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Complexome profiling (CP) is a powerful tool for systematic investigation of protein interactors that has been primarily applied to study the composition and dynamics of mitochondrial protein complexes. Here, we further optimised this method to extend its application to survey mitochondrial DNA- and RNA-interacting protein complexes. We established that high-resolution clear native gel electrophoresis (hrCNE) is a better alternative to preserve DNA- and RNA-protein interactions that are otherwise disrupted when samples are separated by the widely used blue native gel electrophoresis (BNE). In combination with enzymatic digestion of DNA, our CP approach improved the identification of a wide range of protein interactors of the mitochondrial gene expression system without compromising the detection of other multi-protein complexes. The utility of this approach was particularly demonstrated by analysing the complexome changes in human mitochondria with impaired gene expression after transient, chemically-induced mtDNA depletion. Effects of RNase on mitochondrial protein complexes were also evaluated and discussed. Overall, our adaptations significantly improved the identification of mitochondrial DNA- and RNA-protein interactions by CP, thereby unlocking the comprehensive analysis of a near-complete mitochondrial complexome in a single experiment.

Molecular functions of many human proteins remain unstudied, despite the demonstrated association with diseases or pivotal molecular structures, such as mitochondrial DNA (mtDNA). This small genome is crucial for proper functioning of mitochondria, the energy-converting organelles. In mammals, mtDNA is arranged into macromolecular complexes called nucleoids that serve as functional stations for its maintenance and expression. Here, we aimed to explore an uncharacterized protein c17orf80, which was previously detected close to the nucleoid components by proximity-labelling mass spectrometry. To investigate the subcellular localization and function of c17orf80, we took an advantage of immunofluorescence microscopy, interaction proteomics and several biochemical assays. We demonstrate that c17orf80 is a mitochondrial membrane-associated protein that interacts with nucleoids even when mtDNA replication is inhibited. In addition, we show that c17orf80 is not essential for mtDNA maintenance and mitochondrial gene expression in cultured human cells. These results provide a basis for uncovering the molecular function of c17orf80 and the nature of its association with nucleoids, possibly leading to new insights about mtDNA and its expression.

Mitochondrial DNA has been investigated for nearly fifty years, but many aspects of the maintenance of this essential small genome remain unknown. Like any genome, mammalian mitochondrial DNA requires the function of topoisomerases to counter and regulate the topological tension arising during replication, transcription, segregation, and repair. However, the functions of the different mitochondrial topoisomerases are poorly understood. Here, we investigate the role of Topoisomerase 3α (Top3α) in mtDNA replication and transcription, providing evidence that this enzyme, previously reported to act in mtDNA segregation, also participates in mtDNA replication fork progression. Top3α knockdown caused replication fork stalling, increased mtDNA catenation and decreased mtDNA levels. Overexpression in contrast induced abundant double-strand breaks around the replication origin OH and abortion of early replication, while at the same time improving the resolution of mtDNA replication termination intermediates. Both Top3α knockdown and overexpression affected mitochondrial RNA transcription, leading to a decrease in steady-state levels of mitochondrial transcripts. Together, our results indicate that the mitochondrial isoform of Top3α is not only involved in mtDNA segregation, as reported previously, but also supports the progression of the replication fork. Mitochondrial Top3α is also influencing the progression of transcription, with its absence affecting downstream transcript levels.

Contains fulltext : 248221.pdf (Publisher’s version ) (Open Access) SUPV3L1 encodes a helicase that is mainly localized in the mitochondria. It has been shown in vitro to possess both double-stranded RNA and DNA unwinding activity that is ATP-dependent. Here we report the first two patients for this gene who presented with a homozygous preliminary stop codon resulting in a C-terminal truncation of the SUPV3L1 protein. They presented with a characteristic phenotype of neurodegenerative nature with progressive spastic paraparesis, growth restriction, hypopigmentation, and predisposition to autoimmune disease. Ophthalmological examination showed severe photophobia with corneal erosions, optic atrophy, and pigmentary retinopathy, while neuroimaging showed atrophy of the optic chiasm and the pons with calcification of putamina, with intermittent and mild elevation of lactate. We show that the amino acids that are eliminated by the preliminary stop codon are highly conserved and are predicted to form an amphipathic helix. To investigate if the mutation causes mitochondrial dysfunction, we examined fibroblasts of the proband. We observed very low expression of the truncated protein, a reduction in the mature ND6 mRNA species as well as the accumulation of double-stranded RNA. Lentiviral complementation with the full-length SUPV3L1 cDNA partly restored the observed RNA phenotypes, supporting that the SUPV3L1 mutation in these patients is pathogenic and the cause of the disease.

Item does not contain fulltext Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly established. Shotgun proteomics has been used successfully to define whole-cell polyadenylated RNA (poly(A)-RNA) interacting proteomes using the nucleotide analogue 4-thiouridine (4SU) combined with UV crosslinking, poly(A)-RNA isolation and mass spectrometry to identify all poly(A)-RNA bound proteins. Although in this case also a considerable number of mitochondrial proteins were identified, the method was not specifically directed at the mitochondrial poly(A)-RNA bound proteome. Here we describe a method for enrichment of the mitochondrial poly(A)-RNA bound proteome based on 4SU labeling and UV crosslinking. The method can be applied either for isolated mitochondria prior to UV crosslinking or for whole-cell crosslinking followed by mitochondrial isolation.

Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly established. Shotgun proteomics has been used successfully to define whole-cell polyadenylated RNA (poly(A)-RNA) interacting proteomes using the nucleotide analogue 4-thiouridine (4SU) combined with UV crosslinking, poly(A)-RNA isolation and mass spectrometry to identify all poly(A)-RNA bound proteins. Although in this case also a considerable number of mitochondrial proteins were identified, the method was not specifically directed at the mitochondrial poly(A)-RNA bound proteome. Here we describe a method for enrichment of the mitochondrial poly(A)-RNA bound proteome based on 4SU labeling and UV crosslinking. The method can be applied either for isolated mitochondria prior to UV crosslinking or for whole-cell crosslinking followed by mitochondrial isolation.

Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle.

© 2020, Piro-Mégy et al., Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDSs), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in single-strand binding protein 1 (SSBP1) in 5 unrelated families, leading to the R38Q and R107Q amino acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a revised SSBP1 crystal structure. Structural analysis suggested that both mutations affect dimer interactions and presumably distort the DNA-binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication, and induces mtDNA depletion. Our study showing that mutations in SSBP1 cause a form of dominant optic atrophy frequently accompanied with foveopathy brings insights into mtDNA maintenance disorders., This study was supported by the Spanish Ministry of Science, Innovation and Universities, MINECO (BFU2015-70645-R, RTI2018-101015-B-100 to MS), the Generalitat de Catalunya (2014-SGR-997 and 2017-SGR-1192 to MS), and the European Union (FP7-HEALTH-2012-306029-2 to MS). ATS was awarded with an FPU fellowship from the Ministry of Education, Professional Formation (MEFP). The Structural Biology Unit at IBMB-CSIC is a “Maria de Maeztu” Unit of Excellence awarded by MINECO (MDM-2014-0435). This work was supported by the “Prinses Beatrix Spierfonds” and the “Stichting Spieren voor Spieren” (W.OR15-05 to JNS).

Newly synthesized mitochondrial RNA is concentrated in structures juxtaposed to nucleoids, called RNA granules, that have been implicated in mitochondrial RNA processing and ribosome biogenesis. Here we show that two classical mtDNA replication factors, the mtDNA helicase Twinkle and single-stranded DNA-binding protein mtSSB, contribute to RNA metabolism in mitochondria and to RNA granule biology. Twinkle colocalizes with both mitochondrial RNA granules and nucleoids, and it can serve as bait to greatly enrich established RNA granule proteins, such as G-rich sequence factor 1, GRSF1. Likewise, mtSSB also is not restricted to the nucleoids, and repression of either mtSSB or Twinkle alters mtRNA metabolism. Short-term Twinkle depletion greatly diminishes RNA granules but does not inhibit RNA synthesis or processing. Either mtSSB or GRSF1 depletion results in RNA processing defects, accumulation of mtRNA breakdown products as well as increased levels of dsRNA and RNA:DNA hybrids. In particular, the processing and degradation defects become more pronounced with both proteins depleted. These findings suggest that Twinkle is essential for RNA organization in granules, and that mtSSB is involved in the recently proposed GRSF1-mtRNA degradosome pathway, a route suggested to be particularly aimed at degradation of G-quadruplex prone long non-coding mtRNAs., Prinses Beatrix Spierfonds and the Stichting Spieren voor Spieren [W.OR15–05 to J.N.S.]; European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement [721757 to A.P.]; PhD fellowship from the Radboud Institute for Molecular Life Sciences; Radboudumc [R0002792 to S.LvE.]; Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2015-70645-R to M.S.]; Ministry of Education, Culture and Sports [FPU14-06021 to A.T.-S.]; Generalitat de Catalunya [2017-SGR-1192 to M.S.]; European Union [FP7-HEALTH-2012-306029-2 to M.S.]; ITN Fellowship FP7-PEOPLE-2011-290246 to A.C.]; The Structural Biology Unit at IBMB-CSIC is a ‘Maria de Maeztu’ Unit of Excellence awarded by MINECO [MDM-2014-0435].

Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation.

Contains fulltext : 190472.pdf (Publisher’s version ) (Open Access) EXD2 is a recently identified exonuclease that has been implicated in nuclear double-strand break repair. Given our long standing interest in mitochondrial DNA maintenance and indications that EXD2 could also be a mitochondrial protein we sought to determine its cellular localization and possible mitochondrial associated functions. Our results show that EXD2 indeed shows mitochondrial localization, but, surprisingly, is found predominantly associated with the mitochondrial outer-membrane. Gradient purified nuclei show only the faintest hint of EXD2 presence while overexpression of the predicted full-length protein shows exclusive mitochondrial localization. Importantly, induction of double-strand DNA breaks via X-irradiation or Zeocin treatment does not support the notion that EXD2 re-locates to the nucleus following double-strand breaks and thus is unlikely to have a direct role in nuclear DNA repair. Knockdown or overexpression of EXD2 affects the cellular distribution of mitochondria. These results suggest that the reported defects in nuclear DNA repair following EXD2 depletion are likely an indirect consequence of altered mitochondrial dynamics and/or function.

