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A unique ABCB7-FECH-ABCB10 complex
anemia with hypochromia and microcytosis in infancy or early childhood.12-14 Bone marrow examination showed ringed sideroblasts, which give the condition its name. Complementation assays in yeast suggested that each of the human mutations caused a mild partial loss of function.12,13 Conditional gene targeting in mice showed that ABCB7 was essential for hematopoiesis15 and for the development and function of all tissues and organs analyzed.11
Here, we examined the time-dependent consequences of loss of ABCB7 in multiple cell culture models. We found that knockdown (KD) of ABCB7 led to significant loss of mitochondrial iron-sulfur (Fe-S) proteins, which preceded the development of comparatively milder defects in cytosolic Fe-S enzymes. In erythroid cells, loss of Abcb7 caused defective heme biosynthesis and altered cellular iron distribution with mitochondrial iron over- load, which triggered oxidative stress and led to apopto- sis of erythroid progenitors. By combining chemical crosslinking with tandem mass spectrometry and muta- tional analyses, we identified a complex formed of fer- rochelatase (FECH), ABCB7 and ABCB10 and character- ized its overall architecture. Our studies uncovered the importance of ABCB7 for mitochondrial function and iron homeostasis and identified a previously uncharac- terized complex that is required for heme biosynthesis.
Methods
Cell lines and cell culture conditions
HEK293T and HeLa cells were purchased from the American Type Culture Collection (ATCC) and propagated in Dulbecco modified Eagle medium with 4.5 g/L glucose, 10% fetal bovine serum, and 2 mM glutamine at 37°C, 5% CO2 in a humidified incubator. G1E-ER4 cells were maintained in Iscove modified Dulbecco medium with 15% fetal bovine serum, 100 U/mL penicillin-streptomycin, 2 U/mL erythropoietin (Sino Biological Inc.), monothioglycerol (1:10,000), and 50 ng/mL Kit-ligand (R&D Systems). GATA1-mediated differentiation was induced by the addition of 100 nmol of β-estradiol to a cell culture at a density of 2×105 cells/mL. All cell lines were tested for mycoplasma.
Short hairpin and small interfering RNA-mediated knockdown of ABCB7, MFRN2 and IRP2 in HEK293T, HeLa or G1E-ER4 cells
The SMARTvector Inducible Lentiviral short hairpin (sh)RNA system (Dharmacon) was used to generate HEK293T and HeLa stable cell clones with tightly controlled expression of three individual shRNA targeting different regions of the ABCB7 tran- script and a scrambled shRNA, used as negative control.
Knockdown of Abcb7 or Irp2 in G1E-ER4 cells was achieved with the Accell small interfering RNA delivery system (Dharmacon). Further details of the knockdown procedures, together with information on in vitro and in vivo crosslinking and mass spectrometry, in vitro coupled transcription/translation and pull-down assay of 35S-labeled proteins, the dihydropyrimidine assay, gel electrophoresis, complex I, II and III activity assays, iron and heme measurements, histological staining, flow cytometry studies, assays of superoxide dismutase activity, aconitase and catalase, along with polymerase chain reaction studies and other methods are provided in the Online Supplementary Methods.
Results
Knockdown of ABCB7 led to dysfunction
of mitochondrial proteins which preceded
the development of milder defects in cytosolic Fe-Senzymes
We analyzed the effects of progressive loss of ABCB7 in HEK293T, HeLa and G1E-ER4 cells. To interrogate the role of ABCB7 during erythroid differentiation and address the effect of ABCB7 mutations in XLSA patients,12-14 we stud- ied G1E-ER4 cells, a well-validated murine cell model that recapitulates the early stages of terminal red blood cell development.16 We used an inducible shRNA-mediated KD approach to silence the expression of ABCB7 in HEK293T (Online Supplementary Figure S1A) and HeLa cells (Online Supplementary Figure S1B), which enabled us to dis- cern primary from secondary effects by observing when defects appeared during the time-course after induction of KD. KD of ABCB7 resulted in a time-dependent loss of mitochondrial Fe-S proteins, including FECH, glutaredox- in 5 (GLRX5) and multiple subunits of the respiratory complexes I and II (NDUFS1 and NDUFS8 in complex I and SDHB in complex II) in all the cell lines tested (Figure 1A,B, Online Supplementary Figure S2C-E, and S2H). Loss of ABCB7 also elicited profound transcriptional remodeling that included the downregulation of the vast majority of subunits and assembly factors of the mitochondrial respi- ratory chain within 48 h after KD (Figure 1C) in G1E-ER4 cells and profoundly affected cell morphology (Online Supplementary Figure S1C). Loss of the catalytic subunits resulted in a significant decrease in the activities of com- plexes I and II (Figures 1A, B, and D). Mitochondrial aconi- tase (ACO2) activity was also markedly reduced by 80%, whereas cytosolic aconitase (ACO1) decreased to a lesser extent (40%) (Online Supplementary Figure S2H), likely due to the fact that mitochondrial dysfunction altered cytoso- lic iron status and activated the RNA-binding activity of iron-regulatory protein-1 (IRP1) 17 (see later in the paper), without changing IRP1 protein levels (Online Supplementary Figure S2H). Notably, levels of cytosolic Fe- S proteins, including CIAPIN1, GLRX3, POLD1, DPYD, PPAT, ERCC2, ELP3 and ABCE1, were unchanged 3 days after KD of ABCB7 (Figure 1E and Online Supplementary Figure S2B). Activity of the cytosolic Fe-S enzyme DPYD (Online Supplementary Figure S2F) and radioactive iron incorporation into the cytosolic Fe-S protein NUBP2 were also unchanged (Online Supplementary Figure S2G). Decreased stability of the cytosolic Fe-S proteins DPYD, PPAT and POLD1 was observed 5 days after KD of ABCB7 (Online Supplementary Figure S2C,D); however, the extent of loss of mitochondrial Fe-S proteins at day 5 was much more profound than the decrease in cytosolic Fe-S protein levels (Online Supplementary Figure S2C,D). Consistent with the importance of mitochondria in performing a global regulatory role in numerous cellular processes linked to iron homeostasis, we found that KD of ABCB7 stabilized iron-regulatory protein-2 (IRP2), a master regu- lator of iron metabolism18 (Figure 1D and Online Supplementary Figure S2C,D). Levels of the erythroid-spe- cific mitochondrial iron transporter mitoferrin-1 (MFRN1) (Figure 1D) and the ubiquitously expressed mitoferrin-2 (MFRN2) in HEK293T and HeLa cells (Figure 1A and Online Supplementary Figure S2D) increased significantly in ABCB7-KD cells. Overall, stabilization of IRP2 in the
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