Tom70-based transcriptional regulation of mitochondrial biogenesis and aging
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Evaluation Summary:
The authors test the hypothesis that components of the TOM complex regulate efficient mitochondrial biogenesis by coordinating the synthesis (via controlling transcription of the corresponding RNAs) of mitochondrial proteins with the rate of mitochondrial protein import. It has previously been established that failure to import mitochondrial proteins results in the accumulation of toxic protein aggregates in the cytosol. The authors conclude that Tom70 fulfills this role and find that Tom70 expression declines as cells age, which contributes to age-associated mitochondrial dysfunction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Mitochondrial biogenesis has two major steps: the transcriptional activation of nuclear genome-encoded mitochondrial proteins and the import of nascent mitochondrial proteins that are synthesized in the cytosol. These nascent mitochondrial proteins are aggregation-prone and can cause cytosolic proteostasis stress. The transcription factor-dependent transcriptional regulations and the TOM-TIM complex-dependent import of nascent mitochondrial proteins have been extensively studied. Yet, little is known regarding how these two steps of mitochondrial biogenesis coordinate with each other to avoid the cytosolic accumulation of these aggregation-prone nascent mitochondrial proteins. Here, we show that in budding yeast, Tom70, a conserved receptor of the TOM complex, moonlights to regulate the transcriptional activity of mitochondrial proteins. Tom70’s transcription regulatory role is conserved in Drosophila . The dual roles of Tom70 in both transcription/biogenesis and import of mitochondrial proteins allow the cells to accomplish mitochondrial biogenesis without compromising cytosolic proteostasis. The age-related reduction of Tom70, caused by reduced biogenesis and increased degradation of Tom70, is associated with the loss of mitochondrial membrane potential, mtDNA, and mitochondrial proteins. While loss of Tom70 accelerates aging and age-related mitochondrial defects, overexpressing TOM70 delays these mitochondrial dysfunctions and extends the replicative lifespan. Our results reveal unexpected roles of Tom70 in mitochondrial biogenesis and aging.
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Author Response:
Reviewer #1:
The paper by Liu et al investigates the question of whether the mitochondrial protein import component Tom70 might be involved in the coordination of biogenesis and localization of mitochondrial proteins. It follows a smart hypothesis that positions Tom70 in a coordinating role of nuclear-encoded mitochondrial gene expression and subsequent protein incorporation. The paper shows that Tom70 overexpression uniquely promotes the expression of numerous mitochondrial proteins and that Tom70's mitochondrial localization is required for this. The data then suggest that both mtDNA and a combination of transcription factors are involved in Tom70 controlled nuclear gene expression. The authors then find that Tom70 is also required to dampen nuclear mitochondrial gene expression during import stress. Importantly, …
Author Response:
Reviewer #1:
The paper by Liu et al investigates the question of whether the mitochondrial protein import component Tom70 might be involved in the coordination of biogenesis and localization of mitochondrial proteins. It follows a smart hypothesis that positions Tom70 in a coordinating role of nuclear-encoded mitochondrial gene expression and subsequent protein incorporation. The paper shows that Tom70 overexpression uniquely promotes the expression of numerous mitochondrial proteins and that Tom70's mitochondrial localization is required for this. The data then suggest that both mtDNA and a combination of transcription factors are involved in Tom70 controlled nuclear gene expression. The authors then find that Tom70 is also required to dampen nuclear mitochondrial gene expression during import stress. Importantly, Tom70 and numerous other import machinery components become depleted with age in yeast, and the same is true for mitochondrial membrane potential. This is a very strong part of the paper. Tom70 OE rescues this effect, and Tom70 OE extends survival. Finally, suggestive data show that the age-dependent Tom70 depletion is due to reduced expression and enhanced degradation.
This is an interesting study that uses cutting-edge methods. However, a clear focus of the paper is somewhat missing. The paper touches on many topics that remain unresolved. These include the role of CR and the role of LCD-containing TFs as an explanation for the age-dependent decline of Tom70. There is a role of mtDNA in the Tom70 OE but the link to transcription factors remains unaddressed. For example, degradation of Tom70 is investigated via MDCs, but is autophagy involved? There is a large amount of data in the manuscript that cover a lot of territory, but further mechanistic insights would significantly enhance the paper.
The authors appreciate the enthusiasm and comments from the reviewer. The focus of our manuscript is to report the discovery that Tom70 moonlights to regulate the biogenesis of mitochondrial protein and mtDNA (Figure 1-3). After identifying this new role of Tom70, we applied it to understand physiological processes including mitochondrial import-related stress response and aging-related mitochondrial dysfunctions (Figure 4-7). We did these application studies because we think it is necessary to understand the physiological significance of this Tom70’s role, which was strongly encouraged by senior colleagues in the aging field during the progress of our project.
