The effects of inhibition of heme synthesis on the intracellular localization of iron in rat reticulocytes

These studies assessed the fate and localization of incoming iron in 6-8-day rat reticulocytes during inhibition of heme synthesis by succinylacetone. Succinylacetone inhibition of heme synthesis increased iron uptake by increasing the rate of receptor recycling without affecting receptor KD for transferrin, transferrin uptake, or total receptor number. Its net effect was to amplify the number of surface transferrin receptors by recruitment of receptors from an intracellular pool. Despite increased iron influx in inhibited cells, only 2-4% of total incoming iron was diverted into ferritin. The majority of incoming iron (65-80%) in succinylacetone-inhibited cells was recovered in the stroma, where ultrastructural and enzymic analyses revealed it to be accumulated mainly in mitochondria. Intramitochondrial iron (70-75%) was localized mainly in the inner membrane fraction. Removal of succinylacetone restored heme synthesis, utilizing iron accumulated within mitochondria for its support. Thus, inhibition of heme synthesis in rat reticulocytes results in accumulation of incoming iron in a functional mobile intramitochondrial precursor iron pool used directly for heme synthesis. Under normal conditions, there is no significant intracellular or intramitochondrial iron pool in reticulocytes, which are therefore dependent upon continuous delivery of transferrin-bound iron to maintain heme synthesis. Ferritin plays an insignificant role in iron metabolism of reticulocytes.     

The use of exogenous δ-aminolaevulinic acid for the studies of the regulation of haem synthesis in rabbit reticulocytes

δ-Amino [4-¹⁴C]laevulinate added to reticulocytes incubated in vitro is incorporated into haem. Exogenous δ-aminolaevulinate restores the incorporation of ⁵⁹Fe into haem in reticulocytes which had been treated with isonicotinic acid hydrazide (INH) or penicillamine and were hence unable to synthesize δ-aminolaevulinate. On the other hand, the addition of δ-aminolaevulinate does not restore the incorporation of Fe into reticulocytes incubated with haemin. The inhibition of the incorporation of iron is neither restored by δ-aminolaevulinate in reticulocytes incubated with cycloheximide (which inhibits globin synthesis and thus elevates the free intracellular haem pool). These results suggest that in intact reticulocytes haemin does not inhibit δ-aminolaevulinate synthetase. This conclusion is further supported by the finding that the pattern of incorporation of [2-¹⁴C]glycine and δ-amino[4-¹⁴C]-laevulinate into haem differs in reticulocytes incubated with an inhibitor of δ-aminolaevulinate synthetase (INH) and in reticulocytes incubated with haemin and cycloheximide.     

The effect of various chelating agents on the mobilization of iron from reticulocytes in the presence and absence of pyridoxal isonicotinoyl hydrazone

The chelating agent pyridoxal isonicotinoyl hydrazone (PIH) has recently been shown to mobilize ⁵⁹Fe from reticulocytes loaded with non-heme ⁵⁹Fe. In this study, various chelating agents were tested for their ability to effect the mobilization of iron from reticulocytes by PIH. They fall into several groups. The largest group includes chelators such as citrate, ethylenediaminetetracetic acid and desferrioxamine, which fail to affect PIH-induced iron mobilization and do not mobilize iron per se. Either these chelators do not enter reticulocytes or they do not take up iron from PIH-Fe complexes. The second group includes chelators such as 2,2′-bipyridine, 1,10-phenanthroline, bathophenanthroline sulfonate and N,N′-ethylenebis(o-hydroxyphenylglycine) which inhibit PIH-induced iron mobilization from reticulocytes and, when added together with PIH, induce radioiron accumulation in an alcohol-soluble fraction of reticulocytes. It appears that these chelators enter the cell and compete with PIH for ⁵⁹Fe(II), but having bound iron are unable to cross the cell membrane. Spectral analysis suggests that Fe(II) chelators such as 2,2′-bipyridine and 1,10-phenanthroline remove iron from Fe(II)PIH but are not able to do so from Fe(III)PIH. Then there are compounds such as 2,3-dihydroxybenzoic acid and catechol which potentiate PIH-induced iron mobilization although they are unable to mobilize iron from reticulocytes by themselves. Lastly, there is a group of miscellaneous compounds which include chelators that either potentiate the iron-mobilizing effect of PIH as well as mobilizing iron from reticulocytes by themselves (tropolone), or that reduce PIH-induced iron mobilization while themselves having an iron-mobilizing effect (N,N′-bis(2,3-dihydroxybenzoyl)-1,6-diaminohexane). In further experiments, heme was found to stimulate globin synthesis in reticulocytes, the heme synthesis of which was inhibited by PIH, suggesting that PIH is probably not toxic to the cells.     

