Publications by authors named "Peter Rehling"

114 Publications

Monitoring mitochondrial translation in living cells.

EMBO Rep 2021 Apr 15;22(4):e51635. Epub 2021 Feb 15.

Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany.

Mitochondria possess a small genome that codes for core subunits of the oxidative phosphorylation system and whose expression is essential for energy production. Information on the regulation and spatial organization of mitochondrial gene expression in the cellular context has been difficult to obtain. Here we devise an imaging approach to analyze mitochondrial translation within the context of single cells, by following the incorporation of clickable non-canonical amino acids. We apply this method to multiple cell types, including specialized cells such as cardiomyocytes and neurons, and monitor with spatial resolution mitochondrial translation in axons and dendrites. We also show that translation imaging allows to monitor mitochondrial protein expression in patient fibroblasts. Approaching mitochondrial translation with click chemistry opens new avenues to understand how mitochondrial biogenesis is integrated into the cellular context and can be used to assess mitochondrial gene expression in mitochondrial diseases.
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http://dx.doi.org/10.15252/embr.202051635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024989PMC
April 2021

Sulthiame impairs mitochondrial function in vitro and may trigger onset of visual loss in Leber hereditary optic neuropathy.

Orphanet J Rare Dis 2021 Feb 4;16(1):64. Epub 2021 Feb 4.

Interdisciplinary Pediatric Center for Children With Developmental Disabilities and Severe Chronic Disorders, University Medical Center Göttingen, Göttingen, Germany.

Background: Leber hereditary optic neuropathy (LHON) is the most common mitochondrial disorder and characterized by acute or subacute painless visual loss. Environmental factors reported to trigger visual loss in LHON mutation carriers include smoking, heavy intake of alcohol, raised intraocular pressure, and some drugs, including several carbonic anhydrase inhibitors. The antiepileptic drug sulthiame (STM) is effective especially in focal seizures, particularly in benign epilepsy of childhood with centrotemporal spikes, and widely used in pediatric epileptology. STM is a sulfonamide derivate and an inhibitor of mammalian carbonic anhydrase isoforms I-XIV.

Results: We describe two unrelated patients, an 8-year-old girl and an 11-year-old boy, with cryptogenic focal epilepsy, who suffered binocular (subject #1) or monocular (subject #2) visual loss in close temporal connection with starting antiepileptic pharmacotherapy with STM. In both subjects, visual loss was due to LHON. We used real-time respirometry in fibroblasts derived from LHON patients carrying the same mitochondrial mutations as our two subjects to investigate the effect of STM on oxidative phosphorylation. Oxygen consumption rate in fibroblasts from a healthy control was not impaired by STM compared with a vehicle control. In contrast, fibroblasts carrying the m.14484T>C or the m.3460G>A LHON mutation displayed a drastic reduction of the respiration rate when treated with STM compared to vehicle control.

Conclusions: Our observations point to a causal relationship between STM treatment and onset or worsening of visual failure in two subjects with LHON rather than pure coincidence. We conclude that antiepileptic medication with STM may pose a risk for visual loss in LHON mutation carriers and should be avoided in these patients.
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http://dx.doi.org/10.1186/s13023-021-01690-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860214PMC
February 2021

Caveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in Cardiomyocytes.

Circ Res 2021 Mar 25;128(6):e102-e120. Epub 2021 Jan 25.

Cellular Biophysics and Translational Cardiology Section, Heart Research Center Göttingen (J.P., D.K.-D., G.W., S.B., T.K., G.H., J.W., S.E.L.), University Medical Center Göttingen.

[Figure: see text].
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http://dx.doi.org/10.1161/CIRCRESAHA.119.316547DOI Listing
March 2021

Defining the architecture of the human TIM22 complex by chemical crosslinking.

FEBS Lett 2021 Jan 13;595(2):157-168. Epub 2020 Nov 13.

Department of Cellular Biochemistry, University Medical Center Göttingen, Germany.

