Influence of Iron Deprivation on the Membrane Composition of Anacystis nidulans

Cultures of the cyanobacterium Anacystis nidulans were grown under iron-deficient conditions and then restored by the addition of iron. Membrane proteins from iron-deficient and iron-restored cells were analyzed by lithium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The incorporation of [³⁵S]sulfate into membrane proteins and lactoperoxidase-catalyzed ¹²⁵I iodination were used to monitor the rates of polypeptide biosynthesis and surface exposure of membrane proteins, respectively. These polypeptide profiles revealed major differences in the membrane composition of iron-deficient and normal cells. Iron deficiency caused a decrease in the amount of certain important membrane proteins, reflecting a decreased rate of biosynthesis of these peptides. Several photosystem II peptides also showed an increase in surface exposure after iron stress. In addition, iron deficiency led to the synthesis of proteins at 34 and 52 kilodaltons which were not present in normal cells. When iron was restored to a deficient culture, a metabolic sequence was initiated within the first 12 h after the addition of iron which led to phenotypically normal cells. Pulse labeling with [³⁵S]sulfate during this period demonstrated that iron addition initiates a coordinated pattern of synthesis that leads to the assembly of normal membranes.     

Coprophagy in iron-deficient rats: II. 2 novel methods of prevention

2 novel methods of coprophagy prevention were used in iron-deficient rats to measure their effect on haemoglobin regeneration during the feeding of ferrous sulphate or several soya proteins. Neck collars produced considerable weight loss and depression in food intake compared to aluminium anal cups which were tolerated well. Both methods of coprophagy prevention reduced iron availability from a range of soya proteins and ferrous sulphate and it is suggested that the practice of coprophagy in iron-deficient and possibly iron-replete rats may overestimate iron (and possibly other mineral) absorption (retention) studies.     

Involvement of outer membrane proteins in enterochelin-mediated iron uptake in Escherichia coli.

Escherichia coli K-12 grown in iron-deficient media contained a large amount of outer membrane proteins O-2a, O-2b, and O-3, while cells grown in iron-supplemented media contained far smaller amounts of these proteins. The iron uptake by the iron-deficient cells was significantly stimulated in the presence of enterochelin, while that by the iron-rich cells was not. The outer membrane isolated from cells grown in the iron-deficient media showed enterochelin-stimulated binding of iron, while the outer membrane from iron-rich cells and cytoplasmic membranes from both types of cells did not show such binding activity. The amount of iron bound by the outer membrane was almost equivalent to the amount of O-2a, O2b, or O-3, irrespective of the amount of these proteins in the outer membrane, which is controlled by the amount of iron in the medium. Small particles rich in these proteins were prepared from cells by EDTA extraction. The particles were active in enterochelin-mediated iron binding and the amount of iron bound was equivalent to the amount of each of these proteins in the particles. Although the outer membrane of E. coli B was as active in iron binding as that of E. coli K-12, it did not possess an appreciable amount of O-2a. Gel electrophoretic analysis revealed that 9-2b and 9-3 were identical with the proteins missing mutants feuB and feuA, respectively.     

Proteomics, pigment composition, and organization of thylakoid membranes in iron-deficient spinach leaves