© The Author(s) 2015. The mitochondrial replicative helicase Twinkle is involved in strand separation at the replication fork of mitochondrial DNA (mtDNA). Twinkle malfunction is associated with rare diseases that include late onset mitochondrial myopathies, neuromuscular disorders and fatal infantile mtDNA depletion syndrome. We examined its 3D structure by electron microscopy (EM) and small angle X-ray scattering (SAXS) and built the corresponding atomic models, which gave insight into the first molecular architecture of a full-length SF4 helicase that includes an N-terminal zinc-binding domain (ZBD), an intermediate RNA polymerase domain (RPD) and a RecA-like hexamerization C-terminal domain (CTD). The EM model of Twinkle reveals a hexameric two-layered ring comprising the ZBDs and RPDs in one layer and the CTDs in another. In the hexamer, contacts in trans with adjacent subunits occur between ZBDs and RPDs, and between RPDs and CTDs. The ZBDs show important structural heterogeneity. In solution, the scattering data are compatible with a mixture of extended hexa- and heptameric models in variable conformations. Overall, our structural data show a complex network of dynamic interactions that reconciles with the structural flexibility required for helicase activity., MINECO [BFU2009-07134, BFU2012-33516 to M.S., BFU2012-34873 to M.V.]; Generalitat de Catalunya [SGR2009-1366, 2014 SGR 997 to M.S.]; EU [FP7-HEALTH-2010-261460, FP7-PEOPLE-2011-290246, FP7-HEALTH-2012-306029-2 to M.S.]. P.F.-M. held an FPI fellowship from MINECO [BES-2008-005384REF]. Academy of Finland [CoE funding to J.N.S.]; Tampere University Hospital Medical Research Fund [9J119,9K126, 9L097 to J.N.S.]; Netherlands Organization for Scientific Research [NWO: VICI grant 865.10.004 to J.N.S.]; European Molecular Biology Organization [EMBO LTF co-funded by Marie Curie Actions, EMBO LTF1066 2011 (including MCA-EMBOCOFUND2010,GA-2010-267146) to J.M.G.]; Agence Nationale de la Recherche SPIN-HD [CHEX-2011]; ATIP-Avenir. Funding for open access charge: MINECO [BFU2012-33516 to M.S.]

Item does not contain fulltext We studied a group of individuals with elevated urinary excretion of 3-methylglutaconic acid, neutropenia that can develop into leukemia, a neurological phenotype ranging from nonprogressive intellectual disability to a prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, and early death. Exome sequencing of two unrelated individuals and subsequent Sanger sequencing of 16 individuals with an overlapping phenotype identified a total of 14 rare, predicted deleterious alleles in CLPB in 14 individuals from 9 unrelated families. CLPB encodes caseinolytic peptidase B homolog ClpB, a member of the AAA+ protein family. To evaluate the relevance of CLPB in the pathogenesis of this syndrome, we developed a zebrafish model and an in vitro assay to measure ATPase activity. Suppression of clpb in zebrafish embryos induced a central nervous system phenotype that was consistent with cerebellar and cerebral atrophy that could be rescued by wild-type, but not mutant, human CLPB mRNA. Consistent with these data, the loss-of-function effect of one of the identified variants (c.1222A>G [p.Arg408Gly]) was supported further by in vitro evidence with the mutant peptides abolishing ATPase function. Additionally, we show that CLPB interacts biochemically with ATP2A2, known to be involved in apoptotic processes in severe congenital neutropenia (SCN) 3 (Kostmann disease [caused by HAX1 mutations]). Taken together, mutations in CLPB define a syndrome with intellectual disability, congenital neutropenia, progressive brain atrophy, movement disorder, cataracts, and 3-methylglutaconic aciduria.

Contains fulltext : 138677.pdf (Publisher’s version ) (Open Access) Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the "Pattern Finder" G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.

A large number of mutations in the gene encoding the catalytic subunit of mitochondrial DNA polymerase γ (POLγA) cause human disease. The Y955C mutation is common and leads to a dominant disease with progressive external ophthalmoplegia and other symptoms. The biochemical effect of the Y955C mutation has been extensively studied and it has been reported to lower enzyme processivity due to decreased capacity to utilize dNTPs. However, it is unclear why this biochemical defect leads to a dominant disease. Consistent with previous reports, we show here that the POLγA:Y955C enzyme only synthesizes short DNA products at dNTP concentrations that are sufficient for proper function of wild-type POLγA. In addition, we find that this phenotype is overcome by increasing the dNTP concentration, e.g. dATP. At low dATP concentrations, the POLγA:Y955C enzyme stalls at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The POLγA:Y955C enzyme will compete with wild-type POLγA for primer utilization, and this will result in a heterogeneous population of short and long DNA replication products. In addition, there is a possibility that POLγA:Y955C is recruited to nicks of mtDNA and there enters an idling mode preventing ligation. Our results provide a novel explanation for the dominant mtDNA replication phenotypes seen in patients harboring the Y955C mutation, including the existence of site-specific stalling. Our data may also explain why mutations that disturb dATP pools can be especially deleterious for mtDNA synthesis. © The Author 2011. Published by Oxford University Press. All rights reserved.

