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

Bwchamtca et Biophysica Acta, 1012 (1989) 243-253 Elsevier

243

BBAMCR 12493

The effects of inhibition of heme synthesis on the intracellular localization of iron in rat reticulocytes Mary L. Adams, Irene Ostapiuk and Joseph A. Grasso Department of Anatomy, The Umversity of Connecucut Health Center, Farmington, CT (U.S.A.) (Received 25 January 1989)

Key words: Heme synthesis mhibition; Iron metabolism; Iron localization; (~.at reticulocyte)

These studies assessed the fate and localization of incoming iron in 6-8-day rat reticulecytes during inhibition of heine synthes|s by succlnylacetune. Succinylacetone inhibition of heine synthesis increased iron uptake by increasing the rate of receptor recycling without affecting receptor KD for translerrin, transferrin uptake, or total receptor number. Its net effect was to amplify the number of surface transfenrin receptors by recruitment of receptors from an |n~acel|ular pool. Despite increased iron influx in inhibited cells, only 2-4% of total incoming in~n was diverted into fete|tin. The majority of incoming iron (65-80%) in succinylacetone-inhibRod cells was recovered in the stroma, where ultrastructara| and enzymic analyses revealed it to be accumulated mainly in mitechondria, lntramitocbondriaJ iron (70-75%) was localized mainly in the inner membrane fraction. Removal of succiny|acetone restored heine synthesis, utilizing iron accumulated within mitocbondr|a for its support. Thus, inhibition of heine synthesis in rat reticulecytes results in accumulation of incoming iron in a functional mobile intramitochondrial precursor iron pool used directly for heine synthesis. Under normal conditions, there is no significant intracel|ular or intTamitocbondria| iron pool in reficulocytes, which are therefore dependent upon continuous delivery of transfen'in-bound iron to maintain berne synthesis. Ferritin plays an insignificant ro|e in iron metabolism of reticuiocytes.

Introduction The main target of incoming iron in erythroid cells is heine biosynthesis, whose terminal step within mitochondria demands a continuous supply of iron from exogenous transferrin [1,2]. While the mechanisms constituting transferrin-mediated iron delivery to cells are generally understood, the intraceUular pathway involved in iron transit in the cytosol and in targetting of iron into specific metabolic pathways remains obscure. Various low molecular weight components have been isolated from cytosol of several cell types [3-6], but none has been conclusively identified as the elusive cytosolic carrier intermediate presumed to transport iron from the receptor-transferrin complex to sites of utilization. Despite descriptions of ferritin as an intermediate in intracellular iron utilization [7] and as an obligatory iron donor for heine synthesis [8], studies of various

Abbreviations: BSA, bovine serum albumin; PBS, phosphate-buffered saline. Correspondence: J.A. Grasso, Department of Anatomy, University of Connecticut Health Center, Farmington, CT 06032, U.S.A.

erythroid cells fail to support an active role for ferrlt;l~ [5,6,9], indicating instead a principal function of passive uptake and storage of iron exceeding cellular requirements for heme synthesis [9,10]. Moreover, definitive erythroid cells are unable to mobilize intracellular ferritin iron for their own use [6,11]. Inhibition of heme synthesis in erythroid cells may be expected to disturb iron transport equilibria, thereby facilitating detection of the route taken by iron to reach the heme biosynthetic pathway. Earlier studies [5] using |son|cot|nit acid hydrazide (INH) to inhibit heme synthesis reported that approximately half of the incoming iron in rabbit reticulocytes accumulated within mitochondria, but the strong chelating actions of this drug [6,12] complicated the interpretation of these results. In more recent studies using succinylaceto.le (4,6dioxoheptanoic acid), a more specific inhibitor t f heme synthesis [13], evidence was obtained that w~.s t.~,nsistent with mitochondrial accumulation of incoming iron [6]; furthermore, the role of cytosolic ferritin in uptake of excess iron or in provision of iron for heme synthesis was insignificant [6]. The purpose of the present studies was to assess the effects of suceinylacetone inhibition of heine synthesis on the intracellular distribution and fate of incoming iron in rat reticulocytes.

0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

244 Using biochemical and ultrastructural analyses of intact cells and enriched mitochondrial fractions, the results demonstrate that: (1) inhibition of heine synthesis by succinylacetone increases the rate and amount of iron uptake by increasing the rate of receptor recycling and the concentration of transferrin receptors at the cell surface; (2) inhibition of heine synthesis leads to intramitochondrial accumulation of incoming iron, and its locAliTation mainly in the inner mitochondrial membranes; (3) intramitochondrial iron remains stationary as long as inhibition of heme synthesis persists; (4) there is minimal diversion of incoming or pre-existing cellular iron into ferritin. The significance of these results in the interpretation of the functional relationship of the reticulocyte to the total erythron is discussed. Materials and Methods Reticulocytes were obtained from Sprague-Dawley white rats * at 6-8 d after initial injection of acetylphenylhydrazine and were enriched to 77-100~ by centrifugation on an 8-14~ discontinuous Ficoll gradient. Cells were suspended (107-109 cells/ml) in medium containing amino acids, glucose, vitamins, 1-25 defatted, iron-free bovine serum albumiv ;,BSA), and phenol red in phosphate-buffered sql.~te (PBS). The osmolality of this medium was 285-., ~0 mOsmol, which approximated values obtained for rat plasma.

