MITOCH-00556; No of Pages 9 Mitochondrion xxx (2010) xxx–xxx
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Mitochondrion j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o
Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization Jessie M. Cameron a, Valeriy Levandovskiy a, Nevena MacKay a, Cameron Ackerley b, David Chitayat c, Julian Raiman c, W.H. Halliday b, Andreas Schulze c, Brian H. Robinson a,d,e,⁎ a
Genetics and Genome Biology, The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada, M5G 1X8 Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, ON, Canada, M5G 1X8 Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON, Canada, M5G 1X8 d Department of Paediatrics, University of Toronto, ON, 1 King's College Circle, Toronto, Canada, M5S 1A8 e Department of Biochemistry, University of Toronto, ON, 1 King's College Circle, Toronto, Canada, M5S 1A8 b c
a r t i c l e
i n f o
Article history: Received 28 May 2010 Received in revised form 23 August 2010 Accepted 22 September 2010 Available online xxxx Keywords: ATP synthase Mitochondria TMEM70 Nucleoids
a b s t r a c t Mutations in the TMEM70 gene are responsible for a familial form of complex V deficiency presenting with 3methylglutaconic aciduria, lactic acidosis, cardiomyopathy and mitochondrial myopathy. Here we present a case of TMEM70 deficiency due to compound heterozygous mutations, who displayed abnormal mitochondria with whorled cristae in muscle. Immunogold electron microscopy and tomography shows for the first time that nucleoid clusters of mtDNA are disrupted in the abnormal mitochondria, with both nucleoids and mitochondrial respiratory chain complexes confined to the outer rings of the whorls. This could explain the differential effects on the expression and assembly of complex V in different tissues. © 2010 Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.
1. Introduction Mitochondrial morphology in muscle can be strongly influenced by the presence of defects affecting the mitochondrial respiratory chain. The effects vary from mitochondrial proliferation, mitochondria with few cristae, mitochondria with inclusion bodies and mitochondria with concentric layers of membranes. One defect that has been consistently reported as being associated with concentric whorled mitochondrial membranes in familial complex V deficiency, we demonstrate is due to the mutations in the TMEM70 gene. Mutations in the TMEM70 gene were recently identified as the cause of ATP synthase deficiency in patients descended from Roma Gypsies who presented with 3-methylglutaconic aciduria and lactic acidosis (Cizkova et al., 2008). Subsequently several previously published patients with similar clinical features, but no genetic diagnosis, were confirmed to also have TMEM70 mutations (Holme et al., 1992; Mayr et al., 2004; Sperl et al., 2006). We have identified a new patient with a similar presentation of 3-methylglutaconic aciduria, lactic acidosis, hypertrophic cardiomyopathy and developmental delay, that had ATPase (complex V) deficiency reduced to 5% of normal rates in muscle. The patient had the same compound heterozygote mutations in TMEM70, as previously reported (Cizkova et al., 2008). Abbreviations: mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphorylation complexes. ⁎ Corresponding author. Genetics and Genome Biology, The Research Institute, The Hospital for Sick Children, Toronto, ON, Canada, M5G 1X8. Tel.: +1 416 813 5989; fax: +1 416 813 8700. E-mail address:
[email protected] (B.H. Robinson).
