| 1. |
- Singh, Abeer Prakash, 1988, et al.
(författare)
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Molecular Connectivity of Mitochondrial Gene Expression and OXPHOS Biogenesis
- 2020
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 79:6
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria contain their own gene expression systems, including membrane-bound ribosomes dedicated to synthesizing a few hydrophobic subunits of the oxidative phosphorylation (OXPHOS) complexes. We used a proximity-dependent biotinylation technique, BiolD, coupled with mass spectrometry to delineate in baker's yeast a comprehensive network of factors involved in biogenesis of mitochondrial encoded proteins. This mitochondrial gene expression network (MiGENet) encompasses proteins involved in transcription, RNA processing, translation, or protein biogenesis. Our analyses indicate the spatial organization of these processes, thereby revealing basic mechanistic principles and the proteins populating strategically important sites. For example, newly synthesized proteins are directly handed over to ribosomal tunnel exit-bound factors that mediate membrane insertion, co-factor acquisition, or their mounting into OXPHOS complexes in a special early assembly hub. Collectively, the data reveal the connectivity of mitochondrial gene expression, reflecting a unique tailoring of the mitochondrial gene expression system.
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| 2. |
- Aufschnaiter, Andreas, Dr. rer. nat. 1988-, et al.
(författare)
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Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation
- 2023
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Ingår i: The Mitoribosome. - : Humana Press. - 9781071631706 - 9781071631713 ; , s. 119-132
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners.
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| 3. |
- Aufschnaiter, Andreas, Dr. rer. nat. 1988-, et al.
(författare)
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Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation
- 2023
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Ingår i: Methods in Molecular Biology. - : Humana Press. - 1064-3745 .- 1940-6029. ; , s. 119-132, s. 119-132
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners.
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| 4. |
- Kohler, Andreas, Dr. rer. nat. 1988-, et al.
(författare)
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Early fate decision for mitochondrially encoded proteins by a molecular triage
- 2023
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Ingår i: Molecular Cell. - : Cell Press. - 1097-2765 .- 1097-4164. ; 83:19
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Tidskriftsartikel (refereegranskat)abstract
- Folding of newly synthesized proteins poses challenges for a functional proteome. Dedicated protein quality control (PQC) systems either promote the folding of nascent polypeptides at ribosomes or, if this fails, ensure their degradation. Although well studied for cytosolic protein biogenesis, it is not understood how these processes work for mitochondrially encoded proteins, key subunits of the oxidative phosphorylation (OXPHOS) system. Here, we identify dedicated hubs in proximity to mitoribosomal tunnel exits coordinating mitochondrial protein biogenesis and quality control. Conserved prohibitin (PHB)/m-AAA protease supercomplexes and the availability of assembly chaperones determine the fate of newly synthesized proteins by molecular triaging. The localization of these competing activities in the vicinity of the mitoribosomal tunnel exit allows for a prompt decision on whether newly synthesized proteins are fed into OXPHOS assembly or are degraded.
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| 5. |
- Carlström, Andreas, 1988, et al.
(författare)
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Insights into conformational changes in cytochrome b during the early steps of its maturation
- 2024
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Ingår i: FEBS LETTERS. - 0014-5793 .- 1873-3468. ; 598:11, s. 1438-1448
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins carrying redox cofactors are key subunits of respiratory chain complexes, yet the exact path of their folding and maturation remains poorly understood. Here, using cryo-EM and structure prediction via Alphafold2, we generated models of early assembly intermediates of cytochrome b (Cytb), a central subunit of complex III. The predicted structure of the first assembly intermediate suggests how the binding of Cytb to the assembly factor Cbp3-Cbp6 imposes an open configuration to facilitate the acquisition of its heme cofactors. Moreover, structure predictions of the second intermediate indicate how hemes get stabilized by binding of the assembly factor Cbp4, with a concomitant weakening of the contact between Cbp3-Cbp6 and Cytb, preparing for the release of the fully hemylated protein from the assembly factors.
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| 6. |
- Carlström, Andreas, 1988-, et al.