Cellular uptake of iron Iron uptake was measured in cells incubated in the presence and absence of 0.75-1.5 mM succin3lacetone in medium containing 0.1-25 /tM [59Fe]transferrin (35-1005 iron-saturated). Aiiquots were removed at various times of incubation to measure total cell 59Fe uptake; cells were then hemolyzed in 20 mM Hepes (pH 8.0) and centrifuged at 1500 × g for 20 rain to separate stroma from cytosol. Hemoglobin was converted to the cyanmet form by addition of 0.1 voi. of 0.5~ potassium ferricyanide and 1~ potassium cyanide. Heme was extracted from total lysate or cytosol with cyclohexanone after oxidation of hemoglobin to cyanmethemoglobin and after addition of 1 M HCI to 25-40 mM. The yield of heine was 95.3~ + 3.04. Chromatographic analysis of hemolysate or stromafree cytosol was performed on 1.6 × 80 cm. columns of Biogei A-1.5m (Bio-Rad, Richmond, CA) equilibrated with 0.05 M Hepes (pH 8.0), 0.1 M NaC! and eluted at

* Assurance of compfiance. Experimental procedures and use of Sprasue-Dawley white rats were approved by the institutional Animal Care Committee. Animals were mahltained under supervision of the Center for Laboratory Animal Care, University of Connecticut Health Center, according to regulations of the U.S.P.H.S. and U.S. Department of Agriculture.

4°C. at a flow rate of 6.8-10 m l / h per cm2. Chromatographic peaks were identified by comparison with elution profiles of hemoglobin, t~ansferrin, and ferritin standards and by various chemical assays, including Soret band absorbance, iron content, and immunoassay.

Transferrin binding activity and receptor cychng The kinetics of 125I-transferrin binding wet,. analyzed using a commercially-available program (Combicept; Packard Instruments) run on an IBM PC connected to a Packard Model 5000 gamma counter. Ceils were incubated at 0 - 4 ° C and 37°C in medium containing 5-500 nM 1251-diferric transferrin; nonspecific binding was obtained from a parallel set of incubations containing a 50-200-fold excess of non-radioactive transferrin. Incubations were terminated by removal of duplicate aliquots of each sample into ice-cold PBS. Radioactivity in duplicate samples was processed concurrently by Scatchard plot [14] and saturation curve analysis using the Combicept program. The rates of transferrin release and receptor recycling were measured in cells that were pulse-labelled for 35-50 rain with 0.2-0.5 ~tM t251-transferrin in control medium at 37 °C and chased, with and without 1-1.5 mM succinylacetone, in medium containing non-radioactive transferrin. Radioactivity in the cells and medium was measured in aliquots removed at 0-25 rain of chase.

Isolation and fractionation of mitochondria Reticulocytes were incubated for 45 min with [59Fe]transferrin and disrupted by sonication in isolation buffer (0.002 M Hepes (pH 7.4)/0.22 M mannitol/0.07 M sucrose/0.5 m g / m l BSA). The sonicate was centrifuged at 17000 × g for 20 rain at 4 ° C to obtain the stroma, which contained plasma membranes and mitochondria (electron microscopic observations). The stromal pellet was resuspended in 3-5 vol. of 46~ (w/w) sucrose in isolation buffer and layered over a 1-1.5 ml cushion of 60% (w/w) sucrose. A 34-46~ linear sucrose gradient was formed over the sonicate and the tube was topped off with 20~ sucrose to prevent collapse during centrifugation. The gradients were centrifuged at 25000 rpm for 15-18 h at 4 ° C in a Beckman SW27 rotor and 1 ml fractions were collected from the top. Fractions were assayed for radioactivity and total protein was determined by the Bradford assay [15]; cytochrome oxidase [16] and 5'-nucleotidase [17] activities were measured to locate the position of mitochondria and plasma membranes, respectively, in the gradient. Bulk isolation and fractionation of mitochondria were performed with a method modified after Schnaitman and Greenawalt [18]. All centrifugation steps were carried out at 4 ° C. Cells suspended in 2-4 vol. of isolation buffer were disrupted by sonication; the sonicate was

245 diluted with 6 vol. of isolation buffer and centrifuged at 560 × g for 15 rain. The supernatant was removed and centrifuged at 7000 × g for 15 rain. The resulting pellet was resuspended and washed successively in 50% and 25% of the original volume of isolation buffer followed by centrifugation at 7000 X g for 15 min. The final pellet contained enriched mitochondria as confirmed by cytochrome oxidase assay and electron microscopic examination (Fig. 8A). The mitochondrial pellet suspended in isolation buffer minus BSA was solub~,l~ed with 2% digitonin (1-1.35 rag/10 mg protein); the sampl,~ ,~as mixed thoroughly for 20 rain and allowed to stand for 10 rain at 0 - 4 ° C. Then, 10 ml of complete isolation buffer was added and the sample homogenized in a Potter-Elvejhem glass homogenizer with a tight-fitting ?estle. The homogenate was centrifuged at 12000 rpm for 20 rain to obtain a 'low-speed' pellet containing the inner mitochondriai membranes-matrix fraction [18] and a supernatant corresponding to the outer membrane-intracristate fraction. The 'low-speed' pellet was set aside on ice while the supernatant was centrifuged at 144000 × gmax for 1 h to separate the outer mitochondrial membrane fraction from the 'intracristate fraction' [18], or outer mitochondrial compartment. The 'low-speed' pellet was mixed with Lubrol (1 m g / 1 0 mg protein), sonicated to assure total release of 'mitochondrial matrix' from 'inner membranes', and the two fractions were separated by centrifugation at 144000 × g for 1 h. The identity of the different fractions was determined by electron microscopic examination and enzyme assays for cytochrome oxidase [16], malate dehydrogenase [19], and rotenone-insensitive NADH-cytochrome c reductase [20] for the inner membrane, matrix and outer membrane fractions, respectively.

