Ferroportin-mediated mobilization of ferritin iron precedes ferritin degradation by the proteasome

Ferritin is a cytosolic molecule comprised of subunits that self-assemble into a nanocage capable of containing up to 4500 iron atoms. Iron stored within ferritin can be mobilized for use within cells or exported from cells. Expression of ferroportin (Fpn) results in export of cytosolic iron and ferritin degradation. Fpn-mediated iron loss from ferritin occurs in the cytosol and precedes ferritin degradation by the proteasome. Depletion of ferritin iron induces the monoubiquitination of ferritin subunits. Ubiquitination is not required for iron release but is required for disassembly of ferritin nanocages, which is followed by degradation of ferritin by the proteasome. Specific mammalian machinery is not required to extract iron from ferritin. Iron can be removed from ferritin when ferritin is expressed in Saccharomyces cerevisiae, which does not have endogenous ferritin. Expressed ferritin is monoubiquitinated and degraded by the proteasome. Exposure of ubiquitination defective mammalian cells to the iron chelator desferrioxamine leads to degradation of ferritin in the lysosome, which can be prevented by inhibitors of autophagy. Thus, ferritin degradation can occur through two different mechanisms.     

Microgravity mediated changes in phytoferritin accumulation in soybean root cap cells

Phytoferritin is an iron-protein complex analogous to the ferritin found in mammalian, bacteria and fungi cells. Phytoferritin molecules are large proteins, about 10.5 nm in diameter, visualised in an electron microscope as discrete, electron dense particles with iron-containing core, where several thousand atoms of iron lie within the proteinaceous shell (apoferritin). In higher plants, a plastid stroma is the site of phytoferritin storage. Phytoferritin is seen in all types of plastids. It is considered to be a mechanism used by cells to store iron in a non-toxic form. Phytoferritin-bound iron may subsequently be used to form iron-containing components. It was shown that low levels of phytoferritin are synthesised in normal green leaves, whereas chlorotic leaves do not have a measurable amount of phytoferritin and leaves of iron-loaded seedlings contain a high level of total iron, and phytoferritin well-filled by iron. Phytoferritin accumulation was observed in photosynthetic inactivity chloroplasts during senescence and disease. In this study we analised the effects of microgravity and ethylene on production of phytoferritin in the root cap columella cells of soybean seedlings.     

Siderosomal ferritin. The missing link between ferritin and haemosiderin?

A minor electrophoretically fast component was found in ferritin from iron-loaded rat liver in addition to a major electrophoretically slow ferritin similar to that observed in control rats. The electrophoretically fast ferritin showed immunological identity with the slow component, but on electrophoresis in SDS it gave a peptide of 17.3 kDa, in contrast with the electrophoretically slow ferritin, which gave a major band corresponding to the L-subunit (20.7 kDa). Thus the electrophoretically fast ferritin resembles that reported by Massover [(1985) Biochim. Biophys. Acta 829, 377-386] in livers of mice with short-term parenteral iron overload. The electrophoretically fast ferritin had a lower iron content (2000 Fe atoms/molecule) than the electrophoretically slow ferritin (3000 Fe atoms/molecule). Removal and re-incorporation of iron was possible without effect on the electrophoretic mobility of either ferritin species. On subcellular fractionation the electrophoretically fast ferritin was enriched in pellet fractions and was the sole soluble ferritin isolated from iron-laden secondary lysosomes (siderosomes). The amount and relative proportion of the electrophoretically fast species increased with iron loading. Haemosiderin isolated from siderosomes was found to contain a peptide reactive to anti-ferritin serum and corresponding to the 17.3 kDa peptide of the electrophoretically fast ferritin species. Unlike the electrophoretically slow ferritin, the electrophoretically fast ferritin did not become significantly radioactive in a 1 h biosynthetic labelling experiment. We conclude that the minor ferritin is not, as has been suggested for mouse liver ferritin, 'a completely new species of smaller holoferritin that represents a shift in the ferritin phenotype' in response to siderosis, but a precursor of haemosiderin, in agreement with the proposal by Richter [(1984) Lab. Invest. 50, 26-35] concerning siderosomal ferritin.     