(författare)
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Insights into conformational changes occurring during the early steps of cytochrome b maturation
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins carrying redox cofactors are key subunits of respiratory chain complexes, yet the exact path of their folding and maturation remains poorly understood. Here, using cryo-EM and structure prediction via Alphafold2, we generated models of the early assembly intermediates of cytochrome b (Cytb), a central catalytic subunit of complex III, indicating which conformational changes could occur during its assembly. Previous work has shown that newly synthesized Cytb progresses through a series of assembly intermediates that support heme co-factor acquisition, steps that need to occur before the protein is joined with further subunits of the fully mature complex III. The predicted structure of the first assembly intermediate suggests how binding of Cytb to the assembly factor Cbp3-Cbp6 imposes an open configuration, which should facilitate acquisition of its heme cofactors. Moreover, structure predictions of the second intermediate indicate how these hemes get stabilized by binding of the subsequent assembly factor Cbp4, with a concomitant weakening of the contact between Cbp3-Cbp6 and Cytb, preparing a release of the fully hemylated protein from the assembly factors. These results therefore allow to formulate a hypothesis about the structural rearrangements as they occur during biogenesis of Cytb.
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| 7. |
- Carlström, Andreas, 1988-
(författare)
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The mitoribosome as a hub for integrating mitochondrial gene expression : Regulation of translation and early assembly of cytochrome b
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The mitochondrial proteome is a mosaic of proteins from two different genetic systems. The majority of mitochondrial proteins are encoded in the nuclear genome and are synthesized in the cytosol before imported to mitochondria. Interestingly, a handful of mitochondrial proteins are still encoded in the small mitochondrial genome, which is a remnant of its bacterial origin. These proteins are almost exclusively highly hydrophobic core subunits of the respiratory chain and ATP synthase. To synthesize these proteins, mitochondria contain its own complete gene expression system including factors for replication, transcription and translation. During the course of evolution, these machineries have diverged from its origin and have acquired organelle-specific characteristics. Mitochondrially encoded proteins are synthesized on the specialized and membrane-tethered mitoribosome. To avoid stoichiometric problems during assembly, translation in mitochondria needs to be coordinated with import of nuclear encoded subunits. However, the mechanisms behind regulation of mitochondrial protein biogenesis are still largely unclear.My research has focused on mitochondria in the yeast Saccharomyces cerevisiae. This provides a possibility to manipulate genes in both the nuclear and mitochondrial genomes to study effects on mitochondrial translation and respiration. In this thesis, I present results that contributes to our understanding of how translation regulation is connected to early assembly of mitochondrially encoded proteins. Using a proximity labelling technique called Bio-ID, we constructed a proximity network of proteins covering the whole mitochondrial gene expression system. This revealed a close association of factors involved in transcription, translation, membrane insertion and assembly, with the mitoribosome as a central hub. In particular, the mitoribosomal polypeptide tunnel exit was found to host factors involved in both regulation of translation and early assembly of nascent polypeptides. Further analysis of the tunnel exit proximity interactome resulted in identification of the membrane protein Mrx4 as a novel repressor of cytochrome b (Cytb) synthesis. Cytb is a catalytic subunit of the respiratory complex III and is encoded by the gene COB on the mtDNA. Translation of COB is under control of the regulatory proteins Cbp1, Cbs1, Cbs2, and Cbp3-Cbp6. Mrx4 is shown to interact with Cbs2, likely to sequester COB mRNA at the tunnel exit in a repressed state, until binding of Cbp3-Cbp6 releases Cbs2 and triggers activation of COB translation. Cbp3-Cbp6 then interacts with the newly synthesized Cytb, as it emerges from the tunnel exit, to stabilize the nascent polypeptide in an early assembly intermediate. The thesis further describes this chaperone function of Cbp3-Cbp6 on Cytb using structural models and site-specific photo-crosslinking. The results indicated that conformational changes in Cytb, upon incorporation of heme bH, releases Cbp3-Cbp6 to activate a new round of COB translation.In summary, the work presented in this thesis demonstrates a high level of organization and novel connectivity of mitochondrial gene expression. In addition, it expands on our knowledge regarding the intricate feedback loop regulation of Cytb biogenesis and the importance to coordinate mitochondrial translation with import of nuclear encoded subunits.
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