Isolation of transferrin Transferdn was prepared from rat plasma or serum using a procedure modified from Young and Aisen [21]. The main modification was the use of DEAE-Sepharose CL6B as the anion exchanger and elution with a 0.05-0.2 M NaCl gradient. Chromatographic fractionation was repeated until the A4~o/A41o ratio was 1.2 or higher, indicating minimal contamination with heine-containmg compounds. The purity of the transferrin preparation was verified by polyacrylamide gel electrophoresis. Iodination of diferric transferrin was performed with Enzymobead (Bio-Rad), a procedure demonstrated to have no deleterious effects on the physiological ability of iodinated transferrin to deliver iron. Iron loading of transferrin with 56Fe or ~gFe to 35-100% saturation was done by incubating iron-depleted apotransferrin with Fe-nitrilotriacetic acid (NTA) prepared at a molar ratio (Fe/NTA) of 1:2.5. Excess chelate was removed by dialysis against ice-cold 0.9% NaCI. All transferrin preparations were stored in 0.2-0.5 ml aliquots at - 20 ° C. Results

Effects of succinylacetone on iron uptake While consistently inhibiting heine synthesis (Fig. 1), 1.0-1.5 mM succinylacetone exerted variable effects on the rate of iron uptake (Table I), ranging from no change to increases of 10-40% (mean rate change = +18.7%_+ 11.7; n = 7 ) . Increased uptake rates were accompanied by increased cellular iron accumulation (mean = + 28.7% _+ 19.2; n = 7). Augmentation [6,22] or maintenance [23] of the linear rate of iron uptake by succinylacetone has been reported in rabbit reticulocytes but without comment on the extent of variation.

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Fig. l. Effects of succinylacetone on iron uptake and heme synthems. Reticulocytes were incubated with or without 1.5 mM succmylacetone in medium containing 4 #M [59Fe]transferrin (35~ 59Fe(tll)-saturated) and equal aliquots removed at the indicated times. Total won uptake (o) was measured in washed whole cells which were then lysed in 0.02 M Hepes (pH 8.0) and radioactivity in cytosol (4) and stroma (El) determined after separation by centrifugation. Amount of 59Fe(lll) incorporated into heine (O) was obtained by cyclohexanone extraction of cytosols. The stromal 59Fe(lll)/125I-transferrm molar ratio (. . . . . . ) was determined in cells incubated under identical conditions with SgFe- and uSi-labelled transferrin. (A) Untreated, control cells; (B) reticulocytes incubated with 1.5 mM succinylacetone. This figure depicts one of four experiments.

246 TABLE I Effect of succmylacetone on rate of iron uptake Reficulocytes were incubated with 0.5-2.0 pM [59Fe]transferrin (50~$ Fe-saturated) in the presence or absence of 1.25-1.5 mM snccinylacetone. Cell aliquots were withdrawn at 5, 15, 30 and 60 vain of incubation, washed several times with PBS, and total cell-associated 59Fe was measured in a Packard AutoMinaxi Gamma Counter. Rates of iron uptake were obtained by finear regression analysis where r >0`90`

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within 2-5 min, 70-90% of total cell-associated 12Sl-transferrin was inaccessible to mild proteolysis in both control and succinylacetone-treated cells (data not shown). Conversely, exocytic release of internalized 125I transferrin was enhanced in succinylacetone-treated cells; nearly 70~ of the 125I-transferrin internaliTed during a prior pulse was released into the medium during 25 rain of chase, compared to 56~ release from control cells over the same time period (Fig. 2C). Identi-

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The major fraction of SgFe taken up by control reticulocytes was recovered in the cytosol (Fig. IA), with stromal iron accounting for only 8-12~ of total cell 59Fe (Fig. 1A). In contrast, 65-80~ of the incoming 59Fe in succinylacetone-inhibited cells accumulated in the stroma with a correspond~:.~g ~'eduction in recovery of 59Fe in the cytosol (Fig. 113). The stromal 59Fe/125Itransferrin molar ratio in control cells incubated with ~ I - and 59Fe-labelled transferrin containing 50~ 59Fe (expected Fe/transferrin molar ratio = 1.0) remained constant at 1.0-1.1 for 60 rain of incubation (Fig. 1A), indicating that stromal iron in untreated cells was mainly transferrin-bound. In succinylacetone-treated cells, however, the stromal 59Fe/transferrin ratio increased from 2.6 at 15 rain to nearly 10 after 60 rain (Fig. 1B), i.e., the amount of stroma159Fe exceeded the iron-binding capacity of transferrin and, therefore, was not transferrin-bound.

