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

Mutations in the UQCC1-Interacting Protein, UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression

Contains fulltext : 125692.pdf (publisher's version ) (Open Access)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

Item does not contain fulltextThe 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

Proposed model of CIII assembly.

<p>Complex III assembly begins with the translation activation and/or stabilization of cytochrome <i>b</i> (MTCYB) by UQCC1:UQCC2, which then delivers MTCYB to an assembly intermediate containing UQCRQ and UQCRB. This module combines with a module containing CYC1, UQCRH and UQCR10 and a module containing UQCRC2 and UQCRC1. The resulting subcomplex then dimerizes. UQCRFS1 is bound and stabilized by the CIII assembly factor LYRM7, before being incorporated into CIII with the aid of the assembly factor, BCS1L. Finally UQCR11 is added, forming the complete CIII₂. Assembly factors are indicated in gray. Proteins in which mutations are associated with complex III deficiency are bordered in red. The role of TTC19 is yet to be elucidated, although it is likely to be involved in early complex III assembly. Model adapted and updated from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034-FernandezVizarra2" target="_blank">[67]</a>.</p

Depletion of the UQCC2 binding partner, UQCC1, affects complex III assembly.

<p>(A) SDS-PAGE and western blot analysis of mitochondrial extracts from HEK293 cells transfected with <i>UQCC1</i> siRNA shows lower levels of complex III subunits UQCRFS1, UQCRC1 and UQCRC2. Subunits of complex I (ND1), complex II (SDHA), complex IV (COX1) and complex V (ATP5α) are not affected by <i>UQCC1</i> knockdown. (B) BN-PAGE of HEK293 cells transfected with UQCC1 siRNA show reduced levels of holocomplex III (UQCRC2) and a mild effect on complex I in gel activity (IGA) and complex I holocomplex levels (NDUFA9). Levels of other OXPHOS complexes, complex II (SDHB), complex IV (COX2) and complex V (ATP5α) are not affected. Mock transfected cells were used as control). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004034#pgen.1004034.s007" target="_blank">Figure S7B</a>-C for the quantification of the immunoreactive bands. (C) 2D BN-PAGE of HEK293 cells depleted of UQCC1 or cyclophilin B with indicated antibodies. The holocomplex III dimer is indicated with a line labeled CIII₂. To the right are lower molecular weight subcomplexes: UQCRC1-containing subcomplex (1) and, likely, monomeric UQCRFS1 (2). Lauryl maltoside was used to solubilize OXPHOS complexes in parts B and C. (D) Respiratory chain enzyme activity measurements of HEK293 cells transfected with <i>UQCC1</i> and <i>cyclophilin B</i> siRNAs. Mock transfected cells were set at 100%. Error bars indicate one standard deviation. Complex I ubiquinone reducing part (CI-Q), complexes II–V (CII-V) and combined activity of complex II and III (SCC) were measured relative to the activity of citrate synthase (CS).</p

UQCC2 and UQCC1 are involved in cytochrome <i>b</i> translation and/or stability.

<p>(A) SDS-PAGE analysis of ³⁵S-methionine-labeled mtDNA-encoded proteins in patient fibroblasts shows a lack of cytochrome <i>b</i> (MTCYB) protein (even at zero hours chase) suggesting a defect in cytochrome <i>b</i> synthesis or its immediate stability. (B) qRT-PCR shows normal expression of <i>cytochrome b</i> (<i>MTCYB</i>) mRNA in patient fibroblasts. (C) Autoradiogram of single step affinity purified UQCC1-TAP with ³⁵S metabolically labeled mitochondrial translation products shows UQCC1 specifically associates with newly synthesized cytochrome <i>b</i> in HEK293 cells. (D) Inhibition of mitochondrial translation in HEK293 cells results in diminished levels of UQCC1, UQCC2, mtDNA-encoded COX1, but does not affect the SDHA subunit of the nuclear encoded complex II.</p

MitoExome sequencing identifies a homozygous mutation in <i>UQCC2</i> in a patient with complex III deficiency.

<p>(A) The activity of complexes I–IV (CI-IV) as measured by spectrophotometric analysis and normalized to the activity of citrate synthase (CS), expressed as a percentage of control. Values are the average of duplicate assays. (B) Prioritization of single nucleotide variants (SNVs) and small insertion/deletions (indels) identified by MitoExome MPS. (C) Sequence chromatograms of <i>UQCC2</i> in control and patient gDNA validating the c.214-3C>G mutation detected by MitoExome sequencing.</p

<i>UQCC2</i> mutations are responsible for the complex III defect in P^UQCC2.

<p>Fibroblasts from Control, P^UQCC2 with mutations in <i>UQCC2</i> and P^CONTROL with mutations in a complex III subunit gene (and no <i>UQCC2</i> mutation) were transduced with wild-type <i>UQCC2</i> mRNA. (A) Representative SDS-PAGE western blot shows reduced UQCC2 in P^UQCC2 and increased UQCC2 expression following <i>UQCC2</i> transduction. VDAC1 acts as a loading control. UQCRFS1 protein is reduced in both complex III deficient patients and restored in P^UQCC2, but not P^CONTROL, with <i>UQCC2</i> transduction. VDAC1 acts as a loading control. (B) The intensity of immunostained UQCRFS1 relative to VDAC1 before and after transduction with <i>UQCC2</i> was quantified by densitometry. Error bars indicate 1 s.e.m. for 3 independent transductions and the asterisk indicates p<0.05, two way ANOVA.</p