Martin Ott Lab
Research into the molecular mechanisms of mitochondrial gene expression
Research projects
-1-
Regulation and organization of mitochondrial gene expression
We are intrigued by the molecular mechanisms, by which mitochondrial gene expression is regulated. This is particularly important when cells need to produce more respiratory chain complexes, for example due to changed environmental conditions.
-2-
Mitochondrial protein homeostasis

Mitochondrial proteins can also be misfolded and then need to be repaired or turned over. How this works, and which roles mitochondrial gene expression plays for mitochondrial or cellular protein homeostasis, is an exciting area of our research.
-3-
Structural analyses of complexes we like

Single particle cryo electron microscopy has revolutionized modern life sciences. We determine with this technique the three dimensional structure of protein complexes implicated in the processes we study.
A selection of 10 papers
Molecular Connectivity of Mitochondrial Gene Expression and OXPHOS Biogenesis. Singh AP, Salvatori R, Aftab W, Aufschnaiter A, Carlström A, Forne I, Imhof A, Ott M. Mol Cell. 2020;79(6):1051-1065.
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, BioID, 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.
10.1016/j.molcel.2020.07.024
Explore the network here:
https://migenet.shinyapps.io/migenet/
Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes. Toth A, Meyrat A, Stoldt S, Santiago R, Wenzel D, Jakobs S, von Ballmoos C, Ott M. PNAS 2020 ;117(5):2412-2421.
Mitochondria have a characteristic ultrastructure with invaginations of the inner membrane called cristae that contain the protein complexes of the oxidative phosphorylation system. How this particular morphology of the respiratory membrane impacts energy conversion is currently unknown. One proposed role of cristae formation is to facilitate the establishment of local proton gradients to fuel ATP synthesis. Here, we determined the local pH values at defined sublocations within mitochondria of respiring yeast cells by fusing a pH-sensitive GFP to proteins residing in different mitochondrial subcompartments. Only a small proton gradient was detected over the inner membrane in wild type or cristae-lacking cells. Conversely, the obtained pH values did barely permit ATP synthesis in a reconstituted system containing purified yeast F1F0 ATP synthase, although, thermodynamically, a sufficiently high driving force was applied. At higher driving forces, where robust ATP synthesis was observed, a P-side pH value of 6 increased the ATP synthesis rate 3-fold compared to pH 7. In contrast, when ATP synthase was coreconstituted with an active proton-translocating cytochrome oxidase, ATP synthesis readily occurred at the measured, physiological pH values. Our study thus reveals that the morphology of the inner membrane does not influence the subcompartmental pH values and is not necessary for robust oxidative phosphorylation in mitochondria. Instead, it is likely that the dense packing of the oxidative phosphorylation complexes in the cristae membranes assists kinetic coupling between proton pumping and ATP synthesis.
10.1073/pnas.1917968117
Molecular Wiring of a Mitochondrial Translational Feedback Loop. Salvatori R, Kehrein K, Singh AP, Aftab W, Möller-Hergt BV, Forne I, Imhof A, Ott M. Mol Cell. 2020;77(4):887-900.
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.
10.1016/j.molcel.2019.11.019
Cryo-EM structure of the yeast respiratory supercomplex. Rathore S, Berndtsson J, Marin-Buera L, Conrad J, Carroni M, Brzezinski P, Ott M. Nat Struct Mol Biol. 2019;26(1):50-57.
Respiratory chain complexes execute energy conversion by connecting electron transport with proton translocation over the inner mitochondrial membrane to fuel ATP synthesis. Notably, these complexes form multi-enzyme assemblies known as respiratory supercomplexes. Here we used single-particle cryo-EM to determine the structures of the yeast mitochondrial respiratory supercomplexes III2IV and III2IV2, at 3.2-Å and 3.5-Å resolutions, respectively. We revealed the overall architecture of the supercomplex, which deviates from the previously determined assemblies in mammals; obtained a near-atomic structure of the yeast complex IV; and identified the protein-protein and protein-lipid interactions implicated in supercomplex formation. Taken together, our results demonstrate convergent evolution of supercomplexes in mitochondria that, while building similar assemblies, results in substantially different arrangements and structural solutions to support energy conversion.
10.1038/s41594-018-0169-7
Mitochondrial translation efficiency controls cytoplasmic protein homeostasis. Suhm T, Kaimal JM, Dawitz H, Peselj C, Masser AE, Hanzén S, Ambrožič M, Smialowska A, Björck ML, Brzezinski P, Nyström T, Büttner S, Andréasson C, Ott M. Cell Metabol. 2018;27(6):1309-1322.
Cellular proteostasis is maintained via the coordinated synthesis, maintenance, and breakdown of proteins in the cytosol and organelles. While biogenesis of the mitochondrial membrane complexes that execute oxidative phosphorylation depends on cytoplasmic translation, it is unknown how translation within mitochondria impacts cytoplasmic proteostasis and nuclear gene expression. Here we have analyzed the effects of mutations in the highly conserved accuracy center of the yeast mitoribosome. Decreased accuracy of mitochondrial translation shortened chronological lifespan, impaired management of cytosolic protein aggregates, and elicited a general transcriptional stress response. In striking contrast, increased accuracy extended lifespan, improved cytosolic aggregate clearance, and suppressed a normally stress-induced, Msn2/4-dependent interorganellar proteostasis transcription program (IPTP) that regulates genes important for mitochondrial proteostasis. Collectively, the data demonstrate that cytosolic protein homeostasis and nuclear stress signaling are controlled by mitochondrial translation efficiency in an inter-connected organelle quality control network that determines cellular lifespan.
10.1016/j.cmet.2018.04.011
Spatial orchestration of mitochondrial translation and OXPHOS complex assembly. Stoldt S, Wenzel D, Kehrein K, Riedel D, Ott M, Jakobs S. Nature Cell Biol. 2018;20(5):528-534.
Oxidative phosphorylation (OXPHOS) is vital for the regeneration of the vast majority of ATP in eukaryotic cells. OXPHOS is carried out by large multi-subunit protein complexes in the cristae membranes, which are invaginations of the mitochondrial inner membrane. The OXPHOS complexes are a mix of subunits encoded in the nuclear and mitochondrial genomes. Thus, the assembly of these dual-origin complexes is an enormous logistical challenge for the cell. Using super-resolution microscopy (nanoscopy) and quantitative cryo-immunogold electron microscopy, we determined where specific transcripts are translated and where distinct assembly steps of the dual-origin complexes in the yeast Saccharomyces cerevisiae occur. Our data indicate that the mitochondrially encoded proteins of complex III and complex IV are preferentially inserted in different sites of the inner membrane than those of complex V. We further demonstrate that the early, but not the late, assembly steps of complex III and complex IV occur preferentially in the inner boundary membrane. By contrast, all steps of complex V assembly occur mainly in the cristae membranes. Thus, OXPHOS complex assembly is spatially well orchestrated, probably representing an unappreciated regulatory layer in mitochondrial biogenesis.
10.1038/s41556-018-0090-7
Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies. Kehrein K, Schilling R, Möller-Hergt BV, Wurm CA, Jakobs S, Lamkemeyer T, Langer T, Ott M. Cell Rep. 2015;10(6):843–853.
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.
10.1016/j.celrep.2015.01.012
Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation. Hildenbeutel M, Hegg EL, Stephan K, Gruschke S, Meunier B, Ott M. J Cell Biol. 2014; 26;205(4):511-24.
Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3-Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b.
10.1083/jcb.201401009
Cbp3-Cbp6 interacts with the yeast mitochondrial ribosomal tunnel exit and promotes cytochrome b synthesis and assembly. Gruschke S, Kehrein K, Römpler K, Gröne K, Israel L, Imhof A, Herrmann JM, Ott M. J Cell Biol. 2011;193(6):1101-14.
Mitochondria contain their own genetic system to express a small number of hydrophobic polypeptides, including cytochrome b, an essential subunit of the bc(1) complex of the respiratory chain. In this paper, we show in yeast that Cbp3, a bc(1) complex assembly factor, and Cbp6, a regulator of cytochrome b translation, form a complex that associates with the polypeptide tunnel exit of mitochondrial ribosomes and that exhibits two important functions in the biogenesis of cytochrome b. On the one hand, the interaction of Cbp3 and Cbp6 with mitochondrial ribosomes is necessary for efficient translation of cytochrome b transcript [corrected]. On the other hand, the Cbp3-Cbp6 complex interacts directly with newly synthesized cytochrome b in an assembly intermediate that is not ribosome bound and that contains the assembly factor Cbp4. Our results suggest that synthesis of cytochrome b occurs preferentially on those ribosomes that have the Cbp3-Cbp6 complex bound to their tunnel exit, an arrangement that may ensure tight coordination of cytochrome b synthesis and assembly.
10.1083/jcb.201103132
our sponsors
Knut and Alice Wallenberg foundation
The Swedish Research council
ITN remix
the team
the current team
Martin Ott
Professor of Molecular Cellbiology

