision, we will explain this rationale and include new genetic data targeting PA metabolism through the yeast Pah1/Lipin pathway. This provides a way to test the contribution of PA accumulation without simultaneously eliminating cardiolipin synthesis, and our initial results support a role for PA-linked lipid remodeling in partially bypassing the requirement for Yme1. We will also discuss Atp23 as a potentially important regulator of Ups1 and PA metabolism. A full investigation of Atp23 will be an important direction for future work.
(2) I'm not convinced that the data support the notion that Usp2 and MICOS have distinct effects on MDCs. In Figure S3C-D, there is no statistical analysis to indicate whether the small differences between the MICOS-ko and the double knockout are significant. If MICOS-ko and Ups2-ko were acting through different mechanisms, one would expect their combination to be additive; this does not appear to be the case, as both single deletions and the double deletion all cause similar levels of MDCs (~30-40%). Rather, this result is what you would expect if they were working through the same mechanism. There also does not appear to be an additive effect in Figure 4F-G, when using the mic60-ko rather than the complete MICOS-ko. In this regard, the authors note in their discussion that 'loss of MICOS may disrupt membrane associations or alter lipid distribution between mitochondrial subcompartments' (lines 390-392). The latter situation seems like it would be the same mechanism as Usp2 and would more accurately explain their findings.
This is a very good point, and we agree with the reviewer’s interpretation. The lack of strong additivity is consistent with Ups2 and MICOS acting within the same pathway or converging on a shared mechanism, rather than representing two separate mechanisms of MDC regulation. We did not intend to imply that these must be independent pathways. In the revised manuscript, we will ensure that the text reflects this interpretation and will add statistical analyses to the relevant comparisons.
(3) The manuscript is missing key data confirming the re-expression or overexpression of Yme1 protein (Figure 1 E/G and Figure 5A). It is important to know the relative levels of expression of the re-expressed proteins to each other and to endogenous Yme1.
We agree that direct validation of Yme1 protein levels is important. Our quantitative mitochondrial proteomics already confirms strong depletion of Yme1 in yme1Δ cells, and we will also include quantitative proteomics showing increased Yme1 abundance in the overexpression strain. In addition, we have now obtained a Yme1 antibody from a colleague and will add immunoblots validating Yme1 loss, re-expression, catalytic mutant expression, and overexpression.
(4) Some clarification of the details for metabolically restrictive conditions would be helpful.
Thanks for this suggestion. We will clarify these conditions throughout the manuscript and figure legends and will define exactly what we mean by low-amino-acid, amino-acid-free, synthetic, and rich media conditions. More broadly, MDC formation is strongly influenced by media composition and mitochondrial metabolic state. MDCs form less efficiently in synthetic media and do not form under conditions that promote respiratory mitochondrial function (Raghuram and Hughes, 2024).
(5) Beyond just the presence/absence of MDCs, does more detailed quantification of their size/shape reveal any subtle differences between conditions?
This is an interesting question. In our hands, MDC size and shape are variable and appear strongly influenced by mitochondrial fission/fusion state. Conditions that favor more fused mitochondrial networks can produce larger MDC-like structures, whereas fragmented networks can produce smaller structures. So far, we have not found a simple size or shape metric that explains the Yme1/Ups2/MICOS phenotypes better than MDC frequency.
We will clarify this point in the revised manuscript and avoid implying that MDC frequency captures every possible morphological difference. More detailed morphometric analysis of MDC size, topology, and maturation state will be an important future direction, especially as we connect lipid remodeling to membrane curvature and MDC biogenesis.
References
Hughes, A.L., Hughes, C.E., Henderson, K.A., Yazvenko, N., and Gottschling, D.E. 2016. Selective sorting and destruction of mitochondrial membrane proteins in aged yeast. eLife. 5. doi: 10.7554/eLife.13943.
Raghuram, N., and Hughes, A.L. 2024. Amino acids trigger MDC-dependent mitochondrial remodeling by altering mitochondrial function. bioRxiv. 2024.07.09.602707. doi: 10.1101/2024.07.09.602707.
Wang, K., Jin, M., Liu, X., and Klionsky, D.J. 2013. Proteolytic processing of Atg32 by the mitochondrial i-AAA protease Yme1 regulates mitophagy. Autophagy. 9(11):1828–1836. doi: 10.4161/auto.26281.
Xiao, T., English, A.M., Wilson, Z.N., Maschek, J.A., Cox, J.E., and Hughes, A.L. 2024. The phospholipids cardiolipin and phosphatidylethanolamine differentially regulate MDC biogenesis. Journal of Cell Biology. 223(5). doi: 10.1083/jcb.202302069.