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.     

Erythropoietin effects on iron metabolism in rat bone marrow cells

This paper describes a study of the incorporation of ^{5 9}Fe from ^{5 9}Fe-labelled rat transferrin into rat bone marrow cells in culture. ^{5 9}Fe was found in both stroma and cytoplasm of marrow cells, and the cytoplasmic ^{5 9}Fe separated by polyacrylamide gel electrophoresis, into ferritin, haemoglobin and a low molecular weight fraction.The incorporation of ^{5 9}Fe into all three cytoplasmic fractions, but not into the stroma, increased progressively with time. Erythropoietin stimulated the increase of ^{5 9}Fe in ferritin within 1 h, the earliest time examined, and more than 3 h later in the stroma and haemoglobin.A proportion of the ⁵⁹Fe incorporated into the stroma and low molecular weight iron fractions during a 1 h incubation with ⁵⁹Fe-labelled transferrin was mobilised into ferritin and haemoglobin during a subsequent 4-h “cold-chase”. Erythropoietin, when present during the “cold-chase”, did not influence these ⁵⁹Fe fluxes. The erythropoietin stimulation of ⁵⁹Fe incorporation into ferritin, one of the earliest erythropoietin effects to be recorded, was therefore considered to be due to an increase of ⁵⁹Fe uptake by the hormone-responsive cells rather than a direct effect on ferritin synthesis.20-h cultures containing erythropoietin when incubated with ⁵⁹Fe-labelled transferrin for 4 h, showed dose-related erythropoietin stimulation of ⁵⁹Fe incorporation into haemoglobin only.In the presence of 10 mM isonicotinic acid hydrazide, ⁵⁹Fe incorporation into haemoglobin was inhibited, as in reticulocytes (Ponka, P. and Neuwirt, J. (1969) Blood 33, 690–707), while that into the stroma, ferritin and low molecular weight iron fractions, was stimulated; there were no reproducible effects of erythropoietin.     

Ferric-salicylaldehyde isonicotinoyl hydrazone, a synthetic iron chelate, alleviates defective iron utilization by reticulocytes of the Belgrade rat

The Belgrade rat has a hypochromic, microcytic anemia inherited as an autosomal recessive mutation. Although transferrin binds normally to reticulocytes and internalizes normally, iron accumulation into cells and heme is much slower than normal. We have investigated the role of the transferrin cycle in this mutant by bypassing transferrin iron delivery with the iron chelate ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH). Fe-SIH increases iron uptake into heme by Belgrade reticulocytes, restoring it almost to normal levels. This increase indicates that Fe-SIH delivers iron to a step in iron utilization that is after the Belgrade defect. Depleting reticulocytes of transferrin did not alter these observations. Failure to achieve above normal rates of iron incorporation could indicate damage due to chronic intracellular iron deficiency. Also, iron delivery by Fe-SIH restored globin synthesis to near-normal levels in Belgrade reticulocytes. The rates of glycine incorporation into porphyrin and heme in Belgrade reticulocytes incubated with Fe2-transferrin or Fe-SIH paralleled the rates of iron incorporation into heme. These data are consistent with the concept that iron availability limits protoporphyrin formation in rat reticulocytes. The protoporphyrin used for heme synthesis is provided by de novo synthesis and not by a pool of pre-existing protoporphyrin. The Belgrade defect occurs in the movement of iron from transferrin to a step prior to the ferrous state and insertion into heme. This defect diminishes the synthesis of heme and, consequently, that of protoporphyrin and globin.     

Hemin feedback inhibition at reticulocyte δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydratase

Addition of hemin (5–200 μM) to a rabbit reticulocyte iron-free incubation medium, resulted in a progressive inhibition of heme synthesis as measured by incorporation of (¹⁴C)-glycine. In contrast when (¹⁴C) δ-aminolevulinic acid incorporation into heme was studied, significant inhibition below that of the (¹⁴C)-glycine control only occurred with hemin concentrations greater than 100 μM. Hemin progressively inhibited cellular and mitochondrialδ-aminolevulinic acid synthetase activity, as well as cellular δ-aminolevulinic acid dehydratase activity. The results indicated that elevated levels of hemin initially control heme synthesis by feedback inhibition at the rate-limiting enzyme of heme synthesis, δ-aminolevulinic acid synthetase. Hemin inhibition of δ-aminolevulinic acid dehydratase is only significant for the entrire heme synthetic pathway when greater than one-third of this enzyme's activity is inhibited.     