Item does not contain fulltext Pathological mitochondrial DNA (mtDNA) rearrangements have been proposed to result from repair of double-strand breaks caused by blockage of mitochondrial DNA (mtDNA) replication. As mtDNA deletions are seen only in post-mitotic tissues, it has been suggested that they are selected out in actively dividing cells. By electron microscopy we observed rearranged mtDNA molecules in cultured human cells expressing a catalytically impaired helicase. As these molecules were undetectable by PCR, we propose that deleted mtDNA molecules in cultured cells are fragile and sensitive to heating. Further consequences of mtDNA replication stalling are discussed.

Item does not contain fulltext We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.

The accessory subunit of mitochondrial DNA polymerase gamma, POLGbeta, functions as a processivity factor in vitro. Here we show POLGbeta has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGbeta increased nucleoid numbers, whereas over-expression of POLGbeta reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGbeta altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGbeta preferentially bound to plasmids with a short displacement-loop, in contrast to POLGalpha. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGbeta is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.

Mutations in the mitochondrial helicase Twinkle underlie autosomal dominant progressive external ophthalmoplegia (PEO), as well as recessively inherited infantile-onset spinocerebellar ataxia and rare forms of mitochondrial DNA (mtDNA) depletion syndrome. Familial PEO is typically associated with the occurrence of multiple mtDNA deletions, but the mechanism by which Twinkle dysfunction induces deletion formation has been under debate. Here we looked at the effects of Twinkle adPEO mutations in human cell culture and studied the mtDNA replication in the Deletor mouse model, which expresses a dominant PEO mutation in Twinkle and accumulates multiple mtDNA deletions during life. We show that expression of dominant Twinkle mutations results in the accumulation of mtDNA replication intermediates in cell culture. This indicated severe replication pausing or stalling and caused mtDNA depletion. A strongly enhanced accumulation of replication intermediates was evident also in six-week-old Deletor mice compared with wild-type littermates, even though mtDNA deletions accumulate in a late-onset fashion in this model. In addition, our results in cell culture pointed to a problem of transcription that preceded the mtDNA depletion phenotype and might be of relevance in adPEO pathophysiology. Finally, in vitro assays showed functional defects in the various Twinkle mutants and broadly agreed with the cell culture phenotypes such as the level of mtDNA depletion and the level of accumulation of replication intermediates. On the basis of our results we suggest that mtDNA replication pausing or stalling is the common consequence of Twinkle PEO mutations that predisposes to multiple deletion formation.

Infantile-onset spinocerebellar ataxia (IOSCA) is a severe neurodegenerative disorder caused by the recessive mutation in PEO1, leading to an Y508C change in the mitochondrial helicase Twinkle, in its helicase domain. However, no mitochondrial dysfunction has been found in this disease. We studied here the consequences of IOSCA for the central nervous system, as well as the in vitro performance of the IOSCA mutant protein. The results of the mtDNA analyses were compared to findings in a similar juvenile or adult-onset ataxia syndrome, mitochondrial recessive ataxia syndrome (MIRAS), caused by the W748S mutation in the mitochondrial DNA polymerase (POLG). We show here that IOSCA brain does not harbor mtDNA deletions or increased amount of mtDNA point mutations, whereas MIRAS brain shows multiple deletions of mtDNA. However, IOSCA, and to a lesser extent also MIRAS, show mtDNA depletion in the brain and the liver. In both diseases, especially large neurons show respiratory chain complex I (CI) deficiency, but also CIV is decreased in IOSCA. Helicase activity, hexamerization and nucleoid structure of the IOSCA mutant were, however, unaffected. The lack of in vitro helicase defect or cell culture phenotype suggest that Twinkle-Y508C dysfunction affects mtDNA maintenance in a highly context and cell-type specific manner. Our results indicate that IOSCA is a new member of the mitochondrial DNA depletion syndromes.

It has recently been suggested that perhaps as many as 20% of all mitochondrial proteins are regulated through lysine acetylation while SIRT3 has been implicated as an important mitochondrial protein deacetylase. It is therefore of crucial importance that the mitochondrial localization of potential protein deacetylases is unambiguously established. Although mouse SIRT3 was recently shown to be mitochondrial, HsSIRT3 (human SIRT3) was reported to be both nuclear and mitochondrial and to relocate from the nucleus to the mitochondrion upon cellular stress. In the present study we show, using various HsSIRT3 expression constructs and a combination of immunofluorescence and careful subcellular fractionation, that in contrast with earlier reports HsSIRT3 is exclusively mitochondrial. We discuss possible experimental explanations for these discrepancies. In addition we suggest, on the basis of the analysis of public genome databases, that the full-length mouse SIRT3 protein is a 37 kDa mitochondrial precursor protein contrary to the previously suggested 29 kDa protein.

