Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I

Abstract

Complex I of the respiratory chain is composed of at least 45 subunits that assemble together at the mitochondrial inner membrane. Defects in human complex I result in energy generation disorders and are also implicated in Parkinson's disease and altered apoptotic signaling. The assembly of this complex is poorly understood and is complicated by its large size and its regulation by two genomes, with seven subunits encoded by mitochondrial DNA (mtDNA) and the remainder encoded by nuclear genes. Here we analyzed the assembly of a number of mtDNA- and nuclear-gene-encoded subunits into complex I. We found that mtDNA-encoded subunits first assemble into intermediate complexes and require significant chase times for their integration into the holoenzyme. In contrast, a set of newly imported nuclear-gene-encoded subunits integrate with preexisting complex I subunits to form intermediates and/or the fully assembly holoenzyme. One of the intermediate complexes represents a subassembly associated with the chaperone B17.2L. By using isolated patient mitochondria, we show that this subassembly is a productive intermediate in complex I assembly since import of the missing subunit restores complex I assembly. Our studies point to a mechanism of complex I biogenesis involving two complementary processes, (i) synthesis of mtDNA-encoded subunits to seed de novo assembly and (ii) exchange of preexisting subunits with newly imported ones to maintain complex I homeostasis. Subunit exchange may also act as an efficient mechanism to prevent the accumulation of oxidatively damaged subunits that would otherwise be detrimental to mitochondrial oxidative phosphorylation and have the potential to cause disease.

FIG. 1.

Mitochondrially encoded subunits assemble into intermediate subcomplexes. mtDNA-encoded subunits were pulse-chase labeled in wild-type lymphoblasts, followed by mitochondrial isolation. (A) Labeling profile of mtDNA-encoded subunits on SDS-PAGE. (B) BN-PAGE analysis of mitochondria solubilized in Triton X-100. mtDNA-encoded subunits were labeled without (left side) or following (right) CAP pretreatment. The letters a and b indicate regions of potential assembly intermediates. CI, complex I; CIII₂, complex III homodimer; CIV, complex IV; SC, CI/CIII₂ supercomplex. (C) Two-dimensional PAGE analysis of mitochondria isolated from radiolabeled lymphoblasts after 0, 1, 3, and 24 h of chase. For the first-dimension BN-PAGE, mitochondria were solubilized in 1% Triton X-100 as in panel B but at a higher mitochondrial protein/detergent ratio. Regions a and b include complex I intermediate subcomplexes containing subunits ND1, ND2, ND3, and ND6. Subunits CO1 (1), cytochrome b (2), CO3 (3), CO2 (4), ATP6 (5), and ATP8 (6) are indicated. CIV_i, complex IV intermediate.

FIG. 2.

Import and assembly of NDUFV3 into preexisting complex I. ³⁵S-labeled precursor to NDUFV3 (pre-NDUFV3) was incubated for different times with mitochondria isolated from fibroblasts in the presence or absence of a membrane potential (Δψ_m). Samples were treated with or without PK and subjected to SDS-PAGE (A) or solubilized in DDM-containing buffer and then subjected to BN-PAGE (B). Radiolabeled proteins were detected by PhosphorImager analysis. CI, complex I; CIII₂, complex III homodimer; CIII₂/CIV, complex III/complex IV supercomplex; CI/CIII₂, complex I/complex III₂ supercomplex. As shown at the right side of panel B, the migration of complex I (CI) and its supercomplex with dimeric complex III (CI/CIII₂) was identified by Western blot analysis with antibodies to complex I subunit NDUFA9 (α-CI) and the complex III core 1 subunit (α-CIII). (C) ³⁵S-labeled NDUFV3 was incubated for 5 to 45 min with mitochondria isolated from control fibroblasts that had been pretreated with (lanes 4 to 6) or without (lanes 1 to 3) CAP for 24 h. All samples were treated with externally added PK before being solubilized in DDM-containing buffer and subjected to BN-PAGE, Western transfer, and PhosphorImager analysis (top). After the radiolabeled signals were acquired, complex I (CI) and the CI/CIII₂ supercomplex were identified by Western blot analysis with antibodies to complex I subunit NDUFA9 (α-CI). The anti-70-kDa subunit antibody (α-CII) was used to detect complex II (CII) as a loading control (bottom).

FIG. 3.

Import and assembly of complex I subunits. ³⁵S-labeled complex I subunits were individually incubated with isolated fibroblast mitochondria for 60 min in the presence or absence of a membrane potential (Δψ_m). (A) Samples were solubilized in DDM-containing buffer and subjected to BN-PAGE and PhosphorImager analysis. The approximate location of each subunit within complex I is indicated schematically above each pair of lanes. Complex I (CI), complex III (CIII₂), and their supercomplex form (CI/CIII₂) were identified by Western blot analysis with antibodies to complex I subunit NDUFA9 (α-CI) and the core I subunit of complex III (α-CIII; lanes 23 to 24). (B) SDS-PAGE analysis of imported radiolabeled complex I subunits. The precursor (p) and mature (m) forms of the subunits are identified. Samples were imported into mitochondria (mit.) in the presence or absence of a Δψ_m and treated with or without externally added PK. A sample of lysate (representing 20% of the added protein/import) is also shown (lane 4).

FIG. 4.