Evidence for and consequences of chronic heme deficiency in Belgrade rat reticulocytes

The Belgrade rat has a microcytic, hypochromic anemia inherited as an autosomal recessive trait (gene symbol b). Transferrin-dependent iron uptake is defective because of a mutation in Nramp2 (now DMT1, also called DCT1), the protein responsible for endosomal iron efflux. Hence, Belgrade reticulocytes are iron deficient. We show that a chromatographic method is able to measure the amount of 'free' heme in reticulocytes. Most of the 'free' heme is the result of biosynthesis. Succinylacetone, an inhibitor of heme synthesis, decreases the level of 'free' heme and cycloheximide, an inhibitor of globin synthesis, increases the 'free' heme level. In a pulse-chase experiment with 59Fe-transferrin, the 'free' heme pool behaves as an intermediate, with a half-life of just over 2 h. Belgrade reticulocytes contain about 40% as much 'free' heme as do heterozygous or homozygous reticulocytes. This deficiency of 'free' heme slows initiation of translation in Belgrade reticulocytes by increasing the level of an inhibitor of initiation. Thus the Belgrade rat makes a whole animal model available with chronic heme deficiency.     

Transferrin receptors,iron transport and ferritin metabolism in friend erythroleukemia cells

Four aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of erythroid differentiation by dimethyl sulfoxide. (1) The binding of ¹²⁵I-labeled transferrin was determined over a range of transferrin concentrations from 0.5 to 15 μM. Scatchard analysis of the binding curves demonstrated equivalent numbers of transferrin binding sites per cell: 7.78 ± 2.41 · 10⁵ in non-induced cells and 9.28 ± 1.57 · 10⁵ after 4 days of exposure to dimethyl sulfoxide. (2) The rate of iron transport was determined by measuring iron uptake from ⁵⁹Fe-labeled transferrin. Iron uptake in non-induced cells was approx. 17 000 molecules of iron/cell per min; 24 h after addition of dimethyl sulfoxide it increased to 38 000, and it rose to maximal levels of approx. 130 000 at 72 h. (3) Heme synthesis, assayed qualitatively by benzidine staining and measured quantitatively by incorporation of ⁵⁹Fe or [2-¹⁴C]glycine into cyclohexanone-extracted or crystallized heme, was not detected until 3 days after addition of dimethyl sulfoxide, when 12% of the cells were stained by benzidine and 6 pmol ⁵⁹Fe and 32 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. After 4 days, 60% of the cells were benzidine positive and 34 pmol ⁵⁹Fe and 90 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. (4) The rate of incorporation of ⁵⁹Fe into ferritin, measured by immunoprecipitation of ferritin by specific antimouse ferritin immunoglobulin G, rose from 4.4 ± 0.6 cells to 18.4 ± 1.3 pmol ⁵⁹Fe/h per 10⁸ cells 3 days after addition of dimethyl sulfoxide, and then fell to 11.6 ± 3.1 pmol 4 days after dimethyl sulfoxide when heme synthesis was maximal. These studies indicate that one or more steps in cellular iron transport distal to transferrin binding is induced early by dimethyl sulfoxide and that ferritin may play an active role in iron delivery for heme synthesis.     