The changes induced in the photosynthetic apparatus of spinach (Spinacia oleracea L.) seedlings exposed to iron deficiency shortly after germination were characterized with two proteomic approaches coupled with chlorophyll and xanthophyll analysis and in vivo measurements of photosynthesis. During the first 10 d of iron deficiency the concentrations of chlorophyll b and violaxanthin were greatly reduced, but all xanthophylls recovered after 13-17 d of iron deficiency, when both chlorophylls were negatively affected. No new protein was formed in iron-deficient leaves, and no protein disappeared altogether. Photosystem I (PSI) proteins were largely reduced, but the stoichiometry of the antenna composition of PSI was not compromised. On the contrary, PSII proteins were less affected by the stress, but the specific antennae Lhcb4 and Lhcb6, Lhcb2 and its isoform Lhcb1.1 were all reduced, while the concentration of Lhcb3 increased. A strong reduction in thylakoid bending and an altered distribution pattern for the reduced PSI and PSII complexes were observed microscopically in iron-deficient leaves. Supercomplex organization was also affected by the stress. The trimeric organization of Lhcb and the dimerization of Lhca were reduced, while monomerization of Lhcb increased. However, the trimerization of Lhcb was partially recovered after 13-17 d of iron deficiency. In iron-deficient leaves, photosynthesis was strongly inhibited at different light intensities, and a high de-epoxidation status of the xanthophylls was observed, in association with a strong impairment of photochemical efficiency and an increase of heat dissipation as monitored by the non-photochemical quenching of fluorescence. All these negative effects of iron deficiency were attenuated but not fully reversed after again supplying iron to iron-deficient leaves for 7-13 d. These results indicate that iron deficiency has a strong impact on the proteomic structure of spinach photosystems and suggest that, in higher plants, adaptive mechanisms common in lower organisms, which allow rapid changes of the photosystem structure to cope with iron stress, are absent. It is speculated that the observed changes in the monomer-trimer equilibrium of major PSII antennae, which is possibly the result of xanthophyll fluctuations, is a first adaptative adjustment to iron deficiency, and may eventually play a role in light dissipation mechanisms.     

Changes in Thylakoid Galactolipids and Proteins during Iron Nutrition-Mediated Chloroplast Development

Changes in the amounts of thylakoid galactolipids and proteins were monitored for 96 hours following iron resupply to iron-deficient sugar beet (Beta vulgaris L. cv F58-554H1) plants. During this period of iron nutrition-mediated chloroplast development, the amount of galactolipid per leaf area increased linearly with time. Assuming galactolipids are an index for the amount of thylakoids, then there was a linear synthesis of thylakoid membranes during regreening. Total thylakoid protein synthesis, however, lagged behind galactolipid synthesis, suggesting that proteins are inserted secondarily into the galactolipid matrix of the thylakoid membrane during development.

Iron deficiency caused an increase in the free chlorophyll band under the conditions of gel electrophoresis used. Of the chlorophyll proteins resolved, the chlorophyll protein associated with photosystem I was most diminished in iron-deficient tissue, and appeared to recover most rapidly. Changes in the light-harvesting chlorophyll proteins are also discussed.

The number of polypeptides resolved by lithium dodecyl sulfate-polyacrylamide gel electrophoresis was higher in iron-deficient thylakoids. During regreening, the number of resolved polypeptides decreased.

     

The identification of IsiA proteins binding chlorophyll <Emphasis Type="Italic">d</Emphasis> in the cyanobacterium <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

The bioavailable iron in many aquatic ecosystems is extremely low, and limits the growth and photosynthetic activity of phytoplankton. In response to iron limitation, a group of chlorophyll-binding proteins known as iron stress-induced proteins are induced and serve as accessory light-harvesting components for photosystems under iron limitation. In the present study, we investigated physiological features of Acaryochloris marina in response to iron-deficient conditions. The growth doubling time under iron-deficient conditions was prolonged to ~3.4 days compared with 1.9 days under normal culture conditions, accompanied with dramatically decreased chlorophyll content. The isolation of chlorophyll-binding protein complexes using sucrose density gradient centrifugation shows six main green bands and three main fluorescence components of 712, 728, and 748 nm from the iron-deficient culture. The fluorescence components of 712 and 728 nm co-exist in the samples collected from iron-deficient and iron-replete cultures and are attributed to Chl d-binding accessory chlorophyll-binding antenna proteins and also from photosystem II. A new chlorophyll-binding protein complex with its main fluorescence peak at 748 nm was observed and enriched in the heaviest fraction from the samples collected from the iron-deficient culture only. Combining western blotting analysis using antibodies of CP47 (PSII), PsaC (PSI) and IsiA and proteomic analysis on an excised protein band at ~37 kDa, the heaviest fraction (?F6) isolated from iron-deficient culture contained Chl d-bound PSI–IsiA supercomplexes. The PSII-antenna supercomplexes isolated from iron-replete conditions showed two fluorescence peaks of 712 and 728 nm, which can be assigned as 6-transmembrane helix chlorophyll-binding antenna and photosystem II fluorescence, respectively, which is supported by protein analysis of the fractions (F5 and F6).     

