Coiled coil domain-containing protein 56 (CCDC56) is a novel mitochondrial protein essential for cytochrome c oxidase function

Abstract

In Drosophila melanogaster, the mitochondrial transcription factor B1 (d-mtTFB1) transcript contains in its 5'-untranslated region a conserved upstream open reading frame denoted as CG42630 in FlyBase. We demonstrate that CG42630 encodes a novel protein, the coiled coil domain-containing protein 56 (CCDC56), conserved in metazoans. We show that Drosophila CCDC56 protein localizes to mitochondria and contains 87 amino acids in flies and 106 in humans with the two proteins sharing 42% amino acid identity. We show by rapid amplification of cDNA ends and Northern blotting that Drosophila CCDC56 protein and mtTFB1 are encoded on a bona fide bicistronic transcript. We report the generation and characterization of two ccdc56 knock-out lines in Drosophila carrying the ccdc56(D6) and ccdc56(D11) alleles. Lack of the CCDC56 protein in flies induces a developmental delay and 100% lethality by arrest of larval development at the third instar. ccdc56 knock-out larvae show a significant decrease in the level of fully assembled cytochrome c oxidase (COX) and in its activity, suggesting a defect in complex assembly; the activity of the other oxidative phosphorylation complexes remained either unaffected or increased in the ccdc56 knock-out larvae. The lethal phenotype and the decrease in COX were partially rescued by reintroduction of a wild-type UAS-ccdc56 transgene. These results indicate an important role for CCDC56 in the oxidative phosphorylation system and in particular in COX function required for proper development in D. melanogaster. We propose CCDC56 as a candidate factor required for COX biogenesis/assembly.

FIGURE 1.

The protein CCDC56, encoded in a bicistronic transcript together with mt-TFB1 in D. melanogaster, is conserved in metazoans. A, genomic map of CG42630/ccdc56 and mtTFB1. Exons are indicated by boxes, coding regions are colored black for the CCDC56 and mtTFB1 proteins, and untranslated regions are represented in white. B, bicistronic transcript determined by 5′-RACE from control flies (w¹¹¹⁸ and OregonR-C) and cultured Schneider cell cDNAs. The transcription start point identified is depicted as +1. The primers R1, R2, and R3 used are represented in A. C, bicistronic ccdc56-mtTFB1 mRNA detected by Northern blot using 5 μg of RNA from w¹¹¹⁸ (C1) and OregonR-C (C2) control larvae. The signal detected using a specific ccdc56 probe has the same migration as the signal detected when using a probe specific for mtTFB1. D, ClustalW alignment of Drosophila CCDC56 protein with CCDC56 sequences from other metazoan species. Accession numbers are as follows: fly (D. melanogaster), FlyBase annotation CG42630-PA; zebrafish (Danio rerio), UniProtKB accession number A8KB87; western clawed frog (Xenopus tropicalis), UniProtKB accession number A9UMl0-1; mouse (Mus musculus), NCBI accession number NP_080894.1; cow (Bos taurus), UniProtKB accession number Q3T0E3; human (Homo sapiens), NCBI accession number NP_001035521.1. Identical residues in all sequences (*), conserved substitutions (:), and semiconserved substitutions (.) are noted in the alignment. E, schematic diagram of the sequences of the human and D. melanogaster CCDC56 proteins showing the putative transmembrane and protein-protein interaction coiled coil domains. Hu, human; dm, D. melanogaster; aa, amino acids.

FIGURE 2.

CCDC56 protein localizes to mitochondria. Immunocytochemistry of HeLa cells transfected with recombinant d-CCDC56-FLAG is shown. A, MitoTracker (Mtred) staining is shown in red. B, the same cells immunostained for the FLAG epitope (green). The recombinant Drosophila protein colocalizes with the MitoTracker dye in the mitochondrial compartment (C). D, immunoblots of protein extracts (50 μg) from subcellular fractions of control embryos probed with anti-d-CCDC56, anti-porin, and anti-GAPDH antibodies. Total extracts, mitochondrial fraction (Mit), and postmitochondrial supernatant (PMS) are shown.

FIGURE 3.

Molecular characterization of the ccdc56^D6 and ccdc56^D11 alleles. A, genomic map of the ccdc56 and mtTFB1 genes showing the P element insertion (SUPor-P^[kg07792]; triangle). Exons are indicated by boxes, coding regions are colored black for the CCDC56 and mtTFB1 proteins, and untranslated regions are represented in white. 5′-RACE from control (w¹¹¹⁸) and mutant (ccdc56^D6 and ccdc56^D11) homozygous third instar larvae cDNAs identified the transcription start points, which are depicted as +1. The break points of the deletions generated in this work for the alleles ccdc56^D6 and ccdc56^D11 are shown in B and C, respectively. The ratios of the mRNA as determined by 5′-RACE in the clones analyzed are represented (ccdc56^D6/D6, n = 8; ccdc56^D11/D11, n = 10). PCR products amplified with F5 and R4 primers (shown in A) from genomic DNA of third instar larvae are shown in D. Lane 1, DNA from the stock containing the P element SUPor-P^[kg07792] as a negative control; lane 2, DNA from w¹¹¹⁸ control flies; lane 3, DNA from excised flies without any deletion used as an additional control; lane 4, DNA from excised flies of the ccdc56^D6/D6 strain showing a 570-bp deletion; lane 5, DNA from excised flies of the ccdc56^D11/D11 strain showing a 1168-bp deletion. E, transcript levels determined by qRT-PCR in third instar larvae of control and deleted homozygous lines after normalizing to w¹¹¹⁸ flies using 18 S rRNA as an internal control. The two different TaqMan probes used are depicted in B. Control 1, w¹¹¹⁸ flies; Control 2, excised flies without any deletion. Values are mean ± S.E. F, CCDC56 and mtTFB1 protein levels determined by immunoblotting of mitochondrial extracts (30 μg) of control and deleted homozygous larvae. Anti-voltage-dependent anion channel/porin antibody was used as a loading control.

