Publications by authors named "Chris Meisinger"

104 Publications

Monitoring checkpoints of metabolism and protein biogenesis in mitochondria by Phos-tag technology.

J Proteomics 2021 Nov 20:104430. Epub 2021 Nov 20.

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany; CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany. Electronic address:

A role for reversible phosphorylation in regulation of mitochondrial proteins has been neglected for a long time. Particularly, the import machineries that mediate influx of more than 1000 different precursor proteins into the organelle were considered as predominantly constitutively active entities. Only recently, a combination of advanced phosphoproteomic approaches and Phos-tag technology enabled the discovery of several phosphorylation sites at the translocase of the outer membrane TOM and the identification of cellular signalling cascades that allow dynamic adaptation of the protein influx into mitochondria upon changing cellular demands. Here, we present a protocol that allows biochemical and semi-quantitative profiling of intra-mitochondrial protein phosphorylation. We exemplify this with the pyruvate dehydrogenase complex (PDH), which serves as a central metabolic switch in energy metabolism that is based on reversible phosphorylation. Phos-tag technology allows rapid monitoring of the metabolic state via simultaneous detection of phosphorylated and non-phosphorylated species of the PDH core component Pda1. Our protocol can be applied for several further intra-organellar proteins like respiratory chain complexes or protein translocases of the inner membrane. SIGNIFICANCE: Our manuscript describes for the first time how Phos-tag technology can be applied to monitor phosphorylation of intramitochondrial proteins. We exemplify this with the regulation of the pyruvate dehydrogenase complex as central regulatory switch in energy metabolism. We show that our protocol allows a rapid monitoring of the metabolic state of the cell (phosphorylated PDH is inactive while non-phosphorylated PDH is active) and can be applied for rapid profiling of different metabolic conditions as well as for profiling phosphorylation of further intramitochondrial protein (complexes).
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http://dx.doi.org/10.1016/j.jprot.2021.104430DOI Listing
November 2021

Mitochondria as emergency landing for abandoned peroxins.

EMBO Rep 2021 10 19;22(10):e53790. Epub 2021 Aug 19.

CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany.

Zellweger spectrum disorder (ZSD) is the most severe peroxisomal biogenesis disorder (PBD). Why ZSD patients not only loose functional peroxisomes but also present with severe mitochondrial dysfunction was a long-standing mystery. In this issue, Nuebel et al (2021) identified that loss of peroxisomes leads to re-routing of peroxisomal proteins to mitochondria, thereby impairing mitochondrial structure and function. The findings provide the first molecular understanding of the mitochondrial-peroxisomal link in ZSD.
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http://dx.doi.org/10.15252/embr.202153790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8490976PMC
October 2021

Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery.

Nat Commun 2021 07 13;12(1):4284. Epub 2021 Jul 13.

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.
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http://dx.doi.org/10.1038/s41467-021-24426-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8277783PMC
July 2021

Improving Identification of Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker.

Mol Cell Proteomics 2020 04 12;19(4):624-639. Epub 2020 Feb 12.

Department of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada; University of Victoria - Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec, H3T 1E2, Canada; Gerald Bronfman Department of Oncology, Jewish General Hospital, Montreal, Quebec, H3T 1E2, Canada; Department of Data Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Skolkovo Innovation Center, Nobel St., Moscow 143026, Russia. Electronic address:

An experimental and computational approach for identification of protein-protein interactions by chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.
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http://dx.doi.org/10.1074/mcp.RA119.001839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124466PMC
April 2020

An Early mtUPR: Redistribution of the Nuclear Transcription Factor Rox1 to Mitochondria Protects against Intramitochondrial Proteotoxic Aggregates.

Mol Cell 2020 01 17;77(1):180-188.e9. Epub 2019 Oct 17.

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany; CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany. Electronic address:

The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.
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http://dx.doi.org/10.1016/j.molcel.2019.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941230PMC
January 2020

Pptc7 is an essential phosphatase for promoting mammalian mitochondrial metabolism and biogenesis.

Nat Commun 2019 07 19;10(1):3197. Epub 2019 Jul 19.

Morgridge Institute for Research, Madison, WI, 53715, USA.

Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that protein dephosphorylation may be broadly important for calibrating mitochondrial activities. To explore this, we deleted the poorly characterized matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7 mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7 tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Among these, we identify the protein translocase complex subunit Timm50 as a putative Pptc7 substrate whose phosphorylation reduces import activity. We further find that phosphorylation within or near the mitochondrial targeting sequences of multiple proteins could disrupt their import rates and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition.
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http://dx.doi.org/10.1038/s41467-019-11047-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6642090PMC
July 2019

Advanced tools for the analysis of protein phosphorylation in yeast mitochondria.