The majority of mitochondrial proteins are nuclear encoded and imported into mitochondria as precursor proteins via dedicated translocases. The translocase of the inner membrane 22 (TIM22) is a multisubunit molecular machine specialized for the translocation of hydrophobic, multi-transmembrane-spanning proteins with internal targeting signals into the inner mitochondrial membrane. Here, we undertook a crosslinking-mass spectrometry (XL-MS) approach to determine the molecular arrangement of subunits of the human TIM22 complex. Crosslinking of the isolated TIM22 complex using the BS3 crosslinker resulted in the broad generation of crosslinks across the majority of TIM22 components, including the small TIM chaperone complex. The crosslinking data uncovered several unexpected features, opening new avenues for a deeper investigation into the steps required for TIM22-mediated translocation in humans.
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http://dx.doi.org/10.1002/1873-3468.13978DOI Listing
January 2021

MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation.

EMBO J 2020 07 22;39(14):e104105. Epub 2020 Jun 22.

Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Mitochondrial function is critically dependent on the folding of the mitochondrial inner membrane into cristae; indeed, numerous human diseases are associated with aberrant crista morphologies. With the MICOS complex, OPA1 and the F F -ATP synthase, key players of cristae biogenesis have been identified, yet their interplay is poorly understood. Harnessing super-resolution light and 3D electron microscopy, we dissect the roles of these proteins in the formation of cristae in human mitochondria. We individually disrupted the genes of all seven MICOS subunits in human cells and re-expressed Mic10 or Mic60 in the respective knockout cell line. We demonstrate that assembly of the MICOS complex triggers remodeling of pre-existing unstructured cristae and de novo formation of crista junctions (CJs) on existing cristae. We show that the Mic60-subcomplex is sufficient for CJ formation, whereas the Mic10-subcomplex controls lamellar cristae biogenesis. OPA1 stabilizes tubular CJs and, along with the F F -ATP synthase, fine-tunes the positioning of the MICOS complex and CJs. We propose a new model of cristae formation, involving the coordinated remodeling of an unstructured crista precursor into multiple lamellar cristae.
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http://dx.doi.org/10.15252/embj.2019104105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361284PMC
July 2020

A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria.

Mol Cell Proteomics 2020 07 24;19(7):1161-1178. Epub 2020 Apr 24.

Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; Institute of Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany. Electronic address:

Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome oxidase. BN-PAGE analyses of Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome oxidase.
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http://dx.doi.org/10.1074/mcp.RA120.002028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338081PMC
July 2020

Defining the Substrate Spectrum of the TIM22 Complex Identifies Pyruvate Carrier Subunits as Unconventional Cargos.

Curr Biol 2020 03 5;30(6):1119-1127.e5. Epub 2020 Mar 5.

Department of Cellular Biochemistry, University Medical Center Göttingen, 37073 Göttingen, Germany. Electronic address:

In mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1-3]. Tom70, a subunit of TOM complex, represents the major receptor for these precursors [2, 4-6]. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex [7-12]. Tim22 represents the pore-forming core unit of the complex [13, 14]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes.
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http://dx.doi.org/10.1016/j.cub.2020.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090383PMC
March 2020

From TOM to the TIM23 complex - handing over of a precursor.

Biol Chem 2020 05;401(6-7):709-721

Department of Cellular Biochemistry, University Medical Center Göttingen, D-37073 Göttingen, Germany.

Mitochondrial precursor proteins with amino-terminal presequences are imported via the presequence pathway, utilizing the TIM23 complex for inner membrane translocation. Initially, the precursors pass the outer membrane through the TOM complex and are handed over to the TIM23 complex where they are sorted into the inner membrane or translocated into the matrix. This handover process depends on the receptor proteins at the inner membrane, Tim50 and Tim23, which are critical for efficient import. In this review, we summarize key findings that shaped the current concepts of protein translocation along the presequence import pathway, with a particular focus on the precursor handover process from TOM to the TIM23 complex. In addition, we discuss functions of the human TIM23 pathway and the recently uncovered pathogenic mutations in TIM50.
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http://dx.doi.org/10.1515/hsz-2020-0101DOI Listing
May 2020

COA6 Facilitates Cytochrome c Oxidase Biogenesis as Thiol-reductase for Copper Metallochaperones in Mitochondria.

J Mol Biol 2020 03 13;432(7):2067-2079. Epub 2020 Feb 13.

Department of Cellular Biochemistry, University Medical Center Göttingen, D-37073 Göttingen, Germany; Max-Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany. Electronic address:

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the Cu site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6, are required for Cu center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential-driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulfide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CXCXH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2-SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for Cu biogenesis.
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http://dx.doi.org/10.1016/j.jmb.2020.01.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254062PMC
March 2020

MITRAC15/COA1 promotes mitochondrial translation in a ND2 ribosome-nascent chain complex.

