The enlarged lysosomes in beige j cells result from decreased lysosome fission and not increased lysosome fusion.

Chediak-Higashi syndrome is an autosomal recessive disorder that affects vesicle morphology. The Chs1/Lyst protein is a member of the BEige And CHediak family of proteins. The absence of Chs1/Lyst gives rise to enlarged lysosomes. Lysosome size is regulated by a balance between vesicle fusion and fission and can be reversibly altered by acidifying the cytoplasm using Acetate Ringer's or by incubating with the drug vacuolin-1. We took advantage of these procedures to determine rates of lysosome fusion and fission in the presence or absence of Chs1/Lyst. Here, we show by microscopy, flow cytometry and in vitro fusion that the absence of the Chs1/Lyst protein does not increase the rate of lysosome fusion. Rather, our data indicate that loss of this protein decreases the rate of lysosome fission. We further show that overexpression of the Chs1/Lyst protein gives rise to a faster rate of lysosome fission. These results indicate that Chs1/Lyst regulates lysosome size by affecting fission.

Regulation of ribonucleotide reductase during iron limitation.

In this issue of Molecular Cell, Sanvisens et al. (2011) report a new mechanism for regulation of yeast ribonucleotide reductase activity that occurs during iron deprivation.

The role of ubiquitination in hepcidin-independent and hepcidin-dependent degradation of ferroportin.

The iron exporter ferroportin (Fpn) is essential to transfer iron from cells to plasma. Systemic iron homeostasis in vertebrates is regulated by the hepcidin-mediated internalization of Fpn. Here, we demonstrate a second route for Fpn internalization; when cytosolic iron levels are low, Fpn is internalized in a hepcidin-independent manner dependent upon the E3 ubiquitin ligase Nedd4-2 and the Nedd4-2 binding protein Nfdip-1. Retention of cell-surface Fpn through reductions in Nedd4-2 results in cell death through depletion of cytosolic iron. Nedd4-2 is also required for internalization of Fpn in the absence of ferroxidase activity as well as for the entry of hepcidin-induced Fpn into the multivesicular body. C. elegans lacks hepcidin genes, and C. elegans Fpn expressed in mammalian cells is not internalized by hepcidin but is internalized in response to iron deprivation in a Nedd4-2-dependent manner, supporting the hypothesis that Nedd4-2-induced internalization of Fpn is evolutionarily conserved.

Hepcidin and ferroportin: the new players in iron metabolism.

Systemic iron homeostasis is regulated by the interaction of the peptide hormone, hepcidin and the iron exporter, ferroportin. Mutations in FPN1, the gene that encodes ferroportin, result in iron-overload disease that shows dominant inheritance and variation in phenotype. The inheritance of ferroportin-linked disorders can be explained by the finding that ferroportin is a multimer and the product of the mutant allele participates in multimer formation. The nature of the ferroportin mutant can explain the variation in phenotype, which is due to either decreased iron export activity or decreased ability to be downregulated by hepcidin. Iron export through ferroportin is determined by the concentration of ferroportin in plasma membrane, which is the result of both synthetic and degradation events. Ferroportin degradation can occur by hepcidin-dependent and hepcidin-independent internalization. Ferroportin expression is regulated transcriptionally and posttranslationally.

Targeted deletion of the mouse Mitoferrin1 gene: from anemia to protoporphyria.

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show that the activity of mitoferrin1 is required to manage an increase in heme synthesis. The data also show that alterations in heme synthesis within hepatocytes can lead to protoporphyria and hepatotoxicity.

Genetic and biochemical analysis of high iron toxicity in yeast: iron toxicity is due to the accumulation of cytosolic iron and occurs under both aerobic and anaerobic conditions.

