Mutations in C12orf62, a factor that couples COX I synthesis with cytochrome c oxidase assembly, cause fatal neonatal lactic acidosis

Abstract

We investigated a family in which the index subject presented with severe congenital lactic acidosis and dysmorphic features associated with a cytochrome c oxidase (COX)-assembly defect and a specific decrease in the synthesis of COX I, the subunit that nucleates COX assembly. Using a combination of microcell-mediated chromosome transfer, homozygosity mapping, and transcript profiling, we mapped the gene defect to chromosome 12 and identified a homozygous missense mutation (c.88G>A) in C12orf62. C12orf62 was not detectable by immunoblot analysis in subject fibroblasts, and retroviral expression of the wild-type C12orf62 cDNA rescued the biochemical phenotype. Furthermore, siRNA-mediated knockdown of C12orf 62 recapitulated the biochemical defect in control cells and exacerbated it in subject cells. C12orf62 is apparently restricted to the vertebrate lineage. It codes for a very small (6 kDa), uncharacterized, single-transmembrane protein that localizes to mitochondria and elutes in a complex of ∼110 kDa by gel filtration. COX I, II, and IV coimmunoprecipated with an epitope-tagged version of C12orf62, and 2D blue-native-polyacrylamide-gel-electrophoresis analysis of newly synthesized mitochondrial COX subunits in subject fibroblasts showed that COX assembly was impaired and that the nascent enzyme complex was unstable. We conclude that C12orf62 is required for coordination of the early steps of COX assembly with the synthesis of COX I.

Figure 1

Defect in COX I Translation and Normal Levels of Mitochondrial mRNAs in Subject Fibroblasts (A) Pulse-labeled mitochondrial polypeptides were chased for 10 min (PULSE) or overnight (CHASE). The seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX), and two subunits of complex V (ATP) are indicated at the left of the figure. (B) Northern blot analysis of fibroblasts from the subject, TACO1 patient, and controls. Ten micrograms of total RNA was separated on a denaturing formaldehyde agarose gel (MOPS buffer) and transferred to a nylon membrane. We labeled the PCR products (300–500 bp) of individual mitochondrial genes (COX I, COX II, COX III, ND 1, 12S, and 16S) with [α-³²P]-dCTP by using the MegaPrime DNA labeling kit (GE Healthcare). The 12S and 16S mitochondrial rRNAs and actin were used as loading controls.

Figure 2

Mutation Analysis of C12orf62 in the Index Subject (A) Sequence analysis of C12orf62 exon 2 from genomic DNA shows a homozygous c.88G>A missense mutation in the subject fibroblasts. (B) RFLP analysis shows NlaIII of C12orf62 exon 2 amplified from genomic DNA from the subject, TACO1 patient, and three controls fibroblasts. (C) Pedigree of the index subject (box) indicates affected and unaffected individuals. The genotype of individuals is shown in parentheses as follows: Hom mut, homozygous for c.88G>A; and Het, heterozygous for c.88G>A. (D) Protein alignment (MultAlin) of C12orf62 in different species. An arrow indicates the conserved methionine mutated in the subject. The position of the predicted transmembrane domain (predicted with TMHMM Server v. 2.0) is indicated by a gray bar. The color bar reflects the degree of amino acid conservation between species.

Figure 3

Rescue of COX Assembly and COX I Translation Defect in Subject Fibroblasts (A) BN-PAGE analysis of the assembly of individual OXPHOS complexes in control and subject fibroblasts expressing C12orf62 from a retroviral vector (upper panel). With complex II subunit 70 kDa as a loading control, SDS-PAGE analysis of C12orf62 indicates its level of expression (lower panel). (B) Pulse labeling with [³⁵S] methionine and [³⁵S] cysteine of mitochondrial polypeptides and a 10 min chase (PULSE) in control and subject fibroblasts expressing C12orf62 from a retroviral vector. The seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX), and two subunits of complex V (ATP) are indicated at the left of the figure. (C) SDS-PAGE analysis of C12orf62 steady-state levels in two controls, TACO1 patient and subject fibroblasts. Complex II-70kDa subunit and complexI-39kDa subunit were used as loading controls.

Figure 4

Knockdown of C12orf62 in Control and Subject Fibroblasts Results in a Specific Defect in COX Assembly and COX I Translation (A) BN-PAGE analysis of the assembly of individual OXPHOS complexes in control and subject fibroblasts transiently transfected with two different siRNA constructs specific to C12orf62 (KD1 and KD2), with a fluorescent control siRNA (Alexa), and without siRNA (Mock). Complex IV (OE) is a longer exposure of the same blot. (B) Pulse labeling with [³⁵S] methionine and [³⁵S] cysteine of mitochondrial polypeptides of the samples in panel (A). The seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX), and two subunits of complex V (ATP) are indicated at the left of the figure. For quantification of COX I synthesis, COX I labeling was normalized to the labeling of ND3. COX activity was also measured in these samples. The values are shown under the translation gel and are expressed as percentages of the mock control. (C) The mitochondrial translation products were pulse labeled and chased for 10 min (PULSE) or 17 hr (CHASE) in control and subject fibroblasts and in cells that were transfected with either a siRNA construct specific to C12orf62 (KD2) or a fluorescent control siRNA (Alexa). COX I labeling was normalized to the labeling of ND3.

Figure 5

C12orf62-FLAG Coimmunoprecipates with COX I, II, and IV and with LRPPRC, EFTu, and SLIRP (A) Mitochondria from either fibroblasts expressing C12orf62-FLAG or control fibroblasts were extracted and incubated with anti-FLAG agarose beads. Immunoblotting with antibodies against FLAG, COX I, COX II, and COX IV was performed on each fraction. (B) Mitochondria from control fibroblasts expressing C12orf62-FLAG were extracted and incubated with magnetic beads that either were uncoated or were coated with COX I or COX II antibodies. Immunoblotting with antibodies against FLAG, COX I, and COX II was performed on the input, unbound, and eluate fractions. (C) The immunoprecipitation was performed with FLAG beads as described in (A), and immunoblotting with antibodies against FLAG, LRPPRC, EFTu, and SLIRP was performed on each fraction.

Figure 6

Synthesis and Turnover of COX Subunits in Control and Subject Fibroblasts (A) Pulse labeling with [³⁵S] methionine and [³⁵S] cysteine of mitochondrial polypeptides and a 10 min chase (PULSE), as well as the time course of degradation via chases for 8, 17, and 32 hr (CHASE) are shown in control and subject fibroblasts. The seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX), and two subunits of complex V (ATP) are indicated at the left of the figure. (B) Histograms in which the level of the three COX subunits is normalized to the level of ATP6 in the PULSE and CHASE experiments in (A).

Figure 7

COX Subunits II and III Are Not Properly Assembled into the Nascent COX Complex in Subject Fibroblasts 2D BN-PAGE analysis of the sample pulse that was labeled with [³⁵S] methionine and [³⁵S] cysteine and chased for 32 hr. The gels were dried and the labeled mitochondrial translation products were detected through direct autoradiography. The arrows show different mtDNA-encoded polypeptides: one complex III subunit (cyt b), two complex V subunits (ATP6 and ATP8), COX subunit 1 (COX I-S4) in fully assembled complex IV, COX subunit 1 (COX I-S3) in the S3 intermediate of complex IV, COX subunit 2 (COX II), and COX subunit 3 (COX III) in fully assembled COX. The amount of label in each of the identified COX subunits was normalized to the label in ATP6 and is shown beneath the figure for both the 17 and 32 hr chases.