Sideroflexin 4 is a complex I assembly factor that interacts with the MCIA complex and is required for the assembly of the ND2 module

Abstract

SignificanceMitochondria are double-membraned eukaryotic organelles that house the proteins required for generation of ATP, the energy currency of cells. ATP generation within mitochondria is performed by five multisubunit complexes (complexes I to V), the assembly of which is an intricate process. Mutations in subunits of these complexes, or the suite of proteins that help them assemble, lead to a severe multisystem condition called mitochondrial disease. We show that SFXN4, a protein that causes mitochondrial disease when mutated, assists with the assembly of complex I. This finding explains why mutations in SFXN4 cause mitochondrial disease and is surprising because SFXN4 belongs to a family of amino acid transporter proteins, suggesting that it has undergone a dramatic shift in function through evolution.

Keywords: complex assembly; mitochondria; respiratory chain; sideroflexins.

Fig. 1.

SFXN4^KO leads to depletion of complex I subunits in mitochondria. (A) Top 20 most strongly correlated genes with SFXN4. Genes are ranked by Pearson correlation coefficient and color coded by functional category. (B) Genome-wide analysis of SFXN4 coessentiality. Genes are ranked according to correlation with SFXN4. Genes encoding proteins of the OXPHOS system and the mitoribosome are indicated in red on the graph and with black lines on the plot underneath. (C) Mitochondria from control and SFXN4^KO cells were subjected to label free quantitative MS analysis. The volcano plot depicts the levels of proteins in SFXN4^KO mitochondria relative to control (n = 3). Horizontal cutoff represents a P value of 0.05, and vertical cutoffs represent 1.5× fold change. Proteins are color coded according to the complex I assembly module to which they belong. (D) Mitochondrial lysates from control and SFXN4^KO HEK293 cells were analyzed by SDS-PAGE and immunoblotting. (E) Relative abundance of respiratory chain complexes (complexes I–V) in SFXN4^KO cells determined from data in (C) as compared to control. Mean ± 95% CI is depicted. **P < 0.01, ***P < 0.001, ns, not significant. (F) Topographical heatmap showing log₂ fold-change values for complex I subunits from SFXN4^KO mitochondria (PDB 5LDW).

Fig. 2.

SFXN4^KO leads to isolated complex I deficiency. (A and B) Mitochondria isolated from control and SFXN4^KO cells were solubilized in 1% digitonin- (A) or 1% Triton X-100- (B) containing buffer and analyzed by BN-PAGE and immunoblotting. (C) Spectrophotometric measurements of mitochondrial respiratory chain and citrate synthase enzyme activity in enriched mitochondrial fractions prepared from control, SFXN1^KO, and SFXN4^KO cells. Graph depicts mean ± SD (n = 3). Two-sample t test: *P < 0.05, **P < 0.01. (D) Measurement of ATP synthesis rates in digitonin permeabilized cells provided with indicated CI (pyruvate, glutamate, malate), or CII (succinate) substrates or inhibitors (CI, rotenone; CII, malonate). Data are depicted as mean ± SD (n = 3). Two-sample t test: *P < 0.05, **P < 0.01, ***P < 0.001. (E) OCRs in live cells measured using a Seahorse analyzer to monitor mitochondrial stress following injections with indicated inhibitors (Left), with calculations of basal and maximal mitochondrial respiration (Right). One-sample t test (Right): **P < 0.01. (F) Glycolysis stress assay in live cells determined from extracellular acidification rates measured using a Seahorse analyzer following indicated injections (Left). Cellular glycolysis and maximal glycolytic capacity determined following glucose and oligomycin injections, respectively (Middle and Right). Data are depicted as mean ± SD (n = 3). One sample t test (Middle and Right): *P < 0.05.

Fig. 3.

SFXN4 interacts with the MCIA complex and is required for CI assembly. (A) Mitochondria isolated from control, SFXN4^KO, SFXN4^KO + SFXN4, and SFXN4^KO + ^FLAGSFXN4 HEK293 cells were solubilized in 1% digitonin-containing buffer and analyzed by BN-PAGE and immunoblotting. (B) Anti-FLAG IP was performed using mitochondria isolated from control and SFXN4^KO + ^FLAGSFXN4 HEK293 cells. Eluates were analyzed by LFQ proteomics, and significant interacting partners were visualized on a volcano plot. Curved line indicates significance determined using false discovery rate (FDR) statistics (FDR < 5%, S0 = 12). Proteins are colored based on function. (C) Table depicting the 10 most strongly enriched proteins in the SFXN4^KO + ^FLAGSFXN4 eluate relative to the control eluate. Gene names are color coded according to the legend in B. (D) Schematic depiction of the MCIA complex. (E and F) Mitochondria isolated from control and SFXN4^KO cells (E) or control, SFXN4^KO, ACAD9^KO, and NDUFAF1^KO cells (F) were solubilized in 1% digitonin and analyzed by BN-PAGE and immunoblotting. (G and H) Mitochondria isolated from control and SFXN4^KO + ^FLAGSFXN4 cells (G) or control and ACAD9^KO + ACAD9^FLAG cells (H) were solubilized in 1% digitonin and subjected to anti-FLAG IP. Eluates were analyzed by BN-PAGE and immunoblotting.

Fig. 4.

SFXN4 mediates the incorporation of MT-ND6 into complex I. (A) Mitochondrial lysates from control and SFXN4^KO cells were analyzed by SDS-PAGE and immunoblotting. (B) Mitochondria isolated from control and SFXN4^KO + ^FLAGSFXN4 cells were solubilized in 1% digitonin and subjected to anti-FLAG IP. Eluates were analyzed by BN-PAGE and immunoblotting. (C) Mitochondria isolated from NDUFAF1^KO and NDUFAF1^KO + ^FLAGSFXN4 cells were solubilized in 1% digitonin and subjected to anti-FLAG IP. Eluates were analyzed by SDS-PAGE and immunoblotting. (D) Anti-FLAG IP was performed using mitochondria isolated from control (ACAD9^KO + ACAD9^FLAG) and ACAD9 + ACAD9^FLAG SFXN4^KO cells. Eluates were analyzed by label-free quantitative MS. The volcano plot depicts the levels of ACAD9-interacting partners in SFXN4^KO cells relative to control cells (n = 3). Proteins on the right of the volcano were more abundant in the ACAD9^FLAG IP from the SFXN4^KO cell line, and proteins on the left of the volcano were more abundant in the ACAD9^FLAG precipitation from the control cell line. Horizontal cutoff represents a P value of 0.05, and vertical cutoffs represent 1.5× fold change. Proteins are color coded according to the complex I assembly module to which they belong.