The mechanism for the assembly of ATP synthase is not fully understood; only two assembly factors for mammalian ATP synthase have been identified in mammals (ATP11 and ATP12, which assemble components of the F1 head). Only defects in ATP12 have been shown to cause a clinical phenotype in humans (De Meirleir et al., 2004). Several more assembly proteins have been identified in yeast, one of which has a mammalian homologue (ATP23), but would not have the same mode of action as in yeast due to mitochondrial DNA processing differences (Zeng et al., 2007). The specific role of TMEM70 in ATP synthase assembly is still unknown. but using electron microscopy and immunogold labelling, we show that this defect results in a disruption of normal cristae formation. A loss of invaginations and a tendency to form concentric cristae rings actually affect the integrity of mitochondrial nucleoids. Since these are the sites of mtDNA transcription, replication and the likely site of mtDNA driven protein synthesis, this, in turn, affects the distribution of OXPHOS complexes (Bogenhagen et al., 2008). The activity of the OXPHOS complexes other than complex V in muscle appear to be low but not deficient. This illustrates how important the nucleoids are for normal OXPHOS function, and suggests that their disruption is an integral part of the pathogenic process. 2. Methods 2.1. Patient case report The patient was born to a 36-year-old G1P0 mother of Italian descent and a 33-year-old father of Croatian descent. The couple is healthy, non-consanguineous and their family histories were non-
1567-7249/$ – see front matter © 2010 Mitochondria Research Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.mito.2010.09.008
Please cite this article as: Cameron, J.M., et al., Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization, Mitochondrion (2010), doi:10.1016/j.mito.2010.09.008
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contributory. A detailed fetal ultrasound at 20 weeks gestation was interpreted as normal with a normal interval growth. A repeat fetal ultrasound at 27 weeks gestation showed that the heart was abnormally enlarged and occupied more than 1/3 of the fetal thorax. No obvious structural cardiac defects were detected but there was a small pericardial effusion. A fetal echocardiography showed cardiomegaly with moderate bilateral thickening of the ventricular myocardium consistent with hypertrophic cardiomyopathy. Delivery was at 38.6 weeks gestation, vaginal and induced due to poor fetal movements and oligohydramnios. On initial examination the patient had a hoarse cry, and a large anterior fontanelle (3 × 3.5 cm). The facial features were coarse with a high forehead and frontal bossing, bi-temporal narrowing, hypoplasia of the supraorbital ridges, puffy eyelids, bilateral infraorbital creases, broad nasal bridge and broad nasal tip There was a low nuchal hairline, short neck and redundant nuchal skin.. There was diastases recti and mild umbilical hernia. Postnatal echocardiography confirmed hypertrophic cardiomyopathy, narrowing of the aortic arch as well as mild arrhythmia on Holter monitoring. DNA analysis of genes known to be associated with hypertrophic cardiomyopathy showed that the patient is heterozygous for the 690-7 C N T mutation in intron 13 of the TNNT2 gene. Quantitative plasma amino acids were normal but the urinary organic acid analysis revealed trace to small amounts of 3-hydroxy butyric and 3-methylglutaconic acids. The possibility of a mitochondrial disease was thus raised. 2.2. Cell culture Cultured skin fibroblasts were grown from forearm skin biopsy (taken with informed consent) in α-MEM culture medium (11 mM glucose) and 10% fetal calf serum (Wisent, Inc., Quebec, Canada). 2.3. Molecular genetics techniques Genomic DNA was isolated from fibroblasts or blood using Puregene gDNA isolation kit (Qiagen). RNA was isolated using Trizol, and full length TMEM70 cDNA sequence was reverse transcribed using Superscript II reverse transcriptase and amplified using Platinum Hi-Fi Taq polymerase (Invitrogen). Oligonucleotide primers used for RT-PCR: TMEM70F 5´-GTC TCG CAG TCG TGG ACT C-3´ and TMEM70R 5´-TCA CTA ACG GAA TGC AAA AGG-3´ (924 bp); TMEM70ex1Fnew 5´-AGC TCA GCT GGC GGA TAA C-3´ and TMEM70R (1132 bp). Genomic DNA PCR primers: TMEM70 Exon 1: TMEM70PromF 5´-TTT CAG GTA TTT TCC TCC ACT G-3´ and TMEM70Rnew 5´-GTA AGC ACT TTC CAG CCG TTC-3´ (1158 bp); TMEM70 intron 2 and exon 3 was amplified using TMEM70int2F 5´-GCT GAT ACA GTA AGA AGA AAA ATG GA-3´ and TMEM70R (649 bp). PCR products were sequenced directly by fluorescent sequencing methods (ACGT Corporation, Toronto). 2.4. Enzyme assays NADH:cytochrome c reductase (CI + III), citrate synthase, succinate cytochrome c reductase (CII + III) and cytochrome oxidase (CIV) enzyme activities were measured as described (Robinson et al., 1990). 2-14C pyruvate whole cell oxidation and ATP synthesis using various oxidative substrates were also measured (Robinson et al., 1983), (Robinson, 1996). 2.5. Immunoblotting Mitochondria were prepared from fibroblasts (Pitkanen et al., 1996) and 25 μg resolved through a 12.5% stacking SDS/PAGE gel. Membranes were probed with anti-human citrate synthase antibodies (raised in rabbit) or CV antibodies OSCP, delta and F1α (Mitosciences, OR, USA). Immunoreactive proteins were visualized using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer).
