Analysis of the High-Afﬁnity Iron Uptake System at the … · located in the extracellular space. FTR1 contains 7 membrane-span-ning helices with loops 4 and 6 outside and loop

EUKARYOTIC CELL, May 2010, p. 815–826 Vol. 9, No. 51535-9778/10/$12.00 doi:10.1128/EC.00310-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Analysis of the High-Affinity Iron Uptake System at theChlamydomonas reinhardtii Plasma Membrane�

Alaina Terzulli and Daniel J. Kosman*Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214

Received 26 October 2009/Accepted 16 March 2010

Multicopper ferroxidases play a vital role in iron metabolism in bacteria, fungi, algae, and mammals.Saccharomyces cerevisiae utilizes a channeling mechanism to couple the ferroxidase activity of Fet3p to Fe3�

transport into the cell by Ftr1p. In contrast, the mechanisms by which mammals couple the ferroxidasereaction to iron trafficking is unclear. The human ferroxidases ceruloplasmin and hephaestin are twice the sizeof Fet3p and interact with proteins that are not expressed in fungi. Chlamydomonas FOX1 is a homolog of thehuman ferroxidases but likely supports iron uptake in a manner similar to that of yeast, since Chlamydomonasreinhardtii expresses a ferric iron permease homolog, FTR1. The results presented support this hypothesis. Weshow that FOX1 is trafficked to the plasma membrane and is oriented with its multicopper oxidase/ferroxidasedomain in the exocytoplasmic space. Our analysis of FTR1 indicates its topology is similar to that of S.cerevisiae Ftr1p, with a potential exocytoplasmic iron channeling motif and two potential iron permeationmotifs in membrane-spanning regions. We demonstrate that high-affinity iron uptake is dependent on FOX1and the copper status of the cell. Kinetic inhibition of high-affinity iron uptake by a ferric iron chelator doesnot reflect the strength of the chelator, supporting a ferric iron channeling mechanism for high-affinity ironuptake in Chlamydomonas. Last, recombinant FOX1 (rFOX1) has been isolated in a partially holo form thatexhibits the UV-visible absorbance spectrum of a multicopper oxidase and the catalytic activity of a ferroxidase.

Iron is essential to all cells, but its aqueous and redox chem-istry poses a barrier to its cellular metabolism. The ferric spe-cies is the most abundant form of iron in the environment, butthe insolubility of Fe3� makes it relatively biounavailable. Met-alloreductases overcome this problem by presenting the cellwith Fe2�, which has a far more tractable aqueous chemistry(1, 14, 26). The expression of iron binding proteins involved inassimilation, mobilization, and storage then serves to managethis Fe2� (3, 39). Metalloreductases and iron-binding proteinshave been identified in most organisms; however, the mecha-nism by which iron is trafficked between these proteins remainslargely uncharacterized.

Detailed studies of the high-affinity iron uptake system inSaccharomyces cerevisiae have demonstrated that iron traffick-ing is coupled to iron redox chemistry (9, 27–28, 45–46). Thesoluble Fe2� produced by the yeast metalloreductases Fre1pand Fre2p is reoxidized by the plasma membrane multicopperoxidase (MCO) Fet3p; with its MCO domain on the exocyto-plasmic surface, Fet3p is a type Ia membrane protein (25). TheFe3� produced by Fet3p serves as a substrate for permeationof the cell by the ferric permease Ftr1p (46). A key feature ofthis uptake system is that Fe3� alone cannot serve as a sub-strate for uptake by Ftr1p. Iron permeation is coupled toferroxidation via a substrate channeling mechanism in whichthe Fe3� substrate of Ftr1p is handed off directly from Fet3p(27). Residues involved in iron channeling from Fet3p to Ftr1phave been identified in both proteins (27–28, 45). In Fet3p,E185 and D409 participate in Fe2� binding and electron trans-

fer to the type 1 copper site in this copper oxidase and arerequired for Fe trafficking to Ftr1p (27, 41, 47, 50). Ftr1p is atype III membrane protein with seven membrane-spanninghelices; a DASE motif has been identified in extracellular loop6 of Ftr1p that is required for trafficking of Fe3� from Fet3p toFtr1p (45).

Ferroxidases are also expressed by higher eukaryotes (19,38). The mammalian ferroxidases ceruloplasmin (Cp) and hep-haestin (Hp) are critical components of iron metabolism. Forexample, patients lacking functional human Cp (hCp) exhibitneurological disorders associated with maldistribution of sys-temic iron (17, 57). The role that hephaestin plays in ironmetabolism is demonstrated by the severe iron deficiency phe-notype of the sla mouse; this mouse strain expresses a trun-cated murine Hp (mHp) protein that fails to localize to thebasolateral membrane in intestinal enterocytes, where this fer-roxidase activity is required for iron release into circulation(55). That is, Hp (and Cp) appears to play an essential role inthe trafficking of Fe from ferroportin (Fpn), an iron exporter atthe plasma membrane, to transferrin (Tf) for systemic irondelivery (8, 35–36). The oxidation of Fe2� by Cp (or Hp) isessential to the Fe3� loading of Tf in plasma (35), but whetherexport of Fe2� from Fpn or loading of Fe3� onto Tf is coupledto ferroxidation by either of the two proteins remains unclear.