Abeer Prakash Singh
Researcher
Sagar Sridhara
Postdoc
Andreas Carlström
PhD student


Carmela Vázquez Calvo
PhD student
Magdalena Rzepka
PhD student
Sung-jun Jung
Postdoc
Amy Hardy
Technician
alumni
Philippe Ngyuen
Project student 2022
Jens Berndtsson
PhD student 2016-2021
Sorbhi Rathore
PhD student 2018-2020
Katharina Stephan
PhD student 2012-2020
Alexandra Toth
PhD student 2014-2020
Roger Salvatori
PhD student 2014-2020
Anneli Borg
Postdoc 2015-2020
Hannah Dawitz
PhD student 2014-2019
Mama Ndi
PhD student 2014-2019
Tamara Suhm
PhD student 2012-2019
Lorena Marin-Buera
Postdoc 2014-2018
Braulio Vargas Möller-Hergt
PhD student 2013-2018
Markus Hildenbeutel
Postdoc 2011-2014
Manfred Heublein
PhD student 2009-2013
Kirsten Kehrein
PhD student 2009-2014
Steffi Gruschke
PhD student 2008-2012
Martin Prestele
PhD student 2008-2010
our published work
2022
The [PSI+] prion modulates cytochrome c oxidase deficiency caused by deletion of COX12. Saini PK, Dawitz H, Aufschnaiter A, Bondarev S, Thomas J, Amblard A, Stewart J, Thierry-Mieg N, Ott M, Pierrel F. Mol Biol Cell. doi: 10.1091/mbc.E21-10-0499.
Cytochrome c oxidase is a pivotal enzyme of the mitochondrial respiratory chain, which sustains bioenergetics of eukaryotic cells. Cox12, a peripheral subunit of cytochrome c oxidase, is required for full activity of the enzyme, but its exact function is unknown. Here, experimental evolution of a Saccharomyces cerevisiae Δcox12 strain for ∼300 generations allowed to restore the activity of cytochrome c oxidase. In one population, the enhanced bioenergetics was caused by a A375V mutation in the AAA+ disaggregase Hsp104. Deletion or overexpression of HSP104 also increased respiration of the Δcox12 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.
link

Multi-Omic Integration by Machine Learning (MIMaL). Dickinson Q, Aufschnaiter A, Ott M, Meyer JG. Bioinformatics. doi: 10.1093/bioinformatics/btac631.
Motivation: Cells respond to environments by regulating gene expression to exploit resources optimally. Recent advances in technologies allow measuring the abundances of transcripts, proteins, lipids and metabolites. These highly complex datasets reflect the state 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 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 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 protein control over all metabolites enabled prediction of five gene functions, each of which was validated experimentally. Two uncharacterized genes, YJR120W and YLD157C, 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.
Availability: The website for MIMaL is at https://mimal.appCode for the website is at https://github.com/qdickinson/mimal-websiteCode to implement MIMaL is at https://github.com/jessegmeyerlab/MIMaL.
link

Incorporation of reporter genes into mitochondrial DNA in budding yeast. Magdalena Rzepka, Tamara Suhm, Martin Ott STAR Protocols 3(2):101359.
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.
10.1016/j.xpro.2022.101359
Fließbandfertigung von Atmungskettenkomplexen in Mitochondrien. Andreas Aufschnaiter, Martin Ott BIOspektrum 28(4):366-369

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.
link
2021
Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion. Škerlová J, Berndtsson J, Nolte H, Ott M, Stenmark P. Nat Commun. 2021;12(1):5277.
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.
10.1038/s41467-021-25570-y
The Analysis of Yeast Mitochondrial Translation. Carlström A, Rzepka M, Ott M. Methods Mol Bio. 2021;2192:227-242.

The mitochondrial genome encodes only a handful of proteins, but methods to track their synthesis are highly limited. Saccharomyces cerevisiae is a model organism that offers possibilities to expand the classical systems to analyze mitochondrial translation. In this chapter, we present two approaches of monitoring mitochondrial protein synthesis. Labeling of mitochondrially translated products with radioactive amino acids can be performed either in intact cells or in isolated mitochondria. However, these classical methods have disadvantages that can affect cell physiology and hence are not suitable for all types of research questions. Some of these limitations can be overcome by the use of reporter genes that are inserted into yeast genetic screens mitochondrial DNA via biolistic transformation. These reporter genes can be used for yeast genetic screen and to monitor regulation and efficiency of mitochondrial translation with a variety of methods.
10.1007/978-1-0716-0834-0_17
2020
Mapping protein networks in yeast mitochondria using proximity-dependent biotin identification coupled to proteomics. Salvatori R, Aftab W, Forne I, Imhof A, Ott M, Singh AP. STAR Protoc. 2020;1(3):100219.
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.
10.1016/j.xpro.2020.100219

Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance. Berndtsson J, Aufschnaiter A, Rathore S, Marin-Buera L, Dawitz H, Diessl J, Kohler V, Barrientos A, Büttner S, Fontanesi F, Ott M. EMBO Rep. 2020;21(12):e51015.
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 cytochrome c 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 cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms.
10.15252/embr.202051015

