Publications by authors named "David Stroud"

64 Publications

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Mol Cell 2017 Aug 14;67(3):457-470.e5. Epub 2017 Jul 14.

Department of Biochemistry and Molecular Biology and The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia. Electronic address:

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.
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http://dx.doi.org/10.1016/j.molcel.2017.06.014DOI Listing
August 2017

The Arctic in the Twenty-First Century: Changing Biogeochemical Linkages across a Paraglacial Landscape of Greenland.

Bioscience 2017 Feb;67(2):118-133

N. John Anderson is affiliated with the Department of Geography at Loughborough University in Loughborough, UK. Jasmine E. Saros, is affiliated with the School of Biology & Ecology at the University of Maine in Orono, Maine. Joanna E. Bullard, is affiliated with the Department of Geography at Loughborough University in Loughborough, UK. Sean M.P. Cahoon, was at the Department of Biology at Penn State University, in University Park, Pennsylvania. He is presently affiliated with the Environment and Natural Resources Institute at the University of Alaska Anchorage, AK. Suzanne McGowan is affiliated with the School of Geography at the University of Nottingham in Nottingham, UK. Elizabeth A. Bagshaw is affiliated with the Earth and Ocean Sciences at Cardiff University in Cardiff, UK. Christopher D. Barry, is affiliated with the School of Biological Sciences at Queen's University in Belfast, UK. Richard Bindler is affiliated with the Department of Ecology and Environmental Science at Umeå University in Umeå, Sweden. Benjamin T. Burpee is affiliated with the School of Biology & Ecology at the University of Maine in Orono, Maine. Jonathan L. Carrivick, is affiliated with the School of Geography at the University of Leeds in Leeds, UK. Rachel A. Fowler, is affiliated with the School of Biology & Ecology at the University of Maine in Orono, Maine. Anthony D. Fox is affiliated with the Department of Bioscience, at Aarhus University in Rønde, Denmark. Sherilyn C. Fritz is affiliated with the Department of Earth and Atmospheric Sciences at the University of Nebraska in Lincoln, Nebraska. Madeleine E. Giles, is affiliated with the School of Biological Sciences at the University of Essex in Colchester, UK. Ladislav Hamerlik, was affiliated with the Department of Biology and Ecology at Matthias Belius University in Banska Bystrica, Slovakia. He is presently affiliated with the Institute of Geological Sciences, Polish Academy of Sciences, Warsaw, Poland Thomas Ingeman-Nielsen is affiliated with the Department of Civil Engineering at the Technical University of Denmark in Kongens Lyngby, Denmark. Antonia C. Law is affiliated with the Department of Geography, Geology and the Environment at Keele University in Keele, UK. Sebastian H. Mernild is affiliated with the Nansen Environmental and Remote Sensing Center, Bergen, Norway. He also has positions at Faculty of Engineering and Science, Sogn og Fjordane University College, Sogndal, Norway and Antarctic and Sub-Antarctic Program, Universidad de Magallanes, Punta Arenas, Chile. Faculty of Engineering and Science at Sogn og Fjordane University College in Sogndal, Norway. Robert M. Northington is affiliated with the School of Biology & Ecology at the University of Maine in Orono, Maine. Christopher L. Osburn is affiliated with the School of Marine, Earth, and Atmospheric Sciences at NC State University, Raleigh, North Carolina. Sergi Pla-Rabès is affiliated with the Centre de Recerca Ecològica i Aplications Forestals in Cerdanyola del Vallés, Spain. Eric Post is affiliated with the Department of Wildlife, Fish, & Conservation Biology at the University of California in Davis, California. Jon Telling was affiliated with the School of Geographical Sciences at the University of Bristol in Bristol, UK. He is presently affiliated with the School of Civil Engineering and Geosciences, Newcastle University, UK. David A. Stroud is affiliated with the UK Joint Nature Conservation Committee in Peterborough, UK. Erika J. Whiteford is affiliated with the Department of Geography at Loughborough University in Loughborough, UK. Marian L. Yallop is affiliated with the School of Biological Science, at University of Bristol in Bristol, UK. Jacob C. Yde is affiliated with the Faculty of Engineering and Science at Sogn og Fjordane University College in Sogndal, Norway.