Effect of succinylacetone on transfet,'in receptor cycling Correlative measurements of iron uptake and of transferrin uptake and release were performed to de-. terraine the role of receptor.mediated transferrin cycling in increased iron uptake in succinylacetone-inhibited cells. In the illustrated experiment (Fig. 2), 59Fe uptake in succinylacetone treated reticulocytcs was increased by 7-145 over control levels during 60 rain of incubation (Fig. 2A). However, there was no effect on the rate of transferrin binding and uptake: uptake of 12sI-transferrin saturated within 10 rain at a level corresponding to 71960 and 72910 receptors per cell in control and succinylacetone-treated cells, respectively (Fig. 2B), assuming equimolar binding of ligand to receptor. In other experiments, succinylacetone was found to have no effect on the relative rate of t2sI-transferrin internalization as determined by resistance to pronase digestion;

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Fig. 2. Effects of succinylacetone inhibition of heine synthesis on iron uptake (A), transferrin binding and uptake (B). and cellular release of transfertin ((3). (A) Reticulocytes (8.107 cells/mi) were incubated, with or without 1.25 mM succinylacetone as indicated, at 3 7 ° C in medium containing 0.54 FM [SgFe]transferrin. Cell aliquots were removed at the various times for determination of total cell uptake of S9Fe(lll). (B) Cells were incubated as in (A) except for the presence of 0.54 ~M 125I-diferric transferrin. ((3) Reticulocytes were pulse-labelled with 0.2 /tM 12Sl-diferric transferrin for 50 min in control medium and transferred to fresh, non-radioactive medium with or without 1.25 mM suceinylacetone. Cell afiquots were removed at various times for measurement of total radioactivity remaining in cells.

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Fig. 3. Effects of succinylacetoneinhibition of heme synthesis on surface expressionof transfemn receptors. Reticulocyteswere incubated at 37 o C for 3 h in control (o) or in 1.25 mM succinylacetone-containing(ra) medium containing 0.1 tzM [~6Feltransferrin. Cells were washed and transferred to fresh medium and incubated on ice with 0.005-0.75/xM 1251-diferrictransferrin for 3 h. Binding actlv~tyin two separate experiments (A, B and C, D) were analyzed using the Combicept program. (A) Saturation curve analysis of transferrm binding activity m 7-8-day reticulocytes. KD(conlrd) = 24.8 nM, KD (succinylacetone)= 27 nM. Surface receptor concentrations were 0.62 pM and 1.03 pM for control and succmylacetone-treated cells, respectively. (B) Scatchard plots o£ same binding data as in (A) KD (control)= 22 nM, K D (succinylacetone)= 26 nM; Bm~ = 0.58 pM and 1.1 pM in control and succinylacelone-treatedcells, respectively.(C) Saturation curve analysis of transferrm binding activity in 6-day reticulocytes. KD (control) = 22 nM, K D (succmylacetone)= 26 nM; surface receptor concentrations were 2.0 pM and 3.03 pM for control and succinylacelone-lreatedcells, respectively.(D) Scatchard analysis of binding data in (C). KD (control) = 24 nM, Ko(succinylacetone) = 28 nM; Bm~, = 2.1 pM and 3.1 pM in control and succinylacetone-treatedcells, respectwely. cal results were obtained whenever increased iron uptake accompanied succinylacetone treatment. The relative rate o f t25I-transferdn uptake expressed as the ratio of this activity in succinylacetone-treated and control cells ( S A / C ratio) was 0.96 + 0.02 (n = 3), indicating no difference in the rate of transferrin binding and uptake, despite increased iron uptake. However, the ratio between the rates of 125I-transferrin release in treated and control cells was 1.17 _+ 0.06 (n = 3). Thus, where succinylacetone increased iron uptake, exocytic release of transferrin and, accordingly, the recycling of transferrin receptors to the cell surface was augmented. However, there was n o change in receptor affinity (KD) for transferrin (Fig. 3), in the rate o f transferrin binding and uptake (Fig. 2), or in the total number of transferfin receptors (Fig. 2). Since succinylacetone increased the rate of exocytosis and receptor recycling without affecting the rate o f uptake or the total number of receptors, the imbalance between e~ocytosis and uptake should increase the surface density of transferrin receptors. In cells treated with succinylacetone for several hours, to amplify the effects of the inhibitor on receptor cycling, the binding of 125I-diferric transferrin at 0 - 4 ° C increased by 50-65% (Fig. 3), confirming that inhibition of heine

synthesis by succinylacetone increased the number of surface transferrin receptors.