Iron storage in yeast requires the activity of the vacuolar iron transporter Ccc1. Yeast with an intact CCC1 are resistant to iron toxicity, but deletion of CCC1 renders yeast susceptible to iron toxicity. We used genetic and biochemical analysis to identify suppressors of high iron toxicity in Δccc1 cells to probe the mechanism of high iron toxicity. All genes identified as suppressors of high iron toxicity in aerobically grown Δccc1 cells encode organelle iron transporters including mitochondrial iron transporters MRS3, MRS4, and RIM2. Overexpression of MRS3 suppressed high iron toxicity by decreasing cytosolic iron through mitochondrial iron accumulation. Under anaerobic conditions, Δccc1 cells were still sensitive to high iron toxicity, but overexpression of MRS3 did not suppress iron toxicity and did not result in mitochondrial iron accumulation. We conclude that Mrs3/Mrs4 can sequester iron within mitochondria under aerobic conditions but not anaerobic conditions. We show that iron toxicity in Δccc1 cells occurred under both aerobic and anaerobic conditions. Microarray analysis showed no evidence of oxidative damage under anaerobic conditions, suggesting that iron toxicity may not be solely due to oxidative damage. Deletion of TSA1, which encodes a peroxiredoxin, exacerbated iron toxicity in Δccc1 cells under both aerobic and anaerobic conditions, suggesting a unique role for Tsa1 in iron toxicity.

Induction of FPN1 transcription by MTF-1 reveals a role for ferroportin in transition metal efflux.

Ferroportin (Fpn) is the only known iron exporter in vertebrate cells and plays a critical role in iron homeostasis regulating cytosolic iron levels and exporting iron to plasma. Ferroportin1 (FPN1) expression can be transcriptionally regulated by iron as well as other transition metals. Fpn can also be posttranslationally regulated by hepcidin-mediated internalization and degradation. We demonstrate that zinc and cadmium induce FPN1 transcription through the action of Metal Transcription Factor-1 (MTF-1). These transition metals induce MTF-1 translocation into the nucleus. Zinc leads to MTF-1 binding to the FPN1 promoter, while iron does not. Silencing of MTF-1 reduces FPN1 transcription in response to zinc but not in response to iron. The mouse FPN1 promoter contains 2 MTF-1 binding sites and mutation of those sites affects the zinc and cadmium-dependent expression of a FPN1 promoter reporter construct. We demonstrate that Fpn can transport zinc and can protect zinc sensitive cells from high zinc toxicity.

Genetic dissection of a mitochondria-vacuole signaling pathway in yeast reveals a link between chronic oxidative stress and vacuolar iron transport.

Deletion of two homologous genes, MRS3 and MRS4, that encode mitochondrial iron transporters affects the activity of the vacuolar iron importer Ccc1. Ccc1 levels are decreased in Deltamrs3Deltamrs4 cells, but the activity of the transporter is increased, resulting is reduced cytosolic iron. Overexpression of CCC1 in Deltamrs3Deltamrs4 cells results in a severe growth defect due to decreased cytosolic iron, referred to as the mitochondria-vacuole signaling (MVS) phenotype. Mutants were identified that suppress the MVS growth defect, and FRA1 was identified as a gene that suppresses the MVS phenotype. Overexpression of FRA1 suppresses altered transition metal metabolism in Deltamrs3Deltamrs4 cells, whereas deletion of FRA1 is synthetically lethal with Deltamrs3Deltamrs4. Fra1 binds to Tsa1, which encodes a thioredoxin-dependent peroxidase. Deletion of TSA1 or TRR1 is synthetically lethal in Deltamrs3Deltamrs4 cells, suggesting that Deltamrs3Deltamrs4 cells generate reactive oxygen metabolites. The generation of reactive oxygen metabolites in Deltamrs3Deltamrs4 cells was confirmed by use of the reporter molecule 2',7'-dichlorodihydrofluorescein diacetate. These results suggest that mitochondria-induced oxidant damage is responsible for activating Ccc1 and that Fra1 and Tsa1 can reduce oxidant damage.

Specific iron chelators determine the route of ferritin degradation.

Deferoxamine (DFO) is a high-affinity Fe (III) chelator produced by Streptomyces pilosus. DFO is used clinically to remove iron from patients with iron overload disorders. Orally administered DFO cannot be absorbed, and therefore it must be injected. Here we show that DFO induces ferritin degradation in lysosomes through induction of autophagy. DFO-treated cells show cytosolic accumulation of LC3B, a critical protein involved in autophagosomal-lysosomal degradation. Treatment of cells with the oral iron chelators deferriprone and desferasirox did not show accumulation of LC3B, and degradation of ferritin occurred through the proteasome. Incubation of DFO-treated cells with 3-methyladenine, an autophagy inhibitor, resulted in degradation of ferritin by the proteasome. These results indicate that ferritin degradation occurs by 2 routes: a DFO-induced entry of ferritin into lysosomes and a cytosolic route in which iron is extracted from ferritin before degradation by the proteasome.

Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity.

Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn2+, Co2+, Fe2+, Cd2+, Ni2+ and Mn2+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865-33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe2+ could also transport Mn2+. Some Zrc1 mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn2+. All of the amino acid substitutions that resulted in a gain in Fe2+ transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal- binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains.

On the mechanism of iron sensing by IRP2: new players, new paradigms.

Two iron regulatory proteins (IRP1 and IRP2) regulate translation and/or stability of mRNAs encoding proteins required for iron storage, acquisition and utilization. Rather than IRP2 directly sensing iron concentrations, iron has been shown to regulate the level of the SKP1-CUL1-FBXL5 E3 ubiquitin ligase protein complex, which is responsible for IRP2 degradation.

A single amino acid change in the yeast vacuolar metal transporters ZRC1 and COT1 alters their substrate specificity.

Iron is an essential nutrient but in excess may damage cells by generating reactive oxygen species due to Fenton reaction or by substituting for other transition metals in essential proteins. The budding yeast Saccharomyces cerevisiae detoxifies cytosolic iron by storage in the vacuole. Deletion of CCC1, which encodes the vacuolar iron importer, results in high iron sensitivity due to increased cytosolic iron. We selected mutants that permitted Deltaccc1 cells to grow under high iron conditions by UV mutagenesis. We identified a mutation (N44I) in the vacuolar zinc transporter ZRC1 that changed the substrate specificity of the transporter from zinc to iron. COT1, a vacuolar zinc and cobalt transporter, is a homologue of ZRC1 and both are members of the cation diffusion facilitator family. Mutation of the homologous amino acid (N45I) in COT1 results in an increased ability to transport iron and decreased ability to transport cobalt. These mutations are within the second hydrophobic domain of the transporters and show the essential nature of this domain in the specificity of metal transport.

Iron depletion limits intracellular bacterial growth in macrophages.

Many intracellular pathogens infect macrophages and these pathogens require iron for growth. Here we demonstrate in vitro that the intracellular growth of Chlamydia psittaci, trachomatis, and Legionella pneumophila is regulated by the levels of intracellular iron. Macrophages that express cell surface ferroportin, the only known cellular iron exporter, limit the intracellular growth of these bacteria. Hepcidin is an antimicrobial peptide secreted by the liver in response to inflammation. Hepcidin binds to ferroportin mediating its internalization and degradation. Addition of hepcidin to infected macrophages enhanced the intracellular growth of these pathogens. Macrophages from flatiron mice, a strain heterozygous for a loss-of-function ferroportin mutation, showed enhanced intracellular bacterial growth independent of the presence of exogenous hepcidin. Macrophages, from wild-type or flatiron mice, incubated with the oral iron chelator deferriprone or desferasirox showed reduced intracellular bacterial growth suggesting that these chelators might be therapeutic in chronic intracellular bacterial infections.

Chediak-Higashi syndrome.

PURPOSE OF REVIEW: Chediak-Higashi syndrome, a rare autosomal recessive disorder, was described over 50 years ago. Patients show hypopigmentation, recurrent infections, mild coagulation defects and varying neurologic problems. Treatment is bone marrow transplant, which is effective in treating the hematologic and immune defects, however the neurologic problems persist. The CHS1/LYST gene was identified over 10 years ago and homologous CHS1/LYST genes are present in all eukaryotes. This review will discuss the advances made in understanding the clinical aspects of the syndrome and the function of CHS1/LYST/Beige. RECENT FINDINGS: Clinical reports of Chediak-Higashi syndrome have identified mutations throughout the CHS1/LYST gene. The nature of the mutation can be a predictor of the severity of the disease. Over the past decade the CHS1/LYST family of proteins has been analyzed using model organisms, two-hybrid analysis, overexpression phenotypes and dominant negatives. These studies suggest that the CHS1/LYST protein is involved in either vesicle fusion or fission. SUMMARY: Although CHS is a rare disease, the Chediak-like family of proteins is providing insight into the regulation of vesicle trafficking. Understanding the basic mechanisms that govern vesicle trafficking will provide essential information regarding how loss of CHS1/LYST affects hematologic, immunologic and neurologic processes.

Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin.