Impact of iron deficiency and iron re-supply during the early stages of vegetative development in maize (Zea mays L.)

The consequences of iron deficiency and iron re-supply were evaluated during the early stages of growth and development of young maize plantlets grown hydroponically in the absence of iron. Various parameters, such as fresh and dry weights, and the concentration of chlorophylls, iron, copper, manganese, calcium, magnesium and potassium in leaves, were measured at various times during the first 15 d of culture. Ten-day-old maize plantlets grown without iron displayed severe alterations, with a 50% decrease in iron and chlorophyll concentrations in leaves, and serious impairments in mitochondria and chloroplast ultrastructure. In contrast, neither leaf nor root growth, nor other mineral concentrations other than iron were significantly affected at this stage of development. In an attempt to characterize proteins potentially involved in iron nutrition or the adaptative response to iron starvation, comparative 2D-gel electrophoretic analysis of polypeptides was carried out on soluble and membrane fractions prepared from leaves and roots of iron-deficient and iron-sufficient 10-d-old maize plantlets. Two polypeptides (11 and 17 kDa, pI of about 6.8) from the microsomal fraction of leaves were found to be repressed under iron-deficient conditions. Some other polypeptides were found to he induced in microsomal fractions either from roots or leaves. Significant variations in the concentration of most of these polypeptides were observed from one experiment to another. It can be concluded from this study that, at this early stage of maize vegetative growth and development, molecular variations induced by iron deficiency do not affect major house-keeping proteins, but probably affect very specific events depending on low abundance proteins.     

Transferrin and Iron Uptake by the Brain: Effects of Altered Iron Status

Transferrin (Tf) and iron uptake by the brain were measured in rats using 59Fe-125I-Tf and 131I-albumin (to correct for the plasma content of 59Fe and 125I-Tf in the organs). The rats were aged from 15 to 63 days and were fed (a) a low-iron diet (iron-deficient) or, as control, the same diet supplemented with iron, or (b) a chow diet with added carbonyl iron (iron overload), the chow diet alone acting as its control. Iron deficiency was associated with a significant decrease and iron overload with a significant increase in brain nonheme iron concentration relative to the controls. In each dietary treatment group, the uptake of Tf and iron by the brain decreased as the rats aged from 15 to 63 days. Both Tf and iron uptake were significantly greater in the iron-deficient rats than in their controls and lower in the iron-loaded rats than in the corresponding controls. Overall, iron deficiency produced about a doubling and iron overload a halving of the uptake values compared with the controls. In contrast to that in the brain, iron uptake by the femurs did not decrease with age and there was relatively little difference between the different dietary groups. 125I-Tf uptake by the brains of the iron-deficient rats increased very rapidly after injection of the labelled proteins, within 15 min reaching a plateau level which was maintained for at least 6 h. The uptake of 59Fe, however, increased rapidly for 1 h and then more slowly, and in terms of percentage of injected dose reached much higher values than did 125I-Tf uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron Deficiency Alters Discrete Proteins in Rat Caudate Nucleus and Nucleus Accumbens

