Dimeric ferrochelatase bridges ABCB7 and ABCB10 homodimers in an architecturally defined molecular complex required for heme biosynthesis

Abstract

Loss-of-function mutations in the ATP-binding cassette (ABC) transporter of the inner mitochondrial membrane, ABCB7, cause X-linked sideroblastic anemia with ataxia, a phenotype that remains largely unexplained by the proposed role of ABCB7 in exporting a special sulfur species for use in cytosolic iron-sulfur (Fe-S) cluster biogenesis. Here, we generated inducible ABCB7-knockdown cell lines to examine the time-dependent consequences of loss of ABCB7. We found that knockdown of ABCB7 led to significant loss of mitochondrial Fe-S proteins, which preceded the development of milder defects in cytosolic Fe-S enzymes. In erythroid cells, loss of ABCB7 altered cellular iron distribution and caused mitochondrial iron overload due to activation of iron regulatory proteins 1 and 2 in the cytosol and to upregulation of the mitochondrial iron importer, mitoferrin-1. Despite the exceptionally large amount of iron imported into mitochondria, erythroid cells lacking ABCB7 showed a profound hemoglobinization defect and underwent apoptosis triggered by oxidative stress. In ABCB7-depleted cells, defective heme biosynthesis resulted from translational repression of ALAS2 by iron regulatory proteins and from decreased stability of the terminal enzyme ferrochelatase. By combining chemical crosslinking, tandem mass spectrometry and mutational analyses, we characterized a complex formed of ferrochelatase, ABCB7 and ABCB10, and mapped the interfaces of interactions of its components. A dimeric ferrochelatase physically bridged ABCB7 and ABCB10 homodimers by binding near the nucleotide-binding domains of each ABC transporter. Our studies not only underscore the importance of ABCB7 for mitochondrial Fe-S biogenesis and iron homeostasis, but also provide the biochemical characterization of a multiprotein complex required for heme biosynthesis.

From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme

“…In addition, α-ketoglutarate dehydrogenase (KDH) and succinyl-CoA synthase (SUCLA2), which synthesize the heme precursor succinyl-CoA, and TMEM14C, a porphyrinogen transporter [39,82], were also observed in the complex. Independent studies have also shown mitoferrin, a mitochondrial iron importer [84], as well as two ATP binding cassette proteins, ABCB7 [85,86] and ABCB10 [84,86] are protein partners of FECH. These results would suggest that substrate channeling and assembly of the heme metabolon complex may be a key regulatory node for heme synthesis.…”

Systematic Surveys of Iron Homeostasis Mechanisms Reveal Ferritin Superfamily and Nucleotide Surveillance Regulation to be Modified by PINK1 Absence

“…However, Slc25a28 mRNA was diminished under the same treatment, a response that would be in line with an iron export factor. In comparison, Abcb7 and Abcb8 (ATP binding cassette subfamily B member 7/8) are thought to have mitochondrial export functions [ 72 , 80 , 81 , 82 ] and are also important for heme biosynthesis [ 83 ]. As expected after iron depletion, Abcb7 transcript levels were halved, similar to Abcb8 ( Figure 3 ).…”

“…The substrates of ABCB10 transport activity are currently undefined, but its absence was reported to reduce mitoferrin-1 protein levels, iron import into mitochondria, heme biosynthesis, and hemoglobinization, while a role in the export of ALA (5′-aminolevulinic acid) was excluded [ 182 ]. It is difficult to identify the individual substrates for each mitochondrial transporter protein, given that ABCB10, the putative iron-importer mitoferrin, the heme-synthesis factor ferrochelatase, and the ISC-exporter ABCB7 coexist in a protein complex [ 72 , 79 , 83 , 183 ] where the deletion of one member may destabilize also its interactors. Similar to ABCB10, a decrease of iron import and heme biosynthesis was also shown upon deletion of mitoferrin [ 184 , 185 ], leading to universal acceptance of mitoferrin as the main mitochondrial iron importer [ 84 ].…”

Iron Depletion Reduces Abce1 Transcripts While Inducing the Mitophagy Factors Pink1 and Parkin

“…into mitochondria, heme biosynthesis and hemoglobinization, while a role in the export of ALA was excluded [95]. It is difficult to identify the individual substrates for each mitochondrial transporter protein, given that Abcb10, the putative iron-importer mitoferrin, the heme-synthesis factor ferrochelatase, and the ISC-exporter Abcb7 coexist in a protein complex [61,62,66,96] where the deletion of one member may destabilize its interactors. Similar to Abcb10, a decreased iron import and heme biosynthesis was also shown upon deletion of mitoferrin [97,98], leading to universal acceptance of mitoferrin as main mitochondrial iron importer [99].…”

“…and Abcb8 are thought to have mitochondrial export functions [62][63][64][65] and are also important for heme biosynthesis [66]. As expected after iron depletion, Pursuing the heme-and ISC-associated pathways into the cytosol, it is relevant that the putative heme release factor Pgrmc1, as cytosolic factor in association with mitochondrial ferrochelatase, showed a more than two-fold transcriptional induction after iron depletion (DFO: 2.30-fold with p<0.0001; 22BP: 2.16-fold with p<0.0001).…”