2.6. Blue native and clear native gel electrophoresis Blue native gels were prepared using 50 μg fibroblast mitochondria and immunoblotted with F1α antibody (Pitkanen et al., 1996; Xu et al., 2008). High resolution clear native gels and in-gel enzyme activities for CI and CV (ATP hydrolysis) were conducted using 200 μg fibroblast mitochondria (Wittig et al., 2007). 2.7. Electron microscopy and tomography Muscle tissue was minced into mm3 pieces and fixed in 4% paraformaldehyde containing 1% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 for 2–4 h. Tissues were postfixed in 2% osmium tetroxide, dehydrated in acetone and embedded in Embed 812-araldite. Ultrathin sections were cut from these tissues, mounted on grids and stained in uranyl acetate and lead citrate prior to examination in a JEOL JEM 1400 transmission electron microscope (JEOL USA, Peabody, MA). Images were recorded with a CCD camera (AMT Corp., Danvers, MA). Tilt series were collected (±70°), objects of interest aligned and tomograms rendered using a tomography program (Tomography, JEOL System Technology Ltd., Akishima, Japan). 2.8. Immunogold labelling Muscle tissue was fixed in 4% paraformaldehyde, 0.1 M phosphate buffer (pH 7.4) containing 0.1% glutaraldehyde (minimum 4 h) and washed several times with phosphate buffer. They were stored at 4 °C in PBS/20 mM sodium azide. Prior to cryoultramicrotomy, the cells were infused overnight in 2.3 M sucrose. The tissues were frozen in liquid nitrogen on aluminum pins and sections cut on a diamond knife in a cryoultramicrotome (Ultracut R, Leica Canada, Willowdale, ON). Sections were transferred to formvar nickel coated grids in a loop of molten sucrose. The grids were blocked in PBS/0.5% BSA/0.15% glycine. After several rinses in PBS/BSA the sections were incubated in antibody diluted 1:10 in PBS for 1 h. After rinsing the specimens were then incubated with 10 nm gold goat anti-rabbit IgG particles (Amersham, Oakville, ON) for 1 h, rinsed in PBS and distilled water and stabilized in a thin layer of methyl cellulose containing 0.2% uranyl acetate. CIV COXI, CII 70 kDa (SDHA), CV F1α (Mitosciences, Eugene, OR, USA) and anti-DNA monoclonal antibodies (clone AC-3010, Progen Biotechnik, Germany) were used. Controls omitted either primary or secondary antisera. Grids were examined in a transmission electron microscope, and images captured using CCD camera (AMT corp. Danvers, MA). Gold particle density determination was done using Image J (NIH, Bethesda, MD), a morphometry program. Individual particles were counted per linear μm of outer mitochondrial membrane, and inside the mitochondria (particles/μm2). Clusters of mitochondria were also counted (defined as two or more particles in close proximity). For each antibody probing, 250 mitochondria were examined (10 mitochondria from 25 cells). Data was expressed as a mean and standard error. A student's t test was used to determine significance. 3. Results Mutations in TMEM70 were first identified as a cause of ATP synthase deficiency in Roma Gypsies by linkage analysis and homozygosity mapping (Cizkova et al., 2008). Our patient showed a clinical resemblance, with hypertrophic cardiomyopathy and 3methylglutaconic aciduria. Respiratory chain enzyme measurements from fibroblasts and muscle showed a profound reduction in ATPase activity (5% of control mean in muscle) (Table 1). Amplification of MTATP6 and MT-ATP8 showed no mutations. Amplification of the entire coding sequence of the TMEM70 gene from patient fibroblast cDNA identified an apparent homozygous mutation, c.118_119insGT, in exon 1. Based on the genetic diagnosis
Please cite this article as: Cameron, J.M., et al., Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization, Mitochondrion (2010), doi:10.1016/j.mito.2010.09.008
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Table 1 Patient and control enzyme measurements from fibroblasts and muscle. Fibroblast enzyme activities (nmol/mg/min)
Patient Control
Succinate cytochrome c reductase (CII + III)
Cytochrome oxidase (CIV)
ATPase (oligomycin sensitive) (CV)
Citrate synthase
Lactate/pyruvate
Lactate
7.88 ± 0.91 (3)a 5.63 ± 0.50 (3)
8.42 ± 0.76 (3) 5.08 ± 0.06 (3)
123.02 ± 1.17 (2) 422.27 ± 15.64 (5)
72.99 (1) 52.6 ± 4.55 (3)
17.8 ± 3.3 (5) 18.3 ± 3.1 (5)
322.61 (2) 230.62 (1)
Muscle enzyme activities (nmol/mg/min)
Patient Control Control range a
NADH cytochrome c reductase (rotenone sensitive) (CI + III)
Succinate cytochrome c reductase (CII + III)
Cytochrome oxidase (CIV)
ATPase (oligomycin sensitive) (CV)
Citrate synthase
45.76 (1) 94.6 ± 9.5 (17) 45–170
68.64 (1) 102.0 ± 6.9 (17) 56–162
160.16 (1) 119.0 ± 11.3 (17) 51–244
42.46 (1) 849.0 ± 152.0 (8) 394–1750
534.29 (1) 506.5 ± 31.5 (19) 335–767
Values are ± S.E.M. Number of replicates are shown in parentheses.