EMBO Rep 2020 01 13;21(1):e48833. Epub 2019 Nov 13.

Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany.

The mitochondrial genome encodes for thirteen core subunits of the oxidative phosphorylation system. These proteins assemble with imported proteins in a modular manner into stoichiometric enzyme complexes. Assembly factors assist in these biogenesis processes by providing co-factors or stabilizing transient assembly stages. However, how expression of the mitochondrial-encoded subunits is regulated to match the availability of nuclear-encoded subunits is still unresolved. Here, we address the function of MITRAC15/COA1, a protein that participates in complex I biogenesis and complex IV biogenesis. Our analyses of a MITRAC15 knockout mutant reveal that MITRAC15 is required for translation of the mitochondrial-encoded complex I subunit ND2. We find that MITRAC15 is a constituent of a ribosome-nascent chain complex during ND2 translation. Chemical crosslinking analyses demonstrate that binding of the ND2-specific assembly factor ACAD9 to the ND2 polypeptide occurs at the C-terminus and thus downstream of MITRAC15. Our analyses demonstrate that expression of the founder subunit ND2 of complex I undergoes regulation. Moreover, a ribosome-nascent chain complex with MITRAC15 is at the heart of this process.
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http://dx.doi.org/10.15252/embr.201948833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945058PMC
January 2020

Redox signals at the ER-mitochondria interface control melanoma progression.

EMBO J 2019 08 15;38(15):e100871. Epub 2019 Jul 15.

Molecular Physiology, Institute of Cardiovascular Physiology, University Medical Center, Georg-August-University, Göttingen, Germany.

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease.
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http://dx.doi.org/10.15252/embj.2018100871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669928PMC
August 2019

Neuronal Redox-Imbalance in Rett Syndrome Affects Mitochondria as Well as Cytosol, and Is Accompanied by Intensified Mitochondrial O Consumption and ROS Release.

Front Physiol 2019 30;10:479. Epub 2019 Apr 30.

Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Georg-August-University Göttingen, Göttingen, Germany.

Rett syndrome (RTT), an X chromosome-linked neurodevelopmental disorder affecting almost exclusively females, is associated with various mitochondrial alterations. Mitochondria are swollen, show altered respiratory rates, and their inner membrane is leaking protons. To advance the understanding of these disturbances and clarify their link to redox impairment and oxidative stress, we assessed mitochondrial respiration in defined brain regions and cardiac tissue of male wildtype (WT) and MeCP2-deficient ( ) mice. Also, we quantified for the first time neuronal redox-balance with subcellular resolution in cytosol and mitochondrial matrix. Quantitative roGFP1 redox imaging revealed more oxidized conditions in the cytosol of hippocampal neurons than in WT neurons. Furthermore, cytosol and mitochondria of neurons showed exaggerated redox-responses to hypoxia and cell-endogenous reactive oxygen species (ROS) formation. Biochemical analyzes exclude disease-related increases in mitochondrial mass in hippocampus and cortex. Protein levels of complex I core constituents were slightly lower in hippocampus and cortex than in WT; those of complex V were lower in cortex. Respiratory supercomplex-formation did not differ among genotypes. Yet, supplied with the complex II substrate succinate, mitochondria of cortex and hippocampus consumed more O than WT. Furthermore, mitochondria from hippocampus and cortex mediated an enhanced oxidative burden. In conclusion, we further advanced the molecular understanding of mitochondrial dysfunction in RTT. Intensified mitochondrial O consumption, increased mitochondrial ROS generation and disturbed redox balance in mitochondria and cytosol may represent a causal chain, which provokes dysregulated proteins, oxidative tissue damage, and contributes to neuronal network dysfunction in RTT.
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http://dx.doi.org/10.3389/fphys.2019.00479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503037PMC
April 2019

A MICOS-TIM22 Association Promotes Carrier Import into Human Mitochondria.

J Mol Biol 2019 07 17;431(15):2835-2851. Epub 2019 May 17.

Department of Cellular Biochemistry, University Medical Center Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.

