Fig 2: COX16 facilitates integration of COX2 into MITRAC-COX1 modules.(A) Mitochondrial translation products in wild-type (WT) and SURF knockout (SURF–/–) were labeled with [35S]methionine for 1 hr. Whole cell extracts were subjected to immunoprecipitation using anti-COX16, anti-COA6 or control antisera. Eluates were analyzed by digital autoradiography after SDS-PAGE (Total, 5% and Eluate, 100%). Quantification of co-isolated COX2 amounts with the indicated antibodies were performed using ImageJ (mean ± SEM and n = 3). (Β) Immunoprecipitation from wild-type (WT) and FAM36A knockout (FAM36A–/–) mitochondria with anti-MITRAC12 or control antisera. The eluates were analyzed by western blotting after SDS-PAGE with the indicated antibodies (Total 5% and Eluate, 100%). (C) Immunoprecipitation from wild-type (WT) mitochondria with anti-MITRAC12, anti-COA6, anti-FAM36A or control antisera. The eluates were analyzed by western blotting after SDS-PAGE with the indicated antibodies (Total 5% and Eluate, 50%). (D) Mitochondria isolated from induced MITRAC12FLAG cells were solubilized and subjected to anti-FLAG immunoprecipitation and eluates analyzed by SDS-PAGE and western blotting using the indicated antibodies. WT, wild type. (Total 5% and Eluate, 50%). (E) Mitochondria isolated from induced C12ORF62FLAG cells were solubilized and subjected to anti-FLAG immunoprecipitation and eluates analyzed by SDS-PAGE and western blotting using the indicated antibodies. WT, wild type. (Total 3% and Eluate, 100%). (F) Mitochondria isolated from wild-type (WT) and COX16 knockout (COX16–/–) were used for immunoprecipitation with anti-MITRAC12 or control antisera. The eluates were analyzed by western blotting after SDS-PAGE with the indicated antibodies (Total 5% and Eluate, 50%). (G) Antibodies against C12ORF62, MITRAC12 or control antisera were used for immunoisolation after [35S]methionine labeling of mitochondrial translation products in wild-type (WT) and COX16 knockout (COX16–/–) cells and analyzed by SDS-PAGE and digital autoradiography (Total, 5% and Eluate, 100%). Quantification of co-isolated COX1 or COX2 amounts with the indicated antibodies were performed using ImageJ (mean ± SEM and n = 3).

Fig 3: COX16 is required for COX2 assembly.(A) In vivo labeling of mitochondrial translation products with [35S]methionine in wild-type (WT), COX16 knockout (COX16–/–) and COX16 knockout expressing WT COX16 from the T-REx locus (COX16–/– Resc.). Cells were pulsed for 1 hr and analyzed by SDS-PAGE and digital autoradiography. The values represented are quantifications of the indicated mitochondrial translation products normalized to ND1 (mean ± SEM and n = 3). (Β) Mitochondrial translation products in wild-type (WT) and COX16 knockout (COX16–/–) were labeled with [35S]methionine for 1 hr. Subsequently, the medium was replaced and cells were further cultured in standard medium (chase) for 3, 6, 12 and 24 hr. Cell extracts were analyzed by SDS-PAGE and digital autoradiography. (C) Quantifications using ImageQuant software of the indicated mitochondrial translation products from (Β). The values represented were normalized to ND1 (mean ± SEM and n = 3; *p=0.029, **p=0.042, ***p=0.024, ns = non significant). (D) Protein complexes from wild-type (WT) and COX16 knockout (COX16–/–) mitochondria were extracted under non-denaturing conditions and separated by BN-PAGE, followed by a second dimension SDS-PAGE and western blot analysis (top). Mitochondrial translation products were labeled with [35S]methionine, prior whole cell lysis and complexes separation as described above (bottom). The proteins were detected by using indicated antibodies or by digital autoradiography (COX2, ATP6). Intensity curves (right) for COX1 signals from the western blotting and COX2 from the autoradiogram were calculated using ImageJ. Numbers in the gray regions denote area under intensity curves. For COX1 (top), it is represented as percentage of the total signal in CIV and MITRAC and for COX2 (bottom), as arbitrary units. CIV, Monomeric Complex IV; CV, Complex V; RSC, Respiratory Super-Complexes.

Fig 4: Model for the role of COX16.COX1 is assembled and guided through the assembly process through its association with MITRAC, where it awaits the association of COX2. COX2 is initially associated with FAM36A and metallochaperones such as SCO2 and COA6 in the early assembly stages. COX16 acts at the later stages of COX2 assembly. The early assembly factors are apparently no longer associated with the complex at this stage. COX16 facilitates the association of SCO1 and thus probably leads to proper COX2 maturation. It then facilitates the merger of COX1 and COX2 assembly lines after the exit of SCO1.

Fig 5: HBx induces alterations in the mitochondrial dynamics and network structure. Huh7 or HepG2 cells were transiently transfected with HBx plasmid DNA of either GtA, GtB, GtC, GtD, GtE, GtG or empty plasmid DNA as control, and fixed for immunofluorescence or harvested 72 h p.t. (A) Representative confocal microscopy images with 3D high-resolution zoom sections indicated by dashed rectangle. Huh7 cells were immunostained for HBx with HA-specific antibody (green) and TOM20 antibody (red) to image the outer mitochondrial membrane. Nuclei were counterstained with DAPI (blue). Scale bar indicates 20 µm. (B) Quantification of A; mitochondrial footprint indicates the area of mitochondrial pixels in squared micrometer. More than 12 cells per genotype, pcDNA n = 5, were analyzed. (C) Quantification of A; network count mean reflects the mean number of connected mitochondria. More than 12 cells per genotype, pcDNA n = 5, were analyzed. (D) Quantification of the corrected total cell fluorescence of TOM20 staining in panel A. A minimum of 10 cells were analyzed. (E) Huh7 mRNA levels of TOM20, determined by real-time quantitative PCR. Data represent the fold change as compared to pcDNA control from n = 3 independent experiments. (F) Representative Western blot of Huh7 cell lysates, processed for mitochondrial protein levels using TOM20, COX II, and COX IV-specific antibody. β-Actin protein level was used as internal loading control. CCCP, a mitochondrial decoupler, was used as a control to introduce mitochondrial degradation. (G) Western blot quantification of panel F; relative TOM20, COX II, and COX IV protein levels, based on n > 3 independent experiments. All data are indicated as mean ± standard error of the mean. Statistical calculation was performed using unpaired t-test related to the pcDNA sample. *P < 0.05, **P < 0.01, ***P < 0.001. (H) Representative confocal microscopy images with zoom sections indicated by dashed rectangle. HepG2 cells were immunostained for HBx with HA-specific antibody (green) and TOM20 antibody (red) to image the outer mitochondrial membrane. Nuclei were counterstained with DAPI (blue). Scale bar indicates 20 µm. 3D, three-dimensional; CCCP, carbonyl cyanide m-chlorophenyl hydrazine.