Anal Biochem 2018 08 24;554:23-27. Epub 2018 May 24.

Division of Cell Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria. Electronic address:

The biochemical analysis of protein phosphorylation in mitochondria lags behind that of cytosolic signaling events. One reason is the poor stability of many phosphorylation sites during common isolation procedures for mitochondria. We present here an optimized, fast protocol for the purification of yeast mitochondria that greatly increases recovery of phosphorylated mitochondrial proteins. Moreover, we describe improved protocols for the biochemical analysis of mitochondrial protein phosphorylation by Zn-Phos-tag electrophoresis under both denaturing and - for the first time - native conditions, and demonstrate that they outperform previously applied methods.
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http://dx.doi.org/10.1016/j.ab.2018.05.022DOI Listing
August 2018

Guidelines and recommendations on yeast cell death nomenclature.

Microb Cell 2018 Jan 1;5(1):4-31. Epub 2018 Jan 1.

Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, Bari, Italy.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.
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http://dx.doi.org/10.15698/mic2018.01.607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772036PMC
January 2018

Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei.

PLoS Pathog 2017 12 29;13(12):e1006808. Epub 2017 Dec 29.

Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.

Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins-TAC60, TAC42 and TAC40-which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane β-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal β-signals. Thus in trypanosomes the highly conserved β-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.
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http://dx.doi.org/10.1371/journal.ppat.1006808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764417PMC
December 2017

In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy.

Autophagy 2018 1;14(2):311-335. Epub 2018 Feb 1.

a Institute of Biochemistry, Medical Faculty , Friedrich-Alexander-University of Erlangen-Nürnberg , Erlangen , Germany.

In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2β-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.
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http://dx.doi.org/10.1080/15548627.2017.1403716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902202PMC
February 2019

Identification of new channels by systematic analysis of the mitochondrial outer membrane.

J Cell Biol 2017 11 15;216(11):3485-3495. Epub 2017 Sep 15.

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are β-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.
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http://dx.doi.org/10.1083/jcb.201706043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674900PMC
November 2017

Landscape of submitochondrial protein distribution.

Nat Commun 2017 08 18;8(1):290. Epub 2017 Aug 18.

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, 79104, Germany.

The mitochondrial proteome comprises ~1000 (yeast)-1500 (human) different proteins, which are distributed into four different subcompartments. The sublocalization of these proteins within the organelle in most cases remains poorly defined. Here we describe an integrated approach combining stable isotope labeling, various protein enrichment and extraction strategies and quantitative mass spectrometry to produce a quantitative map of submitochondrial protein distribution in S. cerevisiae. This quantitative landscape enables a proteome-wide classification of 986 proteins into soluble, peripheral, and integral mitochondrial membrane proteins, and the assignment of 818 proteins into the four subcompartments: outer membrane, inner membrane, intermembrane space, or matrix. We also identified 206 proteins that were not previously annotated as localized to mitochondria. Furthermore, the protease Prd1, misannotated as intermembrane space protein, could be re-assigned and characterized as a presequence peptide degrading enzyme in the matrix.Protein localization plays an important role in the regulation of cellular physiology. Here the authors use an integrated proteomics approach to localize proteins to the mitochondria and provide a detailed map of their specific localization within the organelle.
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http://dx.doi.org/10.1038/s41467-017-00359-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561175PMC
August 2017

The novel mitochondrial matrix protease Ste23 is required for efficient presequence degradation and processing.

Mol Biol Cell 2017 Apr 22;28(8):997-1002. Epub 2017 Feb 22.

Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, 79104 Freiburg, Germany.

Approximately 70% of mitochondrial precursor proteins are imported from the cytosol via N-terminal presequences, which are cleaved upon exposure to the mitochondrial processing protease MPP in the matrix. Cleaved presequence peptides then need to be efficiently degraded, and impairment of this clearance step, for example, by amyloid β peptides, causes feedback inhibition of MPP, leading ultimately to accumulation of immature precursor proteins within mitochondria. Degradation of mitochondrial peptides is performed by Cym1 in yeast and its homologue, PreP, in humans. Here we identify the novel mitochondrial matrix protease Ste23 in yeast, a homologue of human insulin-degrading enzyme, which is required for efficient peptide degradation. Ste23 and Cym1 tightly cooperate to ensure the correct functioning of the essential presequence processing machinery.
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http://dx.doi.org/10.1091/mbc.E16-10-0732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391191PMC
April 2017

PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy.

