Redefining the roles of mitochondrial DNA-encoded subunits in respiratory Complex I assembly

Abstract

Respiratory Complex I deficiency is implicated in numerous degenerative and metabolic diseases. In particular, mutations in several mitochondrial DNA (mtDNA)-encoded Complex I subunits including ND4, ND5 and ND6 have been identified in several neurological diseases. We previously demonstrated that these subunits played essential roles in Complex I assembly which in turn affected mitochondrial function. Here, we carried out a comprehensive study of the Complex I assembly pathway. We identified a new Complex I intermediate containing both membrane and matrix arms at an early assembly stage. We find that lack of the ND6 subunit does not hinder membrane arm formation; instead it recruits ND1 and ND5 enters the intermediate. While ND4 is important for the formation of the newly identified intermediate, the addition of ND5 stabilizes the complex and is required for the critical transition from Complex I to supercomplex assembly. As a result, the Complex I assembly pathway has been redefined in this study.

Keywords: Assembly; Mitochondrial DNA mutations; ND4; ND5; ND6; Respiratory Complex I.

Figure 1. Early Complex I assembly intermediate in ND6 mutant cells

A) shows the presence of a >950KDa putative Complex I band in A9 and ND6 mutant 4A cells at 0h after ^[35]S methionine labeling. The >950KDa band can also be seen in 4A cells at 24 h, while in A9 cells, the assembled Complex I band along with supercomplexes is seen. B) the chase experiment shows the gradual disappearance of the >950KDa Complex I intermediate band in 4A cells over time. C) ^[35]S labeled pulse chase of wild type A9 cells showing the >950KDa complex at early times and the assembled Complex I, indicated as Complex I*, and supercomplexes at later time point. Complexes III, IV and V are marked for molecular weight reference.

Figure 2. Presence of Complex I subunits in ND6 mutant cells

A) shows the 2D SDS PAGE profile of ND6 mutant 4A and B) shows wild type A9 cells after 2h of ^[35]S labeling and C) shows mutant 4A and D) shows wild type A9 profile after 24 h of ^[35]S labeling. The samples were run on a BNPAGE in the direction indicated and were subsequently run on SDSPAGE in the second dimension. The molecular weights of all subcomplexes observed have been indicated. Complex III, IV and V have been indicated for molecular weight reference. E) shows the formation of a steady state ~400KDa band in 4A cells but no complex I assembly when probed with anti-NDUFA13 antibody. F) The cartoon shows the three subcomplexes observed in 4A and A9 cells and their progress into the >950KDa Complex I intermediate, pink circles indicating presence of assembly factors.

Figure 3. Assembly of mtDNA subunits into Complex I in ND6 mutant cells

A) shows the presence of the >950KDa Complex I band in 1D native gel in 4A-4 and 4A-6 cells at 0 h and 24 h after ^[35]S labeling. B) shows the 2D SDS PAGE profile of 4A-6 cells similar to 4A cells after 2 h of labeling with the >950KDa band containing Complex I subunits. (C-E) shows a 2D SDS PAGE profile of A9 cells and (F-H) shows 2D SDS PAGE profile of 4A cells at the 0 h, 4h and 10 h time points (i.e., 2 h, 6 h and 12 h after ^[35]S labeling begins) respectively. Each observed subcomplex in marked on the SDS PAGE as well as the BNPAGE. The direction for each gel dimension is also indicated by the blue arrows. I) The cartoon shows the kinetic progression of different subcomplexes as observed on the gel.

Figure 4. Early Complex I assembly in ND5 mutant 3A 20-4 cells

A) shows the lack of Complex I in gel activity in 3A 20-4 cells, using A9 cells as wild type controls (left panel) and the lack of assembled Complex I in 3A 20-4 cells using anti-NDUFA13 antibody (right panel). B) shows the 1D native gel profile in 3A20-4 cells at the 0 h time and 24 h and C) shows the 2D SDS PAGE profile and presence of mtDNA-encoded Complex I subunits at the 0 h time point in the >950KDa Complex I intermediate. D) shows the 2D SDS gel profile at 24 h time point of labeling where the presence of the a faint ~800KDa complex can be detected. Complex V has been labeled for molecular weight reference. E) The cartoon shows the presence of the ~400KDa, 700KDa , >950KDa and ~800KDa subcomplexes seen in the 2D SDS profile and 1D Blue native gel in ND5 mutant cells.

Figure 5. Lack of assembly of Complex I in ND4 mutant cells

A) shows the 1D BNPAGE of KB30 and ND4 mutant C4T cells at the 0 h and 24 h time points of ^[35]S labeling. An early Complex I band is seen in KB30 at 0 h but not in C4T cells. B) Shows the 2D SDS PAGE of C4T cells and D) KB30 cells at 0 h time point after ^[35]S labeling and C) shows the 2DSDS PAGE of C4T cells and E) KB30 cells at the 24 h time point after ^[35]S labeling. The cartoon shows the presence of the ~400KDa and ~700KDa subcomplexes seen in the 2DSDS profile in ND4 mutant cells.

Figure 6. Incorporation of nuclear encoded subunits in Complex I assembly

Using chloramphenicol treatment for 6 days followed by recovery at different time points, indicated below each image, to document the incorporation of the following Complex I subunits in Complex I assembly A) anti- NDUFA13 antibody, B) anti-NDUFB6 antibody, C) anti-NDUFS4. Red arrows indicate the position of the >950KDa complex I intermediate while other Complex I subcomplexes have been indicated by their molecular weights. SS denotes steady state controls i.e. Complex I bands observed at steady state only. These samples were not treated with chloramphenicol.

Figure 7. Model for Complex I assembly

A) shows the current hypothesized model for Complex I assembly with the nuclear encoded subunits used in this study B) Stepwise and kinetic assembly of the mitochondrial encoded subunits and some of the nuclear encoded subunits during Complex I assembly. The subunits labeled in red are nuclear encoded subunits while the pink circles are assembly factors.