Intracellular distribution and utilizatton of iron Chromatographic analysis. Most (more than 90%) of the 59Fe entering the cytosol of control cells was recovered in hemoglobin during 60 min of incubation (Figs. 1 and 4). Less than 1% of total cytosolic 59Fe eluted as ferritin (Fig. 4), a result which was confirmed by ferritin immunoprecipitation from total hemolysate. A minor peak of radioactivity subsequently shown to contain mitochondria that were not pelleted by centrifugation at 1500 × g was located in the void volume (exclusion limit = 1.5- 106 Da). Despite the marked suppression of heine synthesis in succinylacetone-inhibited cells, the amount of 59Fe eluted in ferritin was increased only slightly (Fig. 4), representing approx. 2% of total cytosolic 59Fe in cells incubated with 0.2-0.5 t~M transferrin that was 35-50% Fe-saturated. However, radioactivity in the mitochondria-containing void volume peak was considerably increased (Fig. 4). Since the subphysiological concentrations of trans errin used in most experiments could have obscured the role of ferritin in iron metabolism, the effect of increased transferrin concentrations on ferritin

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FRACTIONNUMBER Fig. 4. A representative chromatographic analysis on Bio-Gel A-1.5m of cytosoi from control (o) and succinylacetone-treated (O) reticuIocytes incubated for 60 rain with 0.5 ~tM SgFe transferrin (35~ S~Fe(lll)-saturated). The prominent peak ehiting over fractions 52-60 is hemoglobin. The elution positions of ferritin (Ftn) and transferrin (Tfn) were determined with known standards prepared from rat tissues. Peak X indicates a component(s) characterized b:, absorbance at 280 nm (absorbance profile not shown).

iron uptake was examined. Increasing the concentration of transferrin to approximate physiological levels in vivo increased total 59Fe uptake in succinylacetone-inhibited cells (Fig. 5) but had relatively little effect on the proportion of 59Fe recovered in ferritin (Fig. 5). While 59Fe uptake increased commensurately with the concentration of transferrin (Fig. 5), recovery of 59Fe in ferritin was minimal, accounting for approx. 4~ of total cell ~9Fe at 6 vM but only 2~ at 25 ItM (Fig. 5), even with 100~ Fe-saturated transferrin. These results indicated that ~erritin in rat reticulocytes had a limited capacity to absorb incoming iron, despite inhibition of heme synthesis, and that ferritin storage iron in these cells was quantitatively insignificant. Significant levels of radioactivity in succinylacetoneinifibited cells were recovered in a peak exhibiting absorbance at 280 nm and eluting in the 8-13 kDa region of the chromatogram (Fig. 4). Equivalent amounts of this peak were detected by A,~o absorbance in control cells but containing minimal amounts of S9Fe (Fig. 4). When succinylacetone-inhibited cells were pulse-labelled with 59Fe and chased in control or succinylacetone-containing medium, radioactivity in this peak was chased into hemoglobin and the void volume peak, respectively, i.e., the loss of 59Fe from this peak was independent of the status of heme synthesis during the chase. The absence of heine-specific characteristics excluded the identity of tlus 'low molecular weight peak' as heme-globin mcJnomers or dimers: there was no Soret band absorbance (A416) nor was S9Fe in this peak extractable as heme; furthermore, there was no incorporation of [6J4C]aminolevulinate (ALA) into this peak in succinylacetone-inhibited cells, despite recovery of [14C]ALA in hemoglobin (data not shown). Based upon its apparent precursor-product behavior in pulse-chase

experiments, this component has been tentatively identified as a putative cytosolic iron transporter. Sucrose density gradient analysis. Sucrose density gradient analysis of the stroma yielded three peaks of 59Fe radioactivity (Fig. 6). Peak I, sedimenting between the 34~ and 20% sucrose layers, contained plasma membranes, as indicated by its enrichment for 5'nucleotidase and by electron microscopic examination. Peak II was enriched for cytochrome oxidase and was further identified as mitochondria by electron microscopy (Fig. 8B). Peak III contained mitochondria and soluble proteins, including hemoglobin, which did not rise into the gradient and remained at the interface between the 46% sucrose layer and the 60% sucrose cushion. Approx. 60-75% of the stromal 59Fe in succinylacetone-treated cells was recovered in the main mitochondriai peak (Peak II), with the remaining radioactivity distributed in Peak Ill (approx. 20-35%) and in the Peak I plasma membrane fraction (Fig. 6A). Ferritin accounted for less than 1% of the radioactivity in Peaks I and III, while only 6% of the radioactivity in Peak III was recovered as heine or transferrin. Since mitochondria are a major constituent of Peak III, the major fraction of the radioactivity in this peak was probably intramitochondrial. In control cells, the distribution of stromal sgFe was qualitatively similar, but the size of the peaks was considerably reduced (Fig. 6A). Nearly half of the radioactivity in Peak III of control cells was extracted as heme, presumably from hemoglobin entrapped in the stroma during lysis. Upon removal of succinylacetone, radioactivity in the mitochondrial peak decreased sharply during subse-

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Fig. 5. Effects of increased concentration of [Fe]transferrin on ferritin iron uptake. Reticulocytes were incubated for 50 rain in medium containing 1.5 mM succinylacetone and various concentrations of 100~ 59Fe-saturated transferrin as indicated. Total cell uptake of SgFe (open bars) increased finearly with the concer~tration of tran~ferrin. Amount of SgFe recovered in ferritin is shown by the closed bars; numbers in parentheses indicate percentage of total iron recovered in ferritin. Data were a composite of two experiments.