Specifically, we removed the LCD/proteostasis part according to reviewers’ suggestions. We include caloric restriction (CR) because this Tom70’s role can explain the previously observed phenotypes in the aging field, and it is important to connect our discoveries to the previous works as encouraged by colleagues in the field during the progress of our project. We think this is a strength of our study that generated new knowledge which can be used to address previous questions. Regarding mtDNA, we focused on the biogenesis role of Tom70 on mtDNA as a necessary and immediate extension of our study on the biogenesis of mitochondrial proteins. Indeed, the detailed mechanism of how Tom70 signals through the secondary messengers and multiple TFs to achieve these biogenesis functions is still not clear, which will be our future goal. Similar to our manuscript, the seminal study that identified PGC-1 as a key mitochondrial biogenesis regulator also reported the increase of mitochondrial proteins and mtDNA through the induction of a few TFs by PGC-1 (Wu et al., 1999). Regarding MDCs, we simply applied previous knowledge from Hughes and Gottschling’s study that Fis1/Mdv1 regulate the sorting of Tom70 into MDCs in response to acute vacuole stress (Hughes et al., 2016). They have carefully dissected the role of autophagy downstream of MDC formation. However, our purpose is to see what regulates Tom70’s abundance but not how MDC is disposed during aging.
Reviewer #2:
The authors test the hypothesis that components of the TOM complex regulate efficient mitochondrial biogenesis by coordinating the synthesis of mitochondrial proteins with the rate of mitochondrial protein import. In general, the experiments are well developed and the findings and topic are likely of broad interest. The weaknesses are mainly related to the underdeveloped approaches and the vagaries related to the mechanism(s) by which Tom70 influences transcription of mitochondrial components.
The authors performed a survey of TOM components (proteins required for protein translocation from the cytosol into mitochondria) and found that overexpression of Tom70 was sufficient to increase accumulation of 4 GFP-tagged mitochondrial proteins that localize to each of the four mitochondrial sub-compartments suggesting that Tom70 has a unique role in mitochondrial biogenesis.
Interestingly, the authors demonstrate that Tom70 is required to limit transcription of mitochondrial components when Tim23 is impaired. In doing so, Tom70 prevents the aggregation of mitochondrial proteins that fail to be imported into mitochondria.
The authors demonstrate that mtDNA is required for the increase in mitochondrial component transcription upon Tom70 overexpression. This is an exciting observation. However, experiments to understand the phenomenon are not considered.
The authors appreciate the enthusiasm and comments from the reviewer. The focus of our manuscript is to report the discovery that Tom70 regulates the biogenesis of mitochondrial protein and mtDNA. According to reviewers’ suggestion, we tested several possibilities and the results have been discussed. The detailed mechanism of how Tom70 signals through the secondary messengers to achieve this function will be the next focus of our lab.
Interestingly, overexpression of Tom70 prevents the decline in mitochondrial function typically observed in aging cells, while Tom70-deletion accelerated the loss of mitochondrial function.
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Evaluation Summary:
The authors test the hypothesis that components of the TOM complex regulate efficient mitochondrial biogenesis by coordinating the synthesis (via controlling transcription of the corresponding RNAs) of mitochondrial proteins with the rate of mitochondrial protein import. It has previously been established that failure to import mitochondrial proteins results in the accumulation of toxic protein aggregates in the cytosol. The authors conclude that Tom70 fulfills this role and find that Tom70 expression declines as cells age, which contributes to age-associated mitochondrial dysfunction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the …
Evaluation Summary:
The authors test the hypothesis that components of the TOM complex regulate efficient mitochondrial biogenesis by coordinating the synthesis (via controlling transcription of the corresponding RNAs) of mitochondrial proteins with the rate of mitochondrial protein import. It has previously been established that failure to import mitochondrial proteins results in the accumulation of toxic protein aggregates in the cytosol. The authors conclude that Tom70 fulfills this role and find that Tom70 expression declines as cells age, which contributes to age-associated mitochondrial dysfunction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The paper by Liu et al investigates the question of whether the mitochondrial protein import component Tom70 might be involved in the coordination of biogenesis and localization of mitochondrial proteins. It follows a smart hypothesis that positions Tom70 in a coordinating role of nuclear-encoded mitochondrial gene expression and subsequent protein incorporation. The paper shows that Tom70 overexpression uniquely promotes the expression of numerous mitochondrial proteins and that Tom70's mitochondrial localization is required for this. The data then suggest that both mtDNA and a combination of transcription factors are involved in Tom70 controlled nuclear gene expression. The authors then find that Tom70 is also required to dampen nuclear mitochondrial gene expression during import stress. Importantly, Tom70 …
Reviewer #1 (Public Review):
The paper by Liu et al investigates the question of whether the mitochondrial protein import component Tom70 might be involved in the coordination of biogenesis and localization of mitochondrial proteins. It follows a smart hypothesis that positions Tom70 in a coordinating role of nuclear-encoded mitochondrial gene expression and subsequent protein incorporation. The paper shows that Tom70 overexpression uniquely promotes the expression of numerous mitochondrial proteins and that Tom70's mitochondrial localization is required for this. The data then suggest that both mtDNA and a combination of transcription factors are involved in Tom70 controlled nuclear gene expression. The authors then find that Tom70 is also required to dampen nuclear mitochondrial gene expression during import stress. Importantly, Tom70 and numerous other import machinery components become depleted with age in yeast, and the same is true for mitochondrial membrane potential. This is a very strong part of the paper. Tom70 OE rescues this effect, and Tom70 OE extends survival. Finally, suggestive data show that the age-dependent Tom70 depletion is due to reduced expression and enhanced degradation.