Mobilization of ferritin iron in erythroblasts by chelating agents

Intracellular ferritin in newt (Triturus cristatus) erythroblasts was accessible to the chelating effects of EDTA and pyridoxal phosphate. EDTA (0.5-1 mM) promoted release of radioactive iron from ferritin of pulse-labelled erythroblasts during chase incubation, but its continuous presence was not necessary for ferritin iron mobilization. Brief exposure to EDTA was sufficient to release 60-70% of ferritin 59Fe content during ensuing chase in EDTA-free medium. EDTA also suppressed cellular iron uptake and utilization for heme synthesis, but these activities were restored upon its removal. Pyridoxal-5'-phosphate (0.5-5 mM) also stimulated loss of radioactive iron from ferritin; however, ferritin iron release by pyridoxal phosphate required its continued presence. Unlike EDTA, pyridoxal phosphate did not interfere with iron uptake or its utilization for heme synthesis. Chelator-mobilized ferritin iron accumulated initially in the hemolysate as a low-molecular-weight component and appeared to be eventually released into the medium. No radioactive ferritin was found in the medium of chelator-treated cells, indicating that secretion or loss of ferritin was not responsible for decreasing cellular ferritin 59Fe content. Moreover, there was no transfer of radioactive iron between the low-molecular-weight component released into the medium and plasma transferrin. These results indicate that chelator-released ferritin iron is not available for cellular utilization in heme synthesis and that ferritin iron released by this process is not an alternative or complementary iron source for heme synthesis. Correlation of these data with effects of succinylacetone inhibition of heme synthesis and with previous studies indicates that the main role of erythroid cell ferritin is absorption and storage of excess iron not used for heme synthesis.     

Ferric pyridoxal isonicotinol hydrazone can provide iron for heme synthesis in reticulocytes

We have examined whether reticulocytes depleted of transferrin might incorporate ⁵⁹Fe from ⁵⁹Fe-labelled pyridoxan isonicotinoyl hydrazone (PIH). Transferrin-depleted reticulocytes showed a time-, temperature- and concentration-dependent incorporation of ⁵⁹Fe when incubated with 20–200 μM ⁵⁹Fe-PIH. The amount of ⁵⁹Fe incorporated with 200 μM ⁵⁹Fe-PIH is equal to or higher than that taken up from transferrin at 20 μM ⁵⁹Fe concentration. After 60 min about 60% of the ⁵⁹Fe taken up by the cells is recovered in heme while the remainder is probably still bound to PIH. 1 mM succinyl acetone (a specific inhibitor of heme synthesis) inhibits PIH-mediated incorporation of ⁵⁹Fe into heme by about 79% indicating that ⁵⁹Fe from ⁵⁹Fe-PIH is incorporated into de novo synthesized protoporphyrin. As is the case with transferrin, erythrocytes do not incorporate ⁵⁹Fe from ⁵⁹Fe-PIH. Pretreatment of reticulocytes with pronase does not inhibit their ability to incorporate ⁵⁹Fe from ⁵⁹Fe-PIH, suggesting that, unlike the uptake of Fe from transferrin, membrane receptors are not involved in the uptake of Fe-PIH by the cells.     

Succinylacetone pyrrole,a powerful inhibitor of vitamin B12 biosynthesis: Effect on δ-aminolevulinic acid dehydratase

Succinylacetone, a competitive inhibitor (K_I = 400 μM) of δ-aminolevulinic acid dehydratase of $\text{Clostridium}\text{tetanomorphum}$, is converted non-enzymatically upon incubation with δ-aminolevulinic acid to succinylacetone pyrrole, a much stronger competitive inhibitor (K_I = 5 μM) of the enzyme. A similar effect is seen in vivo: when present in the growth medium at concentrations of about 1 μM, the pyrrole decreases the level of corrinoids produced by this organism by half, while succinylacetone at 200 μM causes only 19 per cent inhibition of corrinoid formation. Levulinic acid is a much weaker inhibitor in vitro and in vivo. The inhibition by succinylacetone pyrrole is considered to be due to its structural resemblance to δ-aminolevulinic acid rather than to porphobilinogen, the reaction product of δ-aminolevulinic acid dehydratase: succinylacetone, succinylacetone pyrrole, and levulinic acid all contain a succinyl group.     

The incorporation of N15 glycine by avian erythrocytes and reticulocytes in vitro

A study of the incorporation of glycine-N¹⁵ by chicken red cells in vitro has shown that: 1. There is no detectable nitrogen turnover in the histone or desoxyribonucleic acid of erythrocytes or reticulocytes. 2. Hemoglobin synthesis in the nucleated reticulocyte proceeds at 2 to 3 times the rate observed in the mature erythrocyte. 3. The uptake of glycine-N¹⁵ by heme is 9 to 14 times the corresponding uptake into hemoglobin, and 12 to 20 times the calculated uptake into globin. 4. Maturation of the red cell results in a decline in the rate of synthesis of both heme and globin, but the deceleration is much more marked in globin. synthesis. 5. No significant differences could be detected in the low N¹⁵ incorporations of nuclear and cytoplasmic hemoglobins.     