of previously published patients with similar clinical history, we sequenced intron 2 from the patient's gDNA and identified a second heterozygous mutation at c.317-2A N G. The apparent homozygosity seen at first was caused by an amplification bias towards the mutant allele. This mutation would be predicted to result in a truncated protein due to the creation of a premature stop codon, p.Ser40Cys.fs. X11. The mother carried the heterozygous c.317-2A N G mutation in intron 2, and the father carried the c.118_119insGT, in exon 1 (Fig. 1). As noted with the proband, there is a strong bias towards amplification of the mutant allele harbouring the c.118_119insGT mutation, and so the normal allele could only be identified by
amplifying from genomic DNA using primers that amplified a larger genomic region: TMEM70-PromF and TMEM70-Rnew (1158 bp). A sibling was tested prenatally and found to have no mutations in TMEM70. Respiratory chain enzyme measurements were made from fibroblasts and muscle, and a complex V (ATPase) defect of 30% and 5% compared to mean control rates was noted respectively (Table 1). Complex V in-gel activity was also carried out on High Resolution Clear Native gels, and negligible ATPase activity was seen in the patient by this method (Fig. 2A). ATP synthesis measurements using digitonin-permeabilized fibroblasts showed a reduction in ATP production with all substrates provided (Fig. 2B). 2-14C pyruvate
Fig. 1. Sequence chromatograms are shown for gDNA regions of TMEM70 flanking the mutations. The mother is heterozygous for c.118_119insGT, and the father is heterozygous for c.317-2A N G. The proband is compound heterozygous for both mutations, and the unaffected sibling has neither.
Please cite this article as: Cameron, J.M., et al., Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization, Mitochondrion (2010), doi:10.1016/j.mito.2010.09.008
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OSCP is in the peripheral stalk connecting the two domains (Fig. 3A). This is corroborated by Blue Native gels, in which the native assembly of complex V is almost undetectable (Fig. 3B). This suggests that the low ATPase activity can be directly attributed to decreased levels of fully assembled complex V enzyme. Electron micrographs of the patient's muscle showed abnormal shaped mitochondria, with circular ‘onion-ring’ cristae and voids in the mitochondrial matrix (Fig. 4A). Electon microscopy tomograms from normal controls show that cristae originate from the inner mitochondrial membrane (not shown), a process which is absent in our patient (Fig. 4B to C). The patient showed apparent whorls of cristae, which in 3-dimensions are the manifestation of a “rolled sock” conformation of the inner mitochondrial membrane (Fig. 4D). Some mitochondria looked relatively normal, with the cristae showing little or no apparent points of origin from inner membrane, suggesting that the cristae because of disorganized folding appeared to have a freefloating existence within the matrix. The mutant mitochondria often have vacant spaces visible. We undertook immunogold labelling in order to define more precisely the distribution of complexes within mitochondria. This was conducted using F1α (a subunit of complex V), COXI (a subunit of complex IV) and SDHA (a subunit of complex II) antibodies. The F1α antibody showed a profound reduction in labelling in the patient muscle compared to the control (Fig. 5A to D and Table 2). COXI labelling on the other hand showed a reduction in number of immunogold particles in the patient compared to the control, with a
Fig. 2. A. 200 μg fibroblast mitochondria was electrophoresed through high resolution clear native gels, and in-gel enzyme activities for complex V (ATP hydrolysis) and complex I were conducted. Ferritin is used as a protein standard, and beef heart mitochondria to show the expected sizes of the five respiratory complexes. B. ATP synthesis assay: digitonin-permeabilized fibroblasts were incubated with various substrates for 1 h, and the amount of ATP produced measured. 4 replicates are shown for the patient, and controls consist of up to 3 replicates each from 8 different controls (n = 15). C. 2-14C pyruvate whole cell oxidation rates are shown for patient (n = 6) and control fibroblasts (n = 4). Incubations were carried out with 10 μg/ml oligomycin for patient (n = 3) and control (n = 3); and with 0.2 mM dinitrophenol for patient (n = 2) and control (n = 2).