Mitochondrial membrane proteins with internal targeting signals are inserted into the inner membrane by the carrier translocase (TIM22 complex). For this, precursors have to be initially directed from the TOM complex in the outer mitochondrial membrane across the intermembrane space toward the TIM22 complex. How these two translocation processes are topologically coordinated is still unresolved. Using proteomic approaches, we find that the human TIM22 complex associates with the mitochondrial contact site and cristae organizing system (MICOS) complex. This association does not appear to be conserved in yeast, whereby the yeast MICOS complex instead interacts with the presequence translocase. Using a yeast mic10Δ strain and a HEK293T MIC10 knockout cell line, we characterize the role of MICOS for protein import into the mitochondrial inner membrane and matrix. We find that a physiological cristae organization promotes efficient import via the presequence pathway in yeast, while in human mitochondria, the MICOS complex is dispensable for protein import along the presequence pathway. However, in human mitochondria, the MICOS complex is required for the efficient import of carrier proteins into the mitochondrial inner membrane. Our analyses suggest that in human mitochondria, positioning of the carrier translocase at the crista junction, and potentially in vicinity to the TOM complex, is required for efficient transport into the inner membrane.
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http://dx.doi.org/10.1016/j.jmb.2019.05.015DOI Listing
July 2019

Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7.

EMBO Mol Med 2019 05;11(5)

Laboratory of Mitochondrial Biogenesis, Centre of New Technologies, University of Warsaw, Warsaw, Poland

Nuclear and mitochondrial genome mutations lead to various mitochondrial diseases, many of which affect the mitochondrial respiratory chain. The proteome of the intermembrane space (IMS) of mitochondria consists of several important assembly factors that participate in the biogenesis of mitochondrial respiratory chain complexes. The present study comprehensively analyzed a recently identified IMS protein cytochrome oxidase assembly factor 7 (COA7), or RESpiratory chain Assembly 1 (RESA1) factor that is associated with a rare form of mitochondrial leukoencephalopathy and complex IV deficiency. We found that COA7 requires the mitochondrial IMS import and assembly (MIA) pathway for efficient accumulation in the IMS We also found that pathogenic mutant versions of COA7 are imported slower than the wild-type protein, and mislocalized proteins are degraded in the cytosol by the proteasome. Interestingly, proteasome inhibition rescued both the mitochondrial localization of COA7 and complex IV activity in patient-derived fibroblasts. We propose proteasome inhibition as a novel therapeutic approach for a broad range of mitochondrial pathologies associated with the decreased levels of mitochondrial proteins.
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http://dx.doi.org/10.15252/emmm.201809561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505684PMC
May 2019

The role of mitochondrial cardiolipin in heart function and its implication in cardiac disease.

Biochim Biophys Acta Mol Basis Dis 2019 04 26;1865(4):810-821. Epub 2018 Aug 26.

Institute of Cellular Biochemistry, University Medical Center Göttingen, D-37073 Göttingen, Germany; Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany. Electronic address:

Mitochondria play an essential role in the energy metabolism of the heart. Many of the essential functions are associated with mitochondrial membranes and oxidative phosphorylation driven by the respiratory chain. Mitochondrial membranes are unique in the cell as they contain the phospholipid cardiolipin. The important role of cardiolipin in cardiovascular health is highlighted by several cardiac diseases, in which cardiolipin plays a fundamental role. Barth syndrome, Sengers syndrome, and Dilated cardiomyopathy with ataxia (DCMA) are genetic disorders, which affect cardiolipin biosynthesis. Other cardiovascular diseases including ischemia/reperfusion injury and heart failure are also associated with changes in the cardiolipin pool. Here, we summarize molecular functions of cardiolipin in mitochondrial biogenesis and morphology. We highlight the role of cardiolipin for the respiratory chain, metabolite carriers, and mitochondrial metabolism and describe links to apoptosis and mitochondria specific autophagy (mitophagy) with possible implications in cardiac disease.
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http://dx.doi.org/10.1016/j.bbadis.2018.08.025DOI Listing
April 2019

O affects mitochondrial functionality ex vivo.

Redox Biol 2019 04 23;22:101152. Epub 2019 Feb 23.