Limited inferences can be drawn from the yeast iron-chan-neling studies in regard to iron-trafficking mechanisms in mam-mals for two reasons. First, structural differences between themammalian and fungal ferroxidases suggest the catalytic fer-roxidase mechanism may be different (25, 31, 49–50, 59). In allferroxidases, the oxidation of 4Fe2� is coupled to the reductionof O2 to 2H2O via an electron transfer pathway that includes aT1 copper site and a trinuclear copper cluster (25). Fet3p iscomprised of three cupredoxin domains and a single T1 Cu

* Corresponding author. Mailing address: Department of Biochem-istry, the University at Buffalo, 140 Farber Hall, 3435 Main St., Buffalo,NY 14214. Phone: (716) 829-2842. Fax: (716) 829-2661. E-mail:[email protected].

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site; as noted, carboxylate side chains at this site directly con-tribute to the enzyme’s specificity toward Fe2� and to thetrafficking of Fe3� to Ftr1p. The mammalian MCOs are doublethe size of Fet3p, containing six cupredoxin domains whichsupport three T1 copper sites in addition to the trinuclear Cucluster (49, 59). All of the T1 sites have carboxylate residuesthat likely contribute to Fe2� binding (31), and thus, Cp andHp (a close Cp paralog) have the potential to oxidize Fe2� atmore than one site. Consequently, Fet3p is inadequate as amodel to suggest how Cp and Hp might “hand off” iron toanother protein. Second, mammals do not express a ferricpermease homologous to yeast Ftr1p. The potential Fe3�-channeling partners to Cp and Hp in mammals likely includethe transmembrane protein, Fp, and the serum-soluble pro-tein, Tf (8, 35), neither of which has Fe-binding motifs com-parable to those found in Ftr1 proteins.

However, a link between iron-trafficking mechanisms inyeast and mammals may be revealed through examination ofiron uptake systems in algae. Chlamydomonas reinhardtii ex-presses FOX1, a homolog of the Cp and Hp ferroxidases, andFTR1, a homolog of the fungal Ftr1 permeases; these twoproteins likely function together as a high-affinity iron importcomplex in this alga (20, 29). Therefore, in this system a “mam-malian-like” ferroxidase potentially traffics iron in a “yeast-like” manner. In addition, the MCO produced in the halotol-erant alga Dunaliella salina interacts with a transferrin-likeprotein (TTf) to support iron uptake (12–13, 37); D-Fox is aFOX1 and hCp homolog. Thus, this “mammalian-like” algalferroxidase appears to traffic iron to a “mammalian-like” ferriciron binding protein. A thorough examination of the iron up-take mechanisms in these algae may provide the bridge be-tween yeast and humans in terms of our understanding of irontrafficking.

A structural homology model indicates FOX1 contains sixcupredoxin domains and three T1 copper sites, each with po-tential iron binding residues within close proximity (51). TheFOX1 gene is coordinately upregulated at the transcript levelunder iron deficiency along with FTR1 and FRE1, genes sonamed for their homology to the yeast ferric permease andreductase, respectively (29). La Fontaine and colleaguesshowed also that FOX1 protein production increased underiron deficiency (29). Growth of a fox1 knockdown strain isslowed in iron-deficient media relative to that of a wild-typestrain, indicating FOX1 plays a direct role in iron metabolism(5). Additionally, wild-type cells grown in copper-depleted me-dia have diminished iron uptake activity, highlighting the linkbetween copper and iron metabolism that is likely due to aferroxidase (20).

Such evidence indirectly points to a role for FOX1 in high-affinity iron uptake, yet there is no evidence that its potentialpartner permease, FTR1, is involved in this pathway asidefrom the fact that the gene is also upregulated under low-ironconditions and has sequence homology to the fungal per-meases. Alignment of FTR1 with fungal Ftr1 homologues in-dicates that the Chlamydomonas permease likely has a com-parable membrane topology and homologous iron traffickingand permeation motifs, although these features have not beenexperimentally confirmed (29). If FOX1 and FTR1 functiontogether to support high-affinity iron import in Chlamydomo-nas, this system would prove to be an excellent model to ex-

amine, since FOX1 shares more similarity to the mammalianferroxidases than to the fungal counterparts. Our hypothesisfor how FOX1 and FTR1 are assembled in the plasma mem-brane of Chlamydomonas is illustrated in Fig. 1.

Here we provide an analysis of the components of the high-affinity iron uptake system in Chlamydomonas and a test of thismodel. First, iron uptake analysis of Chlamydomonas cells un-der three independent conditions in which FOX1 proteinabundance is reduced provides strong evidence that FOX1 isrequired for high-affinity iron uptake. 55Fe uptake kinetic dataare presented that are consistent with a mechanism of high-affinity iron uptake in Chlamydomonas in which FOX1-gener-ated Fe3� is delivered directly to FTR1 for permeation, thusrepresenting another example of coupling between ferroxida-tion and iron trafficking. Indirect immunofluorescence exper-iments indicate both FOX1 and FTR1 are trafficked to theplasma membrane, and the topology of FOX1 places the mul-ticopper oxidase domain in the exocytoplasmic space. The to-pology and orientation of FTR1 are shown to be identical tothose of S. cerevisiae Ftr1p, placing potential iron traffickingand permeation motifs in homologous regions. Finally, a sol-uble (secreted) form of recombinant FOX1 (rFOX1) has beenexpressed in HEK293E cells and purified from the conditionedmedium; this protein exhibits spectral and oxidase character-istics of an MCO and catalyzes the formation of monoferrictransferrin from Fe2� and apo-Tf. Based on the homologybetween FOX1 and the mammalian ferroxidases, these studiesprovide new insight as to how iron trafficking may occur inhigher eukaryotes.

