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Molecular Wiring of a Mitochondrial Translational Feedback Loop
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2020 (English)In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 77, no 4, p. 887-900Article in journal (Refereed) Published
Abstract [en]

The mitochondrial oxidative phosphorylation system comprises complexes assembled from subunits derived from mitochondrial and nuclear gene expression. Both genetic systems are coordinated by feedback loops, which control the synthesis of specific mitochondrial encoded subunits. Here, we studied how this occurs in the case of cytochrome b, a key subunit of mitochondrial complex III. Our data suggest the presence of a molecular rheostat consisting of two translational activators, Cbp3-Cbp6 and Cbs1, which operates at the mitoribosomal tunnel exit to connect translational output with assembly efficiency. When Cbp3-Cbp6 is engaged in assembly of cytochrome b, Cbs1 binds to the tunnel exit to sequester the cytochrome b-encoding mRNA, repressing its translation. After mediating complex III assembly, binding of Cbp3-Cbp6 to the tunnel exit replaces Cbs1 and the bound mRNA to permit cytochrome b synthesis. Collectively, the data indicate the molecular wiring of a feedback loop to regulate synthesis of a mitochondrial encoded protein.

Place, publisher, year, edition, pages
2020. Vol. 77, no 4, p. 887-900
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-159453DOI: 10.1016/j.molcel.2019.11.019ISI: 000519769200017OAI: oai:DiVA.org:su-159453DiVA, id: diva2:1242960
Available from: 2018-08-29 Created: 2018-08-29 Last updated: 2020-04-28Bibliographically approved
In thesis
1. The interactome of the yeast mitochondrial ribosome: Organization of mitochondrial post-transcriptional regulation, membrane protein insertion and quality control
Open this publication in new window or tab >>The interactome of the yeast mitochondrial ribosome: Organization of mitochondrial post-transcriptional regulation, membrane protein insertion and quality control
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The proteins found in mitochondria originate from two different genetic systems. Most mitochondrial proteins are synthesized in the cytosol and post-translationally imported into the organelle. However, a small subset of mitochondrial proteins is encoded in an organelle-resident genome. Mitochondria contain factors responsible for replication, transcription and, most important for this thesis, synthesis of the mitochondrially encoded proteins. In the course of evolution the mitochondria specific ribosomes were extensively remodeled. The reasons for many of these adaptations are currently not well understood. For example, the mitoribosome is less stable and abundant than its bacterial counterpart. Therefore, I contributed in the development of robust biochemical tools in order to isolate and analyze the intact yeast mitoribosome and interaction partners by mass spectrometry. The results revealed a higher order organization of mitochondrial gene expression in complexes that we termed MIOREX (mitochondrial organization of gene expression). Besides the mitoribosome, MIOREX complexes contain factors involved in all steps of gene expression. This study also established many new ribosomal interaction partners, among them some proteins that were previously completely uncharacterized. In order to study these proteins, I refined the mass spectrometry approach, allowing a subunit-specific assignment of ribosomal interaction partners. The Mrx15 protein was determined by this approach as an interactor of the large subunit. I established that Mrx15 has overlapping functions with the ribosome receptor Mba1. Both proteins are necessary for mitoribosome membrane attachment and co-translational Cox2 membrane insertion. In a subsequent study I found a functional interaction of MRX15 and MBA1 with the regulators of the membrane-bound AAA proteases of the mitochondrial quality control system. Furthermore, the absence of Mrx15 leads to increased, the absence of Mba1 to decreased proteotoxic stress resistance of yeast cells. These results demonstrate an interesting connection between the mitochondrial quality control and membrane insertion machineries, suggesting an early quality control step during the biogenesis of mitochondrially encoded proteins. In addition, we could reveal a subunit-specific interaction of translational activators and client mRNAs with the mitochondrial ribosome. This organization demonstrated how cytochrome b synthesis is pre-organized by specific translational activators independently of the COB mRNA. In summary, the work in this thesis showed how the vast and diverse interactome of the yeast mitoribosome organizes and regulates mitochondrial translation. These regulation mechanisms highlighted many organelle specific features. The work presented here will serve as starting point to design future studies aimed at a better understanding on how mitochondria adapted to organize gene expression inside the organelle.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. p. 72
Keywords
Mitochondria, mitochondrial post-transcriptional regulation, mitochondrial ribosome, membrane protein insertion, mitochondrial quality control
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-159455 (URN)978-91-7797-404-8 (ISBN)978-91-7797-405-5 (ISBN)
Public defence
2018-10-19, Magnélisalen Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 4: Manuscript.