Autophagy 2017 Mar 19;13(3):486-505. Epub 2017 Jan 19.

b Department of Pediatrics, Center for Liver, Digestive and Metabolic Diseases , University of Groningen, University Medical Center Groningen , AV Groningen , The Netherlands.

Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1's and PLK1's functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.
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http://dx.doi.org/10.1080/15548627.2016.1263781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5361591PMC
March 2017

Recent advances in mitochondrial biology - integrated aspects.

Cell Tissue Res 2017 Jan;367(1):1-3

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine and BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.

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http://dx.doi.org/10.1007/s00441-016-2533-4DOI Listing
January 2017

MIPEP recessive variants cause a syndrome of left ventricular non-compaction, hypotonia, and infantile death.

Genome Med 2016 11 1;8(1):106. Epub 2016 Nov 1.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.

Background: Mitochondrial presequence proteases perform fundamental functions as they process about 70 % of all mitochondrial preproteins that are encoded in the nucleus and imported posttranslationally. The mitochondrial intermediate presequence protease MIP/Oct1, which carries out precursor processing, has not yet been established to have a role in human disease.

Methods: Whole exome sequencing was performed on four unrelated probands with left ventricular non-compaction (LVNC), developmental delay (DD), seizures, and severe hypotonia. Proposed pathogenic variants were confirmed by Sanger sequencing or array comparative genomic hybridization. Functional analysis of the identified MIP variants was performed using the model organism Saccharomyces cerevisiae as the protein and its functions are highly conserved from yeast to human.

Results: Biallelic single nucleotide variants (SNVs) or copy number variants (CNVs) in MIPEP, which encodes MIP, were present in all four probands, three of whom had infantile/childhood death. Two patients had compound heterozygous SNVs (p.L582R/p.L71Q and p.E602*/p.L306F) and one patient from a consanguineous family had a homozygous SNV (p.K343E). The fourth patient, identified through the GeneMatcher tool, a part of the Matchmaker Exchange Project, was found to have inherited a paternal SNV (p.H512D) and a maternal CNV (1.4-Mb deletion of 13q12.12) that includes MIPEP. All amino acids affected in the patients' missense variants are highly conserved from yeast to human and therefore S. cerevisiae was employed for functional analysis (for p.L71Q, p.L306F, and p.K343E). The mutations p.L339F (human p.L306F) and p.K376E (human p.K343E) resulted in a severe decrease of Oct1 protease activity and accumulation of non-processed Oct1 substrates and consequently impaired viability under respiratory growth conditions. The p.L83Q (human p.L71Q) failed to localize to the mitochondria.

Conclusions: Our findings reveal for the first time the role of the mitochondrial intermediate peptidase in human disease. Loss of MIP function results in a syndrome which consists of LVNC, DD, seizures, hypotonia, and cataracts. Our approach highlights the power of data exchange and the importance of an interrelationship between clinical and research efforts for disease gene discovery.
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http://dx.doi.org/10.1186/s13073-016-0360-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088683PMC
November 2016

Separating mitochondrial protein assembly and endoplasmic reticulum tethering by selective coupling of Mdm10.

Nat Commun 2016 10 10;7:13021. Epub 2016 Oct 10.

Institute of Biochemistry and Molecular Biology, Centre for Biochemistry and Molecular Cell Research, Faculty of Medicine, University of Freiburg, Freiburg D-79104, Germany.

The endoplasmic reticulum-mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 β-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the β-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and β-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.
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http://dx.doi.org/10.1038/ncomms13021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476798PMC
October 2016

ZEB1 turns into a transcriptional activator by interacting with YAP1 in aggressive cancer types.

Nat Commun 2016 Feb 15;7:10498. Epub 2016 Feb 15.

Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany.

