52 results on '"Frazier AE"'
Search Results
2. Land Surface Heterogeneity and Tornado Formation: A Comparison of Tornado Alley and Dixie Alley
- Author
-
Hemingway B, Frazier Ae, and Brasher J
- Subjects
Geography ,Severe weather ,Climatology ,Elevation ,Magnitude (mathematics) ,Terrain ,Storm ,Tornadogenesis ,Tornado ,Alley - Abstract
Tornadoes are among the most destructive hazards to human life and property, and certain areas of the United States are more prone to these events. In particular, scientists use the terms “Tornado Alley” and “Dixie Alley” to refer to two general areas that experience higher incidences of tornadoes. While there is wide recognition that the two regions vary in the number, magnitude, and fatalities caused by tornadoes, more research is needed to better understand the reasons for these differences. The growing recognition that land surface heterogeneity may play a role in tornado formation provides motivation to compare the geographical characteristics of the two regions to determine whether there are significant differences in the landscape characteristics where severe storms form. To investigate the relationship between tornado formation and land surface heterogeneity in these two regions, we first delineate the spatial extent of Tornado Alley and Dixie Alley based on tornadic activity using a statistical test for the detection of significant clusters of spatial association. Next, using severe weather data for tornadoes and storms producing wind/hail (but no tornadoes), we investigate how land surface heterogeneity factors are related to tornado formation of weakly tornadic storms (EF0-EF1) and significantly tornadic storms (EF2-EF5) in each region using binary logistic regression. Lastly, we map probability surfaces for each region to show areas of greater risk. Results show that relationships between land surface heterogeneity and tornado formation vary from region to region. Elevation, slope, and distance to rivers are significant predictors of tornado formation, but the directionality of those relationships varies by region and storm severity. Urban land covers are associated with decreased tornado probability for all storm types in both regions. Spatial trends show an decreasing likelihood for EF0-EF1 from west to east in Tornado Alley but an increasing likelihood in that direction for EF2-EF5 storms.
- Published
- 2017
3. Testicular choriocarcinoma resembling a lipoma.
- Author
-
Chavez-Frazier AE, Whittemore DE, and Rapini RP
- Published
- 2012
4. CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex.
- Author
-
Baker MJ, Blau KU, Anderson AJ, Palmer CS, Fielden LF, Crameri JJ, Milenkovic D, Thorburn DR, Frazier AE, Langer T, and Stojanovski D
- Subjects
- Mitochondria genetics, Proteolysis, Proteomics, Humans, Intracellular Membranes, Membrane Proteins genetics, Endopeptidase Clp genetics
- Abstract
CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we show a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We illustrate an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology., (© 2024 Baker et al.)
- Published
- 2024
- Full Text
- View/download PDF
5. Reduced Protein Import via TIM23 SORT Drives Disease Pathology in TIMM50-Associated Mitochondrial Disease.
- Author
-
Crameri JJ, Palmer CS, Stait T, Jackson TD, Lynch M, Sinclair A, Frajman LE, Compton AG, Coman D, Thorburn DR, Frazier AE, and Stojanovski D
- Subjects
- Humans, Fibroblasts metabolism, HEK293 Cells, Membrane Transport Proteins metabolism, Membrane Transport Proteins genetics, Mitochondrial Membrane Transport Proteins metabolism, Mitochondrial Membrane Transport Proteins genetics, Mitochondrial Membranes metabolism, Mitochondrial Proteins metabolism, Mitochondrial Proteins genetics, Mutation genetics, Proteomics methods, Mitochondria metabolism, Mitochondrial Diseases metabolism, Mitochondrial Diseases pathology, Mitochondrial Diseases genetics, Mitochondrial Precursor Protein Import Complex Proteins metabolism, Oxidative Phosphorylation, Protein Transport
- Abstract
TIMM50 is a core subunit of the TIM23 complex, the mitochondrial inner membrane translocase responsible for the import of pre-sequence-containing precursors into the mitochondrial matrix and inner membrane. Here we describe a mitochondrial disease patient who is homozygous for a novel variant in TIMM50 and establish the first proteomic map of mitochondrial disease associated with TIMM50 dysfunction. We demonstrate that TIMM50 pathogenic variants reduce the levels and activity of endogenous TIM23 complex, which significantly impacts the mitochondrial proteome, resulting in a combined oxidative phosphorylation (OXPHOS) defect and changes to mitochondrial ultrastructure. Using proteomic data sets from TIMM50 patient fibroblasts and a TIMM50 HEK293 cell model of disease, we reveal that laterally released substrates imported via the TIM23
SORT complex pathway are most sensitive to loss of TIMM50. Proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for the specific defects in TIMM50-associated mitochondrial disease patients. These results highlight the power of using proteomics to elucidate molecular mechanisms of disease and uncovering novel features of fundamental biology, with the implication that human TIMM50 may have a more pronounced role in lateral insertion than previously understood.- Published
- 2024
- Full Text
- View/download PDF
6. The Desert Whale: the boom and bust of hemp in Arizona.
- Author
-
Stats AK, Sweat KG, Masson RN, Conrow KD, Frazier AE, and Leung MCK
- Abstract
Background: This paper examines the factors that led to the collapse of hemp grown for cannabidiol (CBD) in Arizona, the United States of America (USA), and particularly in Yuma County, which is a well-established agricultural area in the state., Methods: This research uses a combination of mapping analysis along with a survey of hemp farmers to assess the reasons why the hemp industry collapsed as well as to foster solutions to these problems., Results: In 2019, 5430 acres were sown with hemp seed in Arizona with 3890 acres inspected by the state to determine if they could be harvested. By 2021, there were only 156 acres planted, and only 128 of those acres were inspected by the state for compliance. (Crop mortality accounts for the difference between acres sown and acres inspected.) CONCLUSIONS: A lack of knowledge about the hemp life cycle greatly contributed to the failure of high CBD hemp crops in Arizona. Other problems included noncompliance with tetrahydrocannabinol limits, poor sources for seeds and inconsistent genetics of the hemp varieties sold to farmers, and diseases that hemp plants were susceptible to such as Pythium crown and root rot and beet curly top virus. Addressing these factors will go far in making hemp a profitable and widespread crop in Arizona. Additionally, hemp grown for other traditional uses (e.g., fiber or seed oil) as well as new applications (e.g., microgreens, hempcrete, and phytoremediation) offers other pathways for successful hemp agriculture in this state., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
7. The dynamic relationships between landscape structure and ecosystem services: An empirical analysis from the Wuhan metropolitan area, China.
- Author
-
Ran P, Hu S, Frazier AE, Yang S, Song X, and Qu S
- Subjects
- Agriculture methods, Cities, Carbon Sequestration, Soil, China, Ecosystem, Conservation of Natural Resources methods
- Abstract
Environmental managers have been striving to optimize landscape structure to achieve a sustained supply of ecosystem services (ESs). However, we still lack a full understanding of the relationships between landscape structure and ESs due to the absence of thorough investigations on the variability of these relationships in space and time. To fill this critical gap, we assessed landscape structure alongside four important ESs (agricultural production (AP), carbon sequestration (CS), soil conservation (SC), and water retention (WR)) in the Wuhan metropolitan area (WMA), and then analyzed the spatiotemporal impacts of landscape structure on ESs from 2000 to 2020 using Geographically and Temporally Weighted Regression. The results show only AP maintained a stable growth trend over the past two decades, while the other ESs fluctuated considerably with a noticeable decline in SC and WR. The importance of landscape structure in influencing ESs varies by time and place, depending on the local landscape composition and configuration. In general, landscape composition has a stronger and less temporally stable impact on ESs compared to configuration. Furthermore, increases in landscape diversity, as measured through Shannon's diversity index, and the percentage of woodlands were found to contribute to the simultaneous benefits of multiple ESs, but in most cases the effects of landscape structure on different ESs were different or even opposite, suggesting that trade-offs are critical in landscape management. The findings highlight the complex response of ESs to dramatically changing landscapes in the WMA and can guide decision-makers in precise spatial arrangement and temporal adjustments to improve current landscape management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
8. Mitochondrial biology and dysfunction in secondary mitochondrial disease.
- Author
-
Baker MJ, Crameri JJ, Thorburn DR, Frazier AE, and Stojanovski D
- Subjects
- Humans, Mitochondrial Diseases genetics
- Abstract
Mitochondrial diseases are a broad, genetically heterogeneous class of metabolic disorders characterized by deficits in oxidative phosphorylation (OXPHOS). Primary mitochondrial disease (PMD) defines pathologies resulting from mutation of mitochondrial DNA (mtDNA) or nuclear genes affecting either mtDNA expression or the biogenesis and function of the respiratory chain. Secondary mitochondrial disease (SMD) arises due to mutation of nuclear-encoded genes independent of, or indirectly influencing OXPHOS assembly and operation. Despite instances of novel SMD increasing year-on-year, PMD is much more widely discussed in the literature. Indeed, since the implementation of next generation sequencing (NGS) techniques in 2010, many novel mitochondrial disease genes have been identified, approximately half of which are linked to SMD. This review will consolidate existing knowledge of SMDs and outline discrete categories within which to better understand the diversity of SMD phenotypes. By providing context to the biochemical and molecular pathways perturbed in SMD, we hope to further demonstrate the intricacies of SMD pathologies outside of their indirect contribution to mitochondrial energy generation.