Membrane-tethering of cytochrome c accelerates regulated cell death in yeast. Toth A, Aufschnaiter A, Fedotovskaya O, Dawitz H, Ädelroth P, Büttner S, Ott M. Cell Death Dis. 2020;11(9):722. 10.1038/s41419-020-02920-0.
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.
10.1016/j.celrep.2015.01.012
Molecular Connectivity of Mitochondrial Gene Expression and OXPHOS Biogenesis. Singh AP, Salvatori R, Aftab W, Aufschnaiter A, Carlström A, Forne I, Imhof A, Ott M. Mol Cell. 2020;79(6):1051-1065.
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, BioID, 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.
10.1016/j.molcel.2020.07.024
Explore the network here:
https://migenet.shinyapps.io/migenet/
Timing of dimerization of the bc1 complex during mitochondrial respiratory chain assembly. Stephan K, Ott M. Biochim Biophys Acta Bioenerg. 2020;1861(5-6):148177.
The mitochondrial bc1 complex plays an important role in mitochondrial respiration. It transfers electrons from ubiquinol to the soluble electron shuttle cytochrome c and thereby contributes to the proton motive force across the inner mitochondrial membrane. In the yeast Saccharomyces cerevisiae, each monomer consists of three catalytic and seven accessory subunits. The bc1 complex is an obligate homo-dimer in all systems. It is currently not known when exactly during the assembly dimerization occurs. In this study, we determined that the dimer formation is an early event. Specifically, dimerization is mediated by the interaction of a stable tetramer formed by the two Cor subunits, Cor1 and Cor2, that joins assembly intermediate II, containing the fully hemylated cytochrome b and the two small accessory proteins, Qcr7 and Qcr8. Addition of cytochrome c1 and Qcr6 can either occur concomitantly or independently of dimerization. These results reveal a strict order in assembly, where dimerization occurs after stabilization of co-factor acquisition by cytochrome b. Finally, assembly is completed by addition of the remaining subunits.
10.1016/j.bbabio.2020.148177
Rcf1 Modulates Cytochrome c Oxidase Activity Especially Under Energy-Demanding Conditions. Dawitz H, Schäfer J, Schaart JM, Magits W, Brzezinski P, Ott M. Front Physiol. 2020;10:1555.
The mitochondrial respiratory chain is assembled into supercomplexes. Previously, two respiratory supercomplex-associated proteins, Rcf1 and Rcf2, were identified in Saccharomyces cerevisiae, which were initially suggested to mediate supercomplex formation. Recent evidence suggests that these factors instead are involved in cytochrome c oxidase biogenesis. We demonstrate here that Rcf1 mediates proper function of cytochrome c oxidase, while binding of Rcf2 results in a decrease of cytochrome c oxidase activity. Chemical crosslink experiments demonstrate that the conserved Hig-domain as well as the fungi specific C-terminus of Rcf1 are involved in molecular interactions with the cytochrome c oxidase subunit Cox3. We propose that Rcf1 modulates cytochrome c oxidase activity by direct binding to the oxidase to trigger changes in subunit Cox1, which harbors the catalytic site. Additionally, Rcf1 interaction with cytochrome c oxidase in the supercomplexes increases under respiratory conditions. These observations indicate that Rcf1 could enable the tuning of the respiratory chain depending on metabolic needs or repair damages at the catalytic site.
10.3389/fphys.2019.01555
Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes. Toth A, Meyrat A, Stoldt S, Santiago R, Wenzel D, Jakobs S, von Ballmoos C, Ott M. PNAS 2020;117(5):2412-2421.
Mitochondria have a characteristic ultrastructure with invaginations of the inner membrane called cristae that contain the protein complexes of the oxidative phosphorylation system. How this particular morphology of the respiratory membrane impacts energy conversion is currently unknown. One proposed role of cristae formation is to facilitate the establishment of local proton gradients to fuel ATP synthesis. Here, we determined the local pH values at defined sublocations within mitochondria of respiring yeast cells by fusing a pH-sensitive GFP to proteins residing in different mitochondrial subcompartments. Only a small proton gradient was detected over the inner membrane in wild type or cristae-lacking cells. Conversely, the obtained pH values did barely permit ATP synthesis in a reconstituted system containing purified yeast F1F0 ATP synthase, although, thermodynamically, a sufficiently high driving force was applied. At higher driving forces, where robust ATP synthesis was observed, a P-side pH value of 6 increased the ATP synthesis rate 3-fold compared to pH 7. In contrast, when ATP synthase was coreconstituted with an active proton-translocating cytochrome oxidase, ATP synthesis readily occurred at the measured, physiological pH values. Our study thus reveals that the morphology of the inner membrane does not influence the subcompartmental pH values and is not necessary for robust oxidative phosphorylation in mitochondria. Instead, it is likely that the dense packing of the oxidative phosphorylation complexes in the cristae membranes assists kinetic coupling between proton pumping and ATP synthesis.
10.1073/pnas.1917968117
Molecular Wiring of a Mitochondrial Translational Feedback Loop. Salvatori R, Kehrein K, Singh AP, Aftab W, Möller-Hergt BV, Forne I, Imhof A, Ott M. Mol Cell. 2020;77(4):887-900.
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.
10.1016/j.molcel.2019.11.019
2019
Extracellular Membrane Vesicles from Lactobacilli Dampen IFN-γ Responses in a Monocyte-Dependent Manner. Mata Forsberg M, Björkander S, Pang Y, Lundqvist L, Ndi M, Ott M, Escribá IB, Jaeger MC, Roos S, Sverremark-Ekström E. Sci Rep. 2019;9(1):17109.
Secreted factors derived from Lactobacillus are able to dampen pro-inflammatory cytokine responses. Still, the nature of these components and the underlying mechanisms remain elusive. Here, we aimed to identify the components and the mechanism involved in the Lactobacillus-mediated modulation of immune cell activation. PBMC were stimulated in the presence of the cell free supernatants (CFS) of cultured Lactobacillus rhamnosus GG and Lactobacillus reuteri DSM 17938, followed by evaluation of cytokine responses. We show that lactobacilli-CFS effectively dampen induced IFN-γ and IL-17A responses from T- and NK cells in a monocyte dependent manner by a soluble factor. A proteomic array analysis highlighted Lactobacillus-induced IL-1 receptor antagonist (ra) as a potential candidate responsible for the IFN-γ dampening activity. Indeed, addition of recombinant IL-1ra to stimulated PBMC resulted in reduced IFN-γ production. Further characterization of the lactobacilli-CFS revealed the presence of extracellular membrane vesicles with a similar immune regulatory activity to that observed with the lactobacilli-CFS. In conclusion, we have shown that lactobacilli produce extracellular MVs, which are able to dampen pro-inflammatory cytokine responses in a monocyte-dependent manner.
10.1038/s41598-019-53576-6

The basic machineries for mitochondrial protein quality control. Vazquez-Calvo C, Suhm T, Büttner S, Ott M. Mitochondrion. 2020;50:121-131.
Mitochondria play pivotal roles in cellular energy metabolism, the synthesis of essential biomolecules and the regulation of cell death and aging. The proper folding, unfolding and degradation of the many proteins active within mitochondria is surveyed by the mitochondrial quality control machineries. Here, we describe the principal components of the mitochondrial quality control system and recent developments in the elucidation of the molecular mechanisms maintaining a functional mitochondrial proteome.
10.1016/j.mito.2019.10.003
Structural basis for the interaction of the chaperone Cbp3 with newly synthesized cytochrome b during mitochondrial respiratory chain assembly. Ndi M, Masuyer G, Dawitz H, Carlström A, Michel M, Elofsson A, Rapp M, Stenmark P, Ott M. J Biol Chem. 2019 Nov 8;294(45):16663-16671.
Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3-Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains: an N-terminal domain present in mitochondrial Cbp3 homologs and a highly conserved C-terminal domain comprising a ubiquinol-cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-cross-linking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein.
10.1074/jbc.RA119.010483