whole cell oxidation measurements in fibroblasts showed a low rate compared with controls with no sensitivity to oligomycin. Dinitrophenol, an uncoupler of respiration from ATP synthesis, displays a normal increase in oxidation rate in both patient and control cells (Fig. 2C). Western blots showed a loss of all Complex V subunits in patient mitochondria: subunits α and δ are in the F1 domain, and subunit
Fig. 3. A. Western blots of 25 μg fibroblast mitochondria were immunoblotted with complex V F1α, δ and OSCP antibodies, and citrate synthase. B. blue native gels of 50 μg of lauryl-maltoside solubilized fibroblast mitochondria were immunoblotted with complex V F1α antibody, showing the native assembled complex V at ~ 600 kDa.
Please cite this article as: Cameron, J.M., et al., Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization, Mitochondrion (2010), doi:10.1016/j.mito.2010.09.008
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Fig. 4. A. electron micrograph of skeletal muscle showing abnormal mitochondrial ultrastructure. Black arrows identify free-floating cristae. White arrows identify vacant space devoid of cristae. Scale bar represents 500 nm. B,C. electron tomograms of skeletal muscles from patient. Scale bars represent 1.25 μm and 250 nm. D. cartoon representation of a normal mitochondrion showing outer mitochondrial membrane and inner membrane invaginated to form cristae, and a representation of the TMEM70 mutant mitochondrion shown in C. The cristae form a single whorl, with no connectivity, and vacant spaces.
different localization. The gold particles in the control had a distribution throughout the cristae as expected. However, in the patient, COXI labelling was limited to the immediate periphery of the inner membrane, excluding the cristae and the interior of the mitochondria (Fig. 6A, B).This was especially prominent in mitochondria with concentric whorl configurations. The reduction in the number of particles could be an artifact due to the planar nature of the EM sections, since the enzyme data does not suggest any reduction in complex IV activity (Table 2). Anti-mtDNA antibody labels any DNA in the cell, thus labelling in the nucleus and also mtDNA. In normal muscle, discrete clusters of gold particles are seen in the mitochondria, representing the nucleoids (Fig. 7A, B). These are structures in which the multiple copies of mtDNA and various binding proteins colocalize (Spelbrink, 2010). In the patient mitochondria the single gold particles are located at the superficial peripheral ring of the inner mitochondrial membrane (Fig. 7C and Table 2). This is corroborated by immunogold particle counting, which demonstrates an increase in mtDNA labelling on the periphery of the mitochondria, with a concomitant decrease in the number of internal particles. Similarly, the number of clusters of particles is reduced. Complex II is the only enzyme in the respiratory chain that has no subunits encoded by
mitochondrial DNA. Therefore all are derived from nuclear DNA. Antibody against the SDHA subunit of complex II showed that the number of immunogold particles and the distribution of this enzyme complex was similar in both control and patient, and unlike complex IV which was restricted to the periphery of the mitochondria, complex II showed an even distribution throughout the mitochondria (Fig. 6C, D, E and Table 2). 4. Discussion The role of TMEM70 in ATP synthase assembly is unknown. Both ATP12 and TMEM70 deficiency diseases present with similar clinical symptoms, but the mutations published so far for TMEM70 and ATP12 (ATPAF2), suggest that ATP12 deficiency is fatal at an earlier age. Both assembly defects show low ATP synthase content by BN-PAGE, accompanied by low ATP synthase hydrolysis and synthesis activities. In some TMEM70 patients, sub-assembly products of complex V (specifically the disassociated F1 head) can be detected in conjunction with low levels of fully assembled complex using BN-PAGE gels. This was not the case for our patient, but could be due to the detection threshold of ECL detection. In contrast with assembly defects,
Please cite this article as: Cameron, J.M., et al., Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization, Mitochondrion (2010), doi:10.1016/j.mito.2010.09.008
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Fig. 5. A, B. F1α (complex V) immunogold labelling of control muscle mitochondria (scale bars represent 250 nm). C, D. F1α immunogold labelling of patient muscle mitochondria (scale bars represent 200 nm and 250 nm).