Available from: 2018-09-26 Created: 2018-09-05 Last updated: 2020-05-11Bibliographically approved
2. Organization and regulation of mitochondrial gene expression
Open this publication in new window or tab >>Organization and regulation of mitochondrial gene expression
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mitochondria possess their own genome, remnant of the ancestral eubacterial endosymbiont DNA. This mitochondrial genome encodes mostly few key subunits of the respiratory chain. In order to synthesize these few proteins, mitochondria contain a complete gene expression machinery. Crucially, during the evolution, this apparatus dramatically diverged from its bacterial original counterpart, acquiring unique organellar characteristics. Hence, the mechanisms underlying organization and regulation of mitochondrial gene expression are still enigmatic.

In this thesis, I used the model organism Saccharomyces cerevisiae to reveal few aspects of mitochondrial gene expression. Surprisingly, I report that translation initiation strongly diverged from the bacterial one. In fact, the mitochondrial counterpart of the bacterial translation initiation factor 3 is dispensable in yeast. Furthermore, the research made in this work contributed to establish the proximity labelling technique BioID for yeast mitochondrial proteins. This method permitted to analyse extensively the mitochondrial gene expression milieu, creating a comprehensive proximity-based network of factors involved in biogenesis of mitochondrial synthesized proteins. This protein network revealed a unique organization of factors involved in mitochondrial gene expression, meticulously tailored for the synthesis of few organellar proteins. Crucially, we could identify a clear spatial distribution of factors according to their biological function. Moreover, the thesis describes how the polypeptide tunnel exit hosts proteins involved in multiple functions. First, the results show how factors involved in early maturation of Cox1, the core subunit of complex IV of the respiratory chain, reside at the polypeptide tunnel exit. Second, we demonstrate that the synthesis of cytochrome b, subunit of complex III, is also activated at the polypeptide tunnel exit. In fact, proteins taking part in the regulation of mitochondrial gene expression called translational activators interact with this area in an alternate fashion. When synthesis of cytochrome b is repressed, its coding mRNA COB is sequestered at the polypeptide tunnel exit via the binding to Cbs1, a translational activator. The signal that triggers translation initiation is given by Cbp3-Cbp6, a complex that participates in cytochrome b assembly. When a previously synthesized cytochrome b is correctly assembled into complex III, Cbp3-Cbp6 interacts with the polypeptide tunnel exit, forcing the relocation of Cbs1, and making COB mRNA available for a new round of translation. This mechanism represents a unique form of tuning between mitochondrial and nuclear gene expression systems, essential for the correct assembly of complexes made up by proteins of dual origin.

In summary, the work presented in this thesis reveals novel features of the organization and regulation of the mitochondrial gene expression, highlighting many distinctive organellar features. The concepts and techniques presented here will be a starting point to elucidate many unknown aspects of mitochondrial protein synthesis.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. p. 67
Keywords
Mitochondria, mitochondrial gene expression, post-transcriptional regulation, mitochondrial ribosome, protein-protein proximity, BioID, feedback loop
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-180258 (URN)978-91-7911-094-9 (ISBN)978-91-7911-095-6 (ISBN)
Public defence
2020-05-22, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Submitted.

Available from: 2020-04-27 Created: 2020-03-24 Last updated: 2020-05-26Bibliographically approved

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