A mutation in the human CBP4 ortholog UQCC3 impairs complex III assembly, activity and cytochrome b stability.

Complex III (cytochrome bc1) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c. Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83, hereafter named UQCC3, to be the ortholog of the fungal complex III assembly factor CBP4. We describe a homozygous c.59T>A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T>A mutation has a causal role in complex III deficiency.

Mutations in the UQCC1-interacting protein, UQCC2, cause human complex III deficiency associated with perturbed cytochrome b protein expression.

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.

A mutation in the FAM36A gene, the human ortholog of COX20, impairs cytochrome c oxidase assembly and is associated with ataxia and muscle hypotonia.

The mitochondrial respiratory chain complex IV (cytochrome c oxidase) is a multi-subunit enzyme that transfers electrons from cytochrome c to molecular oxygen, yielding water. Its biogenesis requires concerted expression of mitochondria- and nuclear-encoded subunits and assembly factors. In this report, we describe a homozygous missense mutation in FAM36A from a patient who displays ataxia and muscle hypotonia. The FAM36A gene is a remote, putative ortholog of the fungal complex IV assembly factor COX20. Messenger RNA (mRNA) and protein co-expression analyses support the involvement of FAM36A in complex IV function in mammals. The c.154A>C mutation in the FAM36A gene, a mutation that is absent in sequenced exomes, leads to a reduced activity and lower levels of complex IV and its protein subunits. The FAM36A protein is nearly absent in patient's fibroblasts. Cells affected by the mutation accumulate subassemblies of complex IV that contain COX1 but are almost devoid of COX2 protein. We observe co-purification of FAM36A and COX2 proteins, supporting that the FAM36A defect hampers the early step of complex IV assembly at the incorporation of the COX2 subunit. Lentiviral complementation of patient's fibroblasts with wild-type FAM36A increases the complex IV activity as well as the amount of holocomplex IV and of individual subunits. These results establish the function of the human gene FAM36A/COX20 in complex IV assembly and support a causal role of the gene in complex IV deficiency.

C7orf30 specifically associates with the large subunit of the mitochondrial ribosome and is involved in translation.

In a comparative genomics study for mitochondrial ribosome-associated proteins, we identified C7orf30, the human homolog of the plant protein iojap. Gene order conservation among bacteria and the observation that iojap orthologs cannot be transferred between bacterial species predict this protein to be associated with the mitochondrial ribosome. Here, we show colocalization of C7orf30 with the large subunit of the mitochondrial ribosome using isokinetic sucrose gradient and 2D Blue Native polyacrylamide gel electrophoresis (BN-PAGE) analysis. We co-purified C7orf30 with proteins of the large subunit, and not with proteins of the small subunit, supporting interaction that is specific to the large mitoribosomal complex. Consistent with this physical association, a mitochondrial translation assay reveals negative effects of C7orf30 siRNA knock-down on mitochondrial gene expression. Based on our data we propose that C7orf30 is involved in ribosomal large subunit function. Sequencing the gene in 35 patients with impaired mitochondrial translation did not reveal disease-causing mutations in C7orf30.

BOLA1 is an aerobic protein that prevents mitochondrial morphology changes induced by glutathione depletion.

AIMS: The BolA protein family is widespread among eukaryotes and bacteria. In Escherichia coli, BolA causes a spherical cell shape and is overexpressed during oxidative stress. Here we aim to elucidate the possible role of its human homolog BOLA1 in mitochondrial morphology and thiol redox potential regulation. RESULTS: We show that BOLA1 is a mitochondrial protein that counterbalances the effect of L-buthionine-(S,R)-sulfoximine (BSO)-induced glutathione (GSH) depletion on the mitochondrial thiol redox potential. Furthermore, overexpression of BOLA1 nullifies the effect of BSO and S-nitrosocysteine on mitochondrial morphology. Conversely, knockdown of the BOLA1 gene increases the oxidation of mitochondrial thiol groups. Supporting a role of BOLA1 in controlling the mitochondrial thiol redox potential is that BOLA1 orthologs only occur in aerobic eukaryotes. A measured interaction of BOLA1 with the mitochondrial monothiol glutaredoxin GLRX5 provides hints for potential mechanisms behind BOLA1's effect on mitochondrial redox potential. Nevertheless, we have no direct evidence for a role of GLRX5 in BOLA1's function. INNOVATION: We implicate a new protein, BOLA1, in the regulation of the mitochondrial thiol redox potential. CONCLUSION: BOLA1 is an aerobic, mitochondrial protein that prevents mitochondrial morphology aberrations induced by GSH depletion and reduces the associated oxidative shift of the mitochondrial thiol redox potential.

NDUFB7 and NDUFA8 are located at the intermembrane surface of complex I.

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest protein complex of the oxidative phosphorylation. Crystal structures have elucidated the positions of most subunits of bacterial evolutionary origin in the complex, but the positions of the eukaryotic subunits are unknown. Based on the analysis of sequence conservation we propose intra-molecular disulfide bridges and the inter-membrane space localization of three Cx(9)C-containing subunits in human: NDUFS5, NDUFB7 and NDUFA8. We experimentally confirm the localization of the latter two, while our data are consistent with disulfide bridges in NDUFA8. We propose these subunits stabilize the membrane domain of complex I.

A role for the Rab6B Bicaudal-D1 interaction in retrograde transport in neuronal cells.

The Rab6 subfamily of small GTPases consists of three different isoforms: Rab6A, Rab6A' and Rab6B. Both Rab6A and Rab6A' are ubiquitously expressed whereas Rab6B is predominantly expressed in brain. Recent studies have shown that Rab6A' is the isoform regulating the retrograde transport from late endosomes via the Golgi to the ER and in the transition from anaphase to metaphase during mitosis. Since the role of Rab6B is still ill defined, we set out to characterize its intracellular environment and dynamic behavior. In a Y-2H search for novel Rab6 interacting proteins, we identified Bicaudal-D1, a large coiled-coil protein known to bind to the dynein/dynactin complex and previously shown to be a binding partner for Rab6A/Rab6A'. Co-immunoprecipitation studies and pull down assays confirmed that Bicaudal-D1 also interacts with Rab6B in its active form. Using confocal laser scanning microscopy it was established that Rab6B and Bicaudal-D1 co-localize at the Golgi and vesicles that align along microtubules. Furthermore, both proteins co-localized with dynein in neurites of SK-N-SH cells. Live cell imaging revealed bi-directional movement of EGFP-Rab6B structures in SK-N-SH neurites. We conclude from our data that the brain-specific Rab6B via Bicaudal-D1 is linked to the dynein/dynactin complex, suggesting a regulatory role for Rab6B in the retrograde transport of cargo in neuronal cells.