Mitochondria orchestrate proteostatic and metabolic stress responses. Andréasson C, Ott M, Büttner S. EMBO Rep. 2019;20(10):e47865.
The eukaryotic cell is morphologically and functionally organized as an interconnected network of organelles that responds to stress and aging. Organelles communicate via dedicated signal transduction pathways and the transfer of information in form of metabolites and energy levels. Recent data suggest that the communication between organellar proteostasis systems is a cornerstone of cellular stress responses in eukaryotic cells. Here, we discuss the integration of proteostasis and energy fluxes in the regulation of cellular stress and aging. We emphasize the molecular architecture of the regulatory transcriptional pathways that both sense and control metabolism and proteostasis. A special focus is placed on mechanistic insights gained from the model organism budding yeast in signaling from mitochondria to the nucleus and how this shapes cellular fitness.
10.15252/embr.201947865
Alternative Translation Initiation at a UUG Codon Gives Rise to Two Functional Variants of the Mitochondrial Protein Kgd4. Heublein M, Ndi M, Vazquez-Calvo C, Vögtle FN, Ott M. J Mol Biol. 2019;431(7):1460-1467.
Kgd4 is a novel subunit of the mitochondrial α-ketoglutarate dehydrogenase complex (KGDH). In yeast, the protein is present in two forms of unknown origin, as there is only one open reading frame and no alternative splicing. Here, we show that the two forms of Kgd4 derive from one mRNA that is translated by employing two alternative start sites. The standard, annotated AUG codon gives rise to the short form of the protein, while an upstream UUG codon is utilized to generate the larger form. However, both forms can be efficiently imported into mitochondria and stably incorporate into KGDH to support its activity. Translation of the long variant depends on sequences directly upstream of the alternative initiation site, demonstrating that translation initiation and its efficiency are dictated by the sequence context surrounding a specific codon. In summary, the two forms of Kgd4 follow a very unusual biogenesis pathway, supporting the notion that translation initiation in yeast is more flexible than it is widely recognized.
10.1016/j.jmb.2019.02.023
Cryo-EM structure of the yeast respiratory supercomplex. Rathore S, Berndtsson J, Marin-Buera L, Conrad J, Carroni M, Brzezinski P, Ott M. Nat Struct Mol Biol. 2019;26(1):50-57.
Respiratory chain complexes execute energy conversion by connecting electron transport with proton translocation over the inner mitochondrial membrane to fuel ATP synthesis. Notably, these complexes form multi-enzyme assemblies known as respiratory supercomplexes. Here we used single-particle cryo-EM to determine the structures of the yeast mitochondrial respiratory supercomplexes III2IV and III2IV2, at 3.2-Å and 3.5-Å resolutions, respectively. We revealed the overall architecture of the supercomplex, which deviates from the previously determined assemblies in mammals; obtained a near-atomic structure of the yeast complex IV; and identified the protein-protein and protein-lipid interactions implicated in supercomplex formation. Taken together, our results demonstrate convergent evolution of supercomplexes in mitochondria that, while building similar assemblies, results in substantially different arrangements and structural solutions to support energy conversion.
10.1038/s41594-018-0169-7
2018
Insertion Defects of Mitochondrially Encoded Proteins Burden the Mitochondrial Quality Control System. Vargas Möller-Hergt B, Carlström A, Suhm T, Ott M. Cells. 2018;7(10):172.
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.
10.3390/cells7100172

The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria. Möller-Hergt BV, Carlström A, Stephan K, Imhof A, Ott M. Mol Biol Cell. 2018;29(20):2386-2396.
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.
10.1091/mbc.E18-04-0227
Regulation of cytochrome c oxidase activity by modulation of the catalytic site. Schäfer J, Dawitz H, Ott M, Ädelroth P, Brzezinski P. Sci Rep. 2018;8(1):11397.
The respiratory supercomplex factor 1 (Rcf 1) in Saccharomyces cerevisiae binds to intact cytochrome c oxidase (CytcO) and has also been suggested to be an assembly factor of the enzyme. Here, we isolated CytcO from rcf1Δ mitochondria using affinity chromatography and investigated reduction, inter-heme electron transfer and ligand binding to heme a3. The data show that removal of Rcf1 yields two CytcO sub-populations. One of these sub-populations exhibits the same functional behavior as CytcO isolated from the wild-type strain, which indicates that intact CytcO is assembled also without Rcf1. In the other sub-population, which was shown previously to display decreased activity and accelerated ligand-binding kinetics, the midpoint potential of the catalytic site was lowered. The lower midpoint potential allowed us to selectively reduce one of the two sub-populations of the rcf1Δ CytcO, which made it possible to investigate the functional behavior of the two CytcO forms separately. We speculate that these functional alterations reflect a mechanism that regulates O2 binding and trapping in CytcO, thereby altering energy conservation by the enzyme.
10.1038/s41598-018-29567-4