Ferritin accumulation under iron scarcity in Drosophila iron cells

Ferritins are highly stable, multi-subunit protein complexes with iron-binding capacities that reach 4500 iron atoms per ferritin molecule. The strict dependence of cellular physiology on an adequate supply of iron cofactors has likely been a key driving force in the evolution of ferritins as iron storage molecules. The insect intestine has long been known to contain cells that are responsive to dietary iron levels and a specialized group of “iron cells” that always accumulate iron-loaded ferritin, even when no supplementary iron is added to the diet. Here, we further characterize ferritin localization in Drosophila melanogaster larvae raised under iron-enriched and iron-depleted conditions. High dietary iron intake results in ferritin accumulation in the anterior midgut, but also in garland (wreath) cells and in pericardial cells, which together filter the circulating hemolymph. Ferritin is also abundant in the brain, where levels remain unaltered following dietary iron chelation, a treatment that depletes ferritin from the aforementioned tissues. We attribute the stability of ferritin levels in the brain to the function of the blood-brain barrier that may shield this organ from systemic iron fluctuations. Most intriguingly, our dietary manipulations demonstrably iron-depleted the iron cells without a concomitant reduction in their production of ferritin. Therefore, insect iron cells may constitute an exception from the evolutionary norm with respect to iron-dependent ferritin regulation. It will be of interest to decipher both the physiological purpose served and the mechanism employed to untie ferritin regulation from cellular iron levels in this cell type.     

The role of ferritin in developing primary bean leaves under various light conditions

Ferritin and ferritin-iron in the primary leaves of Phaseolus vulgaris L. were determined during growth in the dark, in the light, and during de-etiolation. The ratio ferritin protein/total protein appeared to be rather constant. In dark-grown leaves maximally 50% of the total extractable iron was found to be present in ferritin. This percentage was lower in deetiolating and light-grown leaves. In ten-day-old green leaves no ferritin-iron could be measured. The translocation of iron from cotyledons to the developing plant appears to be related to the need for iron in the leaves. These results suggest that ferritin acts as a buffer molecule for iron in plants.     

A biochemical comparison of normal human liver and hepatocellular carcinoma ferritins.

1. The iron contents, gel migration rates and isoelectric-focusing patterns of normal liver and hepatocellular carcinoma ferritins from the same patients were compared. 2. Sucrose-density-gradient centrifugation showed that the number of iron atoms per ferritin molecule was decreased to approximately half in carcinoma tissue when compared with normal liver. 3. On electrophoresis, hepatocellular carcinoma ferritin migrates faster and is therefore more negatively charged than normal liver ferritin, thus refuting the general view that the more negatively charged a ferritin molecule the greater its iron content. 4. Comparison of tumour and normal liver ferritin subunit compositions on acid/urea/polyacrylamide gels showed hepatocellular carcinoma ferritin to contain an additional, more negatively charged, subunit to normal liver ferritin. 5. Isoelectric focusing showed that hepatocellular carcinoma tissue contains isoferritins with isoelectric points intermediate between the ranges of normal liver and normal heart isoferritins.     

Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by ⁵⁹Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.     

Biochemistry of nonheme iron in man. I. Iron proteins and cellular iron metabolism

Total plasma iron turnover in man is about 36 mg/day. Transferrin is the iron transport protein of plasma, which can bind 2 atoms of iron per protein molecule, and which interacts with various cell types to provide them with the iron required for their metabolic and proliferative processes. All tissues contain transferrin receptors on their plasma membrane surfaces, which interact preferentially with diferric transferrin. In erythroid cells as well as certain laboratory cell lines, the removal of iron from transferrin apparently proceeds via the receptor-mediated endocytosis process. Transferrin and its receptor are recycled to the cell surface, whereas the iron remains in the cell. The mode of iron uptake in the hepatocyte, the main iron storage tissue, is less certain. The release of iron by hepatocytes, as well as by the reticuloendothelial cells, apparently proceeds nonspecifically. All tissues contain the iron storage protein ferritin, which stores iron in the ferric state, though iron must be in the ferrous state to enter and exit the ferritin molecule. Cellular cytosol also contains a small-molecular-weight ferrous iron pool, which may interact with protoporphyrin to form heme, and which apparently is the form of iron exported by hepatocytes and macrophages. In plasma, the ferrous iron is converted into the ferric form via the action of ceruloplasmin.     

Purification and characterization of an iron-induced ferritin from soybean (Glycine max) cell suspensions.