Young rats (21 days old) made nutritionally iron deficient, by feeding them a semisynthetic diet containing skimmed milk for 5 weeks, had significantly lowered hemoglobin levels (5.2 +/- 4 g/100 ml). The nonheme iron content in caudate nucleus was decreased by 47%. The behavioral response of iron-deficient rats to apomorphine (2 mg/kg) and the density of 3,4-dihydroxyphenylethylamine (dopamine) D2 receptors, as measured by [3H]spiperone binding in caudate nucleus, were significantly reduced by 70 and 53%, respectively. The possibility that nutritional iron deficiency may affect protein content in brain was investigated by measuring the apparent concentration of proteins in caudate nucleus and nucleus accumbens from iron-deficient and control animals using two-dimensional gel electrophoresis. The data indicate that iron deficiency can affect content in these two brain regions. Significant changes in the content of 10 proteins were noted in the caudate nucleus and nucleus accumbens in iron-deficient rats. The albumin level was significantly increased in both regions studied, whereas the neuron-specific enolase level was increased in the nucleus accumbens and the glial fibrillary acidic protein level was reduced in the caudate nucleus. The significance of these protein content changes, as well as a reduction in content of a 94-kilodalton protein (a molecular size similar to that of the D2 dopamine receptor), remains to be established.     

Activation of Nrf2/Keap1 signaling and autophagy induction against oxidative stress in heart in iron deficiency

We investigated the effects of dietary iron deficiency on the redox system in the heart. Dietary iron deficiency increased heart weight and accumulation of carbonylated proteins. However, expression levels of heme oxygenase-1 and LC3-II, an antioxidant enzyme and an autophagic marker, respectively, in iron-deficient mice were upregulated compared to the control group, resulting in a surrogate phenomenon against oxidative stress.     

Iron limitation and its effect on membrane proteins and siderophore transport inNeurospora crassa

Summary Cells of the fungusNeurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. Three strains were studied:N. crassa 74A (wild type), a siderophore-free mutantN. crassa (arg-5 ota aga) as well as a slime variant ofN. crassa which lacks a cell wall. Plasma membranes were purified, solubilized and analyzed by one-dimensional SDS/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecular masses in the range 14–160 kDa. Iron-sufficient and iron-deficient growth resulted in nearly identical plasma membrane protein profiles in all strains. Although minor alterations in the proportion of certain proteins could be detected, significant overproduction of certain membrane proteins during iron limitation could not be observed. Transport of⁵⁵ Fe-labeled siderophores seems to be correlated to the degree of iron limitation. For example, transport rates were enhanced five-fold after 16 h of growth in iron-deficient medium compared to growth in iron-sufficient medium. Extraction and HPLC measurement of siderophores from conidiospores yielded approximately 10^–15 mol/spore, indicating that germination tubes and young cells used for transport measurements are not iron-deficient. It is suggested that the putative transport systems for siderophores in fungal plasma membranes are constitutively expressed and enhanced uptake of siderophores during iron limitation is rather the result of cellular transport regulation mechanisms.     

Identification of differentially expressed genes in response to dietary iron deprivation in rat duodenum

We sought to identify novel genes involved in intestinal iron absorption by inducing iron deficiency in rats during postnatal development from the suckling period through adulthood. We then performed comparative gene chip analyses (RAE230A and RAE230B chips; Affymetrix) with cRNA derived from duodenal mucosa. Real-time PCR was used to confirm changes in gene expression. Genes encoding the apical iron transport-related proteins [divalent metal transporter 1 (DMT1) and duodenal cytochrome b] were strongly induced at all ages studied, whereas increases in mRNA encoding the basolateral proteins iron-regulated gene 1 and hephaestin were observed only by real-time PCR. In addition, transferrin receptor 1 and heme oxygenase 1 were induced. We also identified induction of novel genes not previously associated with intestinal iron transport. The Menkes copper ATPase (ATP7a) and metallothionein were strongly induced at all ages studied, suggesting increased copper absorption by enterocytes during iron deficiency. We also found significantly increased liver copper levels in 7- to 12-wk-old iron-deficient rats. Also upregulated at most ages examined were the sodium-dependent vitamin C transporter, tripartite motif protein 27, aquaporin 4, lipocalin-interacting membrane receptor, and the breast cancer-resistance protein (ABCG2). Some genes also showed decreased expression with iron deprivation, including several membrane transporters, metabolic enzymes, and genes involved in the oxidative stress response. We speculate that dietary iron deprivation leads to increased intestinal copper absorption via DMT1 on the brush-border membrane and the Menkes copper ATPase on the basolateral membrane. These findings may thus explain copper loading in the iron-deficient state. We also demonstrate that many other novel genes may be differentially regulated in the setting of iron deprivation.     