Mitochondrial Translation Efficiency Controls Cytoplasmic Protein Homeostasis. Suhm T, Kaimal JM, Dawitz H, Peselj C, Masser AE, Hanzén S, Ambrožič M, Smialowska A, Björck ML, Brzezinski P, Nyström T, Büttner S, Andréasson C, Ott M. Cell Metab. 2018;27(6):1309-1322.
Cellular proteostasis is maintained via the coordinated synthesis, maintenance, and breakdown of proteins in the cytosol and organelles. While biogenesis of the mitochondrial membrane complexes that execute oxidative phosphorylation depends on cytoplasmic translation, it is unknown how translation within mitochondria impacts cytoplasmic proteostasis and nuclear gene expression. Here we have analyzed the effects of mutations in the highly conserved accuracy center of the yeast mitoribosome. Decreased accuracy of mitochondrial translation shortened chronological lifespan, impaired management of cytosolic protein aggregates, and elicited a general transcriptional stress response. In striking contrast, increased accuracy extended lifespan, improved cytosolic aggregate clearance, and suppressed a normally stress-induced, Msn2/4-dependent interorganellar proteostasis transcription program (IPTP) that regulates genes important for mitochondrial proteostasis. Collectively, the data demonstrate that cytosolic protein homeostasis and nuclear stress signaling are controlled by mitochondrial translation efficiency in an inter-connected organelle quality control network that determines cellular lifespan.
10.1016/j.cmet.2018.04.011
Structural and functional heterogeneity of cytochrome c oxidase in S. cerevisiae. Schäfer J, Dawitz H, Ott M, Ädelroth P, Brzezinski P. Biochim Biophys Acta Bioenerg. 2018;1859(9):699-704.
Respiration in Saccharomyces cerevisiae is regulated by small proteins such as the respiratory supercomplex factors (Rcf). One of these factors (Rcf1) has been shown to interact with complexes III (cyt. bc1) and IV (cytochrome c oxidase, CytcO) of the respiratory chain and to modulate the activity of the latter. Here, we investigated the effect of deleting Rcf1 on the functionality of CytcO, purified using a protein C-tag on core subunit 1 (Cox1). Specifically, we measured the kinetics of ligand binding to the CytcO catalytic site, the O2-reduction activity and changes in light absorption spectra. We found that upon removal of Rcf1 a fraction of the CytcO is incorrectly assembled with structural changes at the catalytic site. The data indicate that Rcf1 modulates the assembly and activity of CytcO by shifting the equilibrium of structural sub-states toward the fully active, intact form.
10.1016/j.bbabio.2018.05.004
Biogenesis of the bc1 Complex of the Mitochondrial Respiratory Chain. Ndi M, Marin-Buera L, Salvatori R, Singh AP, Ott M. J Mol Biol. 2018;430(21):3892-3905.
The oxidative phosphorylation system contains four respiratory chain complexes that connect the transport of electrons to oxygen with the establishment of an electrochemical gradient over the inner membrane for ATP synthesis. Due to the dual genetic source of the respiratory chain subunits, its assembly requires a tight coordination between nuclear and mitochondrial gene expression machineries. In addition, dedicated assembly factors support the step-by-step addition of catalytic and accessory subunits as well as the acquisition of redox cofactors. Studies in yeast have revealed the basic principles underlying the assembly pathways. In this review, we summarize work on the biogenesis of the bc1 complex or complex III, a central component of the mitochondrial energy conversion system.
10.1016/j.jmb.2018.04.036
Rcf1 Modulates Cytochrome c Oxidase Activity Especially Under Energy-Demanding Conditions. Dawitz H, Schäfer J, Schaart JM, Magits W, Brzezinski P, Ott M. Front Physiol. 2020;10:1555.
The mitochondrial respiratory chain is assembled into supercomplexes. Previously, two respiratory supercomplex-associated proteins, Rcf1 and Rcf2, were identified in Saccharomyces cerevisiae, which were initially suggested to mediate supercomplex formation. Recent evidence suggests that these factors instead are involved in cytochrome c oxidase biogenesis. We demonstrate here that Rcf1 mediates proper function of cytochrome c oxidase, while binding of Rcf2 results in a decrease of cytochrome c oxidase activity. Chemical crosslink experiments demonstrate that the conserved Hig-domain as well as the fungi specific C-terminus of Rcf1 are involved in molecular interactions with the cytochrome c oxidase subunit Cox3. We propose that Rcf1 modulates cytochrome c oxidase activity by direct binding to the oxidase to trigger changes in subunit Cox1, which harbors the catalytic site. Additionally, Rcf1 interaction with cytochrome c oxidase in the supercomplexes increases under respiratory conditions. These observations indicate that Rcf1 could enable the tuning of the respiratory chain depending on metabolic needs or repair damages at the catalytic site.
10.3389/fphys.2019.01555
Spatial orchestration of mitochondrial translation and OXPHOS complex assembly. Stoldt S, Wenzel D, Kehrein K, Riedel D, Ott M, Jakobs S. Nat Cell Biol. 2018;20(5):528-534.
Oxidative phosphorylation (OXPHOS) is vital for the regeneration of the vast majority of ATP in eukaryotic cells 1 . OXPHOS is carried out by large multi-subunit protein complexes in the cristae membranes, which are invaginations of the mitochondrial inner membrane. The OXPHOS complexes are a mix of subunits encoded in the nuclear and mitochondrial genomes. Thus, the assembly of these dual-origin complexes is an enormous logistical challenge for the cell. Using super-resolution microscopy (nanoscopy) and quantitative cryo-immunogold electron microscopy, we determined where specific transcripts are translated and where distinct assembly steps of the dual-origin complexes in the yeast Saccharomyces cerevisiae occur. Our data indicate that the mitochondrially encoded proteins of complex III and complex IV are preferentially inserted in different sites of the inner membrane than those of complex V. We further demonstrate that the early, but not the late, assembly steps of complex III and complex IV occur preferentially in the inner boundary membrane. By contrast, all steps of complex V assembly occur mainly in the cristae membranes. Thus, OXPHOS complex assembly is spatially well orchestrated, probably representing an unappreciated regulatory layer in mitochondrial biogenesis.
10.1038/s41556-018-0090-7
A novel system to monitor mitochondrial translation in yeast. Suhm T, Habernig L, Rzepka M, Kaimal JM, Andréasson C, Büttner S, Ott M. Microb Cell. 2018;5(3):158-164.
The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.
10.15698/mic2018.03.621
A caspase-2-RFXANK interaction and its implication for MHC class II expression. Forsberg J, Li X, Akpinar B, Salvatori R, Ott M, Zhivotovsky B, Olsson M. Cell Death Dis. 2018;9(2):80.
Despite recent achievements implicating caspase-2 in tumor suppression, the enzyme stands out from the apoptotic caspase family as a factor whose function requires further clarification. To specify enzyme characteristics through the definition of interacting proteins in apoptotic or non-apoptotic settings, a yeast 2-hybrid (Y2H) screen was performed using the full-length protein as bait. The current report describes the analysis of a captured prey and putative novel caspase-2 interacting factor, the regulatory factor X-associated ankyrin-containing protein (RFXANK), previously associated with CIITA, the transactivator regulating cell-type specificity and inducibility of MHC class II gene expression. The interaction between caspase-2 and RFXANK was verified by co-immunoprecipitations using both exogenous and endogenous proteins, where the latter approach suggested that binding of the components occurs in the cytoplasm. Cellular co-localization was confirmed by transfection of fluorescently conjugated proteins. Enhanced caspase-2 processing in RFXANK-overexpressing HEK293T cells treated with chemotherapeutic agents further supported Y2H data. Yet, no distinct differences with respect to MHC class II expression were observed in plasma membranes of antigen-presenting cells derived from wild type and caspase-2-/- mice. In contrast, increased levels of the total MHC class II protein was evident in protein lysates from caspase-2 RNAi-silenced leukemia cell lines and B-cells isolated from gene-targeted mice. Together, these data identify a novel caspase-2-interacting factor, RFXANK, and indicate a potential non-apoptotic role for the enzyme in the control of MHC class II gene regulation.
10.1038/s41419-017-0144-y
2017
Cofilin1-dependent actin dynamics control DRP1-mediated mitochondrial fission. Rehklau K, Hoffmann L, Gurniak CB, Ott M, Witke W, Scorrano L, Culmsee C, Rust MB. Cell Death Dis. 2017;8(10):e3063.
Mitochondria form highly dynamic networks in which organelles constantly fuse and divide. The relevance of mitochondrial dynamics is evident from its implication in various human pathologies, including cancer or neurodegenerative, endocrine and cardiovascular diseases. Dynamin-related protein 1 (DRP1) is a key regulator of mitochondrial fission that oligomerizes at the mitochondrial outer membrane and hydrolyzes GTP to drive mitochondrial fragmentation. Previous studies demonstrated that DRP1 recruitment and mitochondrial fission is promoted by actin polymerization at the mitochondrial surface, controlled by the actin regulatory proteins inverted formin 2 (INF2) and Spire1C. These studies suggested the requirement of additional actin regulatory activities to control DRP1-mediated mitochondrial fission. Here we show that the actin-depolymerizing protein cofilin1, but not its close homolog actin-depolymerizing factor (ADF), is required to maintain mitochondrial morphology. Deletion of cofilin1 caused mitochondrial DRP1 accumulation and fragmentation, without altering mitochondrial function or other organelles' morphology. Mitochondrial morphology in cofilin1-deficient cells was restored upon (i) re-expression of wild-type cofilin1 or a constitutively active mutant, but not of an actin-binding-deficient mutant, (ii) pharmacological destabilization of actin filaments and (iii) genetic depletion of DRP1. Our work unraveled a novel function for cofilin1-dependent actin dynamics in mitochondrial fission, and identified cofilin1 as a negative regulator of mitochondrial DRP1 activity. We conclude that cofilin1 is required for local actin dynamics at mitochondria, where it may balance INF2/Spire1C-induced actin polymerization.
10.1038/cddis.2017.448