The Kangerlussuaq area of southwest Greenland encompasses diverse ecological, geomorphic, and climate gradients that function over a range of spatial and temporal scales. Ecosystems range from the microbial communities on the ice sheet and moisture-stressed terrestrial vegetation (and their associated herbivores) to freshwater and oligosaline lakes. These ecosystems are linked by a dynamic glacio-fluvial-aeolian geomorphic system that transports water, geological material, organic carbon and nutrients from the glacier surface to adjacent terrestrial and aquatic systems. This paraglacial system is now subject to substantial change because of rapid regional warming since 2000. Here, we describe changes in the eco- and geomorphic systems at a range of timescales and explore rapid future change in the links that integrate these systems. We highlight the importance of cross-system subsidies at the landscape scale and, importantly, how these might change in the near future as the Arctic is expected to continue to warm.
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http://dx.doi.org/10.1093/biosci/biw158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384161PMC
February 2017

Key actions towards the sustainable management of European geese.

Ambio 2017 Mar;46(Suppl 2):328-338

Department of Bioscience, Aarhus University, Kalø, Grenåvej, 8410, Denmark.

Increasing abundance of geese in North America and Europe constitutes a major conservation success, but has caused increasing conflicts with economic, health and safety interests, as well as ecosystem impacts. Potential conflict resolution through a single, 'one size fits all' policy is hindered by differences in species' ecology, behaviour, abundance and population status, and in contrasting political and socio-economic environments across the flyways. Effective goose management requires coordinated application of a suite of tools from the local level to strategic flyway management actions. The European Goose Management Platform, established under the Agreement on the Conservation of African-Eurasian Migratory Waterbirds, aims to harmonise and prioritise management, monitoring and conservation efforts, sharing best practice internationally by facilitating agreed policies, coordinating flyway efforts, and sharing and exchanging experiences and information. This depends crucially upon adequate government financing, the collection of necessary monitoring data (e.g., on distribution, abundance, hunting bags, demography, ecosystem and agricultural damage), the collation and effective use of such data and information, as well as the evaluation of outcomes of existing management measures.
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http://dx.doi.org/10.1007/s13280-017-0903-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316334PMC
March 2017

A novel isoform of the human mitochondrial complex I subunit NDUFV3.

FEBS Lett 2017 Jan 30;591(1):109-117. Epub 2016 Dec 30.

Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia.

Human mitochondrial complex I is the first enzyme of the mitochondrial respiratory chain. Complex I is composed of 45 subunits, seven encoded by mitochondrial DNA, while the remainder are encoded by nuclear DNA. All nuclear-encoded subunits are thought to be expressed as a single isoform. Here we reveal subunit NDUFV3 to be present in both the canonical 10 kDa and a novel 50 kDa isoform, generated through alternative splicing. Both isoforms assemble into complex I and their levels vary in different tissues. While the 50 kDa isoform appears to be dominant in HEK293T cells, we find either isoform alone is sufficient for assembly of mature complex I. NDUFV3 represents the first known complex I subunit present in two functional isoforms.
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http://dx.doi.org/10.1002/1873-3468.12527DOI Listing
January 2017

Accessory subunits are integral for assembly and function of human mitochondrial complex I.

Nature 2016 Oct 14;538(7623):123-126. Epub 2016 Sep 14.

Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, 3800, Melbourne, Australia.

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.
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http://dx.doi.org/10.1038/nature19754DOI Listing
October 2016

Screening Strategies for TALEN-Mediated Gene Disruption.

Methods Mol Biol 2016 ;1419:231-52

Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Wellington Road, Clayton 3800, Melbourne, VIC, Australia.

Targeted gene disruption has rapidly become the tool of choice for the analysis of gene and protein function in routinely cultured mammalian cells. Three main technologies capable of irreversibly disrupting gene-expression exist: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. The desired outcome of the use of any of these technologies is targeted insertions and/or deletions (indels) that result in either a nonsense frame shift or splicing error that disrupts protein expression. Many excellent do-it-yourself systems for TALEN construct assembly are now available at low or no cost to academic researchers. However, for new users, screening for successful gene disruption is still a hurdle. Here, we describe efficient and cost-effective strategies for the generation of gene-disrupted cell lines. Although the focus of this chapter is on the use of TALENs, these strategies can be applied to the use of all three technologies.
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http://dx.doi.org/10.1007/978-1-4939-3581-9_17DOI Listing
December 2017

Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission.

J Cell Sci 2016 06 12;129(11):2170-81. Epub 2016 Apr 12.

Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne 3800, Australia

Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.
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http://dx.doi.org/10.1242/jcs.185165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919635PMC
June 2016

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

FunRich: An open access standalone functional enrichment and interaction network analysis tool.

Proteomics 2015 Aug 17;15(15):2597-601. Epub 2015 Jun 17.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia.

As high-throughput techniques including proteomics become more accessible to individual laboratories, there is an urgent need for a user-friendly bioinformatics analysis system. Here, we describe FunRich, an open access, standalone functional enrichment and network analysis tool. FunRich is designed to be used by biologists with minimal or no support from computational and database experts. Using FunRich, users can perform functional enrichment analysis on background databases that are integrated from heterogeneous genomic and proteomic resources (>1.5 million annotations). Besides default human specific FunRich database, users can download data from the UniProt database, which currently supports 20 different taxonomies against which enrichment analysis can be performed. Moreover, the users can build their own custom databases and perform the enrichment analysis irrespective of organism. In addition to proteomics datasets, the custom database allows for the tool to be used for genomics, lipidomics and metabolomics datasets. Thus, FunRich allows for complete database customization and thereby permits for the tool to be exploited as a skeleton for enrichment analysis irrespective of the data type or organism used. FunRich (http://www.funrich.org) is user-friendly and provides graphical representation (Venn, pie charts, bar graphs, column, heatmap and doughnuts) of the data with customizable font, scale and color (publication quality).
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http://dx.doi.org/10.1002/pmic.201400515DOI Listing
August 2015

Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I.

Hum Mol Genet 2015 May 12;24(10):2952-65. Epub 2015 Feb 12.

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

Human mitochondrial complex I is the largest enzyme of the respiratory chain and is composed of 44 different subunits. Complex I subunits are encoded by both nuclear and mitochondrial (mt) DNA and their assembly requires a number of additional proteins. FAD-dependent oxidoreductase domain-containing protein 1 (FOXRED1) was recently identified as a putative assembly factor and FOXRED1 mutations in patients cause complex I deficiency; however, its role in assembly is unknown. Here, we demonstrate that FOXRED1 is involved in mid-late stages of complex I assembly. In a patient with FOXRED1 mutations, the levels of mature complex I were markedly decreased, and a smaller ∼475 kDa subcomplex was detected. In the absence of FOXRED1, mtDNA-encoded complex I subunits are still translated and transiently assembled into a late stage ∼815 kDa intermediate; but instead of transitioning further to the mature complex I, the intermediate breaks down to an ∼475 kDa complex. As the patient cells contained residual assembled complex I, we disrupted the FOXRED1 gene in HEK293T cells through TALEN-mediated gene editing. Cells lacking FOXRED1 had ∼10% complex I levels, reduced complex I activity, and were unable to grow on galactose media. Interestingly, overexpression of FOXRED1 containing the patient mutations was able to rescue complex I assembly. In addition, FOXRED1 was found to co-immunoprecipitate with a number of complex I subunits. Our studies reveal that FOXRED1 is a crucial component in the productive assembly of complex I and that mutations in FOXRED1 leading to partial loss of function cause defects in complex I biogenesis.
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http://dx.doi.org/10.1093/hmg/ddv058DOI Listing
May 2015

Stalking the mitochondrial ATP synthase: Ina found guilty by association.

EMBO J 2014 Aug 18;33(15):1617-8. Epub 2014 Jun 18.

Department of Biochemistry, La Trobe University, Melbourne, Vic., Australia.

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http://dx.doi.org/10.15252/embj.201489069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4194094PMC
August 2014

Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission.

J Cell Biol 2014 Feb 10;204(4):477-86. Epub 2014 Feb 10.

Department of Biochemistry and 2 Australian Research Council Centre of Excellence in Coherent X-Ray Science, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia.

Mitochondrial fission is important for organelle transport, inheritance, and turnover, and alterations in fission are seen in neurological disease. In mammals, mitochondrial fission is executed by dynamin-related protein 1 (Drp1), a cytosolic guanosine triphosphatase that polymerizes and constricts the organelle. Recruitment of Drp1 to mitochondria involves receptors including Mff, MiD49, and MiD51. MiD49/51 form foci at mitochondrial constriction sites and coassemble with Drp1 to drive fission. Here, we solved the crystal structure of the cytosolic domain of human MiD51, which adopts a nucleotidyltransferase fold. Although MiD51 lacks catalytic residues for transferase activity, it specifically binds guanosine diphosphate and adenosine diphosphate. MiD51 mutants unable to bind nucleotides were still able to recruit Drp1. Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci. MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus.
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http://dx.doi.org/10.1083/jcb.201311014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926961PMC
February 2014

A founder mutation in PET100 causes isolated complex IV deficiency in Lebanese individuals with Leigh syndrome.

