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Mitochondrial translation and its impact on protein homeostasis and aging
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Martin Ott)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2019. , p. 76
Keywords [en]
mitochondrial ribosome, mitochondrial translation accuracy, mitochondrial communication, interorganellar communication, stress signaling, proteostasis, aging, yeast genetics, mitochondrial protein quality control, mitochondrial membrane protein insertion
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-163149ISBN: 978-91-7797-542-7 (print)ISBN: 978-91-7797-543-4 (electronic)OAI: oai:DiVA.org:su-163149DiVA, id: diva2:1271354
Public defence
2019-02-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 09:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2019-01-23 Created: 2018-12-17 Last updated: 2019-01-22Bibliographically approved
List of papers
1. A novel system to monitor mitochondrial translation in yeast
Open this publication in new window or tab >>A novel system to monitor mitochondrial translation in yeast
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2018 (English)In: Microbial Cell, ISSN 2311-2638, Vol. 5, no 3, p. 158-164Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
mitochondrial translation, flow cytometry, superfolder GFP, strain engineering
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-156126 (URN)10.15698/mic2018.03.621 (DOI)000429112200004 ()29487862 (PubMedID)
Available from: 2018-05-03 Created: 2018-05-03 Last updated: 2018-12-18Bibliographically approved
2. Different genetic approaches to mutate the mitochondrial ribosomal protein S12
Open this publication in new window or tab >>Different genetic approaches to mutate the mitochondrial ribosomal protein S12
(English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

Keywords
mitochondrial ribosome, yeast genetics, counter selection
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-163146 (URN)
Available from: 2018-12-17 Created: 2018-12-17 Last updated: 2018-12-18Bibliographically approved
3. Mitochondrial Translation Efficiency Controls Cytoplasmic Protein Homeostasis
Open this publication in new window or tab >>Mitochondrial Translation Efficiency Controls Cytoplasmic Protein Homeostasis
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2018 (English)In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 27, no 6, p. 1309-1322Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biological Sciences
Research subject
Biochemistry; Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-157770 (URN)10.1016/j.cmet.2018.04.011 (DOI)000434480000016 ()29754951 (PubMedID)
Available from: 2018-06-25 Created: 2018-06-25 Last updated: 2019-03-29Bibliographically approved
4. Insertion Defects of Mitochondrially Encoded Proteins Burden the Mitochondrial Quality Control System
Open this publication in new window or tab >>Insertion Defects of Mitochondrially Encoded Proteins Burden the Mitochondrial Quality Control System
2018 (English)In: Cells, ISSN 2073-4409, Vol. 7, no 10, article id 172Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
mitochondria, mitochondrial quality control, membrane protein insertion, membrane-bound AAA proteases
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-162935 (URN)10.3390/cells7100172 (DOI)000448818800030 ()30336542 (PubMedID)
Available from: 2018-12-17 Created: 2018-12-17 Last updated: 2018-12-18Bibliographically approved

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