Reaction of S. cerevisiae mitochondria with ligands: Kinetics of CO and O(2) binding to flavohemoglobin and cytochrome c oxidase. Björck ML, Zhou S, Rydström Lundin C, Ott M, Ädelroth P, Brzezinski P. Biochim Biophys Acta Bioenerg. 2017;1858(2):182-188.
Kinetic methods used to investigate electron and proton transfer within cytochrome c oxidase (CytcO) are often based on the use of light to dissociate small ligands, such as CO, thereby initiating the reaction. Studies of intact mitochondria using these methods require identification of proteins that may bind CO and determination of the ligand-binding kinetics. In the present study we have investigated the kinetics of CO-ligand binding to S. cerevisiae mitochondria and cellular extracts. The data indicate that CO binds to two proteins, CytcO and a (yeast) flavohemoglobin (yHb). The latter has been shown previously to reside in both the cell cytosol and the mitochondrial matrix. Here, we found that yHb resides also in the intermembrane space and binds CO in its reduced state. As observed previously, we found that the yHb population in the mitochondrial matrix binds CO, but only after removal of the inner membrane. The mitochondrial yHb (in both the intermembrane space and the matrix) recombines with CO with τ≅270ms, which is significantly slower than observed with the cytosolic yHb (main component τ≅1.3ms). The data indicate that the yHb populations in the different cell compartments differ in structure.
10.1016/j.bbabio.2016.11.009
Mitochondrial translation and cellular stress response. Suhm T, Ott M. Cell Tissue Res. 2017;367(1):21-31.
Mitochondria are organelles critical for the functionality of eukaryotic cells. One of their most prominent functions is energy conversion, thereby producing most of the cellular ATP. Energy conversion relies on the oxidative phosphorylation system, an ensemble of large protein complexes that include the respiratory chain and the ATP synthase. Biogenesis of this machinery requires the coordination of two separate genetic systems, namely nuclear and mitochondrial gene expression. Recent research into the molecular causes of aging have revealed a prominent contribution of mitochondrial gene expression on many aspects of degenerative processes that typically involve cellular stress signaling pathways. In this review, we summarize recent developments in attempting to identify the molecular mechanism by which dysfunction of mitochondrial gene expression activates cellular stress signaling pathways and how this affects organismal aging. By comparing data obtained in various model organisms, we identify conserved and species-specific aspects of this mitochondria-to-nucleus signaling.
10.1007/s00441-016-2460-4
2016
Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae. Rydström Lundin C, von Ballmoos C, Ott M, Ädelroth P, Brzezinski P. PNAS. 2016;113(31):E4476-85.
The respiratory supercomplex factors (Rcf) 1 and 2 mediate supramolecular interactions between mitochondrial complexes III (ubiquinol-cytochrome c reductase; cyt. bc1) and IV (cytochrome c oxidase; CytcO). In addition, removal of these polypeptides results in decreased activity of CytcO, but not of cyt. bc1 In the present study, we have investigated the kinetics of ligand binding, the single-turnover reaction of CytcO with O2, and the linked cyt. bc1-CytcO quinol oxidation-oxygen-reduction activities in mitochondria in which Rcf1 or Rcf2 were removed genetically (strains rcf1Δ and rcf2Δ, respectively). The data show that in the rcf1Δ and rcf2Δ strains, in a significant fraction of the population, ligand binding occurs over a time scale that is ∼100-fold faster (τ ≅ 100 μs) than observed with the wild-type mitochondria (τ ≅ 10 ms), indicating structural changes. This effect is specific to removal of Rcf and not dissociation of the cyt. bc1-CytcO supercomplex. Furthermore, in the rcf1Δ and rcf2Δ strains, the single-turnover reaction of CytcO with O2 was incomplete. This observation indicates that the lower activity of CytcO is caused by a fraction of inactive CytcO rather than decreased CytcO activity of the entire population. Furthermore, the data suggest that the Rcf1 polypeptide mediates formation of an electron-transfer bridge from cyt. bc1 to CytcO via a tightly bound cyt. c We discuss the significance of the proposed regulatory mechanism of Rcf1 and Rcf2 in the context of supramolecular interactions between cyt. bc1 and CytcO.
10.1073/pnas.1601196113

Cell biology: Choreography of protein synthesis. Ott M. Nature. 2016;533(7604):472-3.
Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1. Bareth B, Nikolov M, Lorenzi I, Hildenbeutel M, Mick DU, Helbig C, Urlaub H, Ott M, Rehling P, Dennerlein S. Mol Biol Cell. 2016;27(10):1570-80.
The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1Δ mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.
10.1091/mbc.E15-12-0811
Organization and Regulation of Mitochondrial Protein Synthesis. Ott M, Amunts A, Brown A. Annu Rev Biochem. 2016;85:77-101.
Mitochondria are essential organelles of endosymbiotic origin that are responsible for oxidative phosphorylation within eukaryotic cells. Independent evolution between species has generated mitochondrial genomes that are extremely diverse, with the composition of the vestigial genome determining their translational requirements. Typically, translation within mitochondria is restricted to a few key subunits of the oxidative phosphorylation complexes that are synthesized by dedicated ribosomes (mitoribosomes). The dramatically rearranged mitochondrial genomes, the limited set of transcripts, and the need for the synthesized proteins to coassemble with nuclear-encoded subunits have had substantial consequences for the translation machinery. Recent high-resolution cryo-electron microscopy has revealed the effect of coevolution on the mitoribosome with the mitochondrial genome. In this review, we place the new structural information in the context of the molecular mechanisms of mitochondrial translation and focus on the novel ways protein synthesis is organized and regulated in mitochondria.
10.1146/annurev-biochem-060815-014334
Aim-less translation: loss of Saccharomyces cerevisiae mitochondrial translation initiation factor mIF3/Aim23 leads to unbalanced protein synthesis. Kuzmenko A, Derbikova K, Salvatori R, Tankov S, Atkinson GC, Tenson T, Ott M, Kamenski P, Hauryliuk V. Sci Rep. 2016;6:18749.
The mitochondrial genome almost exclusively encodes a handful of transmembrane constituents of the oxidative phosphorylation (OXPHOS) system. Coordinated expression of these genes ensures the correct stoichiometry of the system's components. Translation initiation in mitochondria is assisted by two general initiation factors mIF2 and mIF3, orthologues of which in bacteria are indispensible for protein synthesis and viability. mIF3 was thought to be absent in Saccharomyces cerevisiae until we recently identified mitochondrial protein Aim23 as the missing orthologue. Here we show that, surprisingly, loss of mIF3/Aim23 in S. cerevisiae does not indiscriminately abrogate mitochondrial translation but rather causes an imbalance in protein production: the rate of synthesis of the Atp9 subunit of F1F0 ATP synthase (complex V) is increased, while expression of Cox1, Cox2 and Cox3 subunits of cytochrome c oxidase (complex IV) is repressed. Our results provide one more example of deviation of mitochondrial translation from its bacterial origins.
10.1038/srep18749
2015
The MIOREX complex--lean management of mitochondrial gene expression. Kehrein K, Möller-Hergt BV, Ott M. Oncotarget. 2015;6(19):16806-7.
Organization of Mitochondrial Gene Expression in Two Distinct Ribosome-Containing Assemblies. Kehrein K, Schilling R, Möller-Hergt BV, Wurm CA, Jakobs S, Lamkemeyer T, Langer T, Ott M. Cell Rep. 2015;10(6):843-853.
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.
10.1016/j.celrep.2015.01.012
2014
The novel component Kgd4 recruits the E3 subunit to the mitochondrial alpha-ketoglutarate dehydrogenase. Heublein M, Burguillos MA, Vögtle FN, Teixeira PF, Imhof A, Meisinger C, Ott M. Mol Biol Cell. 2014;25(21):3342-9.
The mitochondrial citric acid cycle is a central hub of cellular metabolism, providing intermediates for biosynthetic pathways and channeling electrons to the respiratory chain complexes. In this study, we elucidated the composition and organization of the multienzyme complex α-ketoglutarate dehydrogenase (α-KGDH). In addition to the three classical E1-E3 subunits, we identified a novel component, Kgd4 (Ymr31/MRPS36), which was previously assigned to be a subunit of the mitochondrial ribosome. Biochemical analyses demonstrate that this protein plays an evolutionarily conserved role in the organization of mitochondrial α-KGDH complexes of fungi and animals. By binding to both the E1-E2 core and the E3 subunit, Kgd4 acts as a molecular adaptor that is necessary to a form a stable α-KGDH enzyme complex. Our work thus reveals a novel subunit of a key citric acid-cycle enzyme and shows how this large complex is organized.
10.1091/mbc.E14-07-1178