MATERIALS AND METHODS

Chlamydomonas strains and culture conditions. All Chlamydomonas strainsused in this work were generously provided by Sabeeha Merchant and weremaintained on Tris-acetate-phosphate (TAP) medium plates containing appro-

FIG. 1. Topology model of FOX1 and FTR1 in the Chlamydomo-nas plasma membrane. In this model, FOX1 is anchored in the mem-brane by a single amino-terminal membrane-spanning helix with theMCO domain, containing three T1 Cu sites and a trinuclear Cu cluster,located in the extracellular space. FTR1 contains 7 membrane-span-ning helices with loops 4 and 6 outside and loop 3 and the carboxyterminus located inside the cell; “(HA)2” indicates the location of theepitopes engineered into FTR1 to test this suggested topology. Puta-tive REXXE iron permeation motifs in FTR1 are found within trans-membrane domains 1 and 4; a putative iron-trafficking motif, EPTD,resides in loop 6. The proposed mechanism of iron uptake has Fe3�

produced by FOX1 channeled directly to FTR1 via the EPTD loop 6motif and transmembrane trafficking supported by the REXXE motifs.

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priate selective components. Copper-free and iron-free TAP medium was pre-pared as previously described (40). The arginine-requiring (200 �g/ml) cell wallmutant strain, cc-425, was used for FOX1 indirect immunofluorescence and inthe iron uptake experiments involving chelator inhibition. The wild-type strainNE3 was used in the fox1 knockdown and copper-dependent iron-uptake anal-yses; 34� was used in the crd2 iron uptake experiment. The fox1 knockdownstrain, kd11, was maintained on plates containing 1.5 mM L-tryptophan, 20 �g/mlparomomycin, and 5 �M 5-fluoroindole and kept in a box containing a yellowfilter to prevent degradation of the light-sensitive L-tryptophan and 5-fluoro-indole. For experiments, the knockdown strain was grown in TAP without se-lective components, while cc-425 and the crd2 strain were grown in TAP con-taining 200 �g/ml arginine. For all experiments, cells were inoculated into liquidcultures from plates and grown to approximately 5 � 106 cells/ml in TAPmedium containing 10 �M Fe-EDTA. From these initial cultures, new cultureswere started at 0.5 � 106 cells/ml in the same media and grown to 3 to 5 � 106

cells/ml before induction. To induce expression of FOX1 and FTR1, cells werecollected by centrifugation at 4,000 � g, washed in iron-free TAP medium,resuspended in iron-free TAP medium (with or without arginine), and allowed toshake for 18 h prior to an experiment.

HEK293E cell culture and transfection. HEK 293E cells (10) were used fortopology and localization analysis of FOX1 and FTR1 and for secretion ofsoluble FOX1. The suspension-adapted cells were passaged in Erlenmeyer flasksin low-calcium hybridoma serum-free medium (HSFM) (Invitrogen) containing0.1% Pluronic solution, 10 mM HEPES buffer, and 50 �g/ml Genetecin. Thecells were transiently transfected as previously described (4) with FOX1 or FTR1expression plasmids using linear polyethyleneimine (PEI) (25 kDa) as the trans-fection vehicle. For transfection, cells were collected by centrifugation at 500 �g, resuspended to 0.5 � 106 cells/ml in medium containing the components listedabove, and incubated with shaking for 3 h. The transfection mixture was addeddropwise; it contained 1 �g/ml (of total culture volume, not transfection mixturevolume) plasmid DNA and 2 �g/ml PEI in a volume of medium representing 5%of the total culture. Indirect immunofluorescence analysis was carried out 48 hposttransfection. For soluble rFOX1-secreting cells, the conditioned mediumwas harvested 5 days posttransfection.

Protein reagents. To obtain purified rFOX1, conditioned medium from trans-fected HEK293E cells was diluted 2-fold with 50 mM morpholineethanesulfonicacid (MES) (pH 6.5) and passed over a Mono-Q column equilibrated in the samebuffer. FOX1 was eluted from the column with a 0 to 1.0 M NaCl gradient. Thefraction containing holo-FOX1 as determined by immunoblotting was concen-trated and buffer exchanged into 50 mM MES (pH 6.5). Fet3p was purified aspreviously described (18). Native hCp was purchased from GenWay Biotech.Apo- and holo-Tf were purchased from Sigma.

Plasmid construction. Plasmids containing FOX1 and FTR1 cDNA were giftsfrom Sabeeha Merchant and were used to construct plasmids for expression inHEK293E cells. DNA sequence from the FOX1 open reading frame (ORF)representing residues 42 to 1142 (GenBank accession no. AAM45881.1) wasPCR amplified using primers that added a NotI site upstream of the M42 (start)codon and an EcoRI site downstream of the terminating UAA (29). The PCRproduct was cloned into the mammalian expression plasmid pTT5SH8Q2 (10),using the NotI and EcoRI sites. The FTR1 ORF was subcloned upstream ofgreen fluorescent protein (GFP) in pKS-GFP (15), and this fusion was insertedinto pTT5SH8Q2 at the NotI and BamHI sites. This plasmid was used as atemplate for adding the double hemagglutinin [(HA)2] epitope to FTR1 using acloning strategy previously described (45). Two sets of amplification primers,each containing a single HA epitope, were used to produce two FTR1 transcrip-tion units that when recombined into pTT5SH8Q2 via a three-way ligationproduced a complete FTR1 ORF containing a (HA)2 epitope at the desiredlocation (see Fig. 6 and 7). pTT5SH8Q2 containing a His-tagged hCp ORF wasused to construct the soluble form of FOX1. An EcoRV site was inserted afterhCp codon 24 to allow for insertion of a FOX1 PCR product containing cDNArepresenting residues 86 to 1142 using EcoRV and AgeI sites. All plasmids werepropagated in E. coli strain XL1-blue.