Am J Hum Genet 2014 Feb 23;94(2):209-22. Epub 2014 Jan 23.

Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia; Victorian Clinical Genetics Services, Royal Children's Hospital, Melbourne, VIC 3052, Australia. Electronic address:

Leigh syndrome (LS) is a severe neurodegenerative disorder with characteristic bilateral lesions, typically in the brainstem and basal ganglia. It usually presents in infancy and is genetically heterogeneous, but most individuals with mitochondrial complex IV (or cytochrome c oxidase) deficiency have mutations in the biogenesis factor SURF1. We studied eight complex IV-deficient LS individuals from six families of Lebanese origin. They differed from individuals with SURF1 mutations in having seizures as a prominent feature. Complementation analysis suggested they had mutation(s) in the same gene but targeted massively parallel sequencing (MPS) of 1,034 genes encoding known mitochondrial proteins failed to identify a likely candidate. Linkage and haplotype analyses mapped the location of the gene to chromosome 19 and targeted MPS of the linkage region identified a homozygous c.3G>C (p.Met1?) mutation in C19orf79. Abolishing the initiation codon could potentially still allow initiation at a downstream methionine residue but we showed that this would not result in a functional protein. We confirmed that mutation of this gene was causative by lentiviral-mediated phenotypic correction. C19orf79 was recently renamed PET100 and predicted to encode a complex IV biogenesis factor. We showed that it is located in the mitochondrial inner membrane and forms a ∼300 kDa subcomplex with complex IV subunits. Previous proteomic analyses of mitochondria had overlooked PET100 because its small size was below the cutoff for annotating bona fide proteins. The mutation was estimated to have arisen at least 520 years ago, explaining how the families could have different religions and different geographic origins within Lebanon.
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http://dx.doi.org/10.1016/j.ajhg.2013.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928654PMC
February 2014

Mitochondria: organization of respiratory chain complexes becomes cristae-lized.

Curr Biol 2013 Nov;23(21):R969-71

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia.

For over 100 years mitochondria have been known for their distinctive morphology featuring elaborately folded cristae, and their role as 'the powerhouse of the cell'. New research shows that these two characteristics are more dependent on each other than previously thought.
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http://dx.doi.org/10.1016/j.cub.2013.09.035DOI Listing
November 2013

Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation.

Cell 2013 Aug;154(3):596-608

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.

The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of β-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the β-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring β-barrel formation in vivo and in organello and demonstrated that the β-barrel was formed and membrane inserted while the precursor was bound to SAM. β-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling.
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http://dx.doi.org/10.1016/j.cell.2013.06.033DOI Listing
August 2013

Transport signatures of electronic-nematic stripe phases.

J Phys Condens Matter 2013 May 19;25(20):202201. Epub 2013 Apr 19.

Department of Physics, University of Toronto, Toronto, ON M5S 1A7, Canada.

Electronic-nematic phases are broadly characterized by spontaneously broken rotational symmetry. Although they have been widely recognized in the context of high temperature cuprates, bilayer ruthenates, and iron-based superconductors, the focus so far has been exclusively on the uniform nematic phase. Recently, however, it was proposed that on a square lattice a nematic instability in the d-wave charge channel could lead to a spatially modulated nematic state, where the modulation vector q is determined by the relative location of the Fermi level to the van Hove singularity. Interestingly, this finite-q nematic (nematic stripe) phase has also been identified as an additional leading instability that is as strong as the superconducting instability near the onset of spin density wave order. Here, we study the electrical conductivity tensor in the modulated nematic phase for a general modulation vector. Our results can be used to identify nematic stripe phases in correlated materials.
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http://dx.doi.org/10.1088/0953-8984/25/20/202201DOI Listing
May 2013

Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I.

J Biol Chem 2013 Jan 5;288(3):1685-90. Epub 2012 Dec 5.

Department of Biochemistry, La Trobe Institute for Molecular Science, and ARC Centre of Excellence for Coherent X-ray Science, La Trobe University, Melbourne 3086, Australia.

Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture of NDUFA9 cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9 contained a complex I subcomplex consisting of membrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity.
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http://dx.doi.org/10.1074/jbc.C112.436766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548478PMC
January 2013

Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane.

