Characterization of CIA30. (A) [³⁵S]-labeled precursor forms of CIA30 (pCIA30) and OTC (pOTC) were incubated with mitochondria isolated from HEK-293T at 37°C for the time points indicated in the presence and absence of a membrane potential (Δψ_m). Samples were treated with and without proteinase K (50 μg/ml) before SDS–PAGE and phosphorimage analysis. (B) Isolated mitochondria were subjected to either sonication or alkaline (carbonate, Na₂CO₃) extraction. Following treatments, soluble (S) and insoluble (P) fractions were subjected to SDS–PAGE and Western blot analysis using antibodies against CIA30, mtHsp70 and Tom40. (C) Mitochondria (60 μg protein) isolated from primary fibroblasts were solubilized in 1% (w/v) digitonin, 1% (w/v) DDM or 1% (w/v) Triton X-100 (TX-100) before BN-PAGE and Western blotting with antibodies against CIA30 or subunits of complexes I (CI), or III (CIII). The relative positions of supercomplexes or holoenzymes are indicated.

mtDNA-encoded complex I (CI) subunits associate with CIA30 at early stages in their assembly. (A) BN-PAGE analysis of mitochondria containing radiolabeled mtDNA-encoded subunits (0 h chase) immunodepleted with the αCIA30 antibody (lanes 2, 4 and 6). Preimmune serum was also used for control depletion (lanes 1, 3 and 5). Protein complexes containing radiolabeled mtDNA-encoded subunits were detected by autoradiography (lanes 1 and 2). After this, the lanes were subjected to Western blot analysis using antibodies against CIA30 (lanes 3 and 4) followed by antibodies against CI and complex II (CII) (lanes 5 and 6). (B) After radiolabeling, cells were chased for various times as indicated. Mitochondria were isolated, solubilized in Triton X-100 and incubated with protein A-Sepharose coupled to CIA30 antibodies or preimmune sera. Bound proteins were analyzed by SDS–PAGE and phosphorimaging (lanes 2–7). A labeled subunit profile (control) is shown in lane 1 and represents 16.5% of material used in each co-immunoprecipitation. The mtDNA-encoded subunits are indicated.

Complex I assembly is impaired in patient 1 cells. (A) After radiolabeling of mtDNA-encoded subunits, control or patient 1 fibroblasts were chased for various times as indicated. Mitochondria were isolated, solubilized in Triton X-100 and subjected to BN-PAGE followed by phosphorimage analysis. CI, CIII₂, CIV and CV are indicated. The asterisk (^*) denotes the ∼830 kDa intermediate complex. (B) 2D-PAGE analysis of radiolabeled mtDNA-encoded subunits from both control (left panels) and patient 1 (right panels) fibroblasts at different chase times. Mitochondria were isolated, solubilized in Triton X-100 and subjected to BN-PAGE in the first dimension, followed by SDS–PAGE in the second dimension and phosphorimager analysis. Positions of the ∼460 and ∼830 kDa assembly intermediates and the respiratory chain complexes are shown. The positions of ND1 and ND2 are indicated. CI=complex I, CV=complex V, CIII₂=complex III dimer, CIV=complex IV, CIV_i=complex CIV intermediate, SC=CI/CIII₂ supercomplex.

Expression of wild-type CIA30 in the patient cell line increases complex I levels. Patient 1 fibroblasts (lanes 2–4) and those transfected with wild-type CIA30 using lentiviral (LV) vectors (lanes 5–7) were incubated with 4HT for the indicated times. Untransfected control cells are also shown (lane 1). (A) Mitochondria were isolated and subjected to SDS–PAGE and Western blotting using antibodies against CIA30. (B) The same samples as in A were solubilized in Triton X-100 and subjected to BN-PAGE and immunoblot analysis using antibodies against complex I and complex II subunits. SC=complex I/complex III₂ supercomplex.

