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N. Maio et al.
yielded 147 MS/MS fragmentation spectra corresponding to 29 high-confidence lysine-lysine crosslinks (Online Supplementary Table S1, Figure 5C and Online Supplementary Figure S8A), among which 12 were intersubunit crosslinks between ABCB7 and FECH and ten were crosslinks between endogenous Abcb10 and FECH (Online Supplementary Table S1, Figure 5C and Online Supplementary Figure S8A,B). No crosslinks were detected between ABCB7 and Abcb10, suggesting that FECH mediated an indirect interaction between ABCB7 and ABCB10, as we subse- quently confirmed by co-immunoprecipitation experi- ments. The analysis also identified a high confidence FECH-FECH intersubunit crosslink (K286-K286) (Online Supplementary Table S1). Identification of this crosslinked species is consistent with the distances derived from the crystal structure of FECH, which shows the two lysines at the FECH dimer interface with a Ca-Ca distance of 10.163Å, in agreement with the length of the DSS crosslinker (11.4 Å) (Online Supplementary Figure S9A). The crystal structure of FECH revealed that the PPIX substrate was deeply bound within a pocket that was enclosed by three movable regions34 (Online Supplementary Figure S9B), one of which (residues 90-115 of FECH) was involved in binding the nucleotide-binding domain of ABCB7, according to our data (Online Supplementary Table S1, Figure 5C and Online Supplementary Figure S9C). We performed alanine scanning mutagenesis on the C terminus of ABCB7 (residues V450- C752) (Figure 5D,E), and tested the mutants in vivo and in vitro for their ability to interact with FECH. SDS-PAGE analysis on crosslinked purified ABCB7 and FECH proteins, showed three major bands in the presence of the crosslinker (BS3) with approximate molecular weights of 80, 120 and 230 kDa (Figure 5F and Online Supplementary Figure S10A), which met the expected molecular weights of a dimer of FECH (84 kDa), a dimer of ABCB7 (138.4 kDa), and a het- erotetrameric complex consisting of a dimer of ABCB7 interacting with a dimer of FECH (222.4 kDa). Importantly, the same complexes at 120 and 230 kDa were also detected on SDS-PAGE after in vivo crosslinking on isolated mito- chondria, followed by anti-FLAG immunoprecipitation of ABCB7-FLAG (Figure 5G). We also confirmed the previous- ly reported interaction of ABCB10 with FECH31 in vitro with purified proteins (Online Supplementary Figure S10A,B), which demonstrated that binding of FECH to ABCB10 was direct. BN-PAGE analysis on ABCB7, FECH and ABCB10 purified proteins showed that each ABC transporter dimer- ized when loaded individually (Figure 5H), confirming the results obtained using the crosslinker (Figure 5F and Online Supplementary Figure S10A). Both ABCB7 and ABCB10 were able to interact physically with dimers of FECH and, when combined, ABCB7, FECH and ABCB10 assembled into a complex of approximately 480 kDa (Figure 5H, complex designated with the # symbol), which conformed to the predicted size of a hetero-hexameric complex composed of dimers of ABCB7, FECH, and ABCB10. The ABCB7-FECH interaction was disrupted upon expression of ABCB7Mut1, in which the peptide sequence from Val450 through Leu463 had been replaced by alanines (Figures 5F, H), whereas ABCB7Mut5 (residues L517-K526 replaced by alanines) was able to bind FECH and form a complex with ABCB10 to the same extent as ABCB7-WT (Figure 5H). ABCB7Mut6, in which the peptide sequence between amino acid residues Gly527 and Asp538 were replaced by alanines, also did not form a complex with FECH (Online Supplementary Figure S10A). Immunoprecipitation of recombinantly expressed
FLAG-tagged ABCB7 wildtype or its mutants (Figure 6A,B) in vivo in G1E-ER4 cells, which co-expressed FECH-HA and that had been silenced to KD the expression of endogenous Abcb7, demonstrated that two short sequences in the C ter- minus of ABCB7, residues V450-L463 and G527-D538, were essential molecular mediators of the interaction with FECH (Figure 6C-E and Online Supplementary Figure S10C,D), thereby confirming our results obtained in vitro with purified proteins. Residues between Gln464 and Val504 of ABCB7 were also involved in stabilizing the bind- ing of FECH (mutants 2, 3 and 4) (Figure 6C,D, Online Supplementary Figure S10D), whereas the ABCB7 sequences between G505 and Q525 and downstream of Val539 were not involved in the interaction with FECH (Figure 6C-E and Online Supplementary Figure S10C,D). Endogenous Abcb10 co-immunoprecipitated with the ABCB7/FECH complex (Figure 6C,D), consistent with formation of the native com- plex of 480 kDa (Figure 5H), and the amount recovered in the eluates after immunoprecipitation of ABCB7 decreased upon expression of the ABCB7 mutants, which were defec- tive in binding FECH, suggesting that formation of a multi- meric complex containing ABCB7 and ABCB10 homod- imers was bridged by a dimeric FECH, rather than through direct physical contacts between the two ABC transporters. In vitro pull-down assays with 35S-radiolabeled ABCB7 wild- type or mutants in the presence of FECH confirmed direct binding of FECH to ABCB7 (Figure 6F-H). The half-life of FECH was significantly reduced in cells lacking endogenous Abcb7 and transfected with ABCB7Mut1 (Online Supplementary Figure S11A,B), which was unable to interact with Fech, indicating that formation of a functional ABCB7/FECH complex was required for FECH stability and completion of heme synthesis.
Discussion
Loss-of-function mutations in ABCB7 cause XLSA with ataxia, a recessive disorder characterized by the presence in the patients’ bone marrow of nucleated erythroblasts that exhibit granules of iron accumulated in the mitochondria surrounding the nuclei.
To gain insights into the primary effects of loss of ABCB7, we generated cell lines with inducible silencing of ABCB7. KD of ABCB7 elicited a dramatic loss of multiple mitochon- drial Fe-S enzymes after only 3 days, whereas defects in cytosolic Fe-S proteins were not observed until 5 days after KD. Similarly, studies in Atm1-depleted yeast cells found a severe growth defect caused by mitochondrial dysfunction, which included loss of oxidative respiration and defective heme biosynthesis.35-37 A growing list of human diseases, including sideroblastic anemia, manifest severe mitochon- drial iron overload,17 and many of these disorders affect the core components of the iron-sulfur cluster (ISC) machinery, including frataxin,38 glutaredoxin 522,39 and HSPA9.40 The molecular mechanism underlying the mitochondrial iron accumulation has not been unveiled. Studies in cell culture models have revealed that a feature of defects manifesting mitochondrial iron overload included the activation of the cytosolic iron starvation response,17,38,41 which increased the IRE-binding activities of IRP1 and IRP2, and upregulation of the ubiquitously expressed mitochondrial iron importer MFRN2.41,42 Our studies show that activation of IRP in the cytosol of cells depleted of ABCB7 and upregulation of the mitochondrial iron importers, MFRN1 and MFRN2,
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haematologica | 2019; 104(9)