Early dissemination, metastasis and therapy resistance are central hallmarks of aggressive cancer types and the leading cause of cancer-associated deaths. The EMT-inducing transcriptional repressor ZEB1 is a crucial stimulator of these processes, particularly by coupling the activation of cellular motility with stemness and survival properties. ZEB1 expression is associated with aggressive behaviour in many tumour types, but the potent effects cannot be solely explained by its proven function as a transcriptional repressor of epithelial genes. Here we describe a direct interaction of ZEB1 with the Hippo pathway effector YAP, but notably not with its paralogue TAZ. In consequence, ZEB1 switches its function to a transcriptional co-activator of a 'common ZEB1/YAP target gene set', thereby linking two pathways with similar cancer promoting effects. This gene set is a predictor of poor survival, therapy resistance and increased metastatic risk in breast cancer, indicating the clinical relevance of our findings.
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http://dx.doi.org/10.1038/ncomms10498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756710PMC
February 2016

Peeping at TOMs-Diverse Entry Gates to Mitochondria Provide Insights into the Evolution of Eukaryotes.

Mol Biol Evol 2016 Feb 15;33(2):337-51. Epub 2015 Oct 15.

Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland

Mitochondria are essential for eukaryotic life and more than 95% of their proteins are imported as precursors from the cytosol. The targeting signals for this posttranslational import are conserved in all eukaryotes. However, this conservation does not hold true for the protein translocase of the mitochondrial outer membrane that serves as entry gate for essentially all precursor proteins. Only two of its subunits, Tom40 and Tom22, are conserved and thus likely were present in the last eukaryotic common ancestor. Tom7 is found in representatives of all supergroups except the Excavates. This suggests that it was added to the core of the translocase after the Excavates segregated from all other eukaryotes. A comparative analysis of the biochemically and functionally characterized outer membrane translocases of yeast, plants, and trypanosomes, which represent three eukaryotic supergroups, shows that the receptors that recognize the conserved import signals differ strongly between the different systems. They present a remarkable example of convergent evolution at the molecular level. The structural diversity of the functionally conserved import receptors therefore provides insight into the early evolutionary history of mitochondria.
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http://dx.doi.org/10.1093/molbev/msv219DOI Listing
February 2016

A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling.

Proc Natl Acad Sci U S A 2015 Oct 5;112(42):E5679-88. Epub 2015 Oct 5.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Departments of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) govern cellular homeostasis by inducing signaling. H2O2 modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H2O2 signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H2O2 signaling colocalize as H2O2 induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H2O2 as the respiratory chain, Lyn, and Syk were similarly required for H2O2 signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H2O2 signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.
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http://dx.doi.org/10.1073/pnas.1517932112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620870PMC
October 2015

The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1.

J Cell Biol 2015 Sep;210(6):951-60

Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein.
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http://dx.doi.org/10.1083/jcb.201506085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4576865PMC
September 2015

COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2.

Hum Mol Genet 2015 Oct 9;24(19):5404-15. Epub 2015 Jul 9.

Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Melbourne, Australia,

Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2.
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http://dx.doi.org/10.1093/hmg/ddv265DOI Listing
October 2015

Mitochondrial protein import receptors in Kinetoplastids reveal convergent evolution over large phylogenetic distances.

Nat Commun 2015 Mar 26;6:6646. Epub 2015 Mar 26.

Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, Bern CH-3012, Switzerland.

Mitochondrial protein import is essential for all eukaryotes and mediated by hetero-oligomeric protein translocases thought to be conserved within all eukaryotes. We have identified and analysed the function and architecture of the non-conventional outer membrane (OM) protein translocase in the early diverging eukaryote Trypanosoma brucei. It consists of six subunits that show no obvious homology to translocase components of other species. Two subunits are import receptors that have a unique topology and unique protein domains and thus evolved independently of the prototype receptors Tom20 and Tom70. Our study suggests that protein import receptors were recruited to the core of the OM translocase after the divergence of the major eukaryotic supergroups. Moreover, it links the evolutionary history of mitochondrial protein import receptors to the origin of the eukaryotic supergroups.
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http://dx.doi.org/10.1038/ncomms7646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4389251PMC
March 2015

Metabolic control via the mitochondrial protein import machinery.

Curr Opin Cell Biol 2015 Apr 10;33:42-8. Epub 2014 Dec 10.

Institut für Biochemie und Molekularbiologie, ZBMZ, Germany; BIOSS Centre for Biological Signalling Studies, Germany. Electronic address:

Mitochondria have to import most of their proteins in order to fulfill a multitude of metabolic functions. Sophisticated import machineries mediate targeting and translocation of preproteins from the cytosol and subsequent sorting into their suborganellar destination. The mode of action of these machineries has been considered for long time as a static and constitutively active process. However, recent studies revealed that the mitochondrial protein import machinery is subject to intense regulatory mechanisms that include direct control of protein flux by metabolites and metabolic signalling cascades.
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http://dx.doi.org/10.1016/j.ceb.2014.11.001DOI Listing
April 2015

Mitochondria. Cell cycle-dependent regulation of mitochondrial preprotein translocase.