Indirect immunofluorescence. For FOX1 topology and localization in Chlamy-domonas, two 10-ml cultures of cc-425 were grown for 18 h in TAP mediumcontaining either 10 �M Fe-EDTA to reduce FOX1 expression or no iron plus100 �M ferrozine to maximize FOX1 induction. Cells were collected by centrif-ugation at 4,000 � g for 10 min and resuspended in phosphate-buffered saline(PBS) to a final concentration of 1 � 106 cells/ml. The cells were added topoly-L-lysine-coated coverslips in 35-mm dishes and allowed to adhere for 1 h.The cells were fixed with 3% formaldehyde for 15 min, washed three times withPBS, and blocked for 1 h in 3% bovine serum albumin (BSA). The unperme-abilized cells were probed for FOX1 using a polyclonal anti-FOX1 antibody at a1:500 dilution in 3% BSA overnight at room temperature. An additional set of

cells was permeabilized with 0.2% Triton X-100 in PBS or left unpermeabilizedprior to blocking and probed for ATP synthase (1:500) as a control for mem-brane integrity. After four washes with PBS, the cells were probed with anantirabbit secondary antibody conjugated to Alexa Fluor 488 (MolecularProbes), diluted 1:200 in 3% BSA for 1 h, and then washed four times before thecoverslips were sealed to microscope slides.

For FOX1 and FTR1 localization and topology analysis in HEK293E cells, 10ml of cells was used for transfections for each construct. After 48 h, the cells werecollected by centrifugation at 500 � g for 5 min and resuspended in PBS to a finalconcentration of 1 � 106 cells/ml. Each set of transfected cells was plated on twocoated coverslips, fixed, and washed. For each set, one plate of cells was perme-abilized by adding 3 ml 0.2% Triton X-100 in PBS for 30 min, while the otherplate remained unpermeabilized in PBS. The cells were probed for FOX1 asdescribed above. Rabbit anti-HA (1:500) (Santa Cruz) and mouse anti-GFP(1:500) (Millipore) were used for FTR1 topology analysis. The secondary anti-bodies used were antirabbit Alexa Fluor 488 conjugate and antimouse Cy5conjugate (Jackson Immuno Research), both diluted 1:500 in 3% BSA. All cellswere visualized using a Zeiss Axio Observer fluorescence imaging system. TheApotome structured illumination system was used for visualizing Chlamydomo-nas cells in order to reduce background fluorescence.

Iron uptake assays. Chlamydomonas cultures induced for expression of FOX1and FTR1 were collected by centrifugation at 4,000 � g for 10 min, washed withuptake buffer (100 mM MES, 2 mM potassium acetate, 20 mM sodium citrate,pH 6.0), and resuspended to 108 cells/ml in a minimum of 2.5 ml uptake bufferin a glass scintillation vial. After 15 min of incubation at 30°C with shaking, 20mM ascorbate was added, followed by a 15-min incubation. A prereduced 55Festock was prepared by diluting the 55FeCl3 stock to 50 �M in uptake buffercontaining 20 mM ascorbate. The uptake reaction was initiated by addition of55Fe to 1.0 �M. A time course experiment showed that the uptake reaction waslinear for 25 min, so triplicate samples of 200 �l were taken at 10 s and at 20 min.The 10-s values represent background 55Fe cell association and were subtractedfrom the 20-min time point values in the analyses. Each sample was addeddirectly to 3 ml of ice-cold quench buffer (0.1 M Tris succinate, 1 mM EDTA, pH6.0). Using a vacuum manifold, the samples were filtered over 25-mm type A/Eglass fiber filters and washed three times with 3 ml quench buffer. The filters wereplaced in vials and covered by scintillation fluid (200 �l); the 55Fe in each samplewas quantified by scintillation counting. Statistical analysis of uptake experimentswas performed using the GraphPad Prism4 software program.

Immunoblot analysis. Induced cultures were harvested by centrifugation, andcells were resuspended in 10 mM sodium phosphate (pH 7.0) buffer containing1% Triton X-100, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride [PMSF],and 3 �l protease inhibitor cocktail per 100 �l. An equivalent volume of glassbeads was added to the suspension, and the cells were subjected to 10 cycles of30 s of vortexing then 30 s on ice. The lysate was centrifuged at 14,000 � g at 4°C,and the supernatant extract was separated from the pellet. Total protein wasquantified using the Bio-Rad (Bradford) reagent, and a total of 2 �g of totalprotein was loaded into each lane of an 8% SDS-polyacrylamide gel. Separatedproteins were transferred onto a polyvinylidene difluoride (PVDF) membraneusing a semidry transfer apparatus. The membrane was subsequently blocked for1 h in 5% dry milk in TBST (Tris-buffered saline containing 0.05% Tween 20)and probed for either FOX1 (1:3,300) or ATP synthase (1:3,300; Agrisera)followed by secondary antibody (antirabbit horseradish peroxidase [HRP] con-jugate, 1:4,000; Santa Cruz). Pierce chemiluminescent reagents were applied tothe membrane, and film was used to visualize the immunocomplexes.

Oxidase activity assays. Phenylenediamine (PPD) and Fe2� oxidation assayswere performed in 96-well microtiter plates. For PPD oxidation, each wellcontained 5 mM PPD and 0.06% Triton X-100 in 100 mM sodium acetate, pH5.7. The reaction was initiated by the addition of either 0.5 �M Fet3p, 0.5 �Mnative hCp, or 20 �M FOX1 (assuming �90% purity and containing a mixtureof apo and holo forms of the protein). The absorbance at 530 nm was measuredevery 2 min for 30 min. For Fe2� oxidation, 10 �M ferrous ammonium sulfate in100 mM sodium acetate, pH 5.7, was incubated with 0.5 �M Fet3p, 0.5 �M hCp,or 20 �M FOX1. After 30 min, 100 �M ferrozine was added, and the absorbanceat 550 nm was measured.

