| 1. |
- Rzepka, Magdalena, 1992, et al.
(author)
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Incorporation of reporter genes into mitochondrial DNA in budding yeast
- 2022
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In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
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Journal article (peer-reviewed)abstract
- Many aspects of mitochondrial gene expression are still unknown, which can be attributed to limitations in molecular tools. Here, we present a protocol to introduce reporter genes into the mitochondrial genome of budding yeast, Saccharomyces cerevisiae. Mitochondrially encoded reporter constructs can be used to interrogate various aspects of mitochondrial gene expression. The power of this technique is exemplified by a mitochondrially encoded nanoluciferase, which allows to monitor levels of mitochondrial translation under a variety of growth conditions.
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| 2. |
- Rzepka, Magdalena, et al.
(author)
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Rapid adjustment of mitochondrial function during metabolic switches as revealed by a novel mitochondrially encoded reporter
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Journal article (peer-reviewed)abstract
- Cells need to react to changing environments by adjusting metabolic pathways and gene expression to prepare cellular functions to the new needs. Saccharomyces cerevisiae is a facultative anaerobe that preferentially uses glucose as energy source. During fermentation of this sugar, other metabolic pathways and their genes’ expression are repressed. When glucose is exhausted, the cells activate oxidative phosphorylation to exploit the accumulated ethanol and acetic acid, products of the anaerobic glycolysis in yeast. How these metabolic switches affect mitochondrial function is poorly characterized. Here, we used a mitochondrial encoded nanoluciferase (mtnLuc) to follow adaptation of mitochondrial function during metabolic switches. We find that these switches activate or inhibit mtnLuc activity quickly in a reversible way. The main signaling axis employs components of glucose sensing and impinge on mitochondrial function by modulation of mitochondrial respiration and, consequently, the membrane potential. This study thereby reveals a rapid adjustment of mitochondrial functions during metabolic adaptations, pointing to a yet poorly understood aspect of cytosol-to-mitochondria communication.
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| 3. |
- Suhm, Tamara, et al.
(author)
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A novel system to monitor mitochondrial translation in yeast
- 2018
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In: Microbial Cell. - : Shared Science Publishers OG. - 2311-2638. ; 5:3, s. 158-164
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Journal article (peer-reviewed)abstract
- The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.
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| 4. |
- Suhm, Tamara, 1988-, et al.
(author)
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Different genetic approaches to mutate the mitochondrial ribosomal protein S12
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Other publication (other academic/artistic)abstract
- Over the last decades, an ever-growing number of tools became available to manipulate the genome of the model organism Saccharomyces cerevisiae. The most common approach to study a mutation in a protein is to first replace the native gene with a selection cassette via homologous recombination. In a second step, the mutated gene can be expressed from a plasmid. For certain applications, however, it is necessary to integrate the mutation in the genome. Here we introduced a mutated variant of the mitochondrial ribosomal protein S12 (Mrps12), a protein of the highly conserved accuracy center of the mitochondrial ribosome, using an integrative plasmid. First, we attempted to use a counter-selectable strategy by employing the uracil selection cassette (URA3) in combination with 5-fluoroorotic acid (5-FOA). We observed that this approach is not ideal for mutating certain crucial mitochondrial proteins. In our hands, this method only gave false-positive results. Most likely, deletion of MRPS12 and subsequent loss of mitochondrial DNA caused genome instability. This gave rise to mutated versions of URA3 which could no longer be used for counter selection. Therefore, we eventually introduced the MRPS12* under control of its endogenous promotor and terminator via an integrative plasmid in the deletion strain.
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| 5. |
- Suhm, Tamara, et al.
(author)
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Mitochondrial translation and cellular stress response
- 2017
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In: Cell and Tissue Research. - : Springer Science and Business Media LLC. - 0302-766X .- 1432-0878. ; 367:1, s. 21-31
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Research review (peer-reviewed)abstract
- Mitochondria are organelles critical for the functionality of eukaryotic cells. One of their most prominent functions is energy conversion, thereby producing most of the cellular ATP. Energy conversion relies on the oxidative phosphorylation system, an ensemble of large protein complexes that include the respiratory chain and the ATP synthase. Biogenesis of this machinery requires the coordination of two separate genetic systems, namely nuclear and mitochondrial gene expression. Recent research into the molecular causes of aging have revealed a prominent contribution of mitochondrial gene expression on many aspects of degenerative processes that typically involve cellular stress signaling pathways. In this review, we summarize recent developments in attempting to identify the molecular mechanism by which dysfunction of mitochondrial gene expression activates cellular stress signaling pathways and how this affects organismal aging. By comparing data obtained in various model organisms, we identify conserved and species-specific aspects of this mitochondria-to-nucleus signaling.