249 quent chase in non-radioactive medium (Fig. 6B). This result was consistent with chromatographic analysis which showed that hemoglobin synthesis was restored upon withdrawal of the inhibitor, with quantitative transfer of ~gFe from the mitoehondria-containing void volume peak into hemoglobin (Fig. 7). With continued inhibition of heine synthesis during the chase, 59Fe

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remained in the mitochondfial fraction, with rrfinimal transfer of radioactivity into other components (Fig. 6C). Chromatographic analysis of the corresponding cytosols from cells chased in succinylacetone for up to 20 h confirmed that intrarnJtochondrial ~ F e remained stationary with minimal utilization of SgFe for hemoglobin synthesis and minimal loss of radioactivity from the void volume peak (Fig. 7). Despite persistent inhibition of heme synthesis, there was no significant transfer of 59Fe into ferritin during 20 h of chase (Fig. 7).

lntramitochondrial localization of iron Electron microscopic and enzymic assay revealed optimal disruption of the outer mitochondriai membrane at a digitonin concentration of 1.35 rag/10 mg protein (Fig. 8C, D) and that separation of matrix from inner mitochondrial membranes was facilitated when Lubrol lysis was accompanied by somcation. Clearly, the majority of intramitochondfial 59Fe (70% _ 8.2) in succinylacetone-inhibited cells was localized in the inner membrane fraction (Table If). Exclusion of results obtained with incompletely disrupted mitochondria prior to adoption of modifications cited above increased the proportion of SgFe recovered in the inner membrane fraction to 76% + 3.7 and reduced recovery of iron in the outer membrane fraction, but had no effect on the distribution of 59Fe in the matrix or outer compartment (Table II). The total amount of 59Fe accumulated in the mitochondria was approx. 300 pmol/mg mitochondrial protein (Table If), which was 20-50-fold less than reported for isolated liver mitochondfia iron-loaded with various iron-chelates [24]. However, no attempt was made in the present studies to saturate mitochondrial iron uptake capacity so that after 45 min of incubation iron uptake was still increasing linearly (data not shown). Furthermore, mitochondria were fractionated from cells that were incubated with 0.2-0.5 #M [sgFe]transferrin, or 50-125-times lower than the trans!,:rrin concentration in rive.

C Fig. 6. Sucrose density gradient profile of strom : obtained from reticulocytes which were incubated for 45 rain with 0.5 #M S'~Fe transfernn as md~cated. The stromal suspension was layered over a custuon of 608 (w/w) sucrose cushion and a 34-64~ ( w / / w ) linear gradient formed above it. Gradient was centrifuged at 25000 rpm for 18 h at 4 ° C and fractionated as described in the text. (A) Reticulocytes incubated as indicated with or without succinylacelone for 45 mm w~th 59Fe transferrin. (B) Stroma from cells incubated for 45 mm in medium containing 59Fe-transferrin and 1.5 mM succinylacetone and then chased for 4 h in non-radioactive medium minus succinylacetone. The dashed line represents the d~stnbution of cytochrome oxidase in the gradient. (C) Stroma from cells labelled for ,t5 min m the presence of succmylacetone and [59Fe]transferrir~ (O) and from cells pulsedabelled as described but then chased for 4 h in non-radioactive medium containing succinylacetone (S).

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60

80

Fig, 7. Effects of varying chase conditions on the intracellular iron distribution. A composite chromatographic analysis on Bio-Gel A-1.Sm of total hemolysates from reticulocytes which were pulse-labelled with [SgFeltransferrin for 50 rain (0) and then chased for 20 h in non-radioactive medium without (o) or with ( l ) 1.5 mM succinylacetone.

The amplification of the surface receptor pool was derived from posttranslational redistribution of cellular transferrin receptors. Under steady-state conditions, approx. 80% of the transferrin receptors in rat reticulocytes are located in an intracellular pool or reservoir [25] through which cellular iron uptake can be modulated without further synthesis of receptor. Inhibition of heine synthesis did not ~hange the number of receptors, but stimulated recruitment of transferrin receptor from the intracellular pool to establish a new steady-state distribution of surface and iizternal receptors through which cellular iron uptake was increased. Thus, modulation of the iron deliver pathway in reticulocytes is

Discussion

Inhibition of heme synthesis by succinylacetone has been shown to increase iron uptake in reticulocytes (this reporL and Refs. 6 and 22). In the present study, augmentation of iron uptake was correlated with increased exocytic release of transferrin and concomitant recycling of transferrin receptors to the cell surface. With no change in receptor binding affinity or rate of endocytic uptake of transferrin, the net effect of increased receptor recycling ,~as to amplify the concentration of surface transferrin receptors and, accordingly, to increase cellular accessibility to exogenous transferrin. TABLE !1 Iron localization in succinylacewne-inhibited mitochondrla

Reticulocytes were incubated for 45 rain in medium containing 1.0-1.5 mM succinylacetone and 0.2-0.5/tM [59Fe]transfemn. Mitochondria were isolated by differential centrifugation and fractionated as described by Schnaitman and Greenawalt [18] with modifications as described in text. in each experiment, the total amount of iron (as 59Fe) taken up into intact mitochondria prior to fractionation was determined and expressed as pmol iron per m8 n~tochondrial protein. Recovery of SgFe in each mitochonddal subfraction is expressed as counts per total subfraction. The Intracristate fraction corresponds to the outer mitochondrial compartment [18]. Numbers in parentheses represent the percentage of total mitochondrial r, dioactivity recovered in each fraction. Iron uptake m mitochondria Ipmoi/mg protein) 127.9 325,7 345.8 300.9 317.3