mitochondrial causes of ATP synthase deficiency (usually MT-ATP6) often have decreased complex V catalyzed ATP hydrolysis activities in whole mitochondria but not in broken mitochondria. This happens with no decrease in total amount of the assembled enzyme, and rates of ATP synthesis rates are 60–95% of normal depending on heteroplasmy. Such MT-ATP6 defects can manifest as separate diseases of differing severity depending on the mutation, and the level of mutant heteroplasmy; NARP (Neuropathy, Ataxia, Retinitis Pigmentosa) is Table 2 Immunogold particle counting from EM sections probed with COXI, SDHA, F1α and DNA antibodies. Patienta COXI (Complex IV) SDHA (Complex II) F1α (Complex V) anti-DNA: Particles/linear μm of outer membrane anti-DNA: internal particles (particles/μm2) anti-DNA: clusters/mitochondria a b c d e f g
Control b
2.7 ± 0.5 11 ± 2.7c 0.3 ± 0.05d 8 ± 1.2e 0.2 ± 0.08f 0.1 ± 0.038g
n = 10 mitochondria from 25 cells (250 mitochondria). p N 0.001 between patient and normal (COXI). Not significant between patient and normal (SDHA). p N 0.001 between patient and normal (F1α). p N 0.005 between outer membrane patient and normal. p N 0.001 between internal particles patient and normal. p N 0.002 between clusters patient and normal.
16 ± 3.9 12 ± 1.9 18 ± 4.5 3.2 ± 0.9 16 ± 3.2 2.8 ± 0.4
seen at mutant levels less than 85%, and MILS (Maternally Inherited Leigh Syndrome) is seen at higher mutant levels. MT-ATP6 mutations affects the complex's ability to transport protons from the F0 stalk to the F1 head (Tatuch et al., 1992; Tatuch et al., 1994) preventing transduction of energy from the proton electrochemical gradient to occur normally. This mutation appears to disturb the structural integrity of the complex at the point where the F1 head interacts with F0. The patient in this study showed a severe decrease in oligomycin sensitive ATPase in both fibroblast and muscle mitochondria to 30% and 5% of normal. Complete ablation of ATP hydrolysis activity would not be compatible with life, and it is well known that patients can survive with moderate defects in ATP synthesis. In this patient, ATP synthesis in digitonin-permeabilized fibroblasts was possible at rates of 60–70% of normal. This is in keeping with the T8993G mutation in lymphoblasts in which a 35% decrease in ATPase was responsible for a 65% rate of ATP synthesis in mitochondria from NAD-linked substrates (Tatuch et al., 1994). The patient reported here also showed increases in CII + III, CIV and CS enzyme rates in fibroblasts, and an increase of CIV and CS rates in muscle, all of which can be interpreted as compensatory increases due to the primary CV defect (Table 1). The patient described here only differs from other published TMEM70 deficient patients in that there was no hyperammonemia (Cizkova et al., 2008). The pyruvate whole cell oxidation rate in our patient was lower than control, but was normalized by addition of uncoupling agent 2,4dinitrophenol (Fig. 2C). The patient's fibroblasts also showed little sensitivity to oligomycin suggesting that oxidation was slow and not well coupled. The oxidation rate was therefore only limited while coupled to ATP synthase and showed stimulation with uncoupling agent. Oligomycin is thought to act at subunit a in the F0 domain, encoded by the MT-ATP6 gene. Several mutations in this subunit have been associated with resistance to oligomycin (two in yeast (John and Nagley, 1986); one in hamster (Breen et al., 1986); T8993G in humans shows increased oligomycin sensitivity when grown in medium with galactose as the only carbon source (Manfredi et al., 1999). All these mutations were within the fourth transmembrane domain of the ATP6 protein. Rho0 cells containing no mitochondrial DNA, and therefore no subunits a or A6L (encoded by MT-ATP8), show