This is an interesting study that uses cutting-edge methods. However, a clear focus of the paper is somewhat missing. The paper touches on many topics that remain unresolved. These include the role of CR and the role of LCD-containing TFs as an explanation for the age-dependent decline of Tom70. There is a role of mtDNA in the Tom70 OE but the link to transcription factors remains unaddressed. For example, degradation of Tom70 is investigated via MDCs, but is autophagy involved? There is a large amount of data in the manuscript that cover a lot of territory, but further mechanistic insights would significantly enhance the paper.
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Reviewer #2 (Public Review):
The authors test the hypothesis that components of the TOM complex regulate efficient mitochondrial biogenesis by coordinating the synthesis of mitochondrial proteins with the rate of mitochondrial protein import. In general, the experiments are well developed and the findings and topic are likely of broad interest. The weaknesses are mainly related to the underdeveloped approaches and the vagaries related to the mechanism(s) by which Tom70 influences transcription of mitochondrial components.
The authors performed a survey of TOM components (proteins required for protein translocation from the cytosol into mitochondria) and found that overexpression of Tom70 was sufficient to increase accumulation of 4 GFP-tagged mitochondrial proteins that localize to each of the four mitochondrial sub-compartments …
Reviewer #2 (Public Review):
The authors test the hypothesis that components of the TOM complex regulate efficient mitochondrial biogenesis by coordinating the synthesis of mitochondrial proteins with the rate of mitochondrial protein import. In general, the experiments are well developed and the findings and topic are likely of broad interest. The weaknesses are mainly related to the underdeveloped approaches and the vagaries related to the mechanism(s) by which Tom70 influences transcription of mitochondrial components.
The authors performed a survey of TOM components (proteins required for protein translocation from the cytosol into mitochondria) and found that overexpression of Tom70 was sufficient to increase accumulation of 4 GFP-tagged mitochondrial proteins that localize to each of the four mitochondrial sub-compartments suggesting that Tom70 has a unique role in mitochondrial biogenesis.
Interestingly, the authors demonstrate that Tom70 is required to limit transcription of mitochondrial components when Tim23 is impaired. In doing so, Tom70 prevents the aggregation of mitochondrial proteins that fail to be imported into mitochondria.
The authors demonstrate that mtDNA is required for the increase in mitochondrial component transcription upon Tom70 overexpression. This is an exciting observation. However, experiments to understand the phenomenon are not considered.
Interestingly, overexpression of Tom70 prevents the decline in mitochondrial function typically observed in aging cells, while Tom70-deletion accelerated the loss of mitochondrial function.
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Reviewer #3 (Public Review):
Mitochondria are autonomous double-membrane-bound organelles in eukaryotic cells. They synthesize ATP to meet the energy needs of the organism through oxidative phosphorylation. This cardinal role in energy production makes mitochondria a key player in metabolic, degenerative, and age-related diseases. Dysregulation of mitochondria is ubiquitous in diabetes, obesity, cardiovascular disease, cancer, etc. Research in the past decades have made huge progress in our understanding of mitochondrial biology.
The current manuscript explores the dual role of Tom70 in coupling the highly orchestrated process of the transcriptional activation of nuclear genome-encoded mitochondrial proteins and the import of nascent mitochondrial proteins that are synthesized in the cytosol. With the help of cutting-edge techniques, …
Reviewer #3 (Public Review):
Mitochondria are autonomous double-membrane-bound organelles in eukaryotic cells. They synthesize ATP to meet the energy needs of the organism through oxidative phosphorylation. This cardinal role in energy production makes mitochondria a key player in metabolic, degenerative, and age-related diseases. Dysregulation of mitochondria is ubiquitous in diabetes, obesity, cardiovascular disease, cancer, etc. Research in the past decades have made huge progress in our understanding of mitochondrial biology.
The current manuscript explores the dual role of Tom70 in coupling the highly orchestrated process of the transcriptional activation of nuclear genome-encoded mitochondrial proteins and the import of nascent mitochondrial proteins that are synthesized in the cytosol. With the help of cutting-edge techniques, the authors have demonstrated satisfactorily that Tom70 regulates the transcription of specific mitochondrial proteins and do so through transcription factors. Using quantitative imaging, the authors show that Tom70 regulate the mtDNA content. Further, the Tom70 protein was shown to play a crucial role in the feedback loop that ensures coupling of mitochondrial protein synthesis and import of these proteins into the mitochondria. Tom70 was also shown to have a crucial role in age-related defects in the mitochondria leading to mitochondrial dysfunction which seems to be conserved across various organisms. The conclusions of this paper are mostly well supported by data, but some clarifications are needed.
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