Sideroflexin 4 affects Fe-S cluster biogenesis, iron metabolism, mitochondrial respiration and heme biosynthetic enzymes

Abstract

Sideroflexin4 (SFXN4) is a member of a family of nuclear-encoded mitochondrial proteins. Rare germline mutations in SFXN4 lead to phenotypic characteristics of mitochondrial disease including impaired mitochondrial respiration and hematopoetic abnormalities. We sought to explore the function of this protein. We show that knockout of SFXN4 has profound effects on Fe-S cluster formation. This in turn diminishes mitochondrial respiratory chain complexes and mitochondrial respiration and causes a shift to glycolytic metabolism. SFXN4 knockdown reduces the stability and activity of cellular Fe-S proteins, affects iron metabolism by influencing the cytosolic aconitase-IRP1 switch, redistributes iron from the cytosol to mitochondria, and impacts heme synthesis by reducing levels of ferrochelatase and inhibiting translation of ALAS2. We conclude that SFXN4 is essential for normal functioning of mitochondria, is necessary for Fe-S cluster biogenesis and iron homeostasis, and plays a critical role in mitochondrial respiration and synthesis of heme.

Figure 1

SFXN4 knockout attenuates mitochondrial respiration and affects the steady state of level of respiratory complex proteins. (A) Changes in oxygen consumption rate in response to treatment with indicated metabolic inhibitors in K562 (Scramble/SFXN4 KO) cells. Basal respiration, mitochondrial ATP production, maximal respiration and spare capacity were quantified in 8 replicate experiments (means and standard deviation). (B) The mean mitochondrial DNA copy number in K562 (Scramble/SFXN4 KO) cells from 3 replicate experiments. (C) Immunoblot showing the levels of labile subunits from each of the five mitochondrial respiratory complexes in K562 (Scramble/SFXN4 KO) cells. Means and standard deviations of quantified data from three independent experiments are shown under the blot. Images are representative of three independent experiments.

Figure 2

SFXN4 knockout induces lactate secretion and triggers cell death in cells dependent on galactose as a carbon source. (A) Cell count measurements of K562 (Scramble/SFXN4 KO) cells cultured in glucose and galactose. (B) Percentage of cell death and caspase 3/7 activity in K562 (Scramble/SFXN4 KO) cells cultured in glucose and galactose. (C) BrDU incorporation in K562 (Scramble/SFXN4 KO) cells cultured in glucose and galactose. (D) Levels of lactate and extracellular acidification rate (ECAR) in the spent medium of K562 (Scramble/SFXN4 KO) cells. Shown are means and standard deviations of 4 replicas in a representative experiment of 3 independent experiments. (E) Immunoblot showing the levels of the glucose transporter GLUT1 and indicated glycolytic enzymes in K562 (Scramble/SFXN4 KO) cells.

Figure 3

Depletion of SFXN4 reduces Fe-S cluster assembly and affects the stability of Fe-S cluster proteins. (A) Relative fluorescence of GRX2-Fe-S dependent sensors in siSFXN4 and siNFS1 transfected HEK293 cells compared to siCon. (B) Mean and standard deviation of fold change in fluorescence intensity from three independent experiments. (C) Immunoblot showing the relative levels of Venus N173 and C155 (sensor plasmids) in HEK293 cells that were transfected with a siRNA targeted to SFXN4, NFS1, and a control siRNA. (D) Immunoblot of indicated Fe-S cluster proteins in SFXN4 KO cells and scramble K562 cells. Images are representative of three independent experiments. Means and standard deviations of quantified data from three independent experiments are shown under the blot. SDHB (succininate dehydrogenase complex iron sulfur subunit B), a component of respiratory complex II; ACO2 (mitochondrial aconitase), a citric acid cycle enzyme; PPAT (phosphoribyosyl pyrophosphate amido transferase), an enzyme that catalyzes the first step of the denovo purine nucleotide biosynthetic pathway; POLD1 (large catalytic subunit of DNA polymerase delta), the large catalytic subunit of the DNA polymerase delta (Polδ) complex.

Figure 4

SFXN4 knockout alters cellular iron metabolism. (A) In-gel aconitase activity of mitochondrial and cytosolic aconitase. (B) Cytosolic aconitase activity in scramble and SFXN4 KO K562 cells. (C) Western blot of SFXN4, TFR1 and FTH from scramble and SFXN4 KO K562 cells. (D) IRE binding activity of IRP1 and IRP2 in scramble and SFXN4 KO K562 cells determined by gel retardation assay. (E) Cytosolic labile iron pool in scramble and SFXN4 KO K562 cells. Shown are means and standard deviations of 4 replicas in a representative experiment of 3 independent experiments. Images are representative of three independent experiments.

Figure 5

SFXN4 knockout alters mitochondrial morphology and increases mitochondrial iron load. (A) Representative transmission electron micrographs of mitochondria from K562 (Scramble/SFXN4 KO)) cells. Scale bars represent 500 nm. (B) Measurement of mitochondrial cristae width in K562 (Scramble/SFXN4 KO) cells. (C) Quantification of matrix electron density in scramble and SFXN4 KO K562 cells. (D) Atomic percentage of Fe in mitochondria of K562 (Scramble/SFXN4 KO) cells determined by TEM-EDS. Mitochondria from 10 or more cells were analyzed to quantify cristae width and matrix electron density.

Figure 6

SFXN4 depletion reduces hemoglobinization by destabilizing ferrochelatase (FECH) and by promoting translational inhibition of ALAS2. (A) Hemoglobin in differentiated K562 (Scramble/SFXN4 KO) cell at 3 and 6 days post induction of differentiation with sodium butyrate. (B) Image of cell pellets 6 days post induction of differentiation with sodium butyrate. (C) Western blot of SFXN4, ALAS2 and FECH from K562 (Scramble/SFXN4 KO) cells. (D) Western blot of ALAS2 in differentiated K562 (Scramble/CRISPR clone) cells at different time points. Means and standard deviations of quantified data from three independent experiments are shown under the blot. (E) Quantification of ALAS2 mRNA in scramble and SFXN4 knockout cells at 0, 3, and 6 days after sodium butyrate. Images are representative of three independent experiments.