Complementation of the Complex I Deficiency in Fibroblasts from Patients I-2 and II-1
NI: not infected. Cox8: infected with GFP-tagged COX8 leader sequence. AF3: NDUFAF3-GFP infected.
(A) SDS-PAGE immunodetections of GFP, NDUFS3, and NDUFA9.
(B) Complex I in-gel activity (CI IGA) and BN-PAGE immunodetections of complex I (ND1), complex V (ATPase α), complex III (core II), complex IV (COX5A), and complex II (SDHA).
(C) Enzymatic activities of complexes I (assayed as NADH-ubiquinone reductase), II, and IV after normalization to citrate synthase activity and untreated control (source data in Table S2).
(D) Two-dimensional BN-SDS immunodetections of NDUFS2 and ND1 in control and patient II-1 fibroblasts, before and after complementation (Compl.).
(E and F) RNA interference-mediated knockdowns of NDUFAF3 and NDUFAF4 in HeLa cells. N: not treated. M: mock transfected. #1, #2: targets 1 and 2. (E) SDS-PAGE immunodetection of NDUFAF3, NDUFAF4, complex I subunits NDUFS3 and ND1, and control CII-70 kDa, after each knockdown.
(F) BN-PAGE analysis of complex I in-gel activity (IGA) and immunodetection of complex I subunit NDUFA9, complex II-70 kDa, and complex III core II subunits.

Inhibition of Translation and Proposed Role of NDUFAF3 during Complex I Assembly
HEK293 cells were induced for NDUFS3-, NDUFAF3-, and NDUFAF4-GFP expression with and without prior chloramphenicol treatment (± CAP) for 3 days.
(A) Complex I in-gel activity (CI-IGA) assay.
(B) SDS-PAGE fluorscan for GFP-tagged protein.
(C) BN-PAGE immunodetection with antibody to GFP. Previously described NDUFS3-containing intermediates are indicated on the left as by Vogel et al. 8 M: monomer. Accumulation is indicated with arrows, referring to subcomplexes of NDUFAF3 (AF3) and of NDUFAF4 (AF4).
(D) The conserved gene cluster in alpha-proteobacteria that links orthologs of NDUFAF3 to the bacterial preprotein translocase complex SecD/SecF/YajC, involved in targeting proteins to the membrane, and to CrtB, a phytoene/squalene synthetase of which the human ortholog C8ORF38 has been implicated in complex I assembly. 13 Green: proteins only observed in bacteria. Red: proteins observed in eukaryotes and bacteria.
(E) Tentative model for the role of NDUFAF3 and NDUFAF4 during early complex I assembly. Subcomplex 2 is loosely associated to the mitochondrial inner membrane, as demonstrated by Dieteren et al., 33 by an unknown factor (X). NDUFAF3 and NDUFAF4 are codependent, membrane localized, and present in subcomplex 3, which contains at least subunits NDUFS2 and NDUFS3, and possibly NDUFS8 and NDUFA5. This complex requires mitochondrial translation for progression to higher-molecular-weight subcomplexes. Without mitochondrial translation, NDUFAF3 no longer associates with subcomplex 3 and the complex, with NDUFAF4, accumulates. With mitochondrial translation, both proteins are present until subcomplex 6, close to finalization of holo-complex I. It is yet unknown which factors aid membrane insertion of mitochondrial translation products (indicated with Y). AF3: NDUFAF3. AF4: NDUFAF4. S3: subcomplex 3. X and Y: unknown factors. ND: ND subunits. Green structure: mitochondrial ribosome.

Genetic and Molecular Characterization of the c.229G→C and c.365 G→C Mutations
(A) DNA samples of (a) a patient from family 1, the c.229 G→C mutation indicated by an arrow, (b) an obligate heterozygote from family 1, (c) a control, (d) a patient from family 2, the c.365 G→C mutation indicated by an arrow, (e) an obligate heterozygote from family 2, and (f) a control.
(B) Conservation of the altered amino acids is shown via clustalW alignments. Asterisks (^∗) indicate identical amino acids, colons (:) indicate conserved substitutions, and periods (.) indicate semiconserved substitutions.
(C) Model of NDUFAF3 and the two mutations found in the patients. Middle panel: NDUFAF3 is shown in ribbon visualization and colored by secondary structure element (red = beta strand, blue = alpha helix, green = turn, cyan = coil). The positions of the two mutations found in patients are indicated by the arrows and colored yellow. Side panels: the R122P and G77R mutations shown in more detail. The protein is gray. The side chains of both the wild-type residue (green) and the mutant residue (red) are shown. Both mutations result in modifications at the same side of the protein.

Mitochondrial Localization and High-Molecular-Weight Associations of NDUFAF3
(A) NDUFAF3-GFP, mitotracker, and Hoechst staining of NDUFAF3-GFP-inducible HEK293 Flp-in cells at different magnifications (a and b).
(B) SDS-PAGE immunodetection analysis after subcellular and mitochondrial fractionation of HEK293 cells. Cyto: cytoplasm. Mito: mitochondria. After sonication of purified mitochondria, the pellet (P) contains membrane structures and the supernatant (S) represents the soluble fraction. Carbonate extraction unfolds membrane structures, releasing peripherally associated proteins to the supernatant.
(C) Detection of NDUFS3-GFP (S3-GFP) and NDUFAF3-GFP (AF3-GFP) in native protein complexes via fluorscan of a 5%–15% BN-PAGE gel. Previously identified complex I intermediates are indicated as by Vogel et al. 8
(D) Two-dimensional BN- and SDS-PAGE immunodetection of NDUFAF3 and complex I subunits NDUFS2, NDUFS3, and ND1.
(E) TAP identification of binding partners of NDUFAF3. SDS-PAGE immunodetections of proteins after NDUFS3-, NDUFAF3-, and NDUFAF4-TAP purifications indicated on the left. L: lysate. E: eluate. “−“: without doxycycline. “+”: with 1 μg/ml doxycycline for 24 hr.