Acquisition of iron from transferrin regulates reticulocyte heme synthesis

Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59Fe from [59Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [59Fe]transferrin. Also, Fe-SIH stimulates [2-14C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59Fe incorporation into heme from either [59Fe]transferrin or [59Fe]SIH but does reverse the inhibition of 59Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes.     

Biosynthesis of heme in immature erythroid cells. The regulatory step for heme formation in the human erythron

Heme formation in reticulocytes from rabbits and rodents is subject to end product negative feedback regulation: intracellular "free" heme has been shown to control acquisition of transferrin iron for heme synthesis. To identify the site of control of heme biosynthesis in the human erythron, immature erythroid cells were obtained from peripheral blood and aspirated bone marrow. After incubation with human 59Fe transferrin, 2-[14C]glycine, or 4-[14C]delta-aminolevulinate, isotopic incorporation into extracted heme was determined. Addition of cycloheximide to increase endogenous free heme, reduced incorporation of labeled glycine and iron but not delta-aminolevulinate into cell heme. Incorporation of glycine and iron was also sensitive to inhibition by exogenous hematin (Ki, 30 and 45 microM, respectively) i.e. at concentrations in the range which affect cell-free protein synthesis in reticulocyte lysates. Hematin treatment rapidly diminished incorporation of intracellular 59Fe into heme by human erythroid cells but assimilation of 4-[14C]delta-aminolevulinate into heme was insensitive to inhibition by hematin (Ki greater than 100 microM). In human reticulocytes (unlike those from rabbits), addition of ferric salicylaldehyde isonicotinoylhydrazone, to increase the pre-heme iron pool independently of the transferrin cycle, failed to promote heme synthesis or modify feedback inhibition induced by hematin. In human erythroid cells (but not rabbit reticulocytes) pre-incubation with unlabeled delta-aminolevulinate or protoporphyrin IX greatly stimulated utilization of cell 59Fe for heme synthesis and also attenuated end product inhibition. In human erythroid cells heme biosynthesis is thus primarily regulated by feedback inhibition at one or more steps which lead to delta-aminolevulinate formation. Hence in man the regulatory process affects generation of the first committed precursor of porphyrin biosynthesis by delta-aminolevulinate synthetase, whereas in the rabbit separate regulatory mechanisms exist which control the incorporation of iron into protoporphyrin IX.     

Accumulation of heme in mitochondria from rabbit reticulocytes with inhibited globin synthesis

Incubation of rabbit reticulocytes with cycloheximide and ⁵⁹Fe bound to transferrin in plasma induces excessive non-hemoglobin ⁵⁹Fe-labeled heme accumulation in mitochondria. During incubation of these mitochondria in vitro a part of ⁵⁹Fe-labeled heme is released into the surrounding medium. The addition of globin or bovine serum albumin to the incubation mixture essentially increases the amount of heme released from mitochondria.     

Iron transport mechanisms in reticulocytes and mature erythrocytes

The mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with ⁵⁹Fe-labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration-dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin-bound Fe(II) by two mechanisms. One is a high-affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low-affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly assoicated with exchange of Na⁺. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)-sucrose and Fe(II)-ascorbate using a variety of incubation conditions. No major differences were observed. © 1995 Wiley-Liss, Inc.     

Hepatic subcellular metabolism of heme from heme-hemopexin: incorporation of iron into ferritin

The subcellular distribution and metabolic fate of [⁵⁹Fe]heme-[¹²⁵I]-labeled hemopexin after receptor-mediated interaction with the liver was examined in the rat. After intravenous injection, [⁵⁹Fe]heme from the complex and ⁵⁹Fe from hepatic catabolism of this heme accumulate in the liver and undergo changes in their subcellular distribution over 2 hours. The amounts of [⁵⁹Fe]heme and particularly of ⁵⁹Fe increase in the cytosol while remaining constant or decreasing in membranous fractions. In contrast, [¹²⁵I]-labeled hemopexin associated with the liver during heme transport is always a small fraction of the dose and is not measurably catabolized under these conditions.Gel filtration of the cytosol showed that ⁵⁹Fe increased linearly with time in a high molecular weight fraction which was identified immunologically as ferritin. We conclude that heme transported by hemopexin is metabolized by the liver and the iron conserved.     

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of ⁵⁹Fe²⁺ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was ∼20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH₄Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1. J. Cell. Physiol. 178:349–358, 1999. © 1999 Wiley-Liss, Inc.