Downregulation of Tsx and OmpW and upregulation of OmpX are required for iron homeostasis in Escherichia coli

Upregulation of outer membrane (OM) proteins was systematically investigated in response to poor iron availability in the host and natural environments, but downregulation of OM proteins was ill-defined in this response. We utilized proteomic methodologies to characterize altered OM proteins in the sarcosine-insoluble fraction of Escherichia coli K12 cultured in LB medium with iron limitation. Notably, three novel proteins, Tsx, OmpW, and OmpX, related to iron homeostasis were identified; Tsx and OmpW were downregulated, and OmpX was upregulated. These alterations were functionally validated with the use of gene overexpression and deletion methods. Of the two downregulated proteins, Tsx was more sensitive to an iron-deficient environment than OmpW. In addition, the significantly negative correlation between Tsx with OmpW was achieved when overexpressed strains were used. These findings strongly indicate that the downregulation of Tsx and OmpW and the upregulation of OmpX are required for iron homeostasis in E. coli.     

Olfactory ferric and ferrous iron absorption in iron-deficient rats

The absorption of metals from the nasal cavity to the blood and the brain initiates an important route of occupational exposures leading to health risks. Divalent metal transporter-1 (DMT1) plays a significant role in the absorption of intranasally instilled manganese, but whether iron uptake would be mediated by the same pathway is unknown. In iron-deficient rats, blood (59)Fe levels after intranasal administration of the radioisotope in the ferrous form were significantly higher than those observed for iron-sufficient control rats. Similar results were obtained when ferric iron was instilled intranasally, and blood levels of (59)Fe were even greater in the iron-deficient rats compared with the amount of ferrous iron absorbed. Experiments with Belgrade (b/b) rats showed that DMT1 deficiency limited ferric iron uptake from the nasal cavity to the blood compared with +/b controls matched for iron deficiency. These results indicate that olfactory uptake of ferric iron by iron-deficient rats involves DMT1. Western blot experiments confirmed that DMT1 levels are significantly higher in iron-deficient rats compared with iron-sufficient controls in olfactory tissue. Thus the molecular mechanism of olfactory iron absorption is regulated by body iron status and involves DMT1.     

Influence of intact and partially hydrolysed guar gum on iron utilization in rats fed on iron-deficient diets

Effects of partially hydrolysed guar gum (PHGG) or intact guar gum (GG) on iron utilization in rats fed on several iron-deficient diets were examined. Hemoglobin, serum iron and iron storage in liver of rats fed on iron-deficient diets as a control group (without PHGG and GG) significantly decreased, while those of the test group fed together with PHGG or GG were unchanged. In an iron balance test for 3 days, administration of PHGG or GG caused an increase in iron absorption. The results suggested that PHGG or its metabolites increase the bioavailability of dietary iron in deficiency.     

Long-distance signals positively regulate the expression of iron uptake genes in tobacco roots

Long-distance signals generated in shoots are thought to be associated with the regulation of iron uptake from roots; however, the signaling mechanism is still unknown. To elucidate whether the signal regulates iron uptake genes in roots positively or negatively, we analyzed the expressions of two representative iron uptake genes: NtIRT1 and NtFRO1 in tobacco (Nicotiana tabacum L.) roots, after shoots were manipulated in vitro. When iron-deficient leaves were treated with Fe(II)-EDTA, the expressions of both genes were significantly reduced; nevertheless iron concentration in the roots maintained a similar level to that in roots grown under iron-deficient conditions. Next, all leaves from tobacco plants grown under the iron-deficient condition were excised. The expression of two genes were quickly reduced below half within 2 h after the leaf excision and gradually disappeared by the end of a 24-h period. The NtIRT1 expression was compared among the plants whose leaves were cut off in various patterns. The expression increased in proportion to the dry weight of iron-deficient leaves, although no relation was observed between the gene expression and the position of excised leaves. Interestingly, the NtIRT1 expression in hairy roots increased under the iron-deficient condition, suggesting that roots also have the signaling mechanism of iron status as well as shoots. Taken together, these results indicate that the long-distance signal generated in iron-deficient tissues including roots is a major factor in positive regulation of the expression of NtIRT1 and NtFRO1 in roots, and that the strength of the signal depends on the size of plants.     

