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Different genetic approaches to mutate the mitochondrial ribosomal protein S12
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
(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 [en]
mitochondrial ribosome, yeast genetics, counter selection
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-163146OAI: oai:DiVA.org:su-163146DiVA, id: diva2:1271173
Available from: 2018-12-17 Created: 2018-12-17 Last updated: 2018-12-18Bibliographically approved
In thesis
1. Mitochondrial translation and its impact on protein homeostasis and aging
Open this publication in new window or tab >>Mitochondrial translation and its impact on protein homeostasis and aging
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
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:nbn:se:su:diva-163149 (URN)978-91-7797-542-7 (ISBN)978-91-7797-543-4 (ISBN)
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: 2020-05-11Bibliographically approved

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