Mol Biol Cell 2012 Oct 23;23(20):3948-56. Epub 2012 Aug 23.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.

Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport-associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.
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http://dx.doi.org/10.1091/mbc.E12-04-0295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3469511PMC
October 2012

Role of MINOS in mitochondrial membrane architecture: cristae morphology and outer membrane interactions differentially depend on mitofilin domains.

J Mol Biol 2012 Sep 7;422(2):183-91. Epub 2012 May 7.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.

The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture.
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http://dx.doi.org/10.1016/j.jmb.2012.05.004DOI Listing
September 2012

Dual function of Sdh3 in the respiratory chain and TIM22 protein translocase of the mitochondrial inner membrane.

Mol Cell 2011 Dec;44(5):811-8

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.

The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.
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http://dx.doi.org/10.1016/j.molcel.2011.09.025DOI Listing
December 2011

Composition and topology of the endoplasmic reticulum-mitochondria encounter structure.

J Mol Biol 2011 Nov 16;413(4):743-50. Epub 2011 Sep 16.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.

Eukaryotic cells contain multiple organelles, which are functionally and structurally interconnected. The endoplasmic reticulum-mitochondria encounter structure (ERMES) forms a junction between mitochondria and the endoplasmic reticulum (ER). Four ERMES proteins are known in yeast, the ER-anchored protein Mmm1 and three mitochondria-associated proteins, Mdm10, Mdm12 and Mdm34, with functions related to mitochondrial morphology and protein biogenesis. We mapped the glycosylation sites of ERMES and demonstrate that three asparagine residues in the N‑terminal domain of Mmm1 are glycosylated. While the glycosylation is dispensable, the cytosolic C‑terminal domain of Mmm1 that connects to the Mdm proteins is required for Mmm1 function. To analyze the composition of ERMES, we determined the subunits by quantitative mass spectrometry. We identified the calcium-binding GTPase Gem1 as a new ERMES subunit, revealing that ERMES is composed of five genuine subunits. Taken together, ERMES represents a platform that integrates components with functions in formation of ER-mitochondria junctions, maintenance of mitochondrial morphology, protein biogenesis and calcium binding.
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http://dx.doi.org/10.1016/j.jmb.2011.09.012DOI Listing
November 2011

Biogenesis of mitochondrial β-barrel proteins: the POTRA domain is involved in precursor release from the SAM complex.

Mol Biol Cell 2011 Aug 16;22(16):2823-33. Epub 2011 Jun 16.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.

The mitochondrial outer membrane contains proteinaceous machineries for the translocation of precursor proteins. The sorting and assembly machinery (SAM) is required for the insertion of β-barrel proteins into the outer membrane. Sam50 is the channel-forming core subunit of the SAM complex and belongs to the BamA/Sam50/Toc75 family of proteins that have been conserved from Gram-negative bacteria to mitochondria and chloroplasts. These proteins contain one or more N-terminal polypeptide transport-associated (POTRA) domains. POTRA domains can bind precursor proteins, however, different views exist on the role of POTRA domains in the biogenesis of β-barrel proteins. It has been suggested that the single POTRA domain of mitochondrial Sam50 plays a receptor-like function at the SAM complex. We established a system to monitor the interaction of chemical amounts of β-barrel precursor proteins with the SAM complex of wild-type and mutant yeast in organello. We report that the SAM complex lacking the POTRA domain of Sam50 efficiently binds β-barrel precursors, but is impaired in the release of the precursors. These results indicate the POTRA domain of Sam50 is not essential for recognition of β-barrel precursors but functions in a subsequent step to promote the release of precursor proteins from the SAM complex.
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http://dx.doi.org/10.1091/mbc.E11-02-0148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154879PMC
August 2011

Biogenesis of mitochondria: dual role of Tom7 in modulating assembly of the preprotein translocase of the outer membrane.

J Mol Biol 2011 Jan 6;405(1):113-24. Epub 2010 Nov 6.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.

