Figure 1.
MitoExome sequencing identifies a homozygous mutation in UQCC2 in a patient with complex III deficiency.
(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 UQCC2 in control and patient gDNA validating the c.214-3C>G mutation detected by MitoExome sequencing.
Figure 2.
The c.214-3C>G mutation causes a severe UQCC2 splicing defect.
(A) Gel electrophoresis of full-length UQCC2 RT-PCR products from fibroblasts grown in the absence of cycloheximide. Two prominent bands are seen in PUQCC2 whereas only one is observed in the control. (B) Schematic diagram shows the wild-type (WT) mRNA structure and the two splice variants (1 and 2) observed in PUQCC2. (C) Sequence chromatograms of cloned RT-PCR products show that the upper product in PUQCC2 retains 108 bases of intronic sequence due to the use of a cryptic acceptor site, and that the lower product in PUQCC2 lacks 14 bases of exonic sequence due to the use of an alternative cryptic acceptor site. Splice site prediction scores are from Human Splicing Finder v2.4.1 (http://www.umd.be/HSF/). (D) qRT-PCR analysis using an assay that detects the exon 2/3 junction of UQCC2 (normalized to the endogenous control HPRT1) demonstrates PUQCC2 fibroblasts have only 2% wild-type UQCC2 expression relative to controls (C1–C4). PUQCC2(1) and PUQCC2(2) represent separate fibroblast subcultures.
Figure 3.
UQCC2 and UQCC1 are orthologous to the fungal complex III assembly factors Cbp6p and Cbp3p.
Alignment between fungal and human complex III assembly factors was inferred using iterative orthology pipeline Ortho-Profile [26] and visualized using JalView with the ClustalX color scheme [65]. (A) Alignment of conserved regions among the orthologs of human UQCC2 and fungal Cbp6p. The S. cerevisiae-specific insertion between residues 47 and 96 is replaced with a letter X. The sequences do not have a recognizable targeting signal or additional conserved motifs. Domains were annotated according to PFAM [66]. (B) Alignment of UQCC1 (human) and Cbp3p (yeast) with orthologs in other eukaryotes. Only the conserved part of the sequence is shown in the alignment. Proteins contain the UQCC1-specific domain PF03981. (C and D) Mouse mRNA co-expression of UQCC2 (C) and UQCC1 (D) with other genes across 91 murine cell types and tissues. Black bars represent genes encoding mitochondrial proteins and white bars represent the remaining human genes. Below the chart the co-expression values of complex III subunits are indicated with black dots.
Figure 4.
UQCC2 mutations are responsible for the complex III defect in PUQCC2.
Fibroblasts from Control, PUQCC2 with mutations in UQCC2 and PCONTROL with mutations in a complex III subunit gene (and no UQCC2 mutation) were transduced with wild-type UQCC2 mRNA. (A) Representative SDS-PAGE western blot shows reduced UQCC2 in PUQCC2 and increased UQCC2 expression following UQCC2 transduction. VDAC1 acts as a loading control. UQCRFS1 protein is reduced in both complex III deficient patients and restored in PUQCC2, but not PCONTROL, with UQCC2 transduction. VDAC1 acts as a loading control. (B) The intensity of immunostained UQCRFS1 relative to VDAC1 before and after transduction with UQCC2 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.
Figure 5.
Lack of UQCC2 is associated with aberrant complex III assembly, subunit expression and UQCC1 stability.
(A) BN-PAGE immunoblotting of mitochondria lysed in 1% Triton X-100, using antibodies against the NDUFA9 subunit of complex I, the SDHA subunit of complex II, the UQCRC1 subunit of complex III and the COX1 subunit of complex IV shows reduced complex III assembly in PUQCC2. See Figure S6A for quantification of immunoreactive bands. (B) SDS-PAGE and western blotting of mitochondrial lysates from patient fibroblasts demonstrate a marked deficiency of UQCC2 and UQCC1, a mild deficiency in the UQCRC2 subunit of complex III, and a more pronounced deficiency of the UQCRC1 and UQCRFS1 subunits of complex III. The PCONTROL cell line with mutations in a complex III subunit gene showed a similar profile of complex III subunit instability but had levels of UQCC2 and UQCC1 comparable to the wild-type control. The complex II subunit SDHB and mitochondrial VDAC1 protein act as loading controls. Vertical bars indicate immunoblots performed using the same membrane. See Figure S6A for quantification of immunoreactive bands. (C) Mitochondrial lysates of HEK293 cells transfected with siRNA targeting UQCC2 analyzed by SDS-PAGE and western blotting showed reduced levels of UQCC2 and UQCC1 proteins. As control, cyclophilin B knockdown and mock transfected cells were used. The asterisk indicates a non-specific, cross-reactive species. See Figure S7A for quantification of immunoreactive bands.
Figure 6.
UQCC2 interacts with mitochondrial protein UQCC1.
(A) SDS-PAGE analysis of HEK293 cellular fractions shows that UQCC1 is enriched in the mitochondrial fraction, similar to the mitochondrial protein TOM20. A cytosolic marker creatine kinase B-type (CK-B) was used. TC: Total Cell, Cyt: Cytoplasmic fraction, Mit: Mitochondrial fraction. (B) Proteinase K protection assay performed using mitochondria with digitonin-permeabilized outer membranes shows localization of UQCC1 within the mitochondrial inner membrane. UQCC1, unlike outer membrane localized TOM20 and the inter-membrane localized part of OXA1L, is protected from proteolysis and degraded only after the inner membrane is dissolved with Triton X-100. Western blot analysis of single step affinity purified (C) UQCC2- and (D) UQCC1-TAP from doxycycline-induced HEK293 cells shows that UQCC1 co-purifies with UQCC2-TAP and UQCC2 co-purifies with UQCC1-TAP. Additional probing of the membranes for the complex III structural subunits UQCRC1, UQCRC2, UQCRFS1 and mitochondrial ribosomal subunits MRPS22 and MRPL12 did not reveal co-elution of these proteins. Asterisks with these subunits, including the one with UQCRFS1, correspond to bands at different heights that result from previous incubations. Complex II subunit SDHA was used to rule out non-specific protein binding. Non-induced cells were used as control. Antibodies used are indicated at the left. Arrowheads indicate endogenous UQCC1 and UQCC2.
Figure 7.
Depletion of the UQCC2 binding partner, UQCC1, affects complex III assembly.
(A) SDS-PAGE and western blot analysis of mitochondrial extracts from HEK293 cells transfected with UQCC1 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 UQCC1 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 Figure S7B-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 UQCC1 and cyclophilin B 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).
Figure 8.
UQCC2 and UQCC1 are involved in cytochrome b translation and/or stability.
(A) SDS-PAGE analysis of 35S-methionine-labeled mtDNA-encoded proteins in patient fibroblasts shows a lack of cytochrome b (MTCYB) protein (even at zero hours chase) suggesting a defect in cytochrome b synthesis or its immediate stability. (B) qRT-PCR shows normal expression of cytochrome b (MTCYB) 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 b 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.
Figure 9.
Proposed model of CIII assembly.
Complex III assembly begins with the translation activation and/or stabilization of cytochrome b (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 [67].