Institute for Cardiovascular Physiology, University Medical Center Göttingen, Georg-August-University, Humbdoltallee 23, 37077 Göttingen, Germany. Electronic address:

Mitochondria have originated in eukaryotic cells by endosymbiosis of a specialized prokaryote approximately 2 billion years ago. They are essential for normal cell function by providing energy through their role in oxidizing carbon substrates. Glutathione (GSH) is a major thiol-disulfide redox buffer of the cell including the mitochondrial matrix and intermembrane space. We have generated cardiomyocyte-specific Grx1-roGFP2 GSH redox potential (E) biosensor mice in the past, in which the sensor is targeted to the mitochondrial matrix. Using this mouse model a distinct E of the mitochondrial matrix (-278.9 ± 0.4 mV) in isolated cardiomyocytes is observed. When analyzing the E in isolated mitochondria from the transgenic hearts, however, the E in the mitochondrial matrix is significantly oxidized (-247.7 ± 8.7 mV). This is prevented by adding N-Ethylmaleimide during the mitochondria isolation procedure, which precludes disulfide bond formation. A similar reducing effect is observed by isolating mitochondria in hypoxic (0.1-3% O) conditions that mimics mitochondrial pO levels in cellulo. The reduced E is accompanied by lower ROS production, reduced complex III activity but increased ATP levels produced at baseline and after stimulation with succinate/ADP. Altogether, we demonstrate that oxygenation is an essential factor that needs to be considered when analyzing mitochondrial function ex vivo.
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http://dx.doi.org/10.1016/j.redox.2019.101152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396017PMC
April 2019

Overexpression of branched-chain amino acid aminotransferases rescues the growth defects of cells lacking the Barth syndrome-related gene TAZ1.

J Mol Med (Berl) 2019 02 3;97(2):269-279. Epub 2019 Jan 3.

Interfaculty Institute of Biochemistry, University of Tübingen, 72076, Tübingen, Germany.

The yeast protein Taz1 is the orthologue of human Tafazzin, a phospholipid acyltransferase involved in cardiolipin (CL) remodeling via a monolyso CL (MLCL) intermediate. Mutations in Tafazzin lead to Barth syndrome (BTHS), a metabolic and neuromuscular disorder that primarily affects the heart, muscles, and immune system. Similar to observations in fibroblasts and platelets from patients with BTHS or from animal models, abolishing yeast Taz1 results in decreased total CL amounts, increased levels of MLCL, and mitochondrial dysfunction. However, the biochemical mechanisms underlying the mitochondrial dysfunction in BTHS remain unclear. To better understand the pathomechanism of BTHS, we searched for multi-copy suppressors of the taz1Δ growth defect in yeast cells. We identified the branched-chain amino acid transaminases (BCATs) Bat1 and Bat2 as such suppressors. Similarly, overexpression of the mitochondrial isoform BCAT2 in mammalian cells lacking TAZ improves their growth. Elevated levels of Bat1 or Bat2 did not restore the reduced membrane potential, altered stability of respiratory complexes, or the defective accumulation of MLCL species in yeast taz1Δ cells. Importantly, supplying yeast or mammalian cells lacking TAZ1 with certain amino acids restored their growth behavior. Hence, our findings suggest that the metabolism of amino acids has an important and disease-relevant role in cells lacking Taz1 function. KEY MESSAGES: Bat1 and Bat2 are multi-copy suppressors of retarded growth of taz1Δ yeast cells. Overexpression of Bat1/2 in taz1Δ cells does not rescue known mitochondrial defects. Supplementation of amino acids enhances growth of cells lacking Taz1 or Tafazzin. Altered metabolism of amino acids might be involved in the pathomechanism of BTSH.
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http://dx.doi.org/10.1007/s00109-018-1728-4DOI Listing
February 2019

ROMO1 is a constituent of the human presequence translocase required for YME1L protease import.

J Cell Biol 2019 02 31;218(2):598-614. Epub 2018 Dec 31.

Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany

The mitochondrial presequence translocation machinery (TIM23 complex) is conserved between the yeast and humans; however, functional characterization has been mainly performed in yeast. Here, we define the constituents of the human TIM23 complex using mass spectrometry and identified ROMO1 as a new translocase constituent with an exceptionally short half-life. Analyses of a ROMO1 knockout cell line revealed aberrant inner membrane structure and altered processing of the GTPase OPA1. We show that in the absence of ROMO1, mitochondria lose the inner membrane YME1L protease, which participates in OPA1 processing and ROMO1 turnover. While ROMO1 is dispensable for general protein import along the presequence pathway, we show that it participates in the dynamics of TIM21 during respiratory chain biogenesis and is specifically required for import of YME1L. This selective import defect can be linked to charge distribution in the unusually long targeting sequence of YME1L. Our analyses establish an unexpected link between mitochondrial protein import and inner membrane protein quality control.
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http://dx.doi.org/10.1083/jcb.201806093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363466PMC
February 2019

Combined Use of Unnatural Amino Acids Enables Dual-Color Super-Resolution Imaging of Proteins via Click Chemistry.