Objective Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a clinically and genetically heterogeneous group of autosomal recessive diseases characterized by a reduction in mtDNA copy number. Several nuclear genes have been shown to account for these severe oxidative phosphorylation disorders, but the disease-causing mutations remain largely unknown. Methods By virtue of homozygosity mapping, we tested candidate genes involved in mtDNA maintenance in patients born to consanguineous parents. Results We found homozygosity for microsatellite markers flanking the PEO1 gene, encoding the mitochondrial Twinkle helicase, in two sibs presenting a hepatocerebral form of MDS. Sequencing the PEO1 gene showed a homozygous mutation at a conserved position of the protein in the two patients (T457I). The modeling of the Twinkle protein showed that T457 is located in the interface between two monomers of the hexameric enzyme. Finally, using purified recombinant protein, we demonstrated that the T457I mutant Twinkle has a defective helicase activity. Interpretation Although dominant Twinkle mutations have been previously reported in patients with autosomal dominant progressive external ophthalmoplegia and multiple mtDNA deletions, we report here the first recessive Twinkle mutation in patients with hepatocerebral form of MDS. Identifying other Twinkle mutations in MDS and/or autosomal dominant progressive external ophthalmoplegia and studying their impact on the isolated proteins should help in understanding why some mutations are recessive and others are dominant. Ann Neurol 2007

The mechanism of mitochondrial DNA replication is a subject of intense debate. One model proposes a strand-asynchronous replication in which both strands of the circular genome are replicated semi-independently while the other model proposes both a bidirectional coupled leading- and lagging-strand synthesis mode and a unidirectional mode in which the lagging-strand is initially laid-down as RNA by an unknown mechanism (RITOLS mode). Both the strand-asynchronous and RITOLS model have in common a delayed synthesis of the DNA-lagging strand. Mitochondrial DNA is replicated by a limited set of proteins including DNA polymerase gamma (POLG) and the helicase Twinkle. Here, we report the effects of expression of various catalytically deficient mutants of POLG1 and Twinkle in human cell culture. Both groups of mutants reduced mitochondrial DNA copy number by severe replication stalling. However, the analysis showed that while induction of POLG1 mutants still displayed delayed lagging-strand synthesis, Twinkle-induced stalling resulted in maturated, essentially fully double-stranded DNA intermediates. In the latter case, limited inhibition of POLG with dideoxycytidine restored the delay between leading- and lagging-strand synthesis. The observed cause-effect relationship suggests that Twinkle-induced stalling increases lagging-strand initiation events and/or maturation mimicking conventional strand-coupled replication.

Contains fulltext : 154454.PDF (Publisher’s version ) (Open Access) Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data.

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.

We present a method for automatically detecting the tips of fluorescently labeled mitochondria. The method is based on a Random Forest classifier, which is trained on small patches extracted from confocal microscope images of U2OS human osteosarcoma cells. We then adopt a particle tracking framework for tracking the detected tips, and quantify the tracking accuracy on simulated data. Finally, from images of U2OS cells, we quantify changes in mitochondrial mobility in response to the disassembly of microtubules via treatment with Nocodazole. The results show that our approach provides efficient tracking of the tips of mitochondria, and that it enables the detection of disease-associated changes in mitochondrial motility.

Contains fulltext : 154217.pdf (Publisher’s version ) (Open Access) Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5-5 muM) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies.

Tid1 is a human homolog of bacterial DnaJ and the Drosophila tumor suppressor Tid56 that has two alternatively spliced isoforms, Tid1-long and -short (Tid1-L and -S), which differ only at their carboxyl termini. Although Tid1 proteins localize overwhelmingly to mitochondria, published data demonstrate principally nonmitochondrial protein interactions and activities. This study was undertaken to determine whether Tid1 proteins function as mitochondrial DnaJ-like chaperones and to resolve the paradox of how proteins targeted primarily to mitochondria function in nonmitochondrial pathways. Here we demonstrate that Tid1 isoforms exhibit a conserved mitochondrial DnaJ-like function substituting for the yeast mitochondrial DnaJ-like protein Mdj1p. Like Mdj1p, Tid1 localizes to human mitochondrial nucleoids, which are large protein complexes bound to mitochondrial DNA. Unlike other DnaJs, Tid1-L and -S form heterocomplexes; both unassembled and complexed Tid1 are observed in human cells. Results demonstrate that Tid1-L has a longer residency time in the cytosol prior to mitochondrial import as compared with Tid1-S; Tid1-L is also significantly more stable in the cytosol than Tid1-S, which is rapidly degraded. The longer cytosolic residency time and the half-life of Tid1-L are explained by its interaction with cytosolic Hsc70 and potential protein substrates such as the STAT1 and STAT3 transcription factors. We show that the unique carboxyl terminus of Tid1-L is required for interaction with Hsc70 and STAT1 and -3. We propose that the association of Tid1 with chaperones and/or protein substrates in the cytosol provides a mechanism for the alternate fates and functions of Tid1 in mitochondrial and nonmitochondrial pathways.

The mitochondrial theory of aging proposes that reactive oxygen species (ROS) generated inside the cell will lead, with time, to increasing amounts of oxidative damage to various cell components. The main site for ROS production is the respiratory chain inside the mitochondria and accumulation of mtDNA mutations, and impaired respiratory chain function have been associated with degenerative diseases and aging. The theory predicts that impaired respiratory chain function will augment ROS production and thereby increase the rate of mtDNA mutation accumulation, which, in turn, will further compromise respiratory chain function. Previously, we reported that mice expressing an error-prone version of the catalytic subunit of mtDNA polymerase accumulate a substantial burden of somatic mtDNA mutations, associated with premature aging phenotypes and reduced lifespan. Here we show that these mtDNA mutator mice accumulate mtDNA mutations in an approximately linear manner. The amount of ROS produced was normal, and no increased sensitivity to oxidative stress-induced cell death was observed in mouse embryonic fibroblasts from mtDNA mutator mice, despite the presence of a severe respiratory chain dysfunction. Expression levels of antioxidant defense enzymes, protein carbonylation levels, and aconitase enzyme activity measurements indicated no or only minor oxidative stress in tissues from mtDNA mutator mice. The premature aging phenotypes in mtDNA mutator mice are thus not generated by a vicious cycle of massively increased oxidative stress accompanied by exponential accumulation of mtDNA mutations. We propose instead that respiratory chain dysfunction per se is the primary inducer of premature aging in mtDNA mutator mice.