Co-immunoprecipitation of complex I subunits with CIA30. Mitochondria from HEK-293T cells were solubilized in 1% (w/v) Triton X-100 before immunoprecipitation with antibodies against CIA30 or preimmune serum coupled to protein-A Sepharose. The eluted fraction and a sample of the unbound fraction (2% of total) were then applied to Tris–Tricine SDS–PAGE followed by Western transfer and immunodecoration with antibodies as indicated.

Identification of a complex I-deficient patient cell line with decreased CIA30. (A) Mitochondria isolated from fibroblasts of control (C) and patients (P1–P5) with complex I defects were applied to SDS–PAGE, Western blotted and probed with antibodies against CIA30, Tom40 and mtHsp70 (left panel). Patients had fibroblast complex I activities ranging from 10 to 50% of controls with unknown genetic cause (P2) or with two pathogenic mutations in the complex I subunit genes NDUFV1 (P3), NDUFS4 (P4) or NDUFS6 (P5). The steady-state levels of CIA30 in patient 1 (P1) were quantified against Tom40. Data are mean±s.e.m., n=3 (right panel). (B) Steady-state levels of complex I from patient 1 (P1) fibroblasts were analyzed by BN-PAGE and immunoblotting using antibodies against complex I (CI) and complex II (CII; left panel). SC=complex I/complex III₂ supercomplex. The steady-state levels of complex I were quantified against complex II. Data are mean±s.e.m., n=3 (right panel). (C) Mitochondrial extracts from control (C) and patient 1 (P1) fibroblasts were subjected to SDS–PAGE and Western blot analysis against complex I subunits and the complex II 70 kDa subunit (as control). (D) Respiratory chain enzyme activities in patient 1 (P1) lymphoblasts were measured and standardized against the mitochondrial marker enzyme citrate synthase and shown as a percentage of the control mean. Vertical lines represent the observed range for 6 control lymphoblast cell lines.

Genetic screening of NDUFAF1 in patient 1. (A) Chromatographic trace of relevant NDUFAF1 sequence from control, patient 1 and the patient's parents. The patient is compound heterozygous for a c.1001A>C and a c.1140A>G mutation, the patient's mother was heterozygous for the c.1001A>C mutation while the patient's father was heterozygous for the c.1140A>G mutation. (B) Amino-acid sequence alignment of the mutated region of CIA30 from Homo sapiens (human, accession #Q9Y375), Bos taurus (bovine, accession #XP_615202), Mus musculus (mouse, accession #NP_081451), Xenopus tropicalis (xenopus, accession #NP_001017153.1), Drosophila melanogaster (accession #NP_651718.1), Caenorhabditis elegans (accession #NP_506361.1) and N. crassa (accession #O42636). Shading represents identical residues in four or more species. (C) Predicted NDUFAF1 gene structure showing the location of the mutations within exon 3 (top) and the predicted donor splice sites in control and patient 1 exon 3 (bottom). Predicted percentage scores for splice site probability are shown. SpliceSiteFinder predicts that the c.1140A>G mutation moves the preferred donor splice site 6 bp upstream in patient 1 resulting in the deletion of 6 bp of coding sequence. (D) PCR analysis using primers located in NDUFAF1 exons 2 and 4 of cDNA from control and patient 1 fibroblasts grown without and with cycloheximide (CHX). Patient cDNA shows a second smaller fragment in addition to the expected 333 bp fragment, consistent with the splicing defect predicted by SpliceSiteFinder. (E) Diagrammatic representation of the three sequences identified by cloning the NDUFAF1 cDNA fragment spanning exons 2–4 from patient 1 cDNA. Of 21 sequenced clones, five cDNA sequences represented the expected maternal allele carrying the c.1001A>C (Thr to Pro) mutation. Of 16 paternally derived cDNA sequences (containing c.1001A), four contained the c.1140A>G (Lys to Arg) mutation while 12 lacked the final 6 bp encoded by exon 3 (loss of Val and Lys), consistent with the predicted splicing defect.