ACS Nano 2018 12 13;12(12):12247-12254. Epub 2018 Dec 13.

Institute for Neuro- and Sensory Physiology, Center for Biostructural Imaging of Neurodegeneration , University Medical Center Göttingen, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain , Göttingen , Germany.

Recent advances in optical nanoscopy have brought the imaging resolution to the size of the individual macromolecules, thereby setting stringent requirements for the fluorescent labels. Such requirements are optimally fulfilled by the incorporation of unnatural amino acids (UAAs) in the proteins of interest (POIs), followed by fluorophore conjugation via click chemistry. However, this approach has been limited to single POIs in mammalian cells. Here we solve this problem by incorporating different UAAs in different POIs, which are expressed in independent cell sets. The cells are then fused, thereby combining the different proteins and organelles, and are easily imaged by dual-color super-resolution microscopy. This procedure, which we termed Fuse2Click, is simple, requires only the well-established Amber codon, and allows the use of all previously optimized UAAs and tRNA/RS pairs. This should render it a tool of choice for multicolor click-based imaging.
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http://dx.doi.org/10.1021/acsnano.8b06047DOI Listing
December 2018

Mutations of the mitochondrial carrier translocase channel subunit TIM22 cause early-onset mitochondrial myopathy.

Hum Mol Genet 2018 12;27(23):4135-4144

Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, D-37073, Germany.

Protein import into mitochondria is facilitated by translocases within the outer and the inner mitochondrial membranes that are dedicated to a highly specific subset of client proteins. The mitochondrial carrier translocase (TIM22 complex) inserts multispanning proteins, such as mitochondrial metabolite carriers and translocase subunits (TIM23, TIM17A/B and TIM22), into the inner mitochondrial membrane. Both types of substrates are essential for mitochondrial metabolic function and biogenesis. Here, we report on a subject, diagnosed at 1.5 years, with a neuromuscular presentation, comprising hypotonia, gastroesophageal reflux disease and persistently elevated serum and Cerebrospinal fluid lactate (CSF). Patient fibroblasts displayed reduced oxidative capacity and altered mitochondrial morphology. Using trans-mitochondrial cybrid cell lines, we excluded a candidate variant in mitochondrial DNA as causative of these effects. Whole-exome sequencing identified compound heterozygous variants in the TIM22 gene (NM_013337), resulting in premature truncation in one allele (p.Tyr25Ter) and a point mutation in a conserved residue (p.Val33Leu), within the intermembrane space region, of the TIM22 protein in the second allele. Although mRNA transcripts of TIM22 were elevated, biochemical analyses revealed lower levels of TIM22 protein and an even greater deficiency of TIM22 complex formation. In agreement with a defect in carrier translocase function, carrier protein amounts in the inner membrane were found to be reduced. This is the first report of pathogenic variants in the TIM22 pore-forming subunit of the carrier translocase affecting the biogenesis of inner mitochondrial membrane proteins critical for metabolite exchange.
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http://dx.doi.org/10.1093/hmg/ddy305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240735PMC
December 2018

The codon sequences predict protein lifetimes and other parameters of the protein life cycle in the mouse brain.

Sci Rep 2018 11 15;8(1):16913. Epub 2018 Nov 15.

Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, 37073, Göttingen, Germany.