Tf loading and urea-PAGE. Incorporation of iron into Tf was monitored via apreviously described method (16). Each reaction mixture contained 100 �l of6.25 �M apo-Tf, 200 �M ferrous ammonium sulfate, and 100 �M ascorbate in100 mM sodium acetate, pH 5.0. The reaction was initiated by the addition of 1�M total FOX1, and 15 �l was removed at various time points, immediatelyadded to sample buffer containing 10% glycerol and 0.2% bromophenol blue in1� Tris-borate-EDTA (TBE), and frozen at �20°C. Autooxidation of Fe2� wasmonitored in a reaction mixture containing all components except FOX1. Ad-ditionally, 10 �l of 10-fold-concentrated conditioned medium from mock-trans-

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fected cells was assayed as a negative control. Urea gel electrophoresis wascarried out as previously described (56). One 6.25-ml 6% gel was prepared asfollows: 2.25 g urea, 625 �l 10� TBE (1 M Tris, 0.1 M boric acid, 16 mM EDTA,pH 8.4), 1.35 ml 30% acrylamide (2.6% C), 2.8 ml double-distilled water(ddH2O), 100 �l 10% ammonium persulfate, and 20 �l N,N,N�,N�-tetramethyl-ethylenediamine (TEMED). The separating gel was overlaid with a stacking gelcontaining 867 �l 10� TBE, 1.6 ml H2O, 433 �l acrylamide, 30 �l 10% ammo-nium persulfate, and 16.7 �l TEMED. The samples were electrophoresed for 4 hat 100 V, and the gel was stained with Coomassie R-250.

RESULTS

FOX1 supports high-affinity iron uptake. Initial evidencefor FOX1 involvement in high-affinity iron assimilation was theincrease in FOX1 transcript and protein abundance underlow-iron conditions and the sequence homology that FOX1has to other ferroxidases (29). Subsequently, Chen et al. pro-duced a fox1 knockdown strain of Chlamydomonas that grewpoorly in iron-deficient medium, providing more direct evi-dence of a role for FOX1 in iron metabolism (5). Herbik andcolleagues demonstrated that high-affinity uptake was depen-dent on copper (20); Fe uptake in S. cerevisiae exhibits thissame dependence, because copper-loaded Fet3p is requiredfor cell ferroxidase activity (7, 58). The copper dependence ofiron uptake in Chlamydomonas is consistent with involvementof an MCO but does not require that FOX1 (or any MCO)support iron uptake.

In another study, tetrathiomolybdate (TTM) was shown toreduce iron uptake (21). TTM has been shown to inhibit hCpby reducing the T1 copper site, so TTM inhibition of ironuptake in Chlamydomonas could be the result of FOX1 inhi-bition (6). However, TTM is also a known copper chelator;thus, an alternative model is that TTM inhibits iron uptake inChlamydomonas by altering the copper content of the cell,producing an effect similar to growth in a copper-depletedmedium (34). To demonstrate that FOX1 contributes to high-affinity iron uptake, 55Fe uptake assays were performed underthree separate conditions in which FOX1 protein abundancewas reduced.

Iron uptake analyses were first performed on the fox1 knock-down strain, kd11, and the parental strain, NE3. Initial veloc-ities of 55Fe uptake were quantified as a function of [Fe2�] andfit to the standard Michaelis-Menten equation (Fig. 2A). Theapparent Km values for Fe2� were as follows: NE3 (wild type),4.1 � 0.9 �M; kd11 (knockdown), 4.2 � 0.6 �M. The identicalMichaelis constant for ferrous iron is strong evidence that thesame iron import complex is being quantified in each strain.The Vmax value found for NE3 was 4.0 � 0.4 pmol 55Fe/107

cells/20 min; for the fox1 knockdown strain, kd11, this valuewas 1.1 � 0.1 pmol 55Fe/107 cells/20 min. This 75% decrease inapparent Vmax correlated well with the decrease in the FOX1protein as determined by immunoblot analysis of extracts fromthe cells used in these uptake experiments (Fig. 2A). Theloading control for this analysis was ATP synthase, which isunaffected by cellular iron status (29).

Iron uptake and FOX1 protein abundance was also analyzedin the copper-deprived wild-type strain NE3 (Fig. 2B). Cellswere grown for 5 days in medium containing either 6 �M Cu(the concentration in TAP) or 100 �M bathocuproine disul-fonate (BCS) to deplete the cells of copper before overnightinduction of FOX1 using medium containing no iron. To de-

termine if uptake could be restored, 6 �M Cu was added to thecopper-depleted cells 24 h prior to initiating the 55Fe uptakeexperiments. The immunoblot in Fig. 2B indicates that FOX1protein abundance is reduced in copper-depleted cells. Corre-lated with this reduction, copper-depleted cells exhibited�40% high-affinity iron uptake in comparison to copper-re-plete ones, confirming the results obtained by Herbik et al.(20). Iron uptake is almost completely restored in cells re-supplemented with copper (Fig. 2B); the FOX1 protein recov-ers as well (data not shown).