Intracardiac iron distribution in newborn guinea pigs following isolated and combined fetal hypoxemia and fetal iron deficiency

Myocardial iron deficiency complicates chronic intrauterine hypoxemia during diabetic pregnancies. To understand the effect of both conditions during fetal life on intracardiac iron prioritization, we measured heart myoglobin, cytochrome c, and elemental iron concentrations in six iron-deficient, hypoxic, five iron-sufficient, hypoxic, six iron-deficient, normoxic, and six iron-sufficient, normoxic newborn guinea pigs. The iron-deficient, hypoxic group had lower heart iron (p = 0.03) but higher myoglobin concentration (p < 0.0001) when compared with the iron-sufficient, normoxic group. The percentage of iron incorporated into myoglobin was higher than control in the iron-deficient, hypoxic group (23.2+/-7.2% vs. 5.2+/-0.8%; p < 0.001) and increased as total heart iron decreased (r = 0.97; p < 0.001). In contrast, heart cytochrome c concentration was lower than control in the iron-deficient, hypoxic group (p = 0.01), with equal percentages of heart iron incorporated into cytochrome c. This intracellular prioritization of myocardial iron to myoglobin and away from cytochrome c following combined fetal hypoxemia and iron deficiency may represent an adaptive mechanism to preserve myocardial tissue oxygenation, although at the expense of oxidative phosphorylative capability.     

Deficiency of the mitochondrial electron transport chain in muscle does not cause insulin resistance

Background

It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis.

Methodology/Principal Findings

Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance.

Conclusions/Significance

The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.     

Effect of dietary iron deficiency on mineral levels in tissues of rats

To clarify the influence of iron deficiency on mineral status, the following two synthetic diets were fed to male Wistar rats: a control diet containing 128 micrograms iron/g, and an iron-deficient diet containing 5.9 micrograms iron/g. The rats fed the iron-deficient diet showed pale red conjunctiva and less reactiveness than the rats fed the control diet. The hemoglobin concentration and hematocrit of the rats fed the iron-deficient diet were markedly less than the rats fed the control diet. The changes of mineral concentrations observed in tissues of the rats fed the iron-deficient diet, as compared with the rats fed the control diet, are summarized as follows: . Iron concentrations in blood, brain, lung, heart, liver, spleen, kidney, testis, femoral muscle, and tibia decreased; . Calcium concentrations in blood and liver increased; calcium concentration in lung decreased; . Magnesium concentration in blood increased; . Copper concentrations in blood, liver, spleen and tibia increased; copper concentration in femoral muscle decreased; . Zinc concentration in blood decreased; . Manganese concentrations in brain, heart, kidney, testis, femoral muscle and tibia increased. These results suggest that iron deficiency affects mineral status (iron, calcium, magnesium, copper, zinc, and manganese) in rats.     

Iron homeostasis: recently identified proteins provide insight into novel control mechanisms

Iron is an essential nutrient required for a variety of biochemical processes. It is a vital component of the heme in hemoglobin, myoglobin, and cytochromes and is also an essential cofactor for non-heme enzymes such as ribonucleotide reductase, the limiting enzyme for DNA synthesis. When in excess, iron is toxic because it generates superoxide anions and hydroxyl radicals that react readily with biological molecules, including proteins, lipids, and DNA. As a result, humans possess elegant control mechanisms to maintain iron homeostasis by coordinately regulating iron absorption, iron recycling, and mobilization of stored iron. Disruption of these processes causes either iron-deficient anemia or iron overload disorders. In this minireview, we focus on the roles of recently identified proteins in the regulation of iron homeostasis.