- Published
- 2022
- Full Text
- View/download PDF
9. Sideroflexin 4 is a complex I assembly factor that interacts with the MCIA complex and is required for the assembly of the ND2 module.
- Author
-
Jackson TD, Crameri JJ, Muellner-Wong L, Frazier AE, Palmer CS, Formosa LE, Hock DH, Fujihara KM, Stait T, Sharpe AJ, Thorburn DR, Ryan MT, Stroud DA, and Stojanovski D
- Subjects
- Adenosine Triphosphate metabolism, Humans, Membrane Proteins, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Mutation, Electron Transport Complex I metabolism, Mitochondrial Diseases genetics
- Abstract
SignificanceMitochondria are double-membraned eukaryotic organelles that house the proteins required for generation of ATP, the energy currency of cells. ATP generation within mitochondria is performed by five multisubunit complexes (complexes I to V), the assembly of which is an intricate process. Mutations in subunits of these complexes, or the suite of proteins that help them assemble, lead to a severe multisystem condition called mitochondrial disease. We show that SFXN4, a protein that causes mitochondrial disease when mutated, assists with the assembly of complex I. This finding explains why mutations in SFXN4 cause mitochondrial disease and is surprising because SFXN4 belongs to a family of amino acid transporter proteins, suggesting that it has undergone a dramatic shift in function through evolution.
- Published
- 2022
- Full Text
- View/download PDF
10. High-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content.
- Author
-
Granata C, Caruana NJ, Botella J, Jamnick NA, Huynh K, Kuang J, Janssen HA, Reljic B, Mellett NA, Laskowski A, Stait TL, Frazier AE, Coughlan MT, Meikle PJ, Thorburn DR, Stroud DA, and Bishop DJ
- Subjects
- Adenosine Triphosphate biosynthesis, Adolescent, Adult, Biopsy, Electron Transport physiology, Healthy Volunteers, Humans, Male, Muscle, Skeletal cytology, Oxidative Phosphorylation, Proteome, Quality of Life, Young Adult, Adaptation, Physiological, Energy Metabolism physiology, High-Intensity Interval Training, Mitochondria metabolism, Muscle, Skeletal physiology
- Abstract
Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible, inexpensive therapeutic intervention that can improve mitochondrial bioenergetics and quality of life. By combining multiple omics techniques with biochemical and in silico normalisation, we removed the bias arising from the training-induced increase in mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the overall increase in mitochondrial content. Our findings suggest enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and do not support the hypothesis that training-induced supercomplex formation enhances mitochondrial bioenergetics. Our study provides an analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding, and calling for careful reinterpretation of previous findings., (© 2021. The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
11. Modelling Mitochondrial Disease in Human Pluripotent Stem Cells: What Have We Learned?
- Author
-
McKnight CL, Low YC, Elliott DA, Thorburn DR, and Frazier AE
- Subjects
- Animals, Cell Differentiation genetics, Cell Differentiation physiology, DNA, Mitochondrial genetics, Humans, Phenotype, Mitochondria genetics, Mitochondria pathology, Mitochondrial Diseases genetics, Mitochondrial Diseases pathology, Pluripotent Stem Cells pathology
- Abstract
Mitochondrial diseases disrupt cellular energy production and are among the most complex group of inherited genetic disorders. Affecting approximately 1 in 5000 live births, they are both clinically and genetically heterogeneous, and can be highly tissue specific, but most often affect cell types with high energy demands in the brain, heart, and kidneys. There are currently no clinically validated treatment options available, despite several agents showing therapeutic promise. However, modelling these disorders is challenging as many non-human models of mitochondrial disease do not completely recapitulate human phenotypes for known disease genes. Additionally, access to disease-relevant cell or tissue types from patients is often limited. To overcome these difficulties, many groups have turned to human pluripotent stem cells (hPSCs) to model mitochondrial disease for both nuclear-DNA (nDNA) and mitochondrial-DNA (mtDNA) contexts. Leveraging the capacity of hPSCs to differentiate into clinically relevant cell types, these models permit both detailed investigation of cellular pathomechanisms and validation of promising treatment options. Here we catalogue hPSC models of mitochondrial disease that have been generated to date, summarise approaches and key outcomes of phenotypic profiling using these models, and discuss key criteria to guide future investigations using hPSC models of mitochondrial disease.
- Published
- 2021
- Full Text
- View/download PDF
12. The TIM22 complex mediates the import of sideroflexins and is required for efficient mitochondrial one-carbon metabolism.
- Author
-
Jackson TD, Hock DH, Fujihara KM, Palmer CS, Frazier AE, Low YC, Kang Y, Ang CS, Clemons NJ, Thorburn DR, Stroud DA, and Stojanovski D
- Subjects
- Carbon metabolism, Carrier Proteins metabolism, Cell Culture Techniques, Humans, MCF-7 Cells, Membrane Proteins metabolism, Membrane Transport Proteins physiology, Mitochondria physiology, Mitochondrial Membrane Transport Proteins metabolism, Mitochondrial Membranes metabolism, Mitochondrial Membranes physiology, Mitochondrial Precursor Protein Import Complex Proteins, Mitochondrial Proteins physiology, Mutation, Phenotype, Phosphotransferases (Alcohol Group Acceptor) genetics, Primary Cell Culture, Proteomics methods, Membrane Transport Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism
- Abstract
Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis. We mapped the proteomic changes in Sengers patient fibroblasts and AGK
KO cell lines to understand the effects of AGK dysfunction on mitochondria. This uncovered down-regulation of a number of proteins at the inner mitochondrial membrane, including many SLC25 carrier family proteins, which are predicted substrates of the complex. We also observed down-regulation of SFXN proteins, which contain five transmembrane domains, and show that they represent a novel class of TIM22 complex substrate. Perturbed biogenesis of SFXN proteins in cells lacking AGK reduces the proliferative capabilities of these cells in the absence of exogenous serine, suggesting that dysregulation of one-carbon metabolism is a molecular feature in the biology of Sengers syndrome.- Published
- 2021
- Full Text
- View/download PDF
13. Rothmund-Thomson Syndrome-Like RECQL4 Truncating Mutations Cause a Haploinsufficient Low-Bone-Mass Phenotype in Mice.
- Author
-
Castillo-Tandazo W, Frazier AE, Sims NA, Smeets MF, and Walkley CR
- Abstract
Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by defects in the skeletal system, such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients have germ line compound biallelic protein-truncating mutations of RECQL4 . As existing murine models employ Recql4 null alleles, we have attempted to more accurately model RTS by generating mice with patient-mimicking truncating Recql4 mutations. Truncating mutations impaired the stability and subcellular localization of RECQL4 and resulted in homozygous embryonic lethality and a haploinsufficient low-bone mass phenotype. Combination of a truncating mutation with a conditional Recql4 null allele demonstrated that the skeletal defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis. We utilized murine Recql4 null cells to assess the impact of human RECQL4 mutations using an in vitro complementation assay. While some mutations created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations in mice lead to an osteoporosis-like phenotype through defects in early osteoblast progenitors and identify RECQL4 gene dosage as a novel regulator of bone mass.
