NDUFAF4 variants are associated with Leigh syndrome and cause a specific mitochondrial complex I assembly defect

Abstract

Mitochondrial respiratory chain complex I consists of 44 different subunits and can be subgrouped into three functional modules: the Q-, the P- and the N-module. NDUFAF4 (C6ORF66) is an assembly factor of complex I that associates with assembly intermediates of the Q-module. Via exome sequencing, we identified a homozygous missense variant in a complex I-deficient patient with Leigh syndrome. Supercomplex analysis in patient fibroblasts revealed specifically altered stoichiometry. Detailed assembly analysis of complex I, indicative of all of its assembly routes, showed an accumulation of parts of the P- and the N-module but not the Q-module. Lentiviral complementation of patient fibroblasts with wild-type NDUFAF4 rescued complex I deficiency and the assembly defect, confirming the causal role of the variant. Our report on the second family affected by an NDUFAF4 variant further characterizes the phenotypic spectrum and sheds light into the role of NDUFAF4 in mitochondrial complex I biogenesis.

Figure 1

The NDUFAF4 variant causes isolated complex I deficiency that can be rescued by lentiviral complementation with wild-type NDUFAF4. (a) Complex I enzymatic activity in control (Ctrl) and patient (Pat) cells either transfected with C-terminally V5-tagged wild-type NDUFAF4 (+AF4) or with mock transfected with C-terminally V5-tagged GFP (+GFP). Values are given relative to the activity of citrate synthase (CS) and as average value of two independent experiments. The error bars indicate standard deviation. Lentiviral complementation with wild-type NDUFAF4, but not mock complementation with GFP, increases complex I activity in patient fibroblasts into reference range (163–599 mU/U CS). (b) Non-denaturing BN-PAGE electrophoresis/immunoblotting analysis of mitochondrial protein (lysed with n-dodecyl-β-d-maltoside) from control and patient fibroblasts either transfected with NDUFAF4 or mock transfected with GFP. Holo-complex I (CI, NDUFS3) levels in patient fibroblasts are severely reduced in comparison to control fibroblasts. Complementation with wild-type NDUFAF4 increases holo-complex I levels. The other OXPHOS complexes are not negatively affected: complex V (d subunit, CVdSU), complex III (CIII, UQCRFS1), complex IV (CIV, COX4) and complex II (CII, SDHA). (c) Immunoblotting analysis of total cell lysates after SDS-PAGE with antibodies against subunits representative of the OXPHOS complexes. Levels of complex I subunits representative for each functional module (NDUFV2—N-module, NDUFB11—P-module, NDUFS3—Q-module) are decreased in patient cells and rescued by complementation with wild-type NDUFAF4. Analysis of NDUFAF4, GFP and V5 confirm successful lentiviral complementation. The position of the 25 kDa molecular weight marker is indicated on the right in blots showing the signals of the NDUFAF4 antibody, the V5 antibody and GFP antibody, respectively. Tubulin serves a loading control.

Figure 2

Complex I deficiency in patient fibroblasts carrying the NDUFAF4 variant causes specifically altered supercomplex stoichiometry. Non-denaturing BN-PAGE electrophoresis/immunoblotting analysis of mitochondrial protein (lysed with the mild detergent digitonin) from control (Ctrl) and patient (Pat) fibroblasts either transfected with wild-type NDUFAF4 (+AF4) or mock transfected with GFP (+GFP). Antibody staining was performed to detect (a) complex I (NDUFS3), (b) complex III (holo-CIII) and (c) complex IV (COX4), respectively. 'Holo-CIII' refers to an antibody raised against bovine holo-complex III that detects several different complex III subunits. Detection of complex II (SDHA) served as loading control. Complex I deficiency leads to clearly reduced abundance of the I/III₂- and the I/III₂/IV_n- supercomplexes (SC) and monomeric complex I (CI_M) that is rescued by lentiviral complementation with NDUFAF4. NDUFS3 containing subassemblies (sa) are faintly detectable in control fibroblasts. In patient fibroblasts, complex III dimers (CIII₂) and supercomplexes consisting of a complex III dimer and complex IV (CIII₂/CIV) are increased. Previously described uncharacterized complex IV subunit containing entities (#) behave alike. The levels of monomeric complex IV (CIV_M) remain unchanged.

Figure 3

Patient fibroblasts carrying the NDUFAF4 variant exhibit accumulation of complex I assembly intermediates containing parts of the proximal P-module, the distal P-module and the N-module but not the Q-module. 2D-BN-PAGE/SDS-PAGE and subsequent immunoblotting analysis of mitochondrial protein (lysed with n-dodecyl-β-d-maltoside) from control (Ctrl) and patient (Pat) fibroblasts either transfected with wild-type NDUFAF4 (+AF4) or mock transfected with GFP (+GFP). (a) The upper panel demonstrates simultaneous staining with antibodies against NDUFS3, which is a component of all Q-module subassemblies and fully assembled complex I (CI), and proximal P-module assembly factors ACAD9 and ECSIT. The lower panel demonstrates staining with an antibody against NDUFAF4. In control fibroblasts several NDUFS3 containing Q-module subassemblies (saQ) are detectable. NDUFAF4 is most strongly detectable in a low molecular weight Q-module subassembly (saQ2) but also faintly as a 'smear' reaching into the high molecular weight area (Ctrl+GFP, NDUFAF4). In mock-transfected patient fibroblasts, Q-module subassemblies are not detectable and do not accumulate: subassembly saQ1 is clearly detectable in control fibroblasts and faintly visible in NDUFAF4-transfected patient fibroblasts but not in mock-transfected patient fibroblasts. There is an accumulation of a subassembly containing proximal P-module subassembly components ACAD9 and ECSIT (saP_P). Note that the signal intensity of NDUFS3 at the height of fully assembled complex I (CI) is severely reduced in mock-tranfected patient fibroblasts (Pat+GFP) while the signal intensities of both ACAD9 and ECSIT increase. The arrow indicates the faint signal of the ECSIT antibody in the panel of the mock-transfected control fibroblasts. This ratio is reversed in patient fibroblasts complemented with NDUFAF4 (Pat+AF4). (b) Staining with an antibody against distal P-module subunit, NDUFB11 reveals accumulation of two subassemblies (saP_D2 and saP_D3). Note that, in mock-transfected patient fibroblasts (Pat+GFP), their abundance is higher than that of high molecular weight subassembly saP_D1. This ratio is reversed is patient fibroblasts transfected with wild-type NDUFAF4 (Pat+AF4). (c) Staining with an antibody against N-module subunit, NDUFV2 reveals accumulation of a subassembly (saN2). Note that, in mock-transfected patient fibroblasts (Pat+GFP), its abundance is higher than that of subassembly saN1. This ratio is reversed is patient fibroblasts transfected with wild-type NDUFAF4 (Pat+AF4).