Ferroportin (Fpn), a ferrous iron Fe(II) transporter responsible for the entry of iron into plasma, is regulated post-translationally through internalization and degradation following binding of the hormone hepcidin. Cellular iron export is impaired in mice and humans with aceruloplasminemia, an iron overload disease due to mutations in the ferroxidase ceruloplasmin (Cp). In the absence of Cp Fpn is rapidly internalized and degraded. Depletion of extracellular Fe(II) by the yeast ferroxidase Fet3p or iron chelators can maintain cell surface Fpn in the absence of Cp. Iron remains bound to Fpn in the absence of multicopper oxidases. Fpn with bound iron is recognized by a ubiquitin ligase, which ubiquitinates Fpn on lysine 253. Mutation of lysine 253 to alanine prevents ubiquitination and maintains Fpn-iron on cell surface in the absence of ferroxidase activity. The requirement for a ferroxidase to maintain iron transport activity represents a new mechanism of regulating cellular iron export, a new function for Cp and an explanation for brain iron overload in patients with aceruloplasminemia.

The molecular mechanism of hepcidin-mediated ferroportin down-regulation.

Ferroportin (Fpn) is the only known iron exporter in vertebrates. Hepcidin, a peptide secreted by the liver in response to iron or inflammation, binds to Fpn, inducing its internalization and degradation. We show that after binding of hepcidin, Fpn is tyrosine phosphorylated at the plasma membrane. Mutants of human Fpn that do not get internalized or that are internalized slowly show either absent or impaired phosphorylation. We identify adjacent tyrosines as the phosphorylation sites and show that mutation of both tyrosines prevents hepcidin-mediated Fpn internalization. Once internalized, Fpn is dephosphorylated and subsequently ubiquitinated. An inability to ubiquitinate Fpn does not prevent hepcidin-induced internalization, but it inhibits the degradation of Fpn. Ubiquitinated Fpn is trafficked through the multivesicular body pathway en route to degradation in the late endosome/lysosome. Depletion of proteins involved in multivesicular body trafficking (Endosome Sorting Complex Required for Transport proteins), by small-interfering RNA, reduces the trafficking of Fpn-green fluorescent to the lysosome.

The flatiron mutation in mouse ferroportin acts as a dominant negative to cause ferroportin disease.

Ferroportin disease is caused by mutation of one allele of the iron exporter ferroportin (Fpn/IREG1/Slc40a1/MTP1). All reported human mutations are missense mutations and heterozygous null mutations in mouse Fpn do not recapitulate the human disease. Here we describe the flatiron (ffe) mouse with a missense mutation (H32R) in Fpn that affects its localization and iron export activity. Similar to human patients with classic ferroportin disease, heterozygous ffe/+ mice present with iron loading of Kupffer cells, high serum ferritin, and low transferrin saturation. In macrophages isolated from ffe/+ heterozygous mice and through the use of Fpn plasmids with the ffe mutation, we show that Fpn(ffe) acts as a dominant negative, preventing wild-type Fpn from localizing on the cell surface and transporting iron. These results demonstrate that mutations in Fpn resulting in protein mislocalization act in a dominant-negative fashion to cause disease, and the Fpn(ffe) mouse represents the first mouse model of ferroportin disease.

Evidence for the multimeric structure of ferroportin.

Ferroportin (Fpn) (IREG1, SLC40A1, MTP1) is an iron transporter, and mutations in Fpn result in a genetically dominant form of iron overload disease. Previously, we demonstrated that Fpn is a multimer and that mutations in Fpn are dominant negative. Other studies have suggested that Fpn is not a multimer and that overexpression or epitope tags might affect the localization, topology, or multimerization of Fpn. We generated wild-type Fpn with 3 different epitopes, GFP, FLAG, and c-myc, and expressed these constructs in cultured cells. Co-expression of any 2 different epitope-tagged proteins in the same cell resulted in their quantitative coimmunoprecipitation. Treatment of Fpn-GFP/Fpn-FLAG-expressing cells with crosslinking reagents resulted in the crosslinking of Fpn-GFP and Fpn-FLAG. Western analysis of rat glioma C6 cells or mouse bone marrow macrophages exposed to crosslinking reagents showed that endogenous Fpn is a dimer. These results support the hypothesis that the dominant inheritance of Fpn-iron overload disease is due to the dominant-negative effects of mutant Fpn proteins.