FIG. 2. High-affinity iron uptake in Chlamydomonas is reducedwhen FOX1 protein abundance is reduced. (A) Iron uptake kineticswere analyzed for the wild-type strain, NE3, and for the fox1 knock-down strain, kd11. The Vmax was reduced in kd11 by 75%, but the Kmvalues for ferrous iron were identical. Immunoblot analysis indicatesFOX1 protein abundance is reduced in kd11. The same membrane wasalso probed for ATP synthase as a loading control, since it is unaffectedby the iron status of the cell. (B) Iron uptake activity is reduced undercopper deprivation. Experiments were performed on the wild-typestrain NE3 grown in the presence of 6 �M Cu or no Cu plus 100 �MBCS for 5 days prior to the overnight induction of FOX1. When Cuwas added back to the medium during FOX1 induction, iron uptakewas restored. FOX1 protein abundance is reduced in cells grown in theabsence of copper, as indicated by the immunoblot. (C) The Chla-mydomonas crd2 strain was previously shown to have diminishedFOX1 protein relative to wild-type cells grown under iron deficiency.Iron uptake activity is also reduced in this strain relative to the wild-type strain 34�. The immunoblot confirms the reduction in FOX1protein abundance.



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Iron uptake analysis also was performed on a Chlamydomo-nas strain with a mutation at the CRD2 locus, an allele con-ferring copper-conditional iron deficiency (11). The immuno-blot in Fig. 2C indicates that the crd2 strain produces lessFOX1 protein than to the wild-type strain 34� when cells areiron deprived but grown under normal copper conditions. Adecrease in iron uptake correlates with the decrease in proteinabundance in this strain as well. Thus, the three conditionsevaluated show that a decrease in iron uptake correlates witha decrease in FOX1 protein abundance, providing consistentevidence in support of the conclusion that FOX1 is requiredfor high-affinity Fe uptake in this alga.

Kinetic mechanism of high-affinity iron uptake. Our modelfor high-affinity iron uptake in Chlamydomonas is that Fe3�

produced by FOX1 is supplied to FTR1 for permeation of thecell. The channeling mechanism proposed for Fe uptake in S.cerevisiae, in which the product of Fet3p is handed off directlyto residues on Ftr1p, was demonstrated through the use ofFe3�-specific chelators in kinetic iron uptake analyses (27). IfFOX1 and FTR1 function as do their yeast counterparts, apossible mechanism for iron uptake is one in which Fe3� pro-duced by FOX1 is channeled directly to residues on FTR1. Inorder to test this possibility, the effect of ferric iron chelators asinhibitors of iron uptake in Chlamydomonas was examined.

A chelator can act as an inhibitor of iron uptake in either oftwo ways. If the Fe3� product of FOX1 equilibrates into bulksolvent kinetically much faster than FTR1 uses it as a substratefor permeation, as would be the case for a dissociative, non-channeling mechanism, a Fe3� chelator would bind the FTR1substrate free in solution with an efficacy that reflected thestability constant of the corresponding Fe(III)-chelate com-plex. In the case where iron uptake occurs via a strict channel-ing mechanism, the Fe3� produced by FOX1 dissociates intosolution much more slowly than it traffics to FTR1 for Fe3�

permeation. In this kinetic mechanism, a ferric iron chelatorcould inhibit uptake only through association with the ironuptake complex. A quantitative analysis of the relationshipbetween chelator strength (its stability constant, K1) and theuptake inhibition constant (KI) for each chelator allows fordistinguishing between the two mechanisms. In a standard rate,equilibrium linear free energy plot, where the log KI value isplotted versus the log K1 value, the relationship between che-lator strength and inhibition is represented by the slope of theline, �. This relationship is given by equation 1,

log KI � ��log K1 � C (1)

where the intercept C reflects the hypothetical value of log KI

when the Fe(III)-chelate complex has zero stability. Note thatthe slope of the correlation is negative since K1 is an associa-

tion constant while KI is a dissociation constant. If the mech-anism is a dissociative, nonchanneling one, � equals 1, sincethere would be a 1:1 relationship between chelator strengthand the chelator’s ability to inhibit uptake by scavenging Fe3�

that equilibrates with bulk solvent. A fractional � value indi-cates this 1:1 relationship does not hold and is consistent withan associative, iron channeling mechanism (24, 33).

To examine the kinetic mechanism of high-affinity iron up-take in Chlamydomonas, reductase-independent 55Fe uptakeassays were performed on the cell wall mutant strain cc-425using ferric iron chelators of various strengths. Cells weregrown in TAP medium containing 5 �M iron to a cell densityof 3 � 106 cells/ml and then washed, resuspended, and allowedto shake in TAP containing no iron for 18 h to allow FOX1 andFTR1 induction before an experiment. To ensure the experi-ment was reductase independent, a 55FeCl3 stock was preparedin 20 mM ascorbate. The apparent Km for Fe2� in this strainwas 1.4 � 0.2 �M, while the Vmax was 3.7 � 0.3 pmol 55Fe/107

cells/20 min. To quantify the kinetics of Fe3� chelator inhibi-tion of iron uptake, assays were performed using 1 �M 55Fe atvarious chelator concentrations. The inhibition constants, KIs,for five Fe3� specific chelators with log stability constants rang-ing from 10 to 19, were determined (Table 1). The relationshipbetween chelator strength and the inhibition constant was ex-amined by plotting the log KI value versus the log K1 valueaccording to equation 1 (Fig. 3). This analysis gave a � valueequal to �0.39 � 0.07; this value states that a 1,000-fold in-crease in chelator strength supports only a 15-fold decrease inKI (increase in inhibition), far from the 1:1 relationship (� 1)expected if the chelator were binding FOX1-produced Fe3�

which was released to bulk solvent. This result eliminates thedissociative mechanism as describing the kinetic mechanism ofFe uptake through the FOX1, FTR1 system. This value of �compares favorably with the value derived from a similar studyin yeast, �0.46, suggesting that iron uptake in Chlamydomonasoccurs via a mechanism of iron channeling in a manner similarto that for yeast.