- Published
- 2021
- Full Text
- View/download PDF
14. Fatal perinatal mitochondrial cardiac failure caused by recurrent de novo duplications in the ATAD3 locus.
- Author
-
Frazier AE, Compton AG, Kishita Y, Hock DH, Welch AE, Amarasekera SSC, Rius R, Formosa LE, Imai-Okazaki A, Francis D, Wang M, Lake NJ, Tregoning S, Jabbari JS, Lucattini A, Nitta KR, Ohtake A, Murayama K, Amor DJ, McGillivray G, Wong FY, van der Knaap MS, Jeroen Vermeulen R, Wiltshire EJ, Fletcher JM, Lewis B, Baynam G, Ellaway C, Balasubramaniam S, Bhattacharya K, Freckmann ML, Arbuckle S, Rodriguez M, Taft RJ, Sadedin S, Cowley MJ, Minoche AE, Calvo SE, Mootha VK, Ryan MT, Okazaki Y, Stroud DA, Simons C, Christodoulou J, and Thorburn DR
- Subjects
- ATPases Associated with Diverse Cellular Activities genetics, Australia, Child, Humans, Membrane Proteins genetics, Mitochondrial Proteins genetics, United States, Cardiomyopathies, Heart Failure, Mitochondrial Diseases genetics
- Abstract
Background: In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. The ATAD3 locus is one such region, in which recessive deletions and dominant duplications have recently been reported to cause lethal perinatal mitochondrial diseases characterized by pontocerebellar hypoplasia or cardiomyopathy, respectively., Methods: Whole exome, whole genome and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease., Findings: We report six different de novo duplications in the ATAD3 gene locus causing a distinctive presentation including lethal perinatal cardiomyopathy, persistent hyperlactacidemia, and frequently corneal clouding or cataracts and encephalopathy. The recurrent 68 Kb ATAD3 duplications are identifiable from genome and exome sequencing but usually missed by microarrays. The ATAD3 duplications result in the formation of identical chimeric ATAD3A/ATAD3C proteins, altered ATAD3 complexes and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue., Conclusions: ATAD3 duplications appear to act in a dominant-negative manner and the de novo inheritance infers a low recurrence risk for families, unlike most pediatric mitochondrial diseases. More than 350 genes underlie mitochondrial diseases. In our experience the ATAD3 locus is now one of the five most common causes of nuclear-encoded pediatric mitochondrial disease but the repetitive nature of the locus means ATAD3 diagnoses may be frequently missed by current genomic strategies., Funding: Australian NHMRC, US Department of Defense, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance and Australian Mito Foundation.
- Published
- 2021
- Full Text
- View/download PDF
15. Landscape and Anthropogenic Factors Associated with Adult Aedes aegypti and Aedes albopictus in Small Cities in the Southern Great Plains.
- Author
-
Sanders JD, Talley JL, Frazier AE, and Noden BH
- Abstract
As mosquito-borne diseases are a growing human health concern in the United States, the distribution and potential arbovirus risk from container-breeding Aedes mosquitoes is understudied in the southern Great Plains. The aim of the study was to assess landscape and anthropogenic factors associated with encountering adult container-breeding mosquitoes in small cities in southern Oklahoma. Collections were carried out over a 10 week period from June to August 2017 along two geographical transects, each consisting of three cities, equally distant from the Red River/Texas border. Mosquitoes were collected weekly using two trap types along with data for 13 landscape, vegetation, and anthropogenic variables. After five rounds of collection, 6628 female mosquitoes were collected over 2110 trap-nights involving 242 commercial or residential sites in six cities. Of the mosquitoes collected, 80% consisted of container-breeding species: Aedes albopictus (72%), Culex pipiens complex (16%) and Aedes aegypti (8%). Regionally, Aedes aegypti was more likely present in cities closest to the Texas border while Ae. albopictus was spread throughout the region. In general, Ae. aegypti and Ae. albopictus were significantly more present in sites featuring no or low vegetation and residential sites. Variables associated with Ae. albopictus presence and abundance varied between cities and highlighted the urban nature of the species. The study highlighted the distribution of Ae. aegypti geographically and within the urban context, indicated potential habitat preferences of container-breeding mosquito species in small towns, and demonstrated the usefulness of Gravid Aedes traps (GAT) traps for monitoring Aedes populations in urban habitats in small cities.
- Published
- 2020
- Full Text
- View/download PDF
16. Correction: Function of hTim8a in complex IV assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjærg syndrome.
- Author
-
Kang Y, Anderson AJ, Jackson TD, Palmer CS, De Souza DP, Fujihara KM, Stait T, Frazier AE, Clemons NJ, Tull D, Thorburn DR, McConville MJ, Ryan MT, Stroud DA, and Stojanovski D
- Published
- 2020
- Full Text
- View/download PDF
17. Assessment of mitochondrial respiratory chain enzymes in cells and tissues.
- Author
-
Frazier AE, Vincent AE, Turnbull DM, Thorburn DR, and Taylor RW
- Subjects
- Animals, Electron Transport, Humans, Oxidative Phosphorylation, Enzyme Assays methods, Enzymes metabolism, Mitochondria metabolism, Organ Specificity
- Abstract
Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided. The activities of mitochondrial respiratory chain enzymes are often expressed relative to citrate synthase activity, since these ratios may be more robust in accounting for variability that may arise due to tissue quality, handling and storage, cell growth conditions, or any mitochondrial proliferation that may be present in tissues from patients with mitochondrial disease. Considerations for adaption of these techniques to other cells, tissues, and organisms are presented, as well as comparisons to alternate methods for analysis of respiratory chain function. In this context, a quantitative immunofluorescence protocol is also provided that is suitable for measurement of the amount of multiple respiratory chain complexes in small diagnostic tissue samples. The analysis and interpretation of OXPHOS enzyme activities are then placed in the context of mitochondrial disease tissue pathology and diagnosis., (© 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
18. Function of hTim8a in complex IV assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjærg syndrome.
- Author
-
Kang Y, Anderson AJ, Jackson TD, Palmer CS, De Souza DP, Fujihara KM, Stait T, Frazier AE, Clemons NJ, Tull D, Thorburn DR, McConville MJ, Ryan MT, Stroud DA, and Stojanovski D
- Subjects
- Apoptosis, Apoptosis Regulatory Proteins metabolism, Cell Line, Copper Transport Proteins metabolism, Humans, Membrane Transport Proteins deficiency, Mitochondrial Precursor Protein Import Complex Proteins, Oxidative Stress, Protein Interaction Maps, Deaf-Blind Disorders physiopathology, Dystonia physiopathology, Electron Transport Complex IV metabolism, Intellectual Disability physiopathology, Membrane Transport Proteins metabolism, Neurons metabolism, Optic Atrophy physiopathology, Protein Multimerization
- Abstract
Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS., Competing Interests: YK, AA, TJ, CP, DD, KF, TS, AF, NC, DT, DT, MM, MR, DS, DS No competing interests declared, (© 2019, Kang et al.)
- Published
- 2019
- Full Text
- View/download PDF
19. Intercomparison of Small Unmanned Aircraft System (sUAS) Measurements for Atmospheric Science during the LAPSE-RATE Campaign.
- Author
-
Barbieri L, Kral ST, Bailey SCC, Frazier AE, Jacob JD, Reuder J, Brus D, Chilson PB, Crick C, Detweiler C, Doddi A, Elston J, Foroutan H, González-Rocha J, Greene BR, Guzman MI, Islam ALHA, Kemppinen O, Lawrence D, Pillar-Little EA, Ross SD, Sama M, Schmale DG III, Schuyler TJ, Shankar A, Smith SW, Waugh S, Dixon C, Borenstein S, and Boer G
- Abstract
Small unmanned aircraft systems (sUAS) are rapidly transforming atmospheric research. With the advancement of the development and application of these systems, improving knowledge of best practices for accurate measurement is critical for achieving scientific goals. We present results from an intercomparison of atmospheric measurement data from the Lower Atmospheric Process Studies at Elevation-a Remotely piloted Aircraft Team Experiment (LAPSE-RATE) field campaign. We evaluate a total of 38 individual sUAS with 23 unique sensor and platform configurations using a meteorological tower for reference measurements. We assess precision, bias, and time response of sUAS measurements of temperature, humidity, pressure, wind speed, and wind direction. Most sUAS measurements show broad agreement with the reference, particularly temperature and wind speed, with mean value differences of 1.6 ± 2 . 6 ∘ C and 0.22 ± 0 . 59 m/s for all sUAS, respectively. sUAS platform and sensor configurations were found to contribute significantly to measurement accuracy. Sensor configurations, which included proper aspiration and radiation shielding of sensors, were found to provide the most accurate thermodynamic measurements (temperature and relative humidity), whereas sonic anemometers on multirotor platforms provided the most accurate wind measurements (horizontal speed and direction). We contribute both a characterization and assessment of sUAS for measuring atmospheric parameters, and identify important challenges and opportunities for improving scientific measurements with sUAS.