normal ATP hydrolysis and low ATP synthesis rates, both of which are insensitive to oligomycin (Garcia et al., 2000). Human ATP12 mutant cells show decreased content of fully assembled complex V and no sub-assembly products (De Meirleir et al., 2004; Houstek et al., 1999). In contrast, patients with MT-ATP6 and MT-ATP8 mutations show three sizes of complex V in muscle mitochondria: fully assembled 600 kDa (less than control amounts), as well as 460 kDa and 390 kDa subassembly complexes(Carrozzo et al., 2006; Houstek et al., 1995; Nijtmans et al., 2001). Rho0 cells have no fully assembled complex V, and only the two smaller complexes (Nijtmans et al., 2001). The intermediate subassembly complexes of 390 kDa and 460 kDa, as well as some others, can be seen in patients with various ATP synthase defects. Mitochondrial ultrastructure is modified in a number of genetic defects of both nuclear and MtDNA origin, such as those involved in maintaining outer membrane shape, mitochondrial segregation, fusion/ fission proteins and defects in ATP synthase assembly. It is estimated that ATP synthase constitutes 17–30% of the inner membrane-bound proteins and as such is important for maintaining the integrity of the inner mitochondrial membrane, specifically the formation of the invaginations that form the cristae (Ozawa and Asai, 1973). Mutant yeast cells lacking ATP synthase subunits have been studied using electron microscopy, and strikingly different mitochondrial morphological characteristics can be associated with them. The morphology of mutants ranges from ‘onion ring’-like structures (similar to TMEM70 patients) and large mitochondrial digitations surrounding other organelles. The inner mitochondrial membrane is still present in
Please cite this article as: Cameron, J.M., et al., Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization, Mitochondrion (2010), doi:10.1016/j.mito.2010.09.008
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Fig. 6. A. COXI (complex IV) immunogold labelling of control muscle mitochondria (scale bar represents 250 nm). B. COXI immunogold labelling of patient muscle mitochondria (scale bar represents 200 nm). C. SDHA (complex II) immunogold labelling of control muscle mitochondria (scale bar represents 500 nm). D,E. SDHA (complex II) immunogold labelling of patient muscle mitochondria (scale bars represent 500 nm).
these structures, as immunogold labelling with F1α antibodies identifies the presence of mutant ATP synthase within these structures. Human rho0 cells have small spherical mitochondria with no cristae, and sometimes onion-like structures (Yotsuyanagi, 1962). In contrast, yeast mitochondria with defects in other respiratory chain components such as apocytochrome b and COX, are normal looking, with less cristae (Yotsuyanagi, 1988). No electron micrographs of human ATP12 mutant cells have been published, but mitochondria from yeast deficient of atp12p are small with no cristae with abnormal electron-dense particles in the mitochondrial matrix. Atp12p is the chaperone for F1α subunits, and as a consequence of its loss, both α and β subunits accumulate and aggegate. The electron-dense particles stain with both F1α and β antibodies, suggesting that the subunits co-localize within the inclusion bodies (Lefebvre-Legendre et al., 2005). It is interesting therefore in comparison that TMEM70 patients do not have any such inclusion bodies, and F1α antibody labelling suggests there are no subunits within the mitochondria. It is possible that the F1 head is assembling in these cases (as suggested by 390 kDa band in Blue Native gels in some patients), but is not F1α-immunoreactive possibly because the number of F1 head domains is so low. Both the F1 and F0 subunits are highly susceptible to proteolytic degradation when unassembled.