Biogenesis of the translocase of the outer mitochondrial membrane (TOM complex) involves the assembly of the central β-barrel forming protein Tom40 with six different subunits that are embedded in the membrane via α-helical transmembrane segments. The sorting and assembly machinery (SAM complex) of the outer membrane plays a central role in this process. The SAM complex mediates the membrane integration of β-barrel precursor proteins including Tom40. The small Tom proteins Tom5 and Tom6 associate with the precursor of Tom40 at the SAM complex at an early stage of the assembly process and play a stimulatory role in the formation of the mature TOM complex. A fraction of the SAM components interacts with the outer membrane protein mitochondrial distribution and morphology protein 10 (Mdm10) to form the SAM-Mdm10 machinery; however, different views exist on the function of the SAM-Mdm10 complex. We report here that the third small Tom protein, Tom7, plays an inhibitory role at two distinct steps in the biogenesis of the TOM complex. First, Tom7 plays an antagonistic role to Tom5 and Tom6 at the early stage of Tom40 assembly at the SAM complex. Second, Tom7 interacts with Mdm10 that is not bound to the SAM complex, and thus promotes dissociation of the SAM-Mdm10 complex. Since the SAM-Mdm10 complex is required for the biogenesis of Tom22, Tom7 delays the assembly of Tom22 with Tom40 at a late stage of assembly of the TOM complex. Thus, Tom7 modulates the biogenesis of topologically different proteins, the β-barrel forming protein Tom40 and Tom22 that contains a transmembrane α-helix.
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http://dx.doi.org/10.1016/j.jmb.2010.11.002DOI Listing
January 2011

Assembly of the mitochondrial protein import channel: role of Tom5 in two-stage interaction of Tom40 with the SAM complex.

Mol Biol Cell 2010 Sep 28;21(18):3106-13. Epub 2010 Jul 28.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, Freiburg, Germany.

The preprotein translocase of the outer mitochondrial membrane (TOM) consists of a central β-barrel channel, Tom40, and six proteins with α-helical transmembrane segments. The precursor of Tom40 is imported from the cytosol by a pre-existing TOM complex and inserted into the outer membrane by the sorting and assembly machinery (SAM). Tom40 then assembles with α-helical Tom proteins to the mature TOM complex. The outer membrane protein Mim1 promotes membrane insertion of several α-helical Tom proteins but also affects the biogenesis of Tom40 by an unknown mechanism. We have identified a novel intermediate in the assembly pathway of Tom40, revealing a two-stage interaction of the precursor with the SAM complex. The second SAM stage represents assembly of Tom5 with the precursor of Tom40. Mim1-deficient mitochondria accumulate Tom40 at the first SAM stage like Tom5-deficient mitochondria. Tom5 promotes formation of the second SAM stage and thus suppresses the Tom40 assembly defect of mim1Δ mitochondria. We conclude that the assembly of newly imported Tom40 is directly initiated at the SAM complex by its association with Tom5. The involvement of Mim1 in Tom40 biogenesis can be largely attributed to its role in import of Tom5.
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http://dx.doi.org/10.1091/mbc.E10-06-0518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2938377PMC
September 2010

Biochemistry. Assembling the outer membrane.

Science 2010 May;328(5980):831-2

Institut für Biochemie und Molekularbiologie, ZBMZ, Trinationales Graduiertenkolleg 1478, Fakultät für Biologie, and Centre for Biological Signalling Studies, Universität Freiburg, 79104 Freiburg, Germany.

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http://dx.doi.org/10.1126/science.1190507DOI Listing
May 2010

Two modular forms of the mitochondrial sorting and assembly machinery are involved in biogenesis of alpha-helical outer membrane proteins.

J Mol Biol 2010 Feb 22;396(3):540-9. Epub 2009 Dec 22.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 Freiburg, Germany.

The mitochondrial outer membrane contains two translocase machineries for precursor proteins--the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of beta-barrel proteins of the outer membrane. The SAM complex is required for the assembly of precursors of the TOM complex, including not only the beta-barrel protein Tom40 but also a subset of alpha-helical subunits. While the interaction of beta-barrel proteins with the SAM complex has been studied in detail, little is known about the interaction between the SAM complex and alpha-helical precursor proteins. We report that the SAM is not static but that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of alpha-helical Tom proteins. We found that a subcomplex of TOM, Tom5-Tom40, associates with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the alpha-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the alpha-helical precursor of Tom22 and promotes its membrane integration. We suggest that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes.
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http://dx.doi.org/10.1016/j.jmb.2009.12.026DOI Listing
February 2010

Mitochondrial cardiolipin involved in outer-membrane protein biogenesis: implications for Barth syndrome.

Curr Biol 2009 Dec 3;19(24):2133-9. Epub 2009 Dec 3.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.