- Published
- 2019
- Full Text
- View/download PDF
20. Mitochondrial energy generation disorders: genes, mechanisms, and clues to pathology.
- Author
-
Frazier AE, Thorburn DR, and Compton AG
- Subjects
- Animals, Humans, Energy Metabolism, Genetic Diseases, Inborn genetics, Genetic Diseases, Inborn metabolism, Genetic Diseases, Inborn pathology, Mitochondrial Diseases genetics, Mitochondrial Diseases metabolism, Mitochondrial Diseases pathology, Mutation
- Abstract
Inherited disorders of oxidative phosphorylation cause the clinically and genetically heterogeneous diseases known as mitochondrial energy generation disorders, or mitochondrial diseases. Over the last three decades, mutations causing these disorders have been identified in almost 290 genes, but many patients still remain without a molecular diagnosis. Moreover, while our knowledge of the genetic causes is continually expanding, our understanding into how these defects lead to cellular dysfunction and organ pathology is still incomplete. Here, we review recent developments in disease gene discovery, functional characterization, and shared pathogenic parameters influencing disease pathology that offer promising avenues toward the development of effective therapies., (© 2019 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2019
- Full Text
- View/download PDF
21. Loss of BIM increases mitochondrial oxygen consumption and lipid oxidation, reduces adiposity and improves insulin sensitivity in mice.
- Author
-
Wali JA, Galic S, Tan CY, Gurzov EN, Frazier AE, Connor T, Ge J, Pappas EG, Stroud D, Varanasi LC, Selck C, Ryan MT, Thorburn DR, Kemp BE, Krishnamurthy B, Kay TW, McGee SL, and Thomas HE
- Subjects
- Animals, Bcl-2-Like Protein 11 genetics, Electron Transport Complex IV metabolism, Energy Metabolism, Glucose metabolism, Hepatocytes metabolism, Insulin Resistance, Liver metabolism, Membrane Potential, Mitochondrial, Mice, Oxidation-Reduction, Oxygen Consumption, Weight Loss, Adiposity, Bcl-2-Like Protein 11 physiology, Lipid Metabolism, Mitochondria metabolism
- Abstract
BCL-2 proteins are known to engage each other to determine the fate of a cell after a death stimulus. However, their evolutionary conservation and the many other reported binding partners suggest an additional function not directly linked to apoptosis regulation. To identify such a function, we studied mice lacking the BH3-only protein BIM. BIM
-/- cells had a higher mitochondrial oxygen consumption rate that was associated with higher mitochondrial complex IV activity. The consequences of increased oxygen consumption in BIM-/- mice were significantly lower body weights, reduced adiposity and lower hepatic lipid content. Consistent with reduced adiposity, BIM-/- mice had lower fasting blood glucose, improved insulin sensitivity and hepatic insulin signalling. Lipid oxidation was increased in BIM-/- mice, suggesting a mechanism for their metabolic phenotype. Our data suggest a role for BIM in regulating mitochondrial bioenergetics and metabolism and support the idea that regulation of metabolism and cell death are connected.- Published
- 2018
- Full Text
- View/download PDF
22. Reply: Genotype-phenotype correlation in ATAD3A deletions: not just of scientific relevance.
- Author
-
Frazier AE, Holt IJ, Spinazzola A, and Thorburn DR
- Subjects
- ATPases Associated with Diverse Cellular Activities, Cholesterol, Genetic Association Studies, Humans, Membrane Proteins genetics, Mitochondrial Proteins, Multigene Family, Cerebellar Diseases, DNA, Mitochondrial
- Published
- 2017
- Full Text
- View/download PDF
23. Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.
- Author
-
Kang Y, Stroud DA, Baker MJ, De Souza DP, Frazier AE, Liem M, Tull D, Mathivanan S, McConville MJ, Thorburn DR, Ryan MT, and Stojanovski D
- Subjects
- Cardiomyopathies genetics, Cataract genetics, Citric Acid Cycle, Genetic Predisposition to Disease, HEK293 Cells, HeLa Cells, Humans, Mitochondrial Membrane Transport Proteins genetics, Multiprotein Complexes, Mutation, Phenotype, Phosphotransferases (Alcohol Group Acceptor) genetics, Protein Stability, Protein Transport, Transfection, Cardiomyopathies enzymology, Cataract enzymology, Mitochondria enzymology, Mitochondrial Membrane Transport Proteins metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism
- Abstract
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., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
24. ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.
- Author
-
Desai R, Frazier AE, Durigon R, Patel H, Jones AW, Dalla Rosa I, Lake NJ, Compton AG, Mountford HS, Tucker EJ, Mitchell ALR, Jackson D, Sesay A, Di Re M, van den Heuvel LP, Burke D, Francis D, Lunke S, McGillivray G, Mandelstam S, Mochel F, Keren B, Jardel C, Turner AM, Ian Andrews P, Smeitink J, Spelbrink JN, Heales SJ, Kohda M, Ohtake A, Murayama K, Okazaki Y, Lombès A, Holt IJ, Thorburn DR, and Spinazzola A
- Subjects
- ATPases Associated with Diverse Cellular Activities, Adult, Cerebellum diagnostic imaging, Cerebellum physiopathology, Consanguinity, Developmental Disabilities diagnostic imaging, Developmental Disabilities genetics, Developmental Disabilities physiopathology, Female, Humans, Infant, Infant, Newborn, Male, Mitochondrial Diseases diagnostic imaging, Mitochondrial Diseases physiopathology, Nervous System Malformations diagnostic imaging, Nervous System Malformations physiopathology, Adenosine Triphosphatases genetics, Cerebellum abnormalities, DNA, Mitochondrial genetics, Membrane Proteins genetics, Mitochondrial Diseases genetics, Mitochondrial Proteins genetics, Nervous System Malformations genetics
- Abstract
Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.)
- Published
- 2017
- Full Text
- View/download PDF
25. Accessory subunits are integral for assembly and function of human mitochondrial complex I.
- Author
-
Stroud DA, Surgenor EE, Formosa LE, Reljic B, Frazier AE, Dibley MG, Osellame LD, Stait T, Beilharz TH, Thorburn DR, Salim A, and Ryan MT
- Subjects
- Cell Line, Cell Respiration, Cell Survival genetics, Electron Transport Complex I genetics, Gene Editing, Gene Knockout Techniques, HEK293 Cells, Humans, Membrane Proteins metabolism, Mitochondrial Proteins deficiency, Mitochondrial Proteins genetics, Mitochondrial Proton-Translocating ATPases metabolism, Models, Molecular, Protein Stability, Protein Subunits chemistry, Protein Subunits deficiency, Protein Subunits genetics, Proteomics, Electron Transport Complex I chemistry, Electron Transport Complex I metabolism, Mitochondria chemistry, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Proteins chemistry, Mitochondrial Proteins metabolism, Protein Subunits metabolism
- Abstract
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.
- Published
- 2016
- Full Text
- View/download PDF
26. Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins.
- Author
-
Stiller SB, Höpker J, Oeljeklaus S, Schütze C, Schrempp SG, Vent-Schmidt J, Horvath SE, Frazier AE, Gebert N, van der Laan M, Bohnert M, Warscheid B, Pfanner N, and Wiedemann N
- Subjects
- Cell Nucleus metabolism, Mutation genetics, Saccharomyces cerevisiae Proteins metabolism, Succinate Dehydrogenase metabolism, Electron Transport Complex IV metabolism, Membrane Proteins metabolism, Mitochondria enzymology, Mitochondrial Membranes metabolism, Mitochondrial Proteins metabolism, Nuclear Proteins metabolism, Saccharomyces cerevisiae enzymology
- Abstract
The mitochondrial inner membrane harbors three protein translocases. Presequence translocase and carrier translocase are essential for importing nuclear-encoded proteins. The oxidase assembly (OXA) translocase is required for exporting mitochondrial-encoded proteins; however, different views exist about its relevance for nuclear-encoded proteins. We report that OXA plays a dual role in the biogenesis of nuclear-encoded mitochondrial proteins. First, a systematic analysis of OXA-deficient mitochondria led to an unexpected expansion of the spectrum of OXA substrates imported via the presequence pathway. Second, biogenesis of numerous metabolite carriers depends on OXA, although they are not imported by the presequence pathway. We show that OXA is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase. The export translocase OXA is thus required for the import of metabolite carriers by promoting assembly of the carrier translocase. We conclude that OXA is of central importance for the biogenesis of the mitochondrial inner membrane., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
