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International audience; Mitochondrial iron import is essential for iron-sulfur cluster formation and heme biosynthesis. Two nuclear-encoded vertebrate mitochondrial high-affinity iron importers, mitoferrin1 (Mfrn1) and Mfrn2, have been identified in mammals. In mice, the gene encoding Mfrn1, solute carrier family 25 member 37 (Slc25a37), is highly expressed in sites of erythropoiesis, and whole-body Slc25a37 deletion leads to lethality. Here, we report that mice with a deletion of Slc25a28 (encoding Mfrn2) are born at expected Mendelian ratios, but show decreased male fertility due to reduced sperm numbers and sperm motility. Mfrn2 -/- mice placed on a low-iron diet exhibited reduced mitochondrial manganese, cobalt, and zinc levels, but not reduced iron. Hepatocyte-specific loss of Slc25a37 (encoding Mfrn1) in Mfrn2 -/- mice did not affect animal viability, but resulted in a 40% reduction in mitochondrial iron and reduced levels of oxidative phosphorylation proteins. Placing animals on a low-iron diet exaggerated the reduction in mitochondrial iron observed in liver-specific Mfrn1/2-knockout animals. Mfrn1 -/-/Mfrn2 -/- bone marrow-derived macrophages or skin fibroblasts in vitro were unable to proliferate, and overexpression of Mfrn1-GFP or Mfrn2-GFP prevented this proliferation defect. Loss of both mitoferrins in hepatocytes dramatically reduced regeneration in the adult mouse liver, further supporting the notion that both mitoferrins transport iron and that their absence limits proliferative capacity of mammalian cells. We conclude that Mfrn1 and Mfrn2 contribute to mitochondrial iron homeostasis and are required for high-affinity iron import during active proliferation of mammalian cells.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.

The lysosome is considered the terminal point in the endocytic pathway, as molecules are delivered to the lysosome for degradation (for review see (1)). Fusion of endosomes with lysosomes leads to the intermixing of vesicular contents with lysosomal hydrolases. The addition of vesicular contents and membrane would be expected to increase lysosome size. The steady state median size of lysosomes is constant, however, indicating that lysosomal surface area is regulated by lysosomal membrane efflux. It has been suggested that endocytic membrane influx is transient and that once endosomal contents are transferred to lysosomes, endosomes fission off, a process referred to as “kiss-and-run” (2). Constant fusion and fission of membrane along the endocytic pathway results in remodeling of organelles, which may eventually become lysosomes, a process termed maturation (3). Lysosomes are also capable of homotypic fusion (4–13) yet the number of lysosomes is constant. Therefore, lysosomes must be capable of fission or some other form of membrane recycling, although the machinery required for lysosome fission has not been identified. Malregulation of fusion or fission will result in alterations in lysosome size. This is a hallmark of Chediak Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by enlarged lysosomes or lysosome-related organelles present within all cells (14–17). The mutated gene responsible for CHS (LYST) and the orthologous mouse disorder beige (Lyst) were identified almost 15 years ago (18, 19). The identification of Chs1/Lyst defined a family of proteins termed the BEige And CHediak (BEACH) family (15, 20). The BEACH family protein LvsA in Dictyostelium discoideum regulates the size of the contractile vacuole and is involved in cytokinesis (21–23). Neurobeachin has been characterized to be involved in central synapse formation (24). FAN, the smallest member of the BEACH family, is thought to be an adapter protein linking TNFa signaling to neutral sphingomyelinase. Bph1, the only Saccharomyces cerevisiae homologue is involved in vesicle trafficking, but loss of Bph1 does not affect vacuole morphology (25). The functions of many members of the BEACH family are still unclear. Previously, we suggested that loss of Chs1/Lyst resulted in decreased lysosomal fission based upon the observation that overexpression of Lyst resulted in smaller than wild type lysosomes (26). Fusion of wild type and beigej cells led to complementation of the large lysosome size (27). We noted, however, that complementation of large beigej lysosomes required their fusion with wild type lysosomes, suggesting that the wild type lysosome provided a factor promoting decreased lysosome size. Studies on the D. discoideum homologue LvsB suggested that LvsB was either a negative regulator of lysosome fusion (28, 29) or a positive regulator of “post-lysosome” fission (30). In this study we examine whether the loss of Chs1/Lyst changes the rate of lysosome fusion or lysosome fission by measuring the rate of reformation of lysosomes following treatments that increase or decrease lysosome size. We show that the loss of Chs1/Lyst does not affect lysosome fusion rates but affects the rate of lysosome fission and that it is the defect in fission that gives rise to enlarged lysosomes associated with the loss of Chs1/Lyst.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well understood. To better understand these regulatory mechanisms, we profiled gene expression in EPO-treated fetal liver cells to identify novel iron regulatory genes (Figure A). We determined that FAM210B, a mitochondrial inner membrane protein, was essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation (Figure B). Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation (Figure C). These defects were corrected with a lipid-soluble small molecule iron transporter in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter, but is required for optimal mitochondrial iron import during erythroid differentiation (Figure D). FAM210B is also required for optimal FECH activity in differentiating erythroid cells. As FAM210B interacts with the terminal enzymes of the heme synthesis pathway, we propose that FAM210B functions as an adaptor protein to facilitate the formation of an oligomeric mitochondrial iron transport complex, which is required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis (Figure E). Collectively, our data reveal a novel mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism. Figure. Figure. Disclosures Palis: Rubies Therapeutics: Consultancy.

Lysosomes are membrane-bound organelles that contain hydrolases capable of degrading proteins, lipids, and carbohydrates. They are involved in nutrient sensing and storage and retrieval. Lysosomes are highly dynamic and are capable of fusion and fission events with other organelles and plasma membrane. Higher eukaryotes possess a specialized group of lysosomes termed secretory lysosomes that are involved in pigmentation, coagulation, wound repair, and immunologic functions. Understanding the molecular machinery that regulates lysosome and secretory lysosome homeostasis is underscored by the fact that mutations in the genes that regulate the biogenesis, movement and delivery of lysosome and secretory lysosome contents result in human diseases.