| 1. |
- Aufschnaiter, Andreas, et al.
(author)
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Fließbandfertigung von Atmungskettenkomplexen in Mitochondrien : Assembly line production of respiratory chain complexes in mitochondria
- 2022
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In: BioSpektrum. - : Springer Science and Business Media LLC. - 0947-0867 .- 1868-6249. ; 28:4, s. 366-369
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Research review (peer-reviewed)abstract
- A key function of mitochondria consists of energy conversion, performed with the help of the respiratory chain and the ATP synthase. Biogenesis of these essential molecular machines requires expression of nuclear and mitochondrially encoded genes. We describe our current understanding how these processes are coordinated and how they are organized in specific areas of the inner membrane to facilitate the assembly of these sophisticated complexes.
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| 2. |
- Aufschnaiter, Andreas, Dr. rer. nat. 1988-, et al.
(author)
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Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation
- 2023
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In: Methods in Molecular Biology. - : Humana Press. - 1064-3745 .- 1940-6029. ; , s. 119-132, s. 119-132
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Book chapter (other academic/artistic)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. |
- Berndtsson, Jens, et al.
(author)
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Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance
- 2020
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In: Embo Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 21
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Journal article (peer-reviewed)abstract
- Respiratory chains are crucial for cellular energy conversion and consist of multi-subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high-resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochromec between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochromec. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms.
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| 4. |
- Carlström, Andreas, 1988, et al.
(author)
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Insights into conformational changes in cytochrome b during the early steps of its maturation
- 2024
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In: FEBS LETTERS. - 0014-5793 .- 1873-3468. ; 598:11, s. 1438-1448
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Journal article (peer-reviewed)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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| 5. |
- Dickinson, Q., et al.
(author)
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Multi-omic integration by machine learning (MIMaL)
- 2022
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In: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1367-4811. ; 38:21, s. 4908-4918
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Journal article (peer-reviewed)abstract
- Motivation: Cells respond to environments by regulating gene expression to exploit resources optimally. Recent advances in technologies allow for measuring the abundances of RNA, proteins, lipids and metabolites. These highly complex datasets reflect the states of the different layers in a biological system. Multi-omics is the integration of these disparate methods and data to gain a clearer picture of the biological state. Multi-omic studies of the proteome and metabolome are becoming more common as mass spectrometry technology continues to be democratized. However, knowledge extraction through the integration of these data remains challenging. Results: Connections between molecules in different omic layers were discovered through a combination of machine learning and model interpretation. Discovered connections reflected protein control (ProC) over metabolites. Proteins discovered to control citrate were mapped onto known genetic and metabolic networks, revealing that these protein regulators are novel. Further, clustering the magnitudes of ProC over all metabolites enabled the prediction of five gene functions, each of which was validated experimentally. Two uncharacterized genes, YJR120W and YDL157C, were accurately predicted to modulate mitochondrial translation. Functions for three incompletely characterized genes were also predicted and validated, including SDH9, ISC1 and FMP52. A website enables results exploration and also MIMaL analysis of user-supplied multi-omic data.
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| 6. |
- Diessl, Jutta, 1989-, et al.
(author)
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Manganese-driven CoQ deficiency
- 2022
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Journal article (peer-reviewed)abstract
- Overexposure to manganese disrupts cellular energy metabolism across species, but the molecular mechanism underlying manganese toxicity remains enigmatic. Here, we report that excess cellular manganese selectively disrupts coenzyme Q (CoQ) biosynthesis, resulting in failure of mitochondrial bioenergetics. While respiratory chain complexes remain intact, the lack of CoQ as lipophilic electron carrier precludes oxidative phosphorylation and leads to premature cell and organismal death. At a molecular level, manganese overload causes mismetallation and proteolytic degradation of Coq7, a diiron hydroxylase that catalyzes the penultimate step in CoQ biosynthesis. Coq7 overexpression or supplementation with a CoQ headgroup analog that bypasses Coq7 function fully corrects electron transport, thus restoring respiration and viability. We uncover a unique sensitivity of a diiron enzyme to mismetallation and define the molecular mechanism for manganese-induced bioenergetic failure that is conserved across species. Across phylae, excess manganese disrupts energy metabolism by unclear mechanisms. Here, Diessl et al. report that failure of mitochondrial bioenergetics upon manganese overload is due to mismetallation of a diiron enzyme crucial for CoQ biosynthesis
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| 7. |
- Fomalont, E. B., et al.
