Five entry points of the mitochondrially encoded subunits in mammalian complex I assembly

Abstract

Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.

FIG. 1.

Genetic and biochemical characterization of the ND mutants. (A to C) Chromatograms showing the homoplasmic mutations found in the mt-Nd6 gene (A), the mt-Nd5 gene (B), and the mt-Nd4 gene (C). (D) Allele-specific termination of a primer extension assay to confirm the homoplasmy of the insertion and deletion of a C in the ND6 mutants. (E) Polarographic measurements for the different cell lines employed in the study. Note the difference between the graph corresponding to the control and the rest. All the analyzed ND subunit mutants displayed undetectable NADH-linked substrate (glutamate plus malate) oxygen consumption, while the activities of CII plus CIII (succinate plus glycerol-3-P-linked oxygen consumption) and CIV (TMPD-linked oxygen consumption) were comparable to those of the control cells.

FIG. 2.

Detection of complex I-related species. (A) CI as a “monomer” and associated into supercomplexes was detected in 1D Blue Native gels in digitonin-solubilized mitochondria from control and mutant cells by CI-IGA after 2 h of development of the reaction. (B) WB and immunodetection with specific antibodies against the different respiratory chain complexes after 1D BNGE. Complex I was visualized using a specific antibody against the NDUFB6 subunit that detected mainly the monomeric CI and the CI-containing supercomplexes in the control samples and several CI subcomplexes in the mutant cell lines. (C) Digital composite of the immunodetections for complexes I (blue), III (yellow), and IV (red) performed after 1D BNGE in digitonin-solubilized mitochondria from control and mutant cells, followed by 2D BNGE run with the addition of DDM to the cathode buffer. The association of complexes III and IV is indicated in orange. See the text for a detailed explanation and Fig. S3 in the supplemental material for the original images used to make the composite. The NDUFB6-containing subcomplexes found in the mutant cells are named alphabetically (a to f); higher-molecular-mass associations of CI are indicated as SC1 (supercomplex 1) and SC2. The correspondence of the 2D DDM signals to those observed in the 1D gels (B) is shown.

FIG. 3.

Characterization of the peripheral-arm and membrane arm CI subcomplexes. (A) Complex I was immunodetected by Western blot analysis after Blue Native gel electrophoresis in digitonin-solubilized mitochondria (BN-DIG) and 2D denaturing tricine-SDS-PAGE from the control and the mutant cells. Several antibodies against subunits belonging to different parts of the CI topology were used, as indicated. In the control cells, all the subunits were present in fully assembled CI and in the supercomplexes. In the mutant cells, the presence of these nuclear-encoded subunits in several subassemblies was characterized. The membrane arm NDUFB6-containing subcomplexes were named alphabetically (a to e, or e^WT in the case of the subcomplex detected in the control cells), and the peripheral-arm NDUFS3-containing subcomplexes were named numerically (numbers 1 to 5) according to reference . Antibodies against complex III and complex IV were also used to detect their presence in the analyzed cell lines. (B) Schematic representation of the detected membrane arm (red) and peripheral-arm (green) subassemblies that come together to form the whole CI, in which each topological fraction is represented by a different color.

FIG. 4.

Radiolabeling of the mtDNA-encoded subunits in vivo and their incorporation into the OXPHOS complexes and supercomplexes. Digitonin-solubilized samples from the wild-type and mutant cell lines were run through 1D Blue Native gels (A) and 2D denaturing tricine-SDS-PAGE (B) after a 2-h radioactive pulse and a 14-h chase. The signals from the different subunits were identified and localized in the positions corresponding to the fully assembled isolated complexes and associated into the supercomplexes or with the different subcomplexes: green, complex I subunits (NDL-6 and ND4L); red, complex III₂ subunit cytochrome b (Cytb); purple, complex IV subunits COI, COII, and COIII (cytochrome c oxidase subunits I, II, and III); blue, complex V subunits A6 and A8 (ATPases 6 and 8) (see the text for detailed explanations).

FIG. 5.

Radiolabeling of the mtDNA-encoded subunits in organello and their incorporation into the OXPHOS complexes. Digitonin (A)- and DDM (B)-solubilized mitochondria isolated from mouse liver were run through 1D Blue Native gels and 2D denaturing tricine-SDS-PAGE after a 45-min radioactive pulse and the indicated chase times. Chase was performed in the presence of puromycin in the samples shown in panel A to eliminate the signal corresponding to the labeled peptides associated with the mitoribosomes. Newly synthesized mtDNA-encoded subunits could not be assembled into CI under these conditions; however, the same subcomplexes containing the ND subunits detected in the mutant cell lines (Fig. 4) were also formed in the isolated mitochondria. Moreover, mainly the same subcomplexes were detected independently of the detergent used to solubilize the mitochondrial membranes.

FIG. 6.

Complex I assembly model, including the ND subunit entry points determined in this study. Shown is a complex I assembly model for wild-type cells, based on the refined structure for CI shown by Clason et al. (13). The model unites previous knowledge and the new contributions from the present study.