Radioactivity (cpm) inner membrane

matrix

184170 (62) 137731(59) 218477(72) 292728 (75) 132424(81)

9275 10003 11552 44849 ~

outer membrane (3) (4) (4) (11) ~'

99205 (33) 78239(33) 44856(15) 48415 (12) 7359 (5)

intracristate 6814 (2) 9165(4) 27483(9) 6816 (2) 6330(4)

251

Fig. 8. Electron microscopic visualization of mitochondrial isolation and fractsonation. (A) The ~,na~ pellet obtained after differential centrifugation of reticulocyte sonicate after the method of Schnaitman and Greenawalt [17]. The very dense linear matenal is rat hemoglobm which precipitates at low temperatures. Note enrichment of mitochondria: magnificatior; × 18600. (B) Sample of Peak II obtained after sucrose density gradient analysis of stroma as shown in Fig. 6. The dense material is hemoglobin: ×26370. (C) Contents of the supernatant obtained after centrifugation of the digitonin-solubilized mitochondrial pellet shown in (A). A corresponding afiquot of this subfraction was ~ariched for rotenone-msensmve NADH-cytochrome c reductase: × 43 300. (D) Contents of the low-speed pellet obtained from dsgltonin-solubilized nutochondna following Lubrol solubilization and sonication. This fraction was enriched for cytochrome oxidase and exhibits discoidal, double-membraned profiles reminiscent of intact mitochondrial cristae: × 43 650.

expressed through posttranslational mechanisms acting directly on a pre-existing, finite population of transfertin receptors. Tb~.• stimulation of iron delivery by succinylacetone irddbifion of heine synthesis is consistent with the hypothesis that the availability of intracellular heme is the signal which regulates receptor-mediated transferrin iron delivery [26]. Succinylacetone, acting on 8ALA dehydrase to depress production of porphobilinogen [13], will reduce the amount of heme synthesized in support of hemoglobin assembly and elicit a compensatory increase in iron uptake. The variable effects of succinylacetone inhibition on cellular iron uptake may reflect differences in the extent to which the inhibitor affects heine synthesis and, thus, the amount of intracellular heme a n d / o r the size of the pre-existing heme pool in different experiments. Although direct measurement of intracellular heme was not performed, other data indirectly support a role for free heme as an intracellular signal. Hemin, which inhibits heine synthesis by end-

product inhibition [25~27] and simultaneously provides an alternative source of heine for hemoglobin asqembly, reduced receptor-mediated endocytosis of transferrin and decreased cellular iron uptake [22,23,25]. Furthermore, our data do not supp~,rt a significant role for an intracellular chelatable iron pool as the signal modulating iron delivery in reticulocytes. Treatment of reticulocytes for up to 6 h with desferrioxamiP.c to deplete cellular iron had minimal effects on transferrin-mediated iron delivery [25]. Moreover, the massive increase in intracellular non-heine iron associated with succinylacetone inhibition of heine synthesis should be expected to reduce cellular iron uptake as iron accumulates in the chelatable pool, assuming that the latter ~oes not represent ferritin-bound iron. Instead, despite increased intracellular non-heme iron, the rate of iron uptake was increased, adding to the cellular iron burden and contrary to the effect predicted were the intracellular nonheme iron pool the critical signal regulating the rate of transferrin-iron delivery. Whatever the nature of the

252 signal(s), the iron delivery pathway in reticulocytes responds rapidly to metabolic alterations within the cell, with increased rates of receptor recycling evident within 5-10 rain after exposure of cells to the inhibitor, and progressive amplification of surface transferrin concentration as inhibition of heme synthesis persisted (Figs. 2 and 3). In this regard, the rat reticulocyte responds like various non-erythroid cells in which posttranslational modulation of surface transferrir~ receptor expression by phorbol ester and hormones has been correlated with rapid increases in rates of exocytosis and receptor recycling [28-31]. Iron loading of isolated hepatic mitochondria has been reported to stimulate calcium efflux in vitro [32]. In rat reticulocytes where exocytic release of transferrin and receptor recycling are calcium-dependent [33], it is possible that mitochondrial efflux of calcium resulting from accumulation of intramitochondrial iron may stimulate the increased rate of receptor-mediated iron delivery in succinylacetone-inhibited cells. Me'.s,irements of intracellular calcium levels and flux ~ r i n g succinylacetone inhibition of heme syn:',e~is are in progress. It is unlikely that iron accumulation cause4 ~gnificant disruption of mitochondria, since ATP generation was not affected by succinylacetone over the time periods employed in these studies (data not shown). Several steps in the heine biosynthetic pathway, including the terminal insertion of ferrous iron into protoporphyrin, are compartmentalized within mitochondria [27]. Nearly all of the iron entering the rat reticulocyte is used for heine synthesis (Figs. 1 and 4), leaving minimal amounts of surplus iron within the mitochondria (Figs. 2 and 3A). This utilization of iron for heine synthesis greatly exceeds the capacity of mitochondria to accumulate iron [34,35] and restricts the size of the intramitochondrial iron pool in normal cells [35]. However, in succinylacetone-inhibited cells, the imbalance between the amount of incoming mitochondriai iron and its utilization for heme synthesis leads to accumulation of iron in mitochondria and expansion of the intramitochondrial precursor iron pool. Mitochondrial accumulation of iron has previously been described in isolated reticulocyte mitochondria [36,36a,37] and in intact erythroid cells in which heine synthesis was inhibited [5,6,38]. The most dramatic example is the human sideroblastic anemias, where conspicuous intramitochondrial iron deposits are often associated with extensive degeneration and disruption of mitochondria [39]. In the present studies, the identity of the mitochondria as the site of iron localization has been demonstrated both by specific mitochondrial enzymic markers and ultrastructurai examination. There is no doubt that the cellular compartment into which incoming iron is primarily directed in intact reticulocytes and in which it is accumulated upon impairment of heine synthesis comprises the mitochondria. This