A mutation in the human CBP4 ortholog UQCC3 impairs complex III assembly, activity and cytochrome b stability. Wanschers BF, Szklarczyk R, van den Brand MA, Jonckheere A, Suijskens J, Smeets R, Rodenburg RJ, Stephan K, Helland IB, Elkamil A, Rootwelt T, Ott M, van den Heuvel L, Nijtmans LG, Huynen MA. Hum Mol Genet. 2014;23(23):6356-65.
Complex III (cytochrome bc1) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c. Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83, hereafter named UQCC3, to be the ortholog of the fungal complex III assembly factor CBP4. We describe a homozygous c.59T>A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T>A mutation has a causal role in complex III deficiency.
10.1093/hmg/ddu357
Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation. Hildenbeutel M, Hegg EL, Stephan K, Gruschke S, Meunier B, Ott M. J Cell Biol. 2014;205(4):511-24.
Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3-Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b.
10.1083/jcb.201401009
2013

Mitochondrial protein synthesis: efficiency and accuracy. Kehrein K, Bonnefoy N, Ott M. Antioxid Redox Signal. 2013;19(16):1928-39.
Significance: The mitochondrial genetic system is responsible for the production of a few core-subunits of the respiratory chain and ATP synthase, the membrane protein complexes driving oxidative phosphorylation (OXPHOS). Efficiency and accuracy of mitochondrial protein synthesis determines how efficiently new OXPHOS complexes can be made.
Recent advances: The system responsible for expression of the mitochondrial-encoded subunits developed from that of the bacterial ancestor of mitochondria. Importantly, many aspects of genome organization, transcription, and translation have diverged during evolution. Recent research has provided new insights into the architecture, regulation, and organelle-specific features of mitochondrial translation. Mitochondrial ribosomes contain a number of proteins absent from prokaryotic ribosomes, implying that in mitochondria, ribosomes were tailored to fit the requirements of the organelle. In addition, mitochondrial gene expression is regulated post-transcriptionally by a number of mRNA-specific translational activators. At least in yeast, these factors can regulate translation in respect to OXPHOS complex assembly to adjust the level of newly synthesized proteins to amounts that can be successfully assembled into respiratory chain complexes.
Critical issues: Mitochondrial gene expression is determining aging in eukaryotes, and a number of recent reports indicate that efficiency of translation directly influences this process.
Future directions: Here we will summarize recent advances in our understanding of mitochondrial protein synthesis by comparing the knowledge acquired in the systems most commonly used to study mitochondrial biogenesis. However, many steps have not been understood mechanistically. Innovative biochemical and genetic approaches have to be elaborated to shed light on these important processes.
10.1089/ars.2012.4896
2012
The Cbp3-Cbp6 complex coordinates cytochrome b synthesis with bc(1) complex assembly in yeast mitochondria. Gruschke S, Römpler K, Hildenbeutel M, Kehrein K, Kühl I, Bonnefoy N, Ott M. J Cell Biol. 2012;199(1):137-50.
Respiratory chain complexes in mitochondria are assembled from subunits derived from two genetic systems. For example, the bc(1) complex consists of nine nuclear encoded subunits and the mitochondrially encoded subunit cytochrome b. We recently showed that the Cbp3-Cbp6 complex has a dual function for biogenesis of cytochrome b: it is both required for efficient synthesis of cytochrome b and for protection of the newly synthesized protein from proteolysis. Here, we report that Cbp3-Cbp6 also coordinates cytochrome b synthesis with bc(1) complex assembly. We show that newly synthesized cytochrome b assembled through a series of four assembly intermediates. Blocking assembly at early and intermediate steps resulted in sequestration of Cbp3-Cbp6 in a cytochrome b-containing complex, thereby making Cbp3-Cbp6 unavailable for cytochrome b synthesis and thus reducing overall cytochrome b levels. This feedback loop regulates protein synthesis at the inner mitochondrial membrane by directly monitoring the efficiency of bc(1) complex assembly.
10.1083/jcb.201206040
The membrane insertase Oxa1 is required for efficient import of carrier proteins into mitochondria. Hildenbeutel M, Theis M, Geier M, Haferkamp I, Neuhaus HE, Herrmann JM, Ott M. J Mol Biol. 2012;423(4):590-9.
Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady-state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.
10.1016/j.jmb.2012.07.018
ADF/cofilin proteins translocate to mitochondria during apoptosis but are not generally required for cell death signaling. Rehklau K, Gurniak CB, Conrad M, Friauf E, Ott M, Rust MB. Cell Death Differ. 2012;19(6):958-67.
Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.
10.1038/cdd.2011.180
2011
Cbp3-Cbp6 interacts with the yeast mitochondrial ribosomal tunnel exit and promotes cytochrome b synthesis and assembly. Gruschke S, Kehrein K, Römpler K, Gröne K, Israel L, Imhof A, Herrmann JM, Ott M. J Cell Biol. 2011;193(6):1101-14.
Mitochondria contain their own genetic system to express a small number of hydrophobic polypeptides, including cytochrome b, an essential subunit of the bc(1) complex of the respiratory chain. In this paper, we show in yeast that Cbp3, a bc(1) complex assembly factor, and Cbp6, a regulator of cytochrome b translation, form a complex that associates with the polypeptide tunnel exit of mitochondrial ribosomes and that exhibits two important functions in the biogenesis of cytochrome b. On the one hand, the interaction of Cbp3 and Cbp6 with mitochondrial ribosomes is necessary for efficient translation of cytochrome b transcript [corrected]. On the other hand, the Cbp3-Cbp6 complex interacts directly with newly synthesized cytochrome b in an assembly intermediate that is not ribosome bound and that contains the assembly factor Cbp4. Our results suggest that synthesis of cytochrome b occurs preferentially on those ribosomes that have the Cbp3-Cbp6 complex bound to their tunnel exit, an arrangement that may ensure tight coordination of cytochrome b synthesis and assembly.
10.1083/jcb.201103132