The biogenesis of mitochondria requires the import of a large number of proteins from the cytosol [1, 2]. Although numerous studies have defined the proteinaceous machineries that mediate mitochondrial protein sorting, little is known about the role of lipids in mitochondrial protein import. Cardiolipin, the signature phospholipid of the mitochondrial inner membrane [3-5], affects the stability of many inner-membrane protein complexes [6-12]. Perturbation of cardiolipin metabolism leads to the X-linked cardioskeletal myopathy Barth syndrome [13-18]. We report that cardiolipin affects the preprotein translocases of the mitochondrial outer membrane. Cardiolipin mutants genetically interact with mutants of outer-membrane translocases. Mitochondria from cardiolipin yeast mutants, as well as Barth syndrome patients, are impaired in the biogenesis of outer-membrane proteins. Our findings reveal a new role for cardiolipin in protein sorting at the mitochondrial outer membrane and bear implications for the pathogenesis of Barth syndrome.
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http://dx.doi.org/10.1016/j.cub.2009.10.074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329980PMC
December 2009

Avian influenza surveillance in wild birds in the European Union in 2006.

Influenza Other Respir Viruses 2009 Jan;3(1):1-14

Community Reference Laboratory, Veterinary Laboratories Agency, Surrey, UK.

Background: Infections of wild birds with highly pathogenic avian influenza (AI) subtype H5N1 virus were reported for the first time in the European Union in 2006.

Objectives: To capture epidemiological information on H5N1 HPAI in wild bird populations through large-scale surveillance and extensive data collection.

Methods: Records were analysed at bird level to explore the epidemiology of AI with regard to species of wild birds involved, timing and location of infections as well as the applicability of different surveillance types for the detection of infections.

Results: In total, 120,706 records of birds were sent to the Community Reference Laboratory for analysis. Incidents of H5N1 HPAI in wild birds were detected in 14 EU Member States during 2006. All of these incidents occurred between February and May, with the exception of two single cases during the summer months in Germany and Spain.

Conclusions: For the detection of H5N1 HPAI virus, passive surveillance of dead or diseased birds appeared the most effective approach, whilst active surveillance offered better detection of low pathogenic avian influenza (LPAI) viruses. No carrier species for H5N1 HPAI virus could be identified and almost all birds infected with H5N1 HPAI virus were either dead or showed clinical signs. A very large number of Mallards (Anas platyrhynchos) were tested in 2006 and while a high proportion of LPAI infections were found in this species, H5N1 HPAI virus was rarely identified in these birds. Orders of species that appeared to be very clinically susceptible to H5N1 HPAI virus were swans, diving ducks, mergansers and grebes, supporting experimental evidence. Surveillance results indicate that H5N1 HPAI virus did not establish itself successfully in the EU wild bird population in 2006.
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http://dx.doi.org/10.1111/j.1750-2659.2008.00058.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941908PMC
January 2009

Evolution of mitochondrial protein biogenesis.

Biochim Biophys Acta 2009 Jun 10;1790(6):409-15. Epub 2009 Apr 10.

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.

Mitochondria and the nucleus are key features that distinguish eukaryotic cells from prokaryotic cells. Mitochondria originated from a bacterium that was endosymbiotically taken up by another cell more than a billion years ago. Subsequently, most mitochondrial genes were transferred and integrated into the host cell's genome, making the evolution of pathways for specific import of mitochondrial proteins necessary. The mitochondrial protein translocation machineries are composed of numerous subunits. Interestingly, many of these subunits are at least in part derived from bacterial proteins, although only few of them functioned in bacterial protein translocation. We propose that the primitive alpha-proteobacterium, which was once taken up by the eukaryote ancestor cell, contained a number of components that were utilized for the generation of mitochondrial import machineries. Many bacterial components of seemingly unrelated pathways were integrated to form the modern cooperative mitochondria-specific protein translocation system.
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http://dx.doi.org/10.1016/j.bbagen.2009.04.004DOI Listing
June 2009

Structural and functional requirements for activity of the Tim9-Tim10 complex in mitochondrial protein import.

Mol Biol Cell 2009 Feb 26;20(3):769-79. Epub 2008 Nov 26.

Department of Biochemistry, La Trobe University, Melbourne 3086, Victoria, Australia.

The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9-Tim10 hexameric assembly determined to 2.5 A and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.
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http://dx.doi.org/10.1091/mbc.e08-09-0903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633397PMC
February 2009