Figure 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.