Science 2014 Nov 6;346(6213):1109-13. Epub 2014 Nov 6.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany. BIOSS Centre for Biological Signalling Studies, Universität Freiburg, 79104 Freiburg, Germany.

Mitochondria play central roles in cellular energy conversion, metabolism, and apoptosis. Mitochondria import more than 1000 different proteins from the cytosol. It is unknown if the mitochondrial protein import machinery is connected to the cell division cycle. We found that the cyclin-dependent kinase Cdk1 stimulated assembly of the main mitochondrial entry gate, the translocase of the outer membrane (TOM), in mitosis. The molecular mechanism involved phosphorylation of the cytosolic precursor of Tom6 by cyclin Clb3-activated Cdk1, leading to enhanced import of Tom6 into mitochondria. Tom6 phosphorylation promoted assembly of the protein import channel Tom40 and import of fusion proteins, thus stimulating the respiratory activity of mitochondria in mitosis. Tom6 phosphorylation provides a direct means for regulating mitochondrial biogenesis and activity in a cell cycle-specific manner.
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http://dx.doi.org/10.1126/science.1261253DOI Listing
November 2014

NMR-based detection of hydrogen/deuterium exchange in liposome-embedded membrane proteins.

PLoS One 2014 6;9(11):e112374. Epub 2014 Nov 6.

Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; German Center for Neurodegenerative Diseases (DZNE), Goöttingen, Germany; Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), University Medical Center, Göttingen, Germany.

Membrane proteins play key roles in biology. Determination of their structure in a membrane environment, however, is highly challenging. To address this challenge, we developed an approach that couples hydrogen/deuterium exchange of membrane proteins to rapid unfolding and detection by solution-state NMR spectroscopy. We show that the method allows analysis of the solvent protection of single residues in liposome-embedded proteins such as the 349-residue Tom40, the major protein translocation pore in the outer mitochondrial membrane, which has resisted structural analysis for many years.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112374PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4223039PMC
December 2015

Amyloid-β peptide induces mitochondrial dysfunction by inhibition of preprotein maturation.

Cell Metab 2014 Oct 28;20(4):662-9. Epub 2014 Aug 28.

Institut für Biochemie und Molekularbiologie, ZBMZ, University of Freiburg, 79104 Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany. Electronic address:

Most mitochondrial proteins possess N-terminal presequences that are required for targeting and import into the organelle. Upon import, presequences are cleaved off by matrix processing peptidases and subsequently degraded by the peptidasome Cym1/PreP, which also degrades Amyloid-beta peptides (Aβ). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, Aβ inhibits degradation of presequence peptides by PreP, resulting in accumulation of mitochondrial preproteins and processing intermediates. Dysfunctional preprotein maturation leads to rapid protein degradation and an imbalanced organellar proteome. Our findings reveal a general mechanism by which Aβ peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimer's disease.
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http://dx.doi.org/10.1016/j.cmet.2014.07.024DOI Listing
October 2014

The novel component Kgd4 recruits the E3 subunit to the mitochondrial α-ketoglutarate dehydrogenase.

Mol Biol Cell 2014 Nov 27;25(21):3342-9. Epub 2014 Aug 27.

Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden

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.
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http://dx.doi.org/10.1091/mbc.E14-07-1178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214781PMC
November 2014

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial β-barrel proteins specialized in mitochondrial genome inheritance.

Proc Natl Acad Sci U S A 2014 May 12;111(21):7624-9. Epub 2014 May 12.

Department of Chemistry and Biochemistry, University of Bern, CH-3012 Bern, Switzerland;

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum-mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA-cytoskeleton linkage that is essential for mitochondrial DNA inheritance.
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http://dx.doi.org/10.1073/pnas.1404854111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040615PMC
May 2014

Uniform nomenclature for the mitochondrial contact site and cristae organizing system.

J Cell Biol 2014 Mar;204(7):1083-6

Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung, and 2 BIOSS Centre for Biological Signalling Studies, Universität Freiburg, 79104 Freiburg, Germany.

The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex "mitochondrial contact site and cristae organizing system" and its subunits Mic10 to Mic60.
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http://dx.doi.org/10.1083/jcb.201401006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971754PMC
March 2014
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