Defects of mitochondrial DNA (mtDNA) maintenance have recently been associated with inherited neurodegenerative and muscle diseases and the aging process. Twinkle is a nuclear-encoded mtDNA helicase, dominant mutations of which cause adult-onset progressive external ophthalmoplegia (PEO) with multiple mtDNA deletions. We have generated transgenic mice expressing mouse Twinkle with PEO patient mutations. Multiple mtDNA deletions accumulate in the tissues of these mice, resulting in progressive respiratory dysfunction and chronic late-onset mitochondrial disease starting at 1 year of age. The muscles of the mice faithfully replicate all of the key histological, genetic, and biochemical features of PEO patients. Furthermore, the mice have progressive deficiency of cytochrome c oxidase in distinct neuronal populations. These “deletor” mice do not, however, show premature aging, indicating that subtle accumulation of mtDNA deletions and progressive respiratory chain dysfunction are not sufficient to accelerate aging. This model is a valuable tool for therapy development and testing for adult-onset mitochondrial disorders.

Infantile onset spinocerebellar ataxia (IOSCA) (MIM 271245) is a severe autosomal recessively inherited neurodegenerative disorder characterized by progressive atrophy of the cerebellum, brain stem and spinal cord and sensory axonal neuropathy. We report here the molecular background of this disease based on the positional cloning/candidate approach of the defective gene. Having established the linkage to chromosome 10q24, we restricted the critical DNA region using single nucleotide polymorphism-based haplotypes. After analyzing all positional candidate transcripts, we identified two point mutations in the gene C10orf2 encoding Twinkle, a mitochondrial deoxyribonucleic acid (mtDNA)-specific helicase, and a rarer splice variant Twinky, underlying IOSCA. The founder IOSCA mutation, homozygous in all but one of the patients, leads to a Y508C amino acid change in the polypeptides. One patient, heterozygous for Y508C, carries a silent coding region cytosine to thymine transition mutation in his paternal disease chromosome. This allele is expressed at a reduced level, causing the preponderance of messenger RNAs encoding Y508C polypeptides and thus leads to the IOSCA disease phenotype. Previously, we have shown that different mutations in this same gene cause autosomal dominant progressive external ophthalmoplegia (adPEO) with multiple mtDNA deletions (MIM 606075), a neuromuscular disorder sharing a spectrum of symptoms with IOSCA. IOSCA phenotype is the first recessive one due to Twinkle and Twinky mutations, the dominant PEO mutations affecting mtDNA maintenance, but in IOSCA, mtDNA stays intact. The severe neurological phenotype observed in IOSCA, a result of only a single amino acid substitution in Twinkle and Twinky, suggests that these proteins play a crucial role in the maintenance and/or function of specific affected neuronal subpopulations.

Mechanisms of mitochondrial DNA (mtDNA) maintenance have recently gained wide interest owing to their role in inherited diseases as well as in aging. Twinkle is a new mitochondrial 5'-3' DNA helicase, defects of which we have previously shown to underlie a mitochondrial disease, progressive external ophthalmoplegia with multiple mtDNA deletions. Mouse Twinkle is highly similar to the human counterpart, suggesting conserved function. Here, we have characterized the mouse Twinkle gene and expression profile and report that the expression patterns are not conserved between human and mouse, but are synchronized with the adjacent gene MrpL43, suggesting a shared promoter. To elucidate the in vivo role of Twinkle in mtDNA maintenance, we generated two transgenic mouse lines overexpressing wild-type Twinkle. We could demonstrate for the first time that increased expression of Twinkle in muscle and heart increases mtDNA copy number up to 3-fold higher than controls, more than any other factor reported to date. Additionally, we utilized cultured human cells and observed that reduced expression of Twinkle by RNA interference mediated a rapid drop in mtDNA copy number, further supporting the in vivo results. These data demonstrate that Twinkle helicase is essential for mtDNA maintenance, and that it may be a key regulator of mtDNA copy number in mammals.

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in a variety of tissues during ageing in humans, monkeys and rodents. These mutations are unevenly distributed and can accumulate clonally in certain cells, causing a mosaic pattern of respiratory chain deficiency in tissues such as heart, skeletal muscle and brain. In terms of the ageing process, their possible causative effects have been intensely debated because of their low abundance and purely correlative connection with ageing. We have now addressed this question experimentally by creating homozygous knock-in mice that express a proof-reading-deficient version of PolgA, the nucleus-encoded catalytic subunit of mtDNA polymerase. Here we show that the knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations is associated with reduced lifespan and premature onset of ageing-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement. Our results thus provide a causative link between mtDNA mutations and ageing phenotypes in mammals.

