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The interactome of the yeast mitochondrial ribosome: Organization of mitochondrial post-transcriptional regulation, membrane protein insertion and quality control
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
2018 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2018. , s. 72
Nyckelord [en]
Mitochondria, mitochondrial post-transcriptional regulation, mitochondrial ribosome, membrane protein insertion, mitochondrial quality control
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-159455ISBN: 978-91-7797-404-8 (tryckt)ISBN: 978-91-7797-405-5 (digital)OAI: oai:DiVA.org:su-159455DiVA, id: diva2:1245718
Disputation
2018-10-19, Magnélisalen Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Anmärkning

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted.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

Tillgänglig från: 2018-09-26 Skapad: 2018-09-05 Senast uppdaterad: 2020-05-11Bibliografiskt granskad
Delarbeten
1. Organization of Mitochondrial Gene Expression in Two Distinct Ribosome-Containing Assemblies
Öppna denna publikation i ny flik eller fönster >>Organization of Mitochondrial Gene Expression in Two Distinct Ribosome-Containing Assemblies
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2015 (Engelska)Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 10, nr 6, s. 843-853Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Mitochondria contain their own genetic system that provides subunits of the complexes driving oxidative phosphorylation. A quarter of the mitochondrial proteome participates in gene expression, but how all these factors are orchestrated and spatially organized is currently unknown. Here, we established a method to purify and analyze native and intact complexes of mitochondrial ribosomes. Quantitative mass spectrometry revealed extensive interactions of ribosomes with factors involved in all the steps of posttranscriptional gene expression. These interactions result in large expressosome-like assemblies that we termed mitochondrial organization of gene expression (MIOREX) complexes. Superresolution microscopy revealed that most MIOREX complexes are evenly distributed throughout the mitochondrial network, whereas a subset is present as nucleoid-MIOREX complexes that unite the whole spectrum of organellar gene expression. Our work therefore provides a conceptual framework for the spatial organization of mitochondrial protein synthesis that likely developed to facilitate gene expression in the organelle.

Nationell ämneskategori
Biologiska vetenskaper
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-115276 (URN)10.1016/j.celrep.2015.01.012 (DOI)000349440200001 ()
Anmärkning

AuthorCount:8;

Tillgänglig från: 2015-03-31 Skapad: 2015-03-18 Senast uppdaterad: 2018-09-05Bibliografiskt granskad
2. The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria
Öppna denna publikation i ny flik eller fönster >>The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria
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2018 (Engelska)Ingår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 29, nr 20, s. 2359-2507Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for the production of highly hydrophobic subunits of the oxidative phosphorylation system. Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry-based approach, we discovered the previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria.

Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-159308 (URN)10.1091/mbc.E18-04-0227 (DOI)000451910900004 ()
Tillgänglig från: 2018-08-27 Skapad: 2018-08-27 Senast uppdaterad: 2019-01-07Bibliografiskt granskad
3. Insertion Defects of Mitochondrially Encoded Proteins Burden the Mitochondrial Quality Control System
Öppna denna publikation i ny flik eller fönster >>Insertion Defects of Mitochondrially Encoded Proteins Burden the Mitochondrial Quality Control System
2018 (Engelska)Ingår i: Cells, ISSN 2073-4409, Vol. 7, nr 10, artikel-id 172Artikel i tidskrift (Refereegranskat) 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.

Nyckelord
mitochondria, mitochondrial quality control, membrane protein insertion, membrane-bound AAA proteases
Nationell ämneskategori
Biologiska vetenskaper
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-162935 (URN)10.3390/cells7100172 (DOI)000448818800030 ()30336542 (PubMedID)
Tillgänglig från: 2018-12-17 Skapad: 2018-12-17 Senast uppdaterad: 2018-12-18Bibliografiskt granskad
4. Molecular Wiring of a Mitochondrial Translational Feedback Loop
Öppna denna publikation i ny flik eller fönster >>Molecular Wiring of a Mitochondrial Translational Feedback Loop
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2020 (Engelska)Ingår i: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 77, nr 4, s. 887-900Artikel i tidskrift (Refereegranskat) 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.

Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-159453 (URN)10.1016/j.molcel.2019.11.019 (DOI)000519769200017 ()
Tillgänglig från: 2018-08-29 Skapad: 2018-08-29 Senast uppdaterad: 2020-04-28Bibliografiskt granskad

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