27. Deletion of the Complex I Subunit NDUFS4 Adversely Modulates Cellular Differentiation.
- Author
-
Johnson J, Lee W, Frazier AE, Vaghjiani V, Laskowski A, Rodriguez AL, Cagnone GL, McKenzie M, White SJ, Nisbet DR, Thorburn DR, and St John JC
- Subjects
- Animals, Astrocytes metabolism, Cell Line, Electron Transport Complex I genetics, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Gene Expression Regulation, Developmental, Mice, Mice, Inbred BALB C, Pluripotent Stem Cells cytology, Pluripotent Stem Cells metabolism, Astrocytes cytology, Electron Transport Complex I metabolism, Gene Deletion, Leigh Disease genetics, Neurogenesis
- Abstract
The vast majority of cellular ATP is produced by the oxidative phosphorylation (OXPHOS) system, which comprises the four complexes of the electron transfer chain plus the ATP synthase. Complex I is the largest of the OXPHOS complexes, and mutation of the genes encoding either the subunits or assembly factors of Complex I can result in Complex I deficiency, which is the most common OXPHOS disorder. Mutations in the Complex I gene NDUFS4 lead to Leigh syndrome, which is the most frequent presentation of Complex I deficiency in children presenting with progressive encephalopathy shortly after birth. Symptoms include motor and intellectual retardation, often accompanied by dystonia, ataxia, and growth retardation, and most patients die by 3 years of age. To understand the origins of this disease, we have generated a series of mouse embryonic stem cell lines from blastocysts that were wild type, heterozygous, and homozygous for the deletion of the Ndufs4 gene. We have demonstrated their pluripotency and potential to differentiate into all cell types of the body. Although the loss of Ndufs4 did not affect the stability of the mitochondrial and nuclear genomes, there were significant differences in patterns of chromosomal gene expression following both spontaneous differentiation and directed neural differentiation into astrocytes. The defect also affected the potential of the cells to generate beating embryoid bodies. These outcomes demonstrate that defects associated with Complex I deficiency affect early gene expression patterns, which escalate during early and later stages of differentiation and are mediated by the defect and not other chromosomal or mitochondrial DNA defects.
- Published
- 2016
- Full Text
- View/download PDF
28. COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2.
- Author
-
Stroud DA, Maher MJ, Lindau C, Vögtle FN, Frazier AE, Surgenor E, Mountford H, Singh AP, Bonas M, Oeljeklaus S, Warscheid B, Meisinger C, Thorburn DR, and Ryan MT
- Subjects
- Carrier Proteins metabolism, Copper metabolism, Electron Transport Complex IV metabolism, Fibroblasts cytology, Fibroblasts enzymology, HEK293 Cells, Humans, Infant, Male, Mitochondrial Proteins metabolism, Molecular Chaperones, Cardiomyopathies genetics, Carrier Proteins genetics, Electron Transport Complex IV genetics, Membrane Proteins metabolism, Mitochondrial Proteins genetics
- Abstract
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., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2015
- Full Text
- View/download PDF
29. Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I.
- Author
-
Formosa LE, Mimaki M, Frazier AE, McKenzie M, Stait TL, Thorburn DR, Stroud DA, and Ryan MT
- Subjects
- HEK293 Cells, Humans, Mitochondrial Proteins physiology, Molecular Chaperones genetics, Mutation, Protein Multimerization, Electron Transport Complex I metabolism, Mitochondria metabolism, Mitochondrial Proteins metabolism, Molecular Chaperones physiology
- Abstract
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., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2015
- Full Text
- View/download PDF
30. N-Acetylcysteine improves mitochondrial function and ameliorates behavioral deficits in the R6/1 mouse model of Huntington's disease.
- Author
-
Wright DJ, Renoir T, Smith ZM, Frazier AE, Francis PS, Thorburn DR, McGee SL, Hannan AJ, and Gray LJ
- Subjects
- Animals, Brain drug effects, Brain pathology, Disease Models, Animal, Disease Progression, Excitatory Amino Acid Transporter 2 drug effects, Excitatory Amino Acid Transporter 2 metabolism, Gait drug effects, Mice, Mice, Transgenic, Mitochondria metabolism, Motor Activity drug effects, Organ Size, Acetylcysteine pharmacology, Behavior, Animal drug effects, Free Radical Scavengers pharmacology, Huntington Disease metabolism, Mitochondria drug effects, Oxidative Stress drug effects
- Abstract
Huntington's disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and excitotoxicity have previously been implicated in the pathogenesis of HD. We hypothesized that N-acetylcysteine (NAC) may reduce both excitotoxicity and oxidative stress through its actions on glutamate reuptake and antioxidant capacity. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. It was found that chronic NAC administration delayed the onset and progression of motor deficits in R6/1 mice, while having an antidepressant-like effect on both R6/1 and wild-type mice. A deficit in the astrocytic glutamate transporter protein, GLT-1, was found in R6/1 mice. However, this deficit was not ameliorated by NAC, implying that the therapeutic effect of NAC is not due to rescue of the GLT-1 deficit and associated glutamate-induced excitotoxicity. Assessment of mitochondrial function in the striatum and cortex revealed that R6/1 mice show reduced mitochondrial respiratory capacity specific to the striatum. This deficit was rescued by chronic treatment with NAC. There was a selective increase in markers of oxidative damage in mitochondria, which was rescued by NAC. In conclusion, NAC is able to delay the onset of motor deficits in the R6/1 model of Huntington's disease and it may do so by ameliorating mitochondrial dysfunction. Thus, NAC shows promise as a potential therapeutic agent in HD. Furthermore, our data suggest that NAC may also have broader antidepressant efficacy.
- Published
- 2015
- Full Text
- View/download PDF
31. Neuronal and astrocyte dysfunction diverges from embryonic fibroblasts in the Ndufs4fky/fky mouse.
- Author
-
Bird MJ, Wijeyeratne XW, Komen JC, Laskowski A, Ryan MT, Thorburn DR, and Frazier AE
- Subjects
- Adenosine Triphosphate biosynthesis, Animals, Astrocytes cytology, Blotting, Western, Cells, Cultured, Electron Transport Complex I genetics, Electron Transport Complex II metabolism, Embryo, Mammalian cytology, Fibroblasts cytology, Galactose metabolism, Hydrogen Peroxide metabolism, Membrane Potential, Mitochondrial genetics, Mice, Inbred BALB C, Mice, Knockout, Mitochondria genetics, Mitochondria metabolism, Mitochondria physiology, Necrosis genetics, Neurons cytology, Reactive Oxygen Species metabolism, Rotenone metabolism, Succinates metabolism, Superoxides metabolism, Astrocytes metabolism, Electron Transport Complex I deficiency, Fibroblasts metabolism, Neurons metabolism
- Abstract
Mitochondrial dysfunction causes a range of early-onset neurological diseases and contributes to neurodegenerative conditions. The mechanisms of neurological damage however are poorly understood, as accessing relevant tissue from patients is difficult, and appropriate models are limited. Hence, we assessed mitochondrial function in neurologically relevant primary cell lines from a CI (complex I) deficient Ndufs4 KO (knockout) mouse (Ndufs4fky/fky) modelling aspects of the mitochondrial disease LS (Leigh syndrome), as well as MEFs (mouse embryonic fibroblasts). Although CI structure and function were compromised in all Ndufs4fky/fky cell types, the mitochondrial membrane potential was selectively impaired in the MEFs, correlating with decreased CI-dependent ATP synthesis. In addition, increased ROS (reactive oxygen species) generation and altered sensitivity to cell death were only observed in Ndufs4fky/fky primary MEFs. In contrast, Ndufs4fky/fky primary isocortical neurons and primary isocortical astrocytes displayed only impaired ATP generation without mitochondrial membrane potential changes. Therefore the neurological dysfunction in the Ndufs4fky/fky mouse may partly originate from a more severe ATP depletion in neurons and astrocytes, even at the expense of maintaining the mitochondrial membrane potential. This may provide protection from cell death, but would ultimately compromise cell functionality in neurons and astrocytes. Furthermore, RET (reverse electron transfer) from complex II to CI appears more prominent in neurons than MEFs or astrocytes, and is attenuated in Ndufs4fky/fky cells.
- Published
- 2014
- Full Text
- View/download PDF
32. Functional characterization of Friedreich ataxia iPS-derived neuronal progenitors and their integration in the adult brain.