Ditercalinium chloride was originally synthesized for use as an anticancer drug and was then found to deplete mitochondrial DNA. Ethidium bromide is widely used to deplete mitochondrial DNA and produce mitochondrial DNA-less cell lines. Although ethidium bromide is used in the case of human cell lines, it frequently fails to deplete mitochondrial DNA in mouse cells. In contrast, ditercalinium chloride can deplete mitochondrial DNA in both mouse and human cells. However, little is known of the mechanisms by which ditercalinium chloride depletes mitochondrial DNA. Here, we show that ditercalinium chloride inhibits human DNA polymerase gamma activity as efficiently as does ethidium bromide. Ethidium bromide accumulates much less in mouse B82 cells, as compared with findings in human HeLa cells, whereas ditercalinium chloride accumulates in both to a similar extent. This poor accumulation of ethidium bromide may, in part, account for the resistance. Ethidium bromide distributes diffusely in the mitochondria of HeLa cells, while ditercalinium chloride distributes granularly and hence may be strongly associated with mitochondrial DNA. Each granular spot presumably represents one mitochondrial DNA nucleoid. In support of this idea, ditercalinium chloride co-localizes with Twinkle, a mitochondrial helicase and is assumed to associate with mitochondrial DNA. This close association of ditercalinium chloride with mitochondrial DNA may contribute to the mitochondrial DNA-depleting activity.

The mitochondrial replication machinery includes essential proteins that are similar to T-odd bacteriophages, and thus its mechanism is different from the one in the nucleus. Involved proteins include the replicative helicase Twinkle, which unwinds the DNA at the replication fork. The three-dimensional structure of Twinkle was solved by cryo-electron microscopy (cryo-EM) and small angle X-ray scattering (SAXS), which both showed the presence of heptamers and hexamers. Based on related helicase structures, we built an atomic homology model for the full-length protein, which included, for the first time for an SF4 helicase, an N-terminal zinc-binding domain (ZBD), an intermediate RNA polymerase domain (RPD), and a RecA-like hexamerization C-terminal domain (CTD). The cryo-EM structure of Twinkle reveals two hexameric rings, one comprising the ZBDs and RPDs and the other containing the CTDs. While the CTD ring shows strict six-fold symmetry, the ZBDs in the other ring show structural heterogeneity. We generated models in which the ZBD-RPD linker and the N-terminal segment of the RPD-CTD linker, as well as the N-terminal and the C-terminal tails, were defined as flexible regions with complete conformational freedom. From data measured in solution, we detected that both RPDs and ZBDs protrude considerably from the CTD hexa- and heptameric rings. The structural flexibility observed for the Twinkle N-terminal domains suggests a complex network of interactions consistent with functional requirements of helicase activity.

Item does not contain fulltext Mammalian mtDNA encodes for 13 core proteins of oxidative phosphorylation. Mitochondrial DNA mutations and deletions cause severe myopathies and neuromuscular diseases. Thus, the integrity of mtDNA is pivotal for cell survival and health of the organism. We here discuss the possible impact of mitochondrial fusion and fission on mtDNA maintenance as well as positive and negative selection processes. Our focus is centred on the important question of how the quality of mtDNA nucleoids can be assured when selection and mitochondrial quality control works on functional and physiological phenotypes constituted by oxidative phosphorylation proteins. The organelle control theory suggests a link between phenotype and nucleoid genotype. This is discussed in the light of new results presented here showing that mitochondrial transcription factor A/nucleoids are restricted in their intramitochondrial mobility and probably have a limited sphere of influence. Together with recent published work on mitochondrial and mtDNA heteroplasmy dynamics, these data suggest first, that single mitochondria might well be internally heterogeneous and second, that nucleoid genotypes might be linked to local phenotypes (although the link might often be leaky). We discuss how random or site-specific mitochondrial fission can isolate dysfunctional parts and enable their elimination by mitophagy, stressing the importance of fission in the process of mtDNA quality control. The role of fusion is more multifaceted and less understood in this context, but the mixing and equilibration of matrix content might be one of its important functions.

Contains fulltext : 136701.pdf (Publisher’s version ) (Open Access) Mitochondrial DNA (mtDNA) is organized in discrete protein-DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded DNA-binding protein, mtSSB, co-localize only with a subset of nucleoids. Using nucleotide analogs to identify replicating mtDNA in situ, the fraction of label-positive nucleoids that is Twinkle/mtSSB positive, is highest with the shortest labeling-pulse. In addition, the recruitment of mtSSB is shown to be Twinkle dependent. These proteins thus transiently associate with mtDNA in an ordered manner to facilitate replication. To understand the nature of mtDNA replication complexes, we examined nucleoid protein membrane association and show that endogenous Twinkle is firmly membrane associated even in the absence of mtDNA, whereas mtSSB and other nucleoid-associated proteins are found in both membrane-bound and soluble fractions. Likewise, a substantial amount of mtDNA is found as soluble or loosely membrane bound. We show that, by manipulation of Twinkle levels, mtDNA membrane association is partially dependent on Twinkle. Our results thus show that Twinkle recruits or is assembled with mtDNA at the inner membrane to form a replication platform and amount to the first clear demonstration that nucleoids are dynamic both in composition and concurrent activity.