FIGURE 4.

Lack of CCDC56 causes arrest at the third larval stage. A, size comparison of control (w¹¹¹⁸) and homozygous mutant third instar larvae. Homozygous larvae for both alleles were smaller than control larvae in all cases tested. B, mouth hook morphology of control third instar larvae and mutant larvae 15 days AEL indicating third instar. C, wing imaginal discs from control (w¹¹¹⁸) and homozygous ccdc56^D11 third instar larvae were dissected and immunostained with anti-phosphohistone 3 (PH-3) antibody. Mutant wing discs showed lower cell proliferation levels as compared with controls. D, anti-caspase 3 activated antibody was used to detect apoptotic signals in wing imaginal discs. Increased levels of apoptosis were observed in homozygous ccdc56^D11 flies as compared with control flies. Bar size, 50 μm.

FIGURE 5.

CCDC56 expression partially rescues the mutant lethality phenotype. A, flies homozygous for the ccdc56^D11 allele reach only the pupal stage when they carry the UAS-ccdc56 and the arm-GAL4 transgenes on chromosome II. Larvae and pupae homozygous for the allele ccdc56^D11 are of normal size. Larvae and pupae heterozygous for the deletion (genotype ccdc56^D11/TM6B-Tb) carrying one copy of the Tb marker are smaller. B, qRT-PCR of CCDC56, bicistron, and mtTFB1 mRNA relative to 18 S rRNA from homozygous ccdc56^D11 third instar larvae combined with the different UAS transgenes and with or without the ubiquitous arm-GAL4 driver. The three TaqMan probes used are depicted in the scheme. Data represent the mean ± S.E. of at least three independent determinations (*, p < 0.05; **, p < 0.01; Student's t test). C, immunoblot of mitochondrial extracts (30 μg) from homozygous ccdc56^D11 third instar larvae of the genotypes indicated incubated with polyclonal anti-mtTFB1 and anti-CCDC56 antibodies and with monoclonal anti-voltage-dependent anion channel/porin antibody.

FIGURE 6.

ccd56 knock-out flies exhibit an isolated complex IV enzyme deficiency. A, respiratory chain enzyme activities (complexes I, II, III, and IV) and citrate synthase activity were measured in mitochondrial extracts obtained from control and ccdc56 knock-out third instar larvae 15 days AEL. Both mutant alleles showed a severe decrease in complex IV enzyme activity. B, quantification of relative mtDNA levels by qRT-PCR using ND5 and COXI as mitochondrial gene probes and 18 S rRNA as a nuclear gene probe from control, ccdc56^D6/D6, and ccdc56^D11/D11 third instar larvae 15 days AEL. C, steady-state expression levels of representative genes from polycistronic transcripts from mitochondrial RNA were measured by qRT-PCR from ccdc56^D6/D6 and ccdc56^D11/D11 third instar larvae 15 days AEL and are shown relative to the levels found in control larvae after normalization to 18 S rRNA levels. CONTROL, genotype w¹¹¹⁸; D6/D6, ccdc56^D6/D6; D11/D11, ccdc56^D11/D11. Data shown in A, B, and C represent the mean ± S.E. of at least three independent determinations (*, p < 0.05; **, p < 0.01; ***, p < 0.001; analysis of variance). CYTB, cytochrome b.

FIGURE 7.

Loss of ccdc56 induces a complex IV assembly defect. A, two-dimensional blue native (BN)/SDS-PAGE analysis of mitochondrial extracts immunoblotted with an antibody against the mitochondrially encoded subunit mt-CO3 of the cytochrome c oxidase complex. Mitochondrion-enriched extracts were prepared from third instar larvae of the following genotypes: 1, control w¹¹¹⁸; 2, mutant ccdc56^D11/D11; 3, mutant ubiquitously expressing the UAS-ccdc56 transgene (arm-GAL4/UAS-ccdc56;ccdc56^D11/D11); 4, mutant larvae expressing the UAS-mtTFB1 transgene under the same condition (arm-GAL4/UAS-mtTFB1;ccdc56^D11/D11). The lack of fully assembled holo-COX exhibited by the ccdc56^D11/D11 mutant is rescued partially by overexpression of the UAS-ccdc56 transgene. S4 is the fully assembled complex IV. The previously identified mt-CO3-containing COX subcomplex S3 is indicated. B, first dimension of duplicate blue native PAGE of mitochondrial extracts incubated with a polyclonal antibody against complex V (C-V) as a loading control for the two-dimensional blue native/SDS-PAGE shown in A. C, complex IV enzyme activity measured in mitochondrial extracts from homozygous ccdc56^D11/D11 third instar larvae carrying the indicated constructs on chromosome II. Data were normalized to control larvae (w¹¹¹⁸) and represent the mean S.E. (n = 3; *, p < 0.05; Student's t test).