Ferric citrate induces ferritin synthesis and accumulation in soybean (Glycine max) cell suspension cultures [Proudhon, Briat & Lescure (1989) Plant Physiol. 90, 586-590]. This iron-induced ferritin has been purified from cells grown for 72 h in the presence of either 100 microM- or 500 microM-ferric citrate. It has a molecular mass of about 600 kDa and is built up from a 28 kDa subunit which is recognized by antibodies raised against pea (Pisum sativum) seed ferritin and it has the same N-terminal sequence as this latter, except for residue number 3, which is alanine in pea seed ferritin instead of valine in iron-induced soybean cell ferritin. It contains an average of 1800 atoms of iron per molecule whatever the ferric citrate concentration used to induce its synthesis. It is shown that the presence of 100 microM- or 500 microM-ferric citrate in the culture medium leads respectively to an 11- and 28-fold increase in the total intracellular iron concentration and to a 30- and 60-fold increase in the ferritin concentration. However, the percentage of iron stored in the mineral core of ferritin remains constant whatever the ferric citrate concentration used and represents only 5-6% of cellular iron.     

Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron

Growing human choriocarcinoma BeWo b24 cells contain 1.5 X 10(6) functional cell surface transferrin binding sites and 2.0 X 10(6) intracellular binding sites. These cells rapidly accumulate iron at a rate of 360,000 iron atoms/min/cell. During iron uptake the transferrin and its receptor recycle at least each 19 min. The accumulated iron is released from the BeWo cells at a considerable rate. The time required to release 50% of previously accumulated iron into the extracellular medium is 30 h. This release process is cell line-specific as HeLa cells release very little if any iron. The release of iron by BeWo cells is stimulated by exogenous chelators such as apotransferrin, diethylenetriaminepenta-acetic acid, desferral, and apolactoferrin. The time required to release 50% of the previously accumulated iron into medium supplemented with chelator is 15 h. In the absence of added chelators iron is released as a low molecular weight complex, whereas in the presence of chelator the iron is found complexed to the chelator. Uptake of iron is inhibited by 250 microM primaquine or 2.5 microM monensin. However, the release of iron is not inhibited by these drugs. Intracellular iron is stored bound to ferritin. A model for the release of iron by BeWo cells and its implication for transplacental iron transport is discussed.     

Subcellular Localization and Characterization of Excessive Iron in the Nicotianamine-less Tomato Mutant chloronerva

To understand the function of the Fe2+-complexing compound nicotianamine (NA) in the iron metabolism of plants we have localized iron and other elements in the NA-containing tomato wild type (Lycopersicon esculentum) and its NA-free mutant chloronerva by quantitative x-ray microanalysis. Comparison of element composition of the rhizodermal cell walls indicated that the wild type accumulated considerable amounts of iron and phosphorus in the cell wall, whereas in the mutant iron and phosphorus were detected in the cytoplasm and vacuoles of the rhizodermis. In mutant leaves containing high iron concentrations in the symplast, electron-dense inclusions were detected in chloroplasts and phloem. Such particles, consisting mainly of iron and phosphorus, were never found in the wild type and were very rarely detected in young chlorotic mutant leaves or after treatment of the mutant with NA. For further characterization the electron-dense inclusions in mutant leaves were isolated and compared by sodium dodecyl sulfate-gel electrophoresis and immunoblotting to ferritin from iron-loaded Phaseolus vulgaris leaves. Antibodies raised against purified Phaseolus leaf ferritin were used. Neither in mutant nor in wild type (iron loaded and control) was ferritin protein detected. These results suggest that the electron-dense inclusions in mutant leaves are not identical with ferritin. It is concluded that NA is necessary to complex ferrous iron in a soluble and available form within the cells. In the absence of NA the precipitation of excessive iron in the form of insoluble ferric phosphate compounds could protect the cells from iron overload.     

Mobilization of iron from ferritin by isolated mitochondria effects of species compatibility between ferritin and mitochondria and iron content of ferritin

Mitochondria mobilize iron from ferritin by a mechanism that depends on external FMN. With rat liver mitochondria, the rate of mobilization of iron is higher from rat liver ferritin than from horse spleen ferritin. With horse liver mitochondria, the rate of iron mobilization is higher from horse spleen ferritin than from rat liver ferritin. The results are explained by a higher affinity between mitochondria and ferritins of the same species. The mobilization of iron increases with the iron content of the ferritin and then levels off. A maximum is reached with ferritins containing about 1 200 iron atoms per molecule. The results represent further evidence that ferritin may function as a direct iron donor to the mitochondria.     