The mitochondrial ultrastructure seen in the patient, consisting primarily of concentric whorls of cristae, is not unique to defects in TMEM70. The phenotype is most similar to that seen in yeast cells lacking complex V subunits 4, e or g. These subunits are all present in the peripheral stalk of the ATPase. Subunits e and g facilitate dimer formation of ATPase complexes (Arnold et al., 1998), and it is the rows of dimer ribbons located on the apex of cristae that is thought to produce the steep curvature that appear as cristae invaginations (Strauss et al., 2008). There are phenotypic distinctions between these mutants, as the yeast mitochondria lacking specific complex V subunits appear to only have a few ‘layers’ of onion rings, whereas the TMEM70 patient has continuous concentric layers right to the centre of the mitochondria (see also (Holme et al., 1992)). Rho0 cells also have some ‘onion ring’ mitochondria, but again, the rings are not so densely packed. Another protein defect that produces cristae whorls when defective is mitofilin (IMMT), a protein of unknown function (John et al., 2005). This protein forms a complex with ATPase, which may suggest the mechanism by which its phenotype is manifested (Mun et al., 2010). We suggest therefore that the lack of cristae invaginations in TMEM70 defective patients is a consequence of the loss of ATPase integrity in the inner mitochondrial membrane. Mammals may contain hundreds of nucleoids per cell, each containing several copies of mtDNA (Spelbrink, 2010). Anti-DNA
Please cite this article as: Cameron, J.M., et al., Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization, Mitochondrion (2010), doi:10.1016/j.mito.2010.09.008
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the nucleoids, where mtDNA accumulates with DNA-binding proteins. Complex II has no requirement for mtDNA and so its distribution within the mitochondria is unaffected by the loss of nucleoids. The abnormal concentric whorls of cristae are therefore still able to accommodate respiratory chain complexes in normal numbers, but those requiring mtDNA synthesized proteins are only present around the outer perimeter of the inner mitochondrial membrane where mtDNA is still associated in nucleoids. Complex II has no such restraints and thus its distribution remains unchanged. The fact that nucleoids and complex IV are not present in the inner whorls of the patient mitochondria suggests there are further factors affecting their distribution and limiting the nucleoid tethering to the outer edges of the inner mitochondrial membrane. MtDNA replication and transcription do not appear to be affected in the patient, as the number of nucleoids does not appear to have altered, merely the distribution. The patient does not show a loss of complex IV activity, even though the spatial distribution of the complex is grossly affected. A reduction in complex IV subunits is seen in yeast mutants which have no cristae at all, such as defects of F1α, β, ATP11 or ATP12(Lefebvre-Legendre et al., 2005) or loss of subunit a (MT-ATP6) in which there are cristae, but they are deformed(Rak et al., 2007). 5. Conclusions We have shown for the first time that the concentric whorled cristae seen in the ultrastructure of the mutant mitochondria in TMEM70 defective patients, suggests that loss of ATP synthase compromises not just mitochondrial morphology but nucleoid assembly with a concomitant disruption of mitochondrial function. In future studies it would be interesting to see where TMEM70 localizes in the normal mitochondria; its proposed roles in complex V assembly suggest a transient binding to the complex and therefore some labelling might be noted in the mitochondrial inner membranes and cristae. Acknowledgements We thank “MitoMarch for Kirkland” for funding. Fig. 7. A, B. anti-DNA immunogold labelling of control muscle mitochondria (left) and nucleus (right) (scale bars represent 500 nm and 100 nm). C. anti-DNA immunogold labelling of patient muscle mitochondria (scale bar represents 200 nm).
antibodies reveal mtDNA as punctate foci, and antibodies to some mitochondrial proteins such as POLG show proteins co-exist in the nucleoids (Spelbrink, 2010). For example, the E2 subunit of pyruvate dehydrogenase and α-keto dehydrogenase PDH complexes has been found associated with nucleoids (Bogenhagen et al., 2008; Wang and Bogenhagen, 2006), and the complexes have been shown by immunofluorescence to be distributed in discrete localized areas of the mitochondria, in contrast to complex IV which is distributed continuously throughout the mitochondrial filaments (Margineantu et al., 2002). It is therefore interesting, that the pattern of immunogold labelling of mtDNA in the TMEM70 patient suggest that the punctate labelling in the nucleoids has been lost. Instead labelling only occurs at the periphery of the mitochondria, excluding the inner cristae entirely, even though cristae whorls extend into the centre of the mitochondria. COXI shows a similar labelling pattern which is not shared by SDHA. OXPHOS complexes are normally dispersed throughout the inner mitochondrial membrane, with the majority of labelling in the cristae and less than 7% in the inner boundary membrane (Gilkerson et al., 2003). This suggests that respiratory chain complexes which require mitochondrial DNA encoded subunits are assembled close to
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