(author)
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THE 2014 ALMA LONG BASELINE CAMPAIGN: AN OVERVIEW
- 2015
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In: Astrophysical Journal Letters. - : American Astronomical Society. - 2041-8213 .- 2041-8205. ; 808:1
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Journal article (peer-reviewed)abstract
- A major goal of the Atacama Large Millimeter/submillimeter Array (ALMA) is to make accurate images with resolutions of tens of milliarcseconds, which at submillimeter (submm) wavelengths requires baselines up to similar to 15 km. To develop and test this capability, a Long Baseline Campaign (LBC) was carried out from 2014 September to late November, culminating in end-to-end observations, calibrations, and imaging of selected Science Verification (SV) targets. This paper presents an overview of the campaign and its main results, including an investigation of the short-term coherence properties and systematic phase errors over the long baselines at the ALMA site, a summary of the SV targets and observations, and recommendations for science observing strategies at long baselines. Deep ALMA images of the quasar 3C 138 at 97 and 241 GHz are also compared to VLA 43 GHz results, demonstrating an agreement at a level of a few percent. As a result of the extensive program of LBC testing, the highly successful SV imaging at long baselines achieved angular resolutions as fine as 19 mas at similar to 350 GHz. Observing with ALMA on baselines of up to 15 km is now possible, and opens up new parameter space for submm astronomy.
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| 8. |
- Jung, Sung-Jun, 1987, et al.
(author)
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Early steps in the biogenesis of mitochondrially encoded oxidative phosphorylation subunits
- 2023
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In: IUBMB LIFE. - 1521-6543 .- 1521-6551. ; 76:3, s. 125-139
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Journal article (peer-reviewed)abstract
- The complexes mediating oxidative phosphorylation (OXPHOS) in the inner mitochondrial membrane consist of proteins encoded in the nuclear or the mitochondrial DNA. The mitochondrially encoded membrane proteins (mito-MPs) represent the catalytic core of these complexes and follow complicated pathways for biogenesis. Owing to their overall hydrophobicity, mito-MPs are co-translationally inserted into the inner membrane by the Oxa1 insertase. After insertion, OXPHOS biogenesis factors mediate the assembly of mito-MPs into complexes and participate in the regulation of mitochondrial translation, while protein quality control factors recognize and degrade faulty or excess proteins. This review summarizes the current understanding of these early steps occurring during the assembly of mito-MPs by concentrating on results obtained in the model organism baker's yeast.
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| 9. |
- Kohler, Andreas, Dr. rer. nat. 1988-, et al.
(author)
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Early fate decision for mitochondrially encoded proteins by a molecular triage
- 2023
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In: Molecular Cell. - : Cell Press. - 1097-2765 .- 1097-4164. ; 83:19
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Journal article (peer-reviewed)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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| 10. |
- Kohler, Andreas, Dr. rer. nat. 1988-, et al.
(author)
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The functional significance of mitochondrial respiratory chain supercomplexes
- 2023
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In: EMBO REPORTS. - : EMBO Press. - 1469-221X .- 1469-3178. ; 24:11
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Research review (peer-reviewed)abstract
- The mitochondrial respiratory chain (MRC) is a key energy transducer in eukaryotic cells. Four respiratory chain complexes cooperate in the transfer of electrons derived from various metabolic pathways to molecular oxygen, thereby establishing an electrochemical gradient over the inner mitochondrial membrane that powers ATP synthesis. This electron transport relies on mobile electron carries that functionally connect the complexes. While the individual complexes can operate independently, they are in situ organized into large assemblies termed respiratory supercomplexes. Recent structural and functional studies have provided some answers to the question of whether the supercomplex organization confers an advantage for cellular energy conversion. However, the jury is still out, regarding the universality of these claims. In this review, we discuss the current knowledge on the functional significance of MRC supercomplexes, highlight experimental limitations, and suggest potential new strategies to overcome these obstacles. Mitochondrial respiratory chain complexes can associate into supramolecular structures termed respiratory supercomplexes. This review discusses their structure, assembly and potential physiological functions.image
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| 11. |
- Kohler, Verena, 1992-, et al.
(author)
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Nuclear Hsp104 safeguards the dormant translation machinery during quiescence
- 2024
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 15
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Journal article (peer-reviewed)abstract
- The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.
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| 12. |
- König, Sabine, 1983, et al.
(author)
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Subarcsecond imaging of the water emission in Arp 220
- 2017
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In: Astronomy and Astrophysics. - : EDP Sciences. - 0004-6361 .- 1432-0746. ; 602, s. 42-
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Journal article (peer-reviewed)abstract
- Aims. Extragalactic observations of water emission can provide valuable insight into the excitation of the interstellar medium. In particular they allow us to investigate the excitation mechanisms in obscured nuclei, that is, whether an active galactic nucleus or a starburst dominates.Methods. We use subarcsecond resolution observations to tackle the nature of the water emission in Arp 220. ALMA Band 5 science verification observations of the 183 GHz H2O 313 − 220 line, in conjunction with new ALMA Band 7 H2O 515 − 422 data at 325 GHz, and supplementary 22 GHz H2O 616 − 523 VLA observations, are used to better constrain the parameter space in the excitation modeling of the water lines.Results. We detect 183 GHz H2O and 325 GHz water emission toward the two compact nuclei at the center of Arp 220, being brighter in Arp 220 West. The emission at these two frequencies is compared to previous single-dish data and does not show evidence of variability. The 183 and 325 GHz lines show similar spectra and kinematics, but the 22 GHz profile is significantly different in both nuclei due to a blend with an NH3 absorption line.Conclusions. Our findings suggest that the most likely scenario to cause the observed water emission in Arp 220 is a large number of independent masers originating from numerous star-forming regions.