FOX1 and FTR1 localization and topology. In our model forhigh-affinity iron uptake in Chlamydomonas, the import ofFe3� is coupled directly to the ferroxidase activity of FOX1.This model requires not only FOX1 expression at the plasma

FIG. 3. The linear relationship between the iron uptake inhibitionconstant of a Fe3� chelator and its stability constant represented as astandard rate, equilibrium linear free energy plot (see equation 1). Thefitted KI values determined for each chelator (Table 1) were plottedagainst the corresponding log stability constants. A � value of �0.39,given by the slope of the line, indicates inhibition of uptake by achelator is 1.5% as probable as chelation of aqueous Fe3�. The cor-relation coefficient for this fit was 0.916.

TABLE 1. Ferric iron chelator stability and inhibition constants

Chelator Stability constant(log K1)

Inhibition constant,KI (mM)

IDA 10 13.7 � 2.8EDDP 11 10.9 � 2.5HIDA 13 8.7 � 1.3NTA 15 0.64 � 0.22HEDTA 19 0.005 � 0.001



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membrane but an exocytoplasmic orientation of the protein’sMCO domain. Topology analyses predict that FOX1 containstwo possible transmembrane domains: an amino-terminalplasma membrane targeting sequence that also serves as amembrane anchor and a carboxy-terminal membrane-span-ning region (29). A structural homology model of FOX1 placesthe latter carboxy-terminal portion in the last of the protein’ssix cupredoxin domains, indicating that it is unlikely to be amembrane-spanning region (51). As depicted in Fig. 1, wepredict that FOX1 is anchored in the plasma membrane by asingle amino-terminal membrane-spanning helix (a type Ibmembrane protein lacking a cleavable signal sequence) with allsix cupredoxin domains in the extracellular space.

To demonstrate this localization and orientation of FOX1,indirect immunofluorescence was carried out with the Chla-mydomonas cell wall mutant strain, cc-425, using an antibodydirected against FOX1 residues 394 to 646, which include thesecond, third, and fourth cupredoxin domains (29). Cultureswere grown overnight in medium containing either 10 �M ironto suppress FOX1 expression or 100 �M ferrozine to induce it.Cells were plated, fixed on poly-L-lysine-coated coverslips,blocked, and probed with anti-FOX1 or anti-ATP synthase andAlexa Fluor 488 conjugate secondary antibody and visualizedvia fluorescence microscopy. Cells induced for FOX1 (Fig. 4B)showed distinct plasma membrane fluorescence when probedwith anti-FOX1 relative to the uninduced cells (Fig. 4A), con-firming that FOX1 is trafficked to the plasma membrane inChlamydomonas. The cells were not permeabilized prior toanti-FOX1 treatment, indicating the ferroxidase portion of theprotein recognized by the antibody is located in the exocyto-plasmic space. Permeabilized and unpermeabilized cells wereprobed with anti-ATP synthase as a control for permeabiliza-tion. Chloroplast fluorescence was clearly visualized in thepermeabilized cells (Fig. 4C). Dim fluorescence in the unper-

meabilized cells represents background autofluorescence(Fig. 4D).

FTR1 localization and topology could not be examined inChlamydomonas cells since we did not have the FTR1-specificantibodies directed to different regions needed for a survey ofexocytoplasmic and cytoplasmic epitopes. The mammalianHEK293E cell line was chosen for this topologic analysis ofFTR1 given the homology FOX1 has with respect mammalianferroxidases. To demonstrate that Chlamydomonas proteinstrafficked properly in this heterologous system, indirect immu-nofluorescence against FOX1 was carried out with cells co-transfected with FOX1 and FTR1::GFP (Fig. 5). Consistentwith our findings in Chlamydomonas, the MCO domain ofFOX1 was located extracellularly in HEK293E cells, as indi-cated by the immunofluorescence detected in unpermeabilizedcells (Fig. 5A). This result validated the use of HEK293E cellsfor FTR1 analysis. As shown in Fig. 5B, GFP fused to thecarboxy terminus of FTR1 was visualized at the plasma mem-brane of these cells also. However, in addition to the distinctplasma membrane fluorescence, significant fluorescence (pro-tein) was visualized with a primarily perinuclear localization aswell. This likely is due to a combination of the overexpressionof the protein from the pTT vector used and relatively sluggishprotein trafficking in the heterologous host. The overlay ofFOX1 and FTR1 images shown in Fig. 5C indicates a patternof colocalization of these two proteins. These data do notconfirm FTR1 expression at the plasma membrane of Chla-mydomonas but do support the inference that FTR1 andFOX1 traffic to the same cellular compartment. Although thecells shown in Fig. 5 were cotransfected with FOX1 andFTR1::GFP, the same trafficking was exhibited in cells trans-fected with either gene alone, indicating that FOX1 and FTR1traffic independently of one another in this host (see data inFig. 7 for FTR1; data not shown for FOX1).

FIG. 4. FOX1 localizes to the plasma membrane in iron-deprived Chlamydomonas cells and is oriented with the MCO domain outside the cell.The cell wall mutant strain, cc-425, was grown in TAP medium containing either 10 �M iron for FOX1 repression (A) or no iron plus 100 �Mferrozine for FOX1 induction (B). Cells were plated and left unpermeabilized prior to treatment with anti-FOX1 and Alexa fluor 488 conjugateantibodies. As a control for membrane integrity, permeabilized (C) and unpermeabilized cells (D) were probed for ATP synthase which is localizedto the chloroplast. Dim fluorescence is visualized in the unpermeabilized cells (D), which is likely due to background autofluorescence. The bottompanels represent phase-contrast images of the same cells under each condition.



