Residual enzyme activities of mitochondrial respiratory chain complexes in different tissues from the index patient.

<p>Enzyme activities are expressed as</p><p>*cU/U citrate synthase (CS) and</p><p>**nmol.min⁻¹.mg prot⁻¹. CS activity is expressed as mU/mg protein. Abnormal values are indicated in bold. nd, not determined. Complex I, CI; Complex II, CII; Complex III, CIII; Complex IV, CIV.</p

Genetic and structural analysis of the m.15533A>G mutation.

<p>(A) Electropherogram showing the nucleotide change in patient's muscle DNA, indicated by arrowheads. (B) PCR-RFLP analysis of the <i>MT-CYB</i> mutation. Uncut (wild-type) DNA consists of a 150 bp PCR product. The mutated sequence contains one <i>Dde</i>I restriction site that results in two products of 90 and 60 bp after digestion. The control sequence contains one <i>Mse</i>I restriction site that results in the same two products after digestion. C, wild-type control; F, proband's fibroblasts DNA; M, proband's muscle DNA; Cy, control cybrid; 1, 2 and 3 refer to three independent mutant cybrid clones. (C) Alignment of cytochrome b amino acid sequences from selected species. Asparagine at amino acid position 263 is indicated with an arrow. (D) Partial 3D-images of the cytochrome b protein. The left panel shows the interaction site between the asparagine 263 (indicated in red) of cytochrome b and the aspartate 2 of the cytochrome c1 subunit (coloured in green). This is the closest negatively charged side-chain of cytochrome c1, which is facing the same aqueous pocket at a distance of 11.25 Armstrongs. Asparagine at position 260 of cytochrome b is indicated in blue. The central panel shows the 3D-structure of the asparagine 263 (in blue) in controls. The right panel shows the predicted structural effect of the N263D substitution (in red) in the patient. The images were obtained using the Chimera software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012801#pone.0012801-Pettersen1" target="_blank">[20]</a>.</p

BN-PAGE analysis of mitochondrial respiratory chain complexes in control and mutant fibroblasts and cybrids.

<p>Mitochondrial particles were isolated as described in Methods and 40 µg of protein were analyzed on a 5–15% BN-polyacrylamide gel for the separation of multisubunit complexes. Western-blot analysis was performed using antibodies against the indicated OXPHOS subunits. CI, fully-assembled complex I. CIII₂, complex III dimer. CIV, complex IV. CIII₂+IV indicates the presence of the supercomplex containing complexes III and IV. C1 and C2, control fibroblasts. P, patient's fibroblasts. C, control cybrid. Two independent mutant cybrids are indicated as #1 and #2.</p

Assembly kinetics of respiratory chain complexes in control and mutant cybrids.

<p>Two different control cybrids belonging to haplogroup H, and two independent <i>MT-CYB</i> mutant clones were treated for 6 days with doxycycline (an inhibitor of mitochondrial translation), the medium was replaced by doxycycline-free medium and cells were collected at 0, 6, 15, 24, 48, 72, and 96 hours (indicated as t0–t16). SS indicates the steady-state expression levels of the respiratory chain complexes (A) Example of one control and one mutant clone. 40 µg of crude mitochondrial pellets were analyzed by BN-PAGE in combination with complex I and complex IV-IGA assays. (B) Duplicate gels were blotted and incubated with antibodies against the NDUFA9 complex I subunit, complex III core2 protein, complex IV COX5A subunit and complex II SDHA subunit. (C) The signals from the blots were quantified, expressed as percentage of the untreated cells (SS), normalized with the complex II SDHA subunit and plotted. The restoration curves constitute the mean values ± SD obtained from the two controls and the two independent mutant cybrids. Upper left panel, complex I assembly rates. Upper right panel, complex III assembly rates. Lower left panel, complex IV assembly rates. Lower right panel, supercomplex CIII₂+IV assembly kinetics.</p

Haplogroup affiliation and non-synonymous nucleotide changes of the mtDNA sequence from the index patient.

a<p>rCRS refers to the revised Cambridge reference sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012801#pone.0012801-Andrews1" target="_blank">[16]</a>, which belongs to haplogroup H2a.</p