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| 13. |
- Rzepka, Magdalena, 1992, et al.
(author)
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Incorporation of reporter genes into mitochondrial DNA in budding yeast
- 2022
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In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
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Journal article (peer-reviewed)abstract
- Many aspects of mitochondrial gene expression are still unknown, which can be attributed to limitations in molecular tools. Here, we present a protocol to introduce reporter genes into the mitochondrial genome of budding yeast, Saccharomyces cerevisiae. Mitochondrially encoded reporter constructs can be used to interrogate various aspects of mitochondrial gene expression. The power of this technique is exemplified by a mitochondrially encoded nanoluciferase, which allows to monitor levels of mitochondrial translation under a variety of growth conditions.
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| 14. |
- Saini, P. K., et al.
(author)
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The PSI+ prion modulates cytochrome c oxidase deficiency caused by deletion of COX12
- 2022
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In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 33:14
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Journal article (peer-reviewed)abstract
- Cytochrome c oxidase (CcO) is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of CcO oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here experimental evolution of a Saccharomyces cerevisiae.cox12 strain for similar to 300 generations allowed to restore the activity of CcO oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the cytosolic AAA+ disaggregase Hsp104. Deletion or overexpression of HSP104 also increased respiration of the Delta cox12 ancestor strain. This beneficial effect of Hsp104 was related to the loss of the [PSI+] prion, which forms cytosolic amyloid aggregates of the Sup35 protein. Overall, our data demonstrate that cytosolic aggregation of a prion impairs the mitochondrial metabolism of cells defective for Cox12. These findings identify a new functional connection between cytosolic proteostasis and biogenesis of the mitochondrial respiratory chain.
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| 15. |
- Salvatori, Roger, 1988, et al.
(author)
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Mapping protein networks in yeast mitochondria using proximity-dependent biotin identification coupled to proteomics
- 2020
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In: STAR PROTOCOLS. - : Elsevier BV. - 2666-1667. ; 1:3
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Journal article (peer-reviewed)abstract
- Proximity-dependent biotin identification (BioID) permits biotinylation of proteins interacting directly, indirectly, or just localized in proximity of a protein of interest (bait). Here, we describe how BioID coupled to proteomics and network biology can be used to map protein proximities in yeast mitochondria, aiding in visualization of complex protein-protein interaction landscapes. For complete information on the use and execution of this protocol, please refer to Singh et al., 2020.
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| 16. |
- Singh, Abeer Prakash, 1988, et al.
(author)
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Molecular Connectivity of Mitochondrial Gene Expression and OXPHOS Biogenesis
- 2020
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In: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 79:6
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Journal article (peer-reviewed)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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| 17. |
- Škerlová, Jana, et al.
(author)
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Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion
- 2021
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1
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Journal article (peer-reviewed)abstract
- The pyruvate dehydrogenase complex (PDHc) is a large multienzyme complex that converts pyruvate into acetyl-coenzyme A and in E. coli the core of the PDHc is formed by 24 copies of dihydrolipoyl transacetylase. Here, the authors present the cryo-EM structure of the E. coli dihydrolipoyl transacetylase 24-mer core in a native resting state including lipoyl domains, and discuss the mechanism of substrate shuttling by the lipoyl domains. The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.
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| 18. |
- Vazquez-Calvo, Carmela, 1983-, et al.
(author)
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Newly imported proteins in mitochondria are particularly sensitive to aggregation
- 2023
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In: Acta Physiologica. - : John Wiley & Sons. - 1748-1708 .- 1748-1716. ; 238:3
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Journal article (peer-reviewed)abstract
- Aim: A functional proteome is essential for life and maintained by protein quality control (PQC) systems in the cytosol and organelles. Protein aggregation is an indicator of a decline of PQC linked to aging and disease. Mitochondrial PQC is critical to maintain mitochondrial function and thus cellular fitness. How mitochondria handle aggregated proteins is not well understood. Here we tested how the metabolic status impacts on formation and clearance of aggregates within yeast mitochondria and assessed which proteins are particularly sensitive to denaturation.Methods: Confocal microscopy, electron microscopy, immunoblotting and genetics were applied to assess mitochondrial aggregate handling in response to heat shock and ethanol using the mitochondrial disaggregase Hsp78 as a marker for protein aggregates.Results: We show that aggregates formed upon heat or ethanol stress with different dynamics depending on the metabolic state. While fermenting cells displayed numerous small aggregates that coalesced into one large foci that was resistant to clearance, respiring cells showed less aggregates and cleared these aggregates more efficiently. Acute inhibition of mitochondrial translation had no effect, while preventing protein import into mitochondria by inhibition of cytosolic translation prevented aggregate formation.Conclusion: Collectively, our data show that the metabolic state of the cells impacts the dynamics of aggregate formation and clearance, and that mainly newly imported and not yet assembled proteins are prone to form aggregates. Because mitochondrial functionality is crucial for cellular metabolism, these results highlight the importance of efficient protein biogenesis to maintain the mitochondrial proteome operational during metabolic adaptations and cellular stress.
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