Item does not contain fulltext In the last decade it has become increasingly clear that mitochondrial DNA (mtDNA) is not naked but associated with proteins in poorly defined structures called nucleoids that are essential for mtDNA maintenance. The function of nucleoids is not simply to package mtDNA but also to provide a stable environment for its replication, transcription and repair. Even though their properties and dynamics have begun to be revealed in recent years, their structural and molecular organization remains largely unknown in mammals. Although, there are a number of proteins identified to be nucleoid associated by using several biochemical isolation methods combined with mass spectrometric analysis, the main difficulties in the identification of these proteins are their low abundance and the assumed dynamic composition of nucleoids. Considering various purification methods, there is a thin line between the stringency and specificity in the identification of potential nucleoid associated proteins. In this review, the main focus is to provide a comprehensive comparison of the so far published purification and analysis methods to generate a list of potentially nucleoid associated proteins, but also, to discuss the disadvantages and possible improvements in proteomic analyses.

In this article we develop a model for the organization and maintenance of mitochondrial DNA (mtDNA) in mammalian somatic cells, based on the idea that the unit of genetic function comprises a group of mtDNA molecules that are semi-permanently associated as a mitochondrial nucleoid. Different mtDNA molecules within a nucleoid need not be genetically identical. We propose that nucleoids replicate faithfully via a kind of mitochondrial mitosis, generating daughter nucleoids that are identical copies of each other, but which can themselves segregate freely. This model can account for the very slow rates of mitotic segregation observed in cultured, heteroplasmic cell-lines, and also for the apparently poor complementation observed between different mutant mtDNAs co-introduced into rho(0) cells (cells that lack endogenous mtDNA). It also provides a potential system for maintaining the mitochondrial genetic fitness of stem cells in the face of a presumed high somatic mutation rate of mtDNA and many rounds of cell division in the absence of phenotypic selection. BioEssays 22:564-572, 2000.

The mitochondrial genotype of heteroplasmic human cell lines containing the pathological np 3243 mtDNA mutation, plus or minus its suppressor at np 12300, has been followed over long periods in culture. Cell lines containing various different proportions of mutant mtDNA remained generally at a consistent, average heteroplasmy value over at least 30 wk of culture in nonselective media and exhibited minimal mitotic segregation, with a segregation number comparable with mtDNA copy number (≥1000). Growth in selective medium of cells at 99% np 3243 mutant mtDNA did, however, allow the isolation of clones with lower levels of the mutation, against a background of massive cell death. As a rare event, cell lines exhibited a sudden and dramatic diversification of heteroplasmy levels, accompanied by a shift in the average heteroplasmy level over a short period (

In cybrid cells carrying the mitochondrial A3243G MELAS mutation, which were also heteroplasmic for the G12300A suppressor mutation, we observed a transient episode of heteroplasmic instability, resulting in a wide diversification in G12300A heteroplasmy levels and a shift in the average heteroplasmy level from 11 to 29%. These cells were found to be trisomic for chromosome 9, whereas a minority of cells that retained disomy-9 showed no instability. Coculture experiments implied that trisomy-9 cells exhibited a significant growth advantage, but neither heteroplasmy levels, respiratory phenotype nor trisomy-9 itself had direct selective value under standard culture conditions. Mitochondrial nucleoid number was the same (50-100) in cells that had or had not experienced transient heteroplasmic instability, but 1-2 orders of magnitude less than the segregation number in such cells. These findings support the idea that mtDNA partition is under nuclear genetic control, and implicate a locus on chromosome 9 in this regulation.

Two sons and one daughter of healthy consanguineous parents presented with fatal hepatic failure in association with severe depletion of mitochondrial (mt)DNA in liver; a third son is healthy. Other published cases of mtDNA depletion concern single members of a family, which excludes the use of haplotype analysis. In the family presented here, the inheritance of the genes for mitochondrial transcription factor A (mtTFA), nuclear respiratory factor 1 (NRF-1), mitochondrial single-stranded DNA-binding protein (mtSSBP), and endonuclease G (EndoG) was studied using microsatellite markers linked to these genes. The inheritance of the gene for mtDNA polymerase (pol gamma) was studied using a polymorphic CAG repeat present within the coding region of the gene. EndoG and mtSSBP were excluded, but mtTFA remains a candidate. Pol gamma or NRF-1 involvement would be compatible only with autosomal dominant inheritance. Coding sequence analysis of NRF-1 and mtTFA revealed no novel mutations in affected individuals.

In order to study the still poorly understood dynamics of mitochondrial gene segregation, we attempted to alter the percentage of deleted mtDNA (del-mtDNA) over wild-type mtDNA in cell-culture by manipulating respiratory chain capacity. For this purpose, we used a cell-line harbouring a 6-kb mtDNA-deletion which normally was present in 70% of the molecules. The results show that in the presence of low concentrations of doxycycline (DC), an inhibitor of mitochondrial protein synthesis, the average percentage of del-mtDNA in culture steadily declined. After short-term DC treatment most cells still contained del-mtDNA and removal of DC led to a rapid increase in the proportion of del-mtDNA. In contrast, long-term DC treatment rendered del-mtDNA undetectable by Southern analysis, reflecting the complete absence of del-mtDNA in most cells. In this case, del-mtDNA in culture remained at a constant low level after removal of the drug. The results clearly show the importance of phenotypic selection in the segregation of a deleterious mtDNA mutation.