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| 6. |
- Suhm, Tamara, 1988-
(author)
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Mitochondrial translation and its impact on protein homeostasis and aging
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Besides their famous role as powerhouse of the cell, mitochondria are also involved in many signaling processes and metabolism. Therefore, it is unsurprising that mitochondria are no isolated organelles but are in constant crosstalk with other parts of the cell. Due to the endosymbiotic origin of mitochondria, they still contain their own genome and gene expression machinery. The mitochondrial genome of yeast encodes eight proteins whereof seven are core subunits of the respiratory chain and ATP synthase. These subunits need to be assembled with subunits imported from the cytosol to ensure energy supply of the cell. Hence, coordination, timing and accuracy of mitochondrial gene expression is crucial for cellular energy production and homeostasis. Despite the central role of mitochondrial translation surprisingly little is known about the molecular mechanisms.In this work, I used baker’s yeast Saccharomyces cerevisiae to study different aspects of mitochondrial translation. Exploiting the unique possibility to make directed modifications in the mitochondrial genome of yeast, I established a mitochondrial encoded GFP reporter. This reporter allows monitoring of mitochondrial translation with different detection methods and enables more detailed studies focusing on timing and regulation of mitochondrial translation. Furthermore, employing insights gained from bacterial translation, we showed that mitochondrial translation efficiency directly impacts on protein homeostasis of the cytoplasm and lifespan by affecting stress handling. Lastly, we provided first evidence that mitochondrial protein quality control happens at a very early stage directly after or during protein synthesis at the ribosome. Surveillance of protein synthesis and assembly into complexes is important to avoid accumulation of misfolded or unassembled respiratory chain subunits which would disturb mitochondrial function.
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| 7. |
- Suhm, Tamara, et al.
(author)
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Mitochondrial Translation Efficiency Controls Cytoplasmic Protein Homeostasis
- 2018
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In: Cell Metabolism. - : Elsevier BV. - 1550-4131 .- 1932-7420. ; 27:6
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Journal article (peer-reviewed)abstract
- Cellular proteostasis ismaintained via the coordinated synthesis, maintenance, and breakdown of proteins in the cytosol and organelles. While biogenesis of the mitochondrial membrane complexes that execute oxidative phosphorylation depends on cytoplasmic translation, it is unknown how translation within mitochondria impacts cytoplasmic proteostasis and nuclear gene expression. Here we have analyzed the effects of mutations in the highly conserved accuracy center of the yeast mitoribosome. Decreased accuracy of mitochondrial translation shortened chronological lifespan, impaired management of cytosolic protein aggregates, and elicited a general transcriptional stress response. In striking contrast, increased accuracy extended lifespan, improved cytosolic aggregate clearance, and suppressed a normally stress-induced, Msn2/4-dependent interor-ganellar proteostasis transcription program (IPTP) that regulates genes important for mitochondrial proteostasis. Collectively, the data demonstrate that cytosolic protein homeostasis and nuclear stress signaling are controlled by mitochondrial translation efficiency in an inter-connected organelle quality control network that determines cellular lifespan.
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| 8. |
- Vargas Möller-Hergt, Braulio, et al.
(author)
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Insertion Defects of Mitochondrially Encoded Proteins Burden the Mitochondrial Quality Control System
- 2018
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In: Cells. - : MDPI AG. - 2073-4409. ; 7:10
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Journal article (peer-reviewed)abstract
- The mitochondrial proteome contains proteins from two different genetic systems. Proteins are either synthesized in the cytosol and imported into the different compartments of the organelle or directly produced in the mitochondrial matrix. To ensure proteostasis, proteins are monitored by the mitochondrial quality control system, which will degrade non-native polypeptides. Defective mitochondrial membrane proteins are degraded by membrane-bound AAA-proteases. These proteases are regulated by factors promoting protein turnover or preventing their degradation. Here we determined genetic interactions between the mitoribosome receptors Mrx15 and Mba1 with the quality control system. We show that simultaneous absence of Mrx15 and the regulators of the i-AAA protease Mgr1 and Mgr3 provokes respiratory deficiency. Surprisingly, mutants lacking Mrx15 were more tolerant against proteotoxic stress. Furthermore, yeast cells became hypersensitive against proteotoxic stress upon deletion of MBA1. Contrary to Mrx15, Mba1 cooperates with the regulators of the m-AAA and i-AAA proteases. Taken together, these results suggest that membrane protein insertion and mitochondrial AAA-proteases are functionally coupled, possibly reflecting an early quality control step during mitochondrial protein synthesis.
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| 9. |
- Vazquez-Calvo, Carmela, et al.
(author)
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The basic machineries for mitochondrial protein quality control
- 2020
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In: Mitochondrion (Amsterdam. Print). - : Elsevier BV. - 1567-7249 .- 1872-8278. ; 50, s. 121-131
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Journal article (peer-reviewed)abstract
- Mitochondria play pivotal roles in cellular energy metabolism, the synthesis of essential biomolecules and the regulation of cell death and aging. The proper folding, unfolding and degradation of the many proteins active within mitochondria is surveyed by the mitochondrial quality control machineries. Here, we describe the principal components of the mitochondrial quality control system and recent developments in the elucidation of the molecular mechanisms maintaining a functional mitochondrial proteome.
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