Isolation and characterization of ferritin from the hepatopancreas of the musselMytilus edulis

Summary The main iron-binding protein in the hepatopancreas of the musselMytilus edulis, which had been previously iron-loaded by exposure to carbonyl iron (spheres of elemental iron less than 5 m diameter), has been isolated to electrophoretic purity and identified as ferritin. This ferritin hasM _r, of 480000, pI of 4.7–5.0 and is composed of two subunits,M _r 18500 andM _r 24600. Under the electron microscope, it appears as electron-dense iron cores of average diameter 5 nm surrounded by a polypeptide shell to a final average overall diameter of 11 nm. The purified protein contains, on average, 200 iron atoms/molecule protein. On immunodiffusion,M. edulis hepatopancreas ferritin gives a partial cross-reaction with antiserum to horse spleen ferritin and lamprey (Geotria australis) liver ferritin but does not react with antiserum to chiton (Acanthopleura hirtosa) haemolymph ferritin.     

The binding of ferric iron by ferritin.

Equilibrium-dialysis experiments with 59Fe-labelled Fe(III) chelate solutions show that ferritin is capable of binding a limited number of Fe(III) atoms. Some of this Fe(III) is readily removed, but up to about 200 Fe(III) atoms/molecule remain bound after extensive washing. Some exchange of labelled Fe(III) with endogenous unlabelled ferritin Fe occurs during prolonged dialysis against 59Fe(III)-citrate, but there is a net binding of Fe(III). Bound Fe(III) resembles endogenous Fe(III) in several respects. It appears to be attached to the micelle and not to the protein component of ferritin. Although the physiological mechanism of Fe incorporation into ferritin is unknown, our experiments suggest the possibility that some iron finds its way into ferritin as Fe(III) chelate.     

Ultrastructure of ferritin in the leaves of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> under stress conditions

The location and structure of ferritin in the parenchyma of leaf minor veins of the common ice plant (Mesembryanthemum crystallinum L.) treated with exogenous putrescine under salinity conditions were investigated by electron microscopy. Considerable aggregates of ferritin were detected in the chloroplasts of bundle sheath cells, in companion phloem cells, and other parenchyma cells of leaf minor veins. The structure of ferritin in the vascular parenchyma chloroplasts suggests that it was partially degraded and converted to phytosiderin. This point of view is based on indistinct structure of Fe-containing cores of ferritin molecules, break of distance between the cores, and their pronounced ability to aggregate and produce larger structures. Aggregation of Fe-containing cores apparently pointed to the destruction of ferritin protein envelope or its partial degradation. In a certain stage of ferritin destruction, electron-dense material and the structures resembling small vesicles appeared between the Fe-containing cores. Electron-dense inclusions, whose structure was similar to that of phytosiderin, were also detected in the vacuoles. Examination of the cross sections done without additional staining showed that the same as ferritin, phytosiderin in the chloroplasts and vacuoles was dark-colored against weakly colored cellular structures. In the vascular parenchyma of control plant leaves, the level of ferritin and phytosiderin was greater than in the mesophyll and much lower than in the plants simultaneously treated with NaCl and putrescine. In control material, iron cores of ferritin and phytosiderin were more light-colored and 2–3 times smaller in size than in the experimental treatment. Destruction of ferritin essentially did not occur in the mesophyll but was observed in the chloroplasts of bundle sheath cells on the border between the mesophyll and vascular bundle. The presence of much ferritin and phytosiderin on the border between the mesophyll and the vessels is accounted for by the fact that the vascular parenchyma is a buffer area that maintains a specific concentration of iron in the mesophyll of leaves and other parts of the plant. Within the cell, the role of such a buffer is performed by ferritin and vacuoles. Transformation of ferritin to insoluble hydrophobic phytosiderin is supposed to be an efficient way of withdrawing the excess of active iron from the cellular metabolism and therefore of relaxing oxidative stress. Ferritin and phytosiderin were detected not only in parenchyma cells of leaf minor veins but in sieve tubes as well. This suggests that iron may be transported within the plant as a component of protein complex.     

Ferritin is not a required intermediate for iron utilization in heme synthesis

Pulse-chase analysis of newt (Triturus cristatus) erythroblasts has shown that ferritin is not a primary source of iron for heme synthesis. During chase incubation with and without non-radioactive plasma iron in the medium, no transfer of 59Fe from ferritin to hemoglobin was detected although the integrity of heme synthesis was maintained. In puromycin-inhibited cells where iron uptake was drastically curtailed, heme synthesis continued to occur, though at reduced levels; incorporation of 59Fe from the plasma appeared initially in heme and hemoglobin without any prior labelling of ferritin. These results indicate that ferritin is neither an obligatory iron intermediate in heme synthesis nor a cytosolic transport molecule involved in mobilization of iron from the transferrin-receptor complex. The most likely role for erythroid ferritin is storage of excess iron.