The assembly factor B17.2L is associated with a complex I subassembly in patient and control mitochondria. (A) Mitochondria from control cells (lane 1) and patient cells lacking subunit NDUFS6 (patient B, lane 2) or NDUFS4 (patient A, lane 3) were solubilized in DDM-containing buffer and subjected to BN-PAGE before Western blot analysis with complex I antibodies (anti-NDUFA9). (B) ³⁵S-labeled B17.2L was incubated with mitochondria isolated from control or patient cells for increasing times in the presence or absence of a Δψ_m. Half of each sample was treated with external PK before being split in two and subjected to BN-PAGE (protease-treated samples only) or (C) SDS-PAGE and phosphorimaging. As shown in lane 1 of panel B, ³⁵S-labeled NDUFV3 was imported into control mitochondria to illustrate the fully assembled forms of complex I. CI/CIII₂, complex I/complex III₂ supercomplex; CI_i/CIII₂, complex I intermediate/complex III₂ supercomplex; CI_i, complex I intermediate; CI, complex I.

FIG. 5.

Import of NDUFS4 into patient mitochondria restores complex I assembly. Mitochondria from control or NDUFS4-deficient patient A cells were incubated with ³⁵S-labeled NDUFV3 (as a control) or ³⁵S-labeled NDUFS4 for 5 to 45 min as indicated before BN-PAGE analysis. (A) Western blot analysis of complex I (probed with NDUFA9 antibodies) and (B) PhosphorImager analysis of import and assembly. Schematic models depicting complex I forms are shown. CI/CIII₂, complex I/complex III₂ supercomplex; CI_i/CIII₂, complex I intermediate/complex III₂ supercomplex; CI_i, complex I intermediate; CI, complex I. The asterisk denotes a nonspecific complex.

FIG. 6.

Import of NDUFS6 into patient mitochondria restores complex I assembly. Mitochondria from control or NDUFS6-deficient patient B cells were incubated with ³⁵S-labeled NDUFV3 (as a control) or ³⁵S-labeled NDUFS6 for 5 to 45 min as indicated before BN-PAGE analysis. (A) Western blot analysis of complex I (probed with NDUFA9 antibodies) and (B) PhosphorImager analysis of import and assembly. Schematic models depicting complex I forms are shown. CI/CIII₂, complex I/complex III₂ supercomplex; CI_i/CIII₂, complex I intermediate/complex III₂ supercomplex; CI_i, complex I intermediate; CI, complex I. The asterisk denotes a nonspecific complex.

FIG. 7.

Import and assembly of complex I subunits into patient mitochondria lacking NDUFS4. ³⁵S-labeled complex I subunits were individually incubated for 60 min with mitochondria isolated from patient A fibroblasts. (A) Samples were treated with or without externally added PK before being solubilized in DDM-containing buffer and subjected to BN-PAGE and PhosphorImager analysis. The approximate location of each subunit is indicated schematically above each pair of lanes. The migration of the ∼800-kDa complex I intermediate (CI_i), complex III (CIII₂), and their supercomplex form (CI_i/CIII₂) were identified by Western blot analysis with antibodies to complex I subunit NDUFA9 (α-CI, lanes 9 and 23) and the core I subunit of complex III (α-CIII, lanes 10 and 24). (B) SDS-PAGE analysis of the import of a selection of radiolabeled complex I subunits into patient A mitochondria. The precursor (p) and mature (m) forms of the proteins are identified. Samples were imported into mitochondria (mit.) in the presence or absence of a Δψ_m and treated with or without externally added PK. A sample of lysate (representing 20% of added protein/import) is also shown (lane 4).

FIG. 8.

Import and assembly of complex I subunits into patient mitochondria lacking NDUFS6. ³⁵S-labeled complex I subunits were individually incubated for 60 min with mitochondria isolated from patient B fibroblasts. (A) Samples were treated with or without externally added PK before being solubilized in DDM-containing buffer and subjected to BN-PAGE and PhosphorImager analysis. The approximate location of each subunit is indicated schematically above each pair of lanes. The migration of the ∼800-kDa complex I intermediate (CI_i), complex III (CIII₂), and their supercomplex form (CI_i/CIII₂) was identified by Western blot analysis with antibodies to complex I subunit NDUFA9 (α-CI, lanes 9 and 23) and the core I subunit of complex III (α-CIII, lanes 10 and 24). (B) SDS-PAGE analysis of the import of a selection of radiolabeled complex I subunits into patient B mitochondria. The precursor (p) and mature (m) forms of the proteins are identified. Samples were imported into mitochondria (mit.) in the presence or absence of a Δψ_m and treated with or without externally added PK. A sample of lysate (representing 20% of added protein/import) is also shown (lane 4).

FIG. 9.

Model depicting the assembly of complex I. De novo assembly occurs via the seeding of intermediate complexes through the synthesis of mitochondrially encoded (ND) subunits, followed by recruitment of other nuclear-gene-encoded subunits. ND1 is found in a separate, smaller complex before associating with other ND subunits. An intermediate containing the assembly factor B17.2L is present in the absence of NDUFS4 or NDUFS6. Their incorporation into the complex drives complete assembly and releases B17.2L. Complex I homeostasis can also occur by competition of newly imported subunits with preexisting ones for assembly into complex I. Complex I intermediates may be found in supercomplexes that include complex III.