intramitochondrial iron is preferentially localized in the inner membrane fraction, with minimal amounts recovered in the matrix. This pattern of distribution was confirmed by electron microscopic examination: the specific activity of iron was highest in a subfraction exhibiting the morphological characteristics of inner membranes of cristae and enrichment for cytochrome oxidase. Following removal of succinylacetone, intramitochondrial iron was promptly used for heine synthesis, released from the mitochondria, and recovered in hemoglobin. Thus, incoming iron is primarily directed into a functionally mobile intramitochondrial pool that is localized in the inner membranes and which provides iron for heine synthesis. The properties of this pool resemble the 'non-heine, non-Fe-S iron' recovered in inner membranes and matrix of liver mitochondria [40] which consists of ferrous iron and serves as the immediate donor of ~ron for heine synthesis by ferrochelatase [41,42]. Release of cells from inhibition of heine synthesis invariably resulted in utilization of most (80~ or more) of the intramitochondrial iron for heme synthesis (Fig. 6B), a result which contrasts with the efficiency of recovery reported elsewhere [6]. The reasons for these apparently dissimilar findings are not immediately obvious, but probably reflect significant differences in experimental procedures used in these studies. In our study, nonspecific binding of iron to stroma was kept to a minimum by stringent preparation of 59Fe-saturated transferrin; chase periods were prolonged to as much as 4-20 h; and, iron localization and efficiency of its utilization were assessed specifically in isolated mitochondria. In rat reticulocytes, the demands of heme synthesis leave relatively little surplus iron that requires uptake and storage in ferritin, a conclusion based upon our consistent finding that essentially none of the incoming iron in these cells is recovered in ferritin. More significant for iron metabolism in these cells, the apparent balance between the amount of inco:ang iron and its consumption for heme synthesis implies that the size of the intracellular non-heme iron pool in reticulocytes is negligible. The reticulocyte, then, is optimally specialized to accommodate virtually all of the incoming iron for its immediate metabolic needs and, therefore, diverts little or none of it to ferritin stores or to an intracellular non-heine iron pool. An unexpected result of these studies was the insignificant role of ferritin in rat reticulocytes, even in the presence of an added iron burden associated with inhibition of heme synthesis. Administration of iron to various cells has been shown to increase cellular ferritin levels [7,43-45] by stimulating translational expression of pre-formed ferritin mRNA [44,45]. This ability to respond to an iron burden with increased ferritin production reinforces the protective capacity of ferritin

253

against the potentially toxic effects of excess iron. Even in other erythropoietic cells where iron is mainly targetted into the stroma, presumably into mitochondria, continued inhibition of heme synthesis leads to release of stromal iron and its capture and storage in cytosoric ferritin [9,11]. In rat reticulocytes, however, prolonged inhibition of heine synthesis results in continuous accumulation and retenti~ of incoming iron in mitochondria, with minimal diversion of unusable iron into ferritin. Despite the potential toxicity of intramitochondrial iron, rat reticulocytes cannot respond to the stimulus of surplus iron with increased ferritin synthesis, as occurs in the intact erythron [46] and liver cells [43]. Several features of reticulocytes account for their inability to accumulate excess iron in ferritin: (1) the amount of endogenous ferritin is very low in these cells [47], thus severely restricting their iron storage capacity; (2) reticulocytes lack the transcriptional and, apparently, the translational machinery needed to enable them to respond to an increased iron burden. Either they contain no pre-existing ferritin mRNA or efficient translation of any stored ferritin mRNA cannot be induced. In either case, there is no recruitment of ferritin to compensate for the increased cellular iron levels produced by succinylacetone. Reticulocytes, a transient, near-terminal phase in the rife cycle of the erythron, are at a distinct metabolic disadvantage since they lack the plasticity to react to drastic environmental and metaboric variables. These cells are devoid of transcriptional machinery and contain a residual translational apparatus which is only sufficient to support the final stages of hemoglobin synthesis. Acknowledgement These studies were supported by a grant (DK 19167) from the National Institutes of Health. References 1 Ponka, P., Neuwirt, J., Borova, J. and Fuchs, O. (1977) Ciba Found. Symp. 51 (new series), pp. 167-188, Excerpta Medica, Amsterdam. 2 Tangeras, A. (1986) Blechem. J. 235, 671-675. 3 Romslo, I. (1980) in iron in Biochemistry and Medicine, II (Jacobs, A. and Worwood, M., eds.) pp. 325-362, Academic Press, New York.

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