Evolution of YidC/Oxa1/Alb3 insertases: three independent gene duplications followed by functional specialization in bacteria, mitochondria and chloroplasts. Funes S, Kauff F, van der Sluis EO, Ott M, Herrmann JM. Biol Chem. 2011;392(1-2):13-9.
Members of the YidC/Oxa1/Alb3 protein family facilitate the insertion, folding and assembly of proteins of the inner membranes of bacteria and mitochondria and the thylakoid membrane of plastids. All homologs share a conserved hydrophobic core region comprising five transmembrane domains. On the basis of phylogenetic analyses, six subgroups of the family can be distinguished which presumably arose from three independent gene duplications followed by functional specialization. During evolution of bacteria, mitochondria and chloroplasts, subgroup-specific regions were added to the core domain to facilitate the association with ribosomes or other components contributing to the substrate spectrum of YidC/Oxa1/Alb3 proteins.
10.1515/BC.2011.013
2010
The polypeptide tunnel exit of the mitochondrial ribosome is tailored to meet the specific requirements of the organelle. Gruschke S, Ott M. Bioessays. 2010;32(12):1050-7.
The ribosomal polypeptide tunnel exit is the site where a variety of factors interact with newly synthesized proteins to guide them through the early steps of their biogenesis. In mitochondrial ribosomes, this site has been considerably modified in the course of evolution. In contrast to all other translation systems, mitochondrial ribosomes are responsible for the synthesis of only a few hydrophobic membrane proteins that are essential subunits of the mitochondrial respiratory chain. Membrane insertion of these proteins occurs co-translationally and is connected to a sophisticated assembly process that not only includes the assembly of the different subunits but also the acquisition of redox co-factors. Here, we describe how mitochondrial translation is organized in the context of respiratory chain assembly and speculate how alteration of the ribosomal tunnel exit might allow the establishment of a subset of specialized ribosomes that individually organize the early steps in the biogenesis of distinct mitochondrially-encoded proteins.
10.1002/bies.201000081
Ribosome-binding proteins Mdm38 and Mba1 display overlapping functions for regulation of mitochondrial translation. Bauerschmitt H, Mick DU, Deckers M, Vollmer C, Funes S, Kehrein K, Ott M, Rehling P, Herrmann JM. Mol Biol Cell. 2010;21(12):1937-44.
Biogenesis of respiratory chain complexes depends on the expression of mitochondrial-encoded subunits. Their synthesis occurs on membrane-associated ribosomes and is probably coupled to their membrane insertion. Defects in expression of mitochondrial translation products are among the major causes of mitochondrial disorders. Mdm38 is related to Letm1, a protein affected in Wolf-Hirschhorn syndrome patients. Like Mba1 and Oxa1, Mdm38 is an inner membrane protein that interacts with ribosomes and is involved in respiratory chain biogenesis. We find that simultaneous loss of Mba1 and Mdm38 causes severe synthetic defects in the biogenesis of cytochrome reductase and cytochrome oxidase. These defects are not due to a compromised membrane binding of ribosomes but the consequence of a mis-regulation in the synthesis of Cox1 and cytochrome b. Cox1 expression is restored by replacing Cox1-specific regulatory regions in the mRNA. We conclude, that Mdm38 and Mba1 exhibit overlapping regulatory functions in translation of selected mitochondrial mRNAs.
10.1091/mbc.e10-02-0101


Proteins at the polypeptide tunnel exit of the yeast mitochondrial ribosome. Gruschke S, Gröne K, Heublein M, Hölz S, Israel L, Imhof A, Herrmann JM, Ott M. J Biol Chem. 2010;285(25):19022-8.
Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.
10.1074/jbc.M110.113837

Co-translational membrane insertion of mitochondrially encoded proteins. Ott M, Herrmann JM. Biochim Biophys Acta. 2010;1803(6):767-75.
The components of the mitochondrial proteome represent a mosaic of dual genetic origin: while most mitochondrial proteins are encoded by nuclear genes and imported into the organelle following synthesis in the cytosol, a small number of proteins is encoded by the mitochondrial genome. Though small in number, mitochondrial translation products are vital for cellular functionality as these proteins represent the core subunits of the respiratory chain and the ATPase which produce the vast majority of the cellular ATP. Mitochondrial translation products are almost exclusively highly hydrophobic polypeptides which are inserted into the inner membrane in the course of their synthesis. The machinery that mediates membrane insertion in mitochondria is deduced from that of their bacterial ancestors and hence shows profound similarities to the insertion machinery of prokaryotes. However, the specialization on the production of a very small set of translation products drove a severe reduction in the complexity of this system. The insertase Oxa1 forms the central component of the insertion machinery. Oxa1 directly binds to mitochondrial ribosomes and, together with the inner membrane protein Mba1, aligns the polypeptide exit tunnel of the ribosome with the insertion site at the inner membrane. The specific hallmarks and the critical components of this machinery are discussed in this review article.
10.1016/j.bbamcr.2009.11.010
2009
Mrpl36 is important for generation of assembly competent proteins during mitochondrial translation. Prestele M, Vogel F, Reichert AS, Herrmann JM, Ott M. Mol Biol Cell. 2009;20(10):2615-25.
The complexes of the respiratory chain represent mosaics of nuclear and mitochondrially encoded components. The processes by which synthesis and assembly of the various subunits are coordinated remain largely elusive. During evolution, many proteins of the mitochondrial ribosome acquired additional domains pointing at specific properties or functions of the translation machinery in mitochondria. Here, we analyzed the function of Mrpl36, a protein associated with the large subunit of the mitochondrial ribosome. This protein, homologous to the ribosomal protein L31 from bacteria, contains a mitochondria-specific C-terminal domain that is not required for protein synthesis per se; however, its absence decreases stability of Mrpl36. Cells lacking this C-terminal domain can still synthesize proteins, but these translation products fail to be properly assembled into respiratory chain complexes and are rapidly degraded. Surprisingly, overexpression of Mrpl36 seems to even increase the efficiency of mitochondrial translation. Our data suggest that Mrpl36 plays a critical role during translation that determines the rate of respiratory chain assembly. This important function seems to be carried out by a stabilizing activity of Mrpl36 on the interaction between large and small ribosomal subunits, which could influence accuracy of protein synthesis.
10.1091/mbc.e08-12-1162

some lab pics
Martin Ott
Professor of Molecular Cell Biology at Gothenburg University
Professor of Biochemistry at Stockholm University
Visiting address:
Department of Medical Biochemistry and Cell Biology
Institute for Biomedicine
Sahlgrenska academy
University of Gothenburg
Medicinaregatan 9A
41390 Göteborg

Phone: +46722612871
E-mail: martin.ott@gu.se
Vacancies
We typically have possibilities for Master projects and ERASMUS internships, but we are always interested in applications for PhD positions or postdoc projects.

Currently we have one opening for a PhD student, see here at Nature Careers

If you are interested in joining us, please contact us by Email: martin.ott@gu.se




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