- Author
-
Bird MJ, Needham K, Frazier AE, van Rooijen J, Leung J, Hough S, Denham M, Thornton ME, Parish CL, Nayagam BA, Pera M, Thorburn DR, Thompson LH, and Dottori M
- Subjects
- Adult, Animals, Cell Death, Cell Differentiation, Cell Line, Cell Survival, Female, Gene Expression Regulation, Humans, Iron-Binding Proteins metabolism, Mitochondria metabolism, Neural Stem Cells metabolism, Rats, Frataxin, Cerebellum cytology, Friedreich Ataxia pathology, Induced Pluripotent Stem Cells cytology, Neural Stem Cells cytology
- Abstract
Friedreich ataxia (FRDA) is an autosomal recessive disease characterised by neurodegeneration and cardiomyopathy that is caused by an insufficiency of the mitochondrial protein, frataxin. Our previous studies described the generation of FRDA induced pluripotent stem cell lines (FA3 and FA4 iPS) that retained genetic characteristics of this disease. Here we extend these studies, showing that neural derivatives of FA iPS cells are able to differentiate into functional neurons, which don't show altered susceptibility to cell death, and have normal mitochondrial function. Furthermore, FA iPS-derived neural progenitors are able to differentiate into functional neurons and integrate in the nervous system when transplanted into the cerebellar regions of host adult rodent brain. These are the first studies to describe both in vitro and in vivo characterization of FA iPS-derived neurons and demonstrate their capacity to survive long term. These findings are highly significant for developing FRDA therapies using patient-derived stem cells.
- Published
- 2014
- Full Text
- View/download PDF
33. Modelling biochemical features of mitochondrial neuropathology.
- Author
-
Bird MJ, Thorburn DR, and Frazier AE
- Subjects
- Animals, Biomarkers analysis, Biomarkers metabolism, DNA, Mitochondrial genetics, Humans, Mitochondria pathology, Organelle Shape physiology, Oxidative Phosphorylation, Oxidative Stress physiology, Brain Diseases, Metabolic metabolism, Disease Models, Animal, Mitochondrial Diseases metabolism, Models, Neurological
- Abstract
Background: The neuropathology of mitochondrial disease is well characterised. However, pathophysiological mechanisms at the level of biochemistry and cell biology are less clear. Progress in this area has been hampered by the limited accessibility of neurologically relevant material for analysis., Scope of Review: Here we discuss the recent development of a variety of model systems that have greatly extended our capacity to understand the biochemical features associated with mitochondrial neuropathology. These include animal and cell based models, with mutations in both nuclear and mitochondrial DNA encoded genes, which aim to recapitulate the neuropathology and cellular biochemistry of mitochondrial diseases., Major Conclusions: Analysis of neurological tissue and cells from these models suggests that although there is no unifying mode of pathogenesis, dysfunction of the oxidative phosphorylation (OXPHOS) system is often central. This can be associated with altered reactive oxygen species (ROS) generation, disruption of the mitochondrial membrane potential (ΔΨm) and inadequate ATP synthesis. Thus, other cellular processes such as calcium (Ca(2+)) homeostasis, cellular signaling and mitochondrial morphology could be altered, ultimately compromising viability of neuronal cells., General Significance: Mechanisms of neuronal dysfunction in mitochondrial disease are only just beginning to be characterised, are system dependent and complex, and not merely driven by energy deficiency. The diversity of pathogenic mechanisms emphasises the need for characterisation in a wide range of models, as different therapeutic strategies are likely to be needed for different diseases. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research., (© 2013.)
- Published
- 2014
- Full Text
- View/download PDF
34. Mutations in the UQCC1-interacting protein, UQCC2, cause human complex III deficiency associated with perturbed cytochrome b protein expression.
- Author
-
Tucker EJ, Wanschers BF, Szklarczyk R, Mountford HS, Wijeyeratne XW, van den Brand MA, Leenders AM, Rodenburg RJ, Reljić B, Compton AG, Frazier AE, Bruno DL, Christodoulou J, Endo H, Ryan MT, Nijtmans LG, Huynen MA, and Thorburn DR
- Subjects
- Consanguinity, Cytochromes b genetics, Electron Transport Complex III metabolism, Fibroblasts metabolism, Fibroblasts pathology, Gene Expression Regulation, Homozygote, Humans, Membrane Proteins metabolism, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Diseases pathology, Mitochondrial Diseases therapy, Mitochondrial Proteins genetics, Molecular Chaperones genetics, Molecular Chaperones metabolism, Mutation, Oxidative Phosphorylation, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Cytochromes b biosynthesis, Electron Transport Complex III genetics, Membrane Proteins genetics, Mitochondrial Diseases genetics
- Abstract
Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2013
- Full Text
- View/download PDF
35. Biochemical analyses of the electron transport chain complexes by spectrophotometry.
- Author
-
Frazier AE and Thorburn DR
- Subjects
- Cells, Cultured, Citrate (si)-Synthase metabolism, Humans, Mitochondria enzymology, Mitochondria metabolism, Oxidative Phosphorylation, Electron Transport Chain Complex Proteins metabolism, Enzyme Assays methods, Spectrophotometry methods
- Abstract
In the diagnostic work-up of patients with suspected mitochondrial disease, evaluating the activity of the individual oxidative phosphorylation (OXPHOS) complexes is crucial. Here, we describe spectrophotometric assays for OXPHOS enzymology that can be applied to both tissue samples and cultured cells. These assays are designed to assess the enzymatic activity of the individual OXPHOS complexes I-V, along with the Krebs cycle enzyme citrate synthase as a mitochondrial control. As well, we include an assay for the coupled energy transfer between complexes II and III. Determining the enzymatic activities can be valuable in defining isolated or multicomplex disorders and may be relevant to the design of future molecular investigations.
- Published
- 2012
- Full Text
- View/download PDF
36. MiD49 and MiD51, new components of the mitochondrial fission machinery.
- Author
-
Palmer CS, Osellame LD, Laine D, Koutsopoulos OS, Frazier AE, and Ryan MT
- Subjects
- Actins metabolism, Amino Acid Sequence, Animals, COS Cells, Carbonyl Cyanide m-Chlorophenyl Hydrazone pharmacology, Cell Line, Chlorocebus aethiops, HeLa Cells, Humans, Membrane Potential, Mitochondrial drug effects, Membrane Proteins genetics, Mice, Microtubule-Associated Proteins metabolism, Mitochondria pathology, Mitochondrial Proteins genetics, Molecular Sequence Data, Peptide Elongation Factors genetics, Protein Transport genetics, RNA Interference, Receptors, Cytoplasmic and Nuclear genetics, Sequence Alignment, Uncoupling Agents pharmacology, Membrane Proteins metabolism, Mitochondria metabolism, Mitochondrial Membranes metabolism, Mitochondrial Proteins metabolism, Peptide Elongation Factors metabolism, Receptors, Cytoplasmic and Nuclear metabolism
- Abstract
Mitochondria form intricate networks through fission and fusion events. Here, we identify mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) anchored in the mitochondrial outer membrane. MiD49/51 form foci and rings around mitochondria similar to the fission mediator dynamin-related protein 1 (Drp1). MiD49/51 directly recruit Drp1 to the mitochondrial surface, whereas their knockdown reduces Drp1 association, leading to unopposed fusion. Overexpression of MiD49/51 seems to sequester Drp1 from functioning at mitochondria and cause fused tubules to associate with actin. Thus, MiD49/51 are new mediators of mitochondrial division affecting Drp1 action at mitochondria.
- Published
- 2011
- Full Text
- View/download PDF
37. Inhibition of Bak activation by VDAC2 is dependent on the Bak transmembrane anchor.
- Author
-
Lazarou M, Stojanovski D, Frazier AE, Kotevski A, Dewson G, Craigen WJ, Kluck RM, Vaux DL, and Ryan MT
- Subjects
- Animals, Apoptosis, Cell Membrane Permeability, Cells, Cultured, Embryo, Mammalian cytology, Fibroblasts cytology, HeLa Cells, Humans, Immunoblotting, Membrane Proteins metabolism, Mice, Mice, Knockout, Mitochondrial Proteins metabolism, Embryo, Mammalian metabolism, Fibroblasts metabolism, Mitochondrial Membranes metabolism, Voltage-Dependent Anion Channel 2 physiology, bcl-2 Homologous Antagonist-Killer Protein antagonists & inhibitors, bcl-2 Homologous Antagonist-Killer Protein physiology, bcl-2-Associated X Protein physiology
- Abstract
Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.