The homeostasis of the proteome depends on the tight regulation of the mRNA and protein abundances, of the translation rates, and of the protein lifetimes. Results from several studies on prokaryotes or eukaryotic cell cultures have suggested that protein homeostasis is connected to, and perhaps regulated by, the protein and the codon sequences. However, this has been little investigated for mammals in vivo. Moreover, the link between the coding sequences and one critical parameter, the protein lifetime, has remained largely unexplored, both in vivo and in vitro. We tested this in the mouse brain, and found that the percentages of amino acids and codons in the sequences could predict all of the homeostasis parameters with a precision approaching experimental measurements. A key predictive element was the wobble nucleotide. G-/C-ending codons correlated with higher protein lifetimes, protein abundances, mRNA abundances and translation rates than A-/U-ending codons. Modifying the proportions of G-/C-ending codons could tune these parameters in cell cultures, in a proof-of-principle experiment. We suggest that the coding sequences are strongly linked to protein homeostasis in vivo, albeit it still remains to be determined whether this relation is causal in nature.
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http://dx.doi.org/10.1038/s41598-018-35277-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237891PMC
November 2018

Defective Mitochondrial Cardiolipin Remodeling Dampens HIF-1α Expression in Hypoxia.

Cell Rep 2018 10;25(3):561-570.e6

Department of Cellular Biochemistry, University Medical Center Göttingen, GZMB, 37073 Göttingen, Germany.

Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs, integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin remodeling, upon loss of the cardiolipin acyl transferase tafazzin, decreases HIF-1α signaling in hypoxia. Tafazzin deficiency does not affect posttranslational HIF-1α regulation but rather HIF-1α gene expression, a dysfunction recapitulated in iPSC-derived cardiomyocytes from Barth syndrome patients with tafazzin deficiency. RNA-seq analyses confirmed drastically altered signaling in tafazzin mutant cells. In hypoxia, tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-κB activation and concomitantly HIF-1α gene expression. Tafazzin-deficient mice hearts display reduced HIF-1α levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin remodeling dampens HIF-1α signaling due to a lack of NF-κB activation through reduced mitochondrial ROS production, decreasing HIF-1α transcription.
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http://dx.doi.org/10.1016/j.celrep.2018.09.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205837PMC
October 2018

Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions.

Nat Commun 2018 10 12;9(1):4230. Epub 2018 Oct 12.

Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, 37073, Göttingen, Germany.

The turnover of brain proteins is critical for organism survival, and its perturbations are linked to pathology. Nevertheless, protein lifetimes have been difficult to obtain in vivo. They are readily measured in vitro by feeding cells with isotopically labeled amino acids, followed by mass spectrometry analyses. In vivo proteins are generated from at least two sources: labeled amino acids from the diet, and non-labeled amino acids from the degradation of pre-existing proteins. This renders measurements difficult. Here we solved this problem rigorously with a workflow that combines mouse in vivo isotopic labeling, mass spectrometry, and mathematical modeling. We also established several independent approaches to test and validate the results. This enabled us to measure the accurate lifetimes of ~3500 brain proteins. The high precision of our data provided a large set of biologically significant observations, including pathway-, organelle-, organ-, or cell-specific effects, along with a comprehensive catalog of extremely long-lived proteins (ELLPs).
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http://dx.doi.org/10.1038/s41467-018-06519-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185916PMC
October 2018

Motor recruitment to the TIM23 channel's lateral gate restricts polypeptide release into the inner membrane.

Nat Commun 2018 10 2;9(1):4028. Epub 2018 Oct 2.

Department of Cellular Biochemistry, University Medical Center Göttingen, GZMB, 37073, Göttingen, Germany.

The presequence translocase of the mitochondrial inner membrane (TIM23 complex) facilitates anterograde precursor transport into the matrix and lateral release of precursors with stop-transfer signal into the membrane (sorting). Sorting requires precursor exit from the translocation channel into the lipid phase through the lateral gate of the TIM23 complex. How the two transport modes are regulated and balanced against each other is unknown. Here we show that the import motor J-protein Pam18, which is essential for matrix import, controls lateral protein release into the lipid bilayer. Constitutively translocase-associated Pam18 obstructs lateral precursor transport. Concomitantly, Mgr2, implicated in precursor quality control, is displaced from the translocase. We conclude that during motor-dependent matrix protein transport, the transmembrane segment of Pam18 closes the lateral gate to promote anterograde polypeptide movement. This finding explains why a motor-free form of the translocase facilitates the lateral movement of precursors with a stop-transfer signal.
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http://dx.doi.org/10.1038/s41467-018-06492-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168564PMC
October 2018

COX16 promotes COX2 metallation and assembly during respiratory complex IV biogenesis.

Elife 2018 01 30;7. Epub 2018 Jan 30.

Department of Cellular Biochemistry, University Medical Centre Göttingen, Göttingen, Germany.