- Published
- 2010
- Full Text
- View/download PDF
38. Human Miltons associate with mitochondria and induce microtubule-dependent remodeling of mitochondrial networks.
- Author
-
Koutsopoulos OS, Laine D, Osellame L, Chudakov DM, Parton RG, Frazier AE, and Ryan MT
- Subjects
- Adaptor Proteins, Vesicular Transport genetics, Animals, COS Cells, Chlorocebus aethiops, Cloning, Molecular, Humans, Immunoprecipitation, Microscopy, Fluorescence, Mitochondrial Proteins metabolism, rho GTP-Binding Proteins metabolism, Adaptor Proteins, Vesicular Transport metabolism, Microtubules metabolism, Mitochondria metabolism, Nerve Tissue Proteins metabolism
- Abstract
Proper mitochondrial distribution is crucial for cell function. In Drosophila, mitochondrial transport is facilitated by Miro and Milton, which regulate mitochondrial attachment to microtubules via kinesin heavy chain. Mammals contain two sequence orthologs of Milton however, they have been ascribed various functions in intracellular transport. In this report, we show that the human Miltons target to mitochondria irrespective of whether they are linked to GFP at their C- or N-termini. Their ectopic expression induces the formation of extended mitochondrial tubules as well as large bulbous-like mitochondria with narrow tubular membrane necks that connect them to the mitochondrial mass. The mitochondrial extensions appear highly dynamic and their formation relies on the presence of microtubules. Using the photoswitchable fluorescent protein Dendra2 targeted to the mitochondrial matrix, we found that the mitochondrial extensions and bulbous mitochondria are fused with neighboring regions of the network. Truncation analysis of huMilton1 revealed that the N-terminal region, inclusive of the coiled-coil segment could localize to microtubules, suggesting that Milton attachment to kinesin occurs independent of Miro or mitochondrial attachment. In addition, we show that the huMiltons have the capacity to self-interact and can also facilitate mitochondrial recruitment of a cytosolic Miro mutant. We conclude that the human Miltons are important mediators of the mitochondrial trafficking machinery., (Copyright 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
39. Structural and functional requirements for activity of the Tim9-Tim10 complex in mitochondrial protein import.
- Author
-
Baker MJ, Webb CT, Stroud DA, Palmer CS, Frazier AE, Guiard B, Chacinska A, Gulbis JM, and Ryan MT
- Subjects
- Cross-Linking Reagents pharmacology, Crystallography, X-Ray, Microbial Viability drug effects, Mitochondria drug effects, Mitochondrial Membrane Transport Proteins, Mitochondrial Precursor Protein Import Complex Proteins, Mutant Proteins chemistry, Mutant Proteins metabolism, Mutation genetics, Protein Structure, Secondary, Protein Transport drug effects, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae enzymology, Solubility drug effects, Tetrahydrofolate Dehydrogenase metabolism, Membrane Proteins chemistry, Membrane Proteins metabolism, Membrane Transport Proteins chemistry, Membrane Transport Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins chemistry, Mitochondrial Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism
- Abstract
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.
- Published
- 2009
- Full Text
- View/download PDF
40. Introducing the string-of-beads biopsy: solving diagnostic challenges of the skin with punch biopsies for multiple studies, simple closure.
- Author
-
Chavez-Frazier AE, Wanitphakdeedecha R, Nguyen TH, and Chen TM
- Subjects
- Biopsy methods, Humans, Skin pathology
- Published
- 2008
- Full Text
- View/download PDF
41. Shy1 couples Cox1 translational regulation to cytochrome c oxidase assembly.
- Author
-
Mick DU, Wagner K, van der Laan M, Frazier AE, Perschil I, Pawlas M, Meyer HE, Warscheid B, and Rehling P
- Subjects
- DNA, Mitochondrial metabolism, Electron Transport, Genes, Fungal, Membrane Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins metabolism, Models, Biological, Protein Binding, Protein Interaction Mapping, Electron Transport Complex IV metabolism, Gene Expression Regulation, Fungal, Membrane Proteins physiology, Protein Biosynthesis, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins physiology
- Abstract
Cytochrome c oxidase (complex IV) of the respiratory chain is assembled from nuclear and mitochondrially-encoded subunits. Defects in the assembly process lead to severe human disorders such as Leigh syndrome. Shy1 is an assembly factor for complex IV in Saccharomyces cerevisiae and mutations of its human homolog, SURF1, are the most frequent cause for Leigh syndrome. We report that Shy1 promotes complex IV biogenesis through association with different protein modules; Shy1 interacts with Mss51 and Cox14, translational regulators of Cox1. Additionally, Shy1 associates with the subcomplexes of complex IV that are potential assembly intermediates. Formation of these subcomplexes depends on Coa1 (YIL157c), a novel assembly factor that cooperates with Shy1. Moreover, partially assembled forms of complex IV bound to Shy1 and Cox14 can associate with the bc1 complex to form transitional supercomplexes. We suggest that Shy1 links Cox1 translational regulation to complex IV assembly and supercomplex formation.
- Published
- 2007
- Full Text
- View/download PDF
42. Mitochondrial protein-import machinery: correlating structure with function.
- Author
-
Baker MJ, Frazier AE, Gulbis JM, and Ryan MT
- Subjects
- Animals, HSP70 Heat-Shock Proteins metabolism, Humans, Membrane Transport Proteins, Mitochondria ultrastructure, Mitochondrial Membrane Transport Proteins chemistry, Mitochondrial Precursor Protein Import Complex Proteins, Mitochondrial Proteins metabolism, Models, Molecular, Molecular Chaperones metabolism, Multiprotein Complexes, Protein Conformation, Receptors, Cell Surface, Receptors, Cytoplasmic and Nuclear metabolism, Mitochondria metabolism, Mitochondrial Membrane Transport Proteins metabolism, Protein Precursors chemistry, Protein Precursors metabolism, Protein Transport physiology
- Abstract
Most mitochondrial proteins are synthesized in the cytosol, translocated into the organelle and directed along specific sorting pathways. Over the past 20 years, >30 proteins have been identified as having key roles in mitochondrial protein import. Recently, the elucidation of the structures of several import components has provided fresh insight into the import process. Here, we review the different pathways involved in sorting proteins into mitochondrial subcompartments. Along the way, we highlight the available structural information about the protein-import machinery and discuss how these structures correlate with previously ascribed functions. Future challenges for the cell biologists will be to use this structural information to test specific hypotheses addressing the molecular mechanisms of mitochondrial protein import.
- Published
- 2007
- Full Text
- View/download PDF
43. Mitochondrial morphology and distribution in mammalian cells.
- Author
-
Frazier AE, Kiu C, Stojanovski D, Hoogenraad NJ, and Ryan MT
- Subjects
- Animals, Apoptosis, Humans, Mammals, Mitochondria ultrastructure
- Abstract
It is now appreciated that mitochondria form tubular networks that adapt to the requirements of the cell by undergoing changes in their shape through fission and fusion. Proper mitochondrial distribution also appears to be required for ATP delivery and calcium regulation, and, in some cases, for cell development. While we now realise the great importance of mitochondria for the cell, we are only beginning to work out how these organelles undergo the drastic morphological changes that are essential for cellular function. Of the few known components involved in shaping mitochondria, some have been found to be essential to life and their gene mutations are linked to neurological disorders, while others appear to be recruited in the activation of cell death pathways. Here we review our current understanding of the functions of the main players involved in mitochondrial fission, fusion and distribution in mammalian cells.
- Published
- 2006
- Full Text
- View/download PDF
44. Mdm38 interacts with ribosomes and is a component of the mitochondrial protein export machinery.
- Author
-
Frazier AE, Taylor RD, Mick DU, Warscheid B, Stoepel N, Meyer HE, Ryan MT, Guiard B, and Rehling P
- Subjects
- Calcium-Binding Proteins metabolism, Cytochromes b metabolism, Electron Transport Complex IV metabolism, Humans, Membrane Proteins genetics, Mitochondrial Proton-Translocating ATPases metabolism, Nuclear Proteins metabolism, Protein Transport physiology, Saccharomyces cerevisiae Proteins genetics, Signal Transduction, Membrane Proteins metabolism, Mitochondria metabolism, Mitochondrial Membranes metabolism, Mitochondrial Proteins metabolism, Ribosomes metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Saccharomyces cerevisiae Mdm38 and Ylh47 are homologues of human Letm1, a protein implicated in Wolf-Hirschhorn syndrome. We analyzed the function of Mdm38 and Ylh47 in yeast mitochondria to gain insight into the role of Letm1. We find that mdm38Delta mitochondria have reduced amounts of certain mitochondrially encoded proteins and low levels of complex III and IV and accumulate unassembled Atp6 of complex V of the respiratory chain. Mdm38 is especially required for efficient transport of Atp6 and cytochrome b across the inner membrane, whereas Ylh47 plays a minor role in this process. Both Mdm38 and Ylh47 form stable complexes with mitochondrial ribosomes, similar to what has been reported for Oxa1, a central component of the mitochondrial export machinery. Our results indicate that Mdm38 functions as a component of an Oxa1-independent insertion machinery in the inner membrane and that Mdm38 plays a critical role in the biogenesis of the respiratory chain by coupling ribosome function to protein transport across the inner membrane.