Cytochrome oxidase of the mitochondrial oxidative phosphorylation system reduces molecular oxygen with redox equivalent-derived electrons. The conserved mitochondrial-encoded COX1- and COX2-subunits are the heme- and copper-center containing core subunits that catalyze water formation. COX1 and COX2 initially follow independent biogenesis pathways creating assembly modules with subunit-specific, chaperone-like assembly factors that assist in redox centers formation. Here, we find that COX16, a protein required for cytochrome oxidase assembly, interacts specifically with newly synthesized COX2 and its copper center-forming metallochaperones SCO1, SCO2, and COA6. The recruitment of SCO1 to the COX2-module is COX16- dependent and patient-mimicking mutations in SCO1 affect interaction with COX16. These findings implicate COX16 in Cu-site formation. Surprisingly, COX16 is also found in COX1-containing assembly intermediates and COX2 recruitment to COX1. We conclude that COX16 participates in merging the COX1 and COX2 assembly lines.
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http://dx.doi.org/10.7554/eLife.32572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809144PMC
January 2018

The mitochondrial TMEM177 associates with COX20 during COX2 biogenesis.

Biochim Biophys Acta Mol Cell Res 2018 Feb 16;1865(2):323-333. Epub 2017 Nov 16.

Department of Cellular Biochemistry, University Medical Centre Göttingen, GZMB, D-37073 Göttingen, Germany; Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany. Electronic address:

The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the Cu-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network. Loss or increase in the amount of TMEM177 affects COX20 abundance leading to reduced or increased COX20 levels respectively. TMEM177 associates with newly synthesized COX2 and SCO2 in a COX20-dependent manner. Our data shows that by unbalancing the amount of TMEM177, newly synthesized COX2 accumulates in a COX20-associated state. We conclude that TMEM177 promotes assembly of COX2 at the level of Cu-site formation.
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http://dx.doi.org/10.1016/j.bbamcr.2017.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764226PMC
February 2018

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.

EMBO J 2018 01 16;37(1):139-159. Epub 2017 Nov 16.

Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.
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http://dx.doi.org/10.15252/embj.201695709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753035PMC
January 2018

INA complex liaises the FF-ATP synthase membrane motor modules.

Nat Commun 2017 11 1;8(1):1237. Epub 2017 Nov 1.

Department of Cellular Biochemistry, University Medical Center Göttingen, GZMB, D-37073, Göttingen, Germany.

The FF-ATP synthase translates a proton flux across the inner mitochondrial membrane into a mechanical rotation, driving anhydride bond formation in the catalytic portion. The complex's membrane-embedded motor forms a proteinaceous channel at the interface between Atp9 ring and Atp6. To prevent unrestricted proton flow dissipating the H-gradient, channel formation is a critical and tightly controlled step during ATP synthase assembly. Here we show that the INA complex (INAC) acts at this decisive step promoting Atp9-ring association with Atp6. INAC binds to newly synthesized mitochondrial-encoded Atp6 and Atp8 in complex with maturation factors. INAC association is retained until the F-portion is built on Atp6/8 and loss of INAC causes accumulation of the free F. An independent complex is formed between INAC and the Atp9 ring. We conclude that INAC maintains assembly intermediates of the F F-ATP synthase in a primed state for the terminal assembly step-motor module formation.
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http://dx.doi.org/10.1038/s41467-017-01437-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665977PMC
November 2017

Cation selectivity of the presequence translocase channel Tim23 is crucial for efficient protein import.

Elife 2017 08 31;6. Epub 2017 Aug 31.

Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany.

Virtually all mitochondrial matrix proteins and a considerable number of inner membrane proteins carry a positively charged, N-terminal presequence and are imported by the TIM23 complex (presequence translocase) located in the inner mitochondrial membrane. The voltage-regulated Tim23 channel constitutes the actual protein-import pore wide enough to allow the passage of polypeptides with a secondary structure. In this study, we identify amino acids important for the cation selectivity of Tim23. Structure based mutants show that selectivity is provided by highly conserved, pore-lining amino acids. Mutations of these amino acid residues lead to reduced selectivity properties, reduced protein import capacity and they render the Tim23 channel insensitive to substrates. We thus show that the cation selectivity of the Tim23 channel is a key feature for substrate recognition and efficient protein import.
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http://dx.doi.org/10.7554/eLife.28324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578737PMC
August 2017