- Published
- 2006
- Full Text
- View/download PDF
45. Taz1, an outer mitochondrial membrane protein, affects stability and assembly of inner membrane protein complexes: implications for Barth Syndrome.
- Author
-
Brandner K, Mick DU, Frazier AE, Taylor RD, Meisinger C, and Rehling P
- Subjects
- Acyltransferases metabolism, Amino Acid Sequence, Electron Transport, Genes, X-Linked, Humans, Membrane Proteins metabolism, Membrane Transport Proteins metabolism, Mitochondrial Diseases genetics, Mitochondrial Membrane Transport Proteins, Mitochondrial Membranes metabolism, Mitochondrial Precursor Protein Import Complex Proteins, Mitochondrial Proteins metabolism, Molecular Sequence Data, Mutation, Myocardium pathology, Saccharomyces cerevisiae Proteins metabolism, Syndrome, Acyltransferases physiology, Membrane Proteins physiology, Mitochondria metabolism, Saccharomyces cerevisiae Proteins physiology
- Abstract
The Saccharomyces cerevisiae Taz1 protein is the orthologue of human Tafazzin, a protein that when inactive causes Barth Syndrome (BTHS), a severe inherited X-linked disease. Taz1 is a mitochondrial acyltransferase involved in the remodeling of cardiolipin. We show that Taz1 is an outer mitochondrial membrane protein exposed to the intermembrane space (IMS). Transport of Taz1 into mitochondria depends on the receptor Tom5 of the translocase of the outer membrane (TOM complex) and the small Tim proteins of the IMS, but is independent of the sorting and assembly complex (SAM). TAZ1 deletion in yeast leads to growth defects on nonfermentable carbon sources, indicative of a defect in respiration. Because cardiolipin has been proposed to stabilize supercomplexes of the respiratory chain complexes III and IV, we assess supercomplexes in taz1delta mitochondria and show that these are destabilized in taz1Delta mitochondria. This leads to a selective release of a complex IV monomer from the III2IV2 supercomplex. In addition, assembly analyses of newly imported subunits into complex IV show that incorporation of the complex IV monomer into supercomplexes is affected in taz1Delta mitochondria. We conclude that inactivation of Taz1 affects both assembly and stability of respiratory chain complexes in the inner membrane of mitochondria.
- Published
- 2005
- Full Text
- View/download PDF
46. Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17.
- Author
-
Chacinska A, Lind M, Frazier AE, Dudek J, Meisinger C, Geissler A, Sickmann A, Meyer HE, Truscott KN, Guiard B, Pfanner N, and Rehling P
- Subjects
- Amino Acid Sequence, Mitochondrial Membrane Transport Proteins metabolism, Mitochondrial Precursor Protein Import Complex Proteins, Molecular Sequence Data, Protein Binding, Protein Isoforms metabolism, Protein Transport, Saccharomyces cerevisiae metabolism, Carrier Proteins metabolism, Membrane Transport Proteins metabolism, Mitochondria metabolism, Repressor Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The presequence translocase of the inner mitochondrial membrane (TIM23 complex) operates at a central junction of protein import. It accepts preproteins from the outer membrane TOM complex and directs them to inner membrane insertion or, in cooperation with the presequence translocase-associated motor (PAM), to the matrix. Little is known of how the TIM23 complex coordinates these tasks. We have identified Tim21 (YGR033c) that interacts with the TOM complex. Tim21 is specific for a TIM23 form that cooperates with TOM and promotes inner membrane insertion. Protein translocation into the matrix requires a switch to a Tim21-free, PAM bound presequence translocase. Tim17 is crucial for the switch by performing two separable functions: promotion of inner membrane insertion and binding of Pam18 to form the functional TIM-PAM complex. Thus, the presequence translocase is not a static complex but switches between TOM tethering and PAM binding in a reaction cycle involving Tim21 and Tim17.
- Published
- 2005
- Full Text
- View/download PDF
47. The protein import machinery of mitochondria.
- Author
-
Wiedemann N, Frazier AE, and Pfanner N
- Subjects
- Active Transport, Cell Nucleus, Animals, Cell Nucleus metabolism, Fungal Proteins physiology, Humans, Intracellular Membranes metabolism, Membrane Proteins chemistry, Mitochondria metabolism, Models, Biological, Mitochondria physiology, Protein Transport
- Published
- 2004
- Full Text
- View/download PDF
48. Pam16 has an essential role in the mitochondrial protein import motor.
- Author
-
Frazier AE, Dudek J, Guiard B, Voos W, Li Y, Lind M, Meisinger C, Geissler A, Sickmann A, Meyer HE, Bilanchone V, Cumsky MG, Truscott KN, Pfanner N, and Rehling P
- Subjects
- Carrier Proteins metabolism, HSP70 Heat-Shock Proteins metabolism, Intracellular Membranes metabolism, Membrane Proteins metabolism, Membrane Transport Proteins metabolism, Mitochondria metabolism, Mitochondria ultrastructure, Mitochondrial Membrane Transport Proteins, Mitochondrial Precursor Protein Import Complex Proteins, Mitochondrial Proteins metabolism, Molecular Motor Proteins metabolism, Protein Binding, Protein Precursors metabolism, Protein Transport, Saccharomyces cerevisiae Proteins metabolism, Saccharomyces cerevisiae Proteins physiology, Yeasts chemistry, Yeasts metabolism, Yeasts ultrastructure, Membrane Transport Proteins physiology, Mitochondrial Proteins physiology
- Abstract
Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70-Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.
- Published
- 2004
- Full Text
- View/download PDF
49. A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria.
- Author
-
Truscott KN, Voos W, Frazier AE, Lind M, Li Y, Geissler A, Dudek J, Müller H, Sickmann A, Meyer HE, Meisinger C, Guiard B, Rehling P, and Pfanner N
- Subjects
- Amino Acid Sequence genetics, Base Sequence genetics, Carrier Proteins metabolism, Cells, Cultured, DNA, Complementary analysis, DNA, Complementary genetics, HSP70 Heat-Shock Proteins metabolism, Heat-Shock Proteins metabolism, Macromolecular Substances, Membrane Proteins genetics, Membrane Proteins metabolism, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Mitochondrial Precursor Protein Import Complex Proteins, Molecular Chaperones, Molecular Sequence Data, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Membrane Proteins isolation & purification, Membrane Transport Proteins isolation & purification, Mitochondria metabolism, Mitochondrial Membrane Transport Proteins, Molecular Motor Proteins metabolism, Protein Transport physiology, Saccharomyces cerevisiae Proteins isolation & purification
- Abstract
Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase-associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.
- Published
- 2003
- Full Text
- View/download PDF
50. Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space.
- Author
-
Frazier AE, Chacinska A, Truscott KN, Guiard B, Pfanner N, and Rehling P
- Subjects
- Electron Transport Complex IV, Fungal Proteins genetics, Fungal Proteins metabolism, HSP70 Heat-Shock Proteins metabolism, Macromolecular Substances, Membrane Potentials, Membrane Proteins genetics, Mitochondrial Membrane Transport Proteins, Mitochondrial Proteins genetics, Nuclear Proteins genetics, Nuclear Proteins metabolism, Protein Precursors genetics, Protein Precursors metabolism, Protein Transport, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Membrane Proteins metabolism, Membrane Transport Proteins, Mitochondria metabolism, Mitochondrial Proteins metabolism, Receptors, Cell Surface
- Abstract
The mitochondrial inner membrane contains numerous multispanning integral proteins. The precursors of these hydrophobic proteins are synthesized in the cytosol and therefore have to cross the mitochondrial outer membrane and intermembrane space to reach the inner membrane. While the import pathways of noncleavable multispanning proteins, such as the metabolite carriers, have been characterized in detail by the generation of translocation intermediates, little is known about the mechanism by which cleavable preproteins of multispanning proteins, such as Oxa1, are transferred from the outer membrane to the inner membrane. We have identified a translocation intermediate of the Oxa1 preprotein in the translocase of the outer membrane (TOM) and found that there are differences from the import mechanisms of carrier proteins. The intermembrane space domain of the receptor Tom22 supports the stabilization of the Oxa1 intermediate. Transfer of the Oxa1 preprotein to the inner membrane is not affected by inactivation of the soluble TIM complexes. Both the inner membrane potential and matrix heat shock protein 70 are essential to release the preprotein from the TOM complex, suggesting a close functional cooperation of the TOM complex and the presequence translocase of the inner membrane. We conclude that mitochondria employ different mechanisms for translocation of multispanning proteins across the aqueous intermembrane space.
- Published
- 2003
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.