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Based on sequence homology to the S. cerevisiae ferric per-mease Ftr1p, we proposed that FTR1 has a similar topology(Fig. 1). Ftr1p is a 7-transmembrane protein in which the twoREXLE motifs involved in iron permeation are located in thefirst and fourth transmembrane domains (45). The DASE mo-tif located in extracellular loop 6 is involved in the hand-off ofiron from Fet3p to Ftr1p (27, 45). Three of six topology pre-diction programs (HMMTOP, TMHMM, and TMPRED) pre-dicted FTR1 contains seven transmembrane domains (22–23,43, 52–54). The ClustalW alignment of FTR1 with Ftr1p,shown in Fig. 6, reveals that the seven transmembrane domainsof FTR1 predicted by these programs align well with the struc-turally homologous domains of Ftr1p (30). Furthermore, thisalignment shows that the two REXXE motifs in FTR1 alignwith their yeast counterparts, placing them in transmembranedomains 1 and 4. There is no DASE motif in FTR1; however,we propose that the EPTD motif within loop 6 of FTR1 ishomologous to the DASE motif in S. cerevisiae Ftr1p (ScFtr1p)and may play a role in iron trafficking between FOX1 andFTR1 (Fig. 1).

This FTR1 model was tested using a series of (HA)2-taggedspecies as shown in Fig. 1 and 6. An (HA)2-encoding sequencewas cloned into FTR1 cDNA to be located after residue 131,171, or 390, corresponding to predicted loop 3, 4, or 6, respec-tively (noted in Fig. 6). Indirect immunofluorescence was per-formed on HEK293E cells expressing each of the tagged FTR1proteins using an anti-HA antibody. To determine the orien-tation of the carboxy terminus, indirect immunofluorescencewas performed on the GFP-tagged version of FTR1 probedwith a GFP-specific antibody. For each set of transfections,cells were plated on coverslips, fixed, and either permeabilizedwith Triton X-100 or left untreated prior to probing with pri-mary and secondary antibodies. Figure 7 reveals that the131(HA)2-tagged and GFP-tagged versions of FTR1 were ac-cessible to antibody only if the cells were permeabilized. Notethat one cell in the 131(HA)2-tagged unpermeabilized samplewas fluorescent, which was possibly due to slight permeabili-zation in the membrane of that cell. In contrast, the 171 and390(HA)2-tagged versions were accessible regardless of thestate of the membrane, indicating these epitopes are located

extracellularly. We conclude that loop 3 and the carboxy-ter-minal domain are oriented toward the inside of the cell whileloops 4 and 6 are oriented exocytoplasmically. Since loop 6contains the potential iron trafficking motif EPTD, the topol-ogy determined here is consistent with our model that irontransfer from FOX1 to FTR1 is a key aspect of the mechanismof iron import in this alga.

FOX1 is a ferroxidase that catalyzes holotransferrin forma-tion. Based on the similarity between FOX1 and hCp, wepredicted that a soluble form of FOX1 could be produced inHEK293E cells and secreted into the medium, allowing puri-fication of rFOX1. The coding sequence of FOX1 correspond-ing to residues 86 to 1142 was cloned into the pTT5SH8Q2vector downstream of the hCp coding sequence correspondingto residues 1 to 25, which include the cleavable, hCp signalpeptide, residues 1 to 19. A 100-ml culture of HEK293E cellswas transfected with this hCp-FOX1 fusion construct, and af-ter 5 days, the conditioned medium was harvested for purifi-cation over Mono-Q equilibrated in 50 mM MES, pH 6.5. Thecolumn was eluted with a gradient from 0 to 1 M NaCl.

The resulting fractions from the Mono-Q column were an-alyzed by SDS-PAGE and immunoblotting for rFOX1. Previ-ous data indicate that copper-loaded hCp migrates at �70 kDawhen the sample is left unheated prior to electrophoresis (44).Apo-hCp (heated or unheated) and heated hCp (heated andthus Cu depleted) migrate according to the protein’s molecularmass, 125 kDa. We considered that rFOX1 produced fromHEK293E cells might be a mixture of metalated forms fromfully apo to fully copper loaded and would exhibit similarelectrophoretic behavior; thus, the fractions eluted from themono-Q resin were left unheated prior to electrophoretic anal-ysis. The rFOX1 protein migrating at the expected mass of 125kDa was detectable in several fractions from the Mono-Qcolumn, as shown in the immunoblot in Fig. 8A, indicating thatmuch of the protein secreted was in the apo form. However,fractions 4 and 5 contained a species recognized by anti-FOX1that migrated as a lower-molecular-mass protein, indicatingthat it could be Cu replete. The slower-migrating species wasmore abundant, however, and as a result, the longer film ex-

FIG. 5. FOX1 and FTR1 localization in HEK293E cells. (A) FOX1 localization was analyzed by indirect immunofluorescence in cellscotransfected with FOX1 and FTR1-GFP using the anti-FOX1 antibody. The cells were unpermeabilized prior to antibody treatment, indicatingthat the MCO domain resides outside the cell. (B) GFP fluorescence reveals localization of FTR1-GFP at the plasma membrane. (C) Thefluorescence overlay indicates the colocalization of FOX1 and FTR1-GFP at the plasma membrane. (D) Differential interference contrast imageof cells shown in panels A to C.



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posure required to detect the lower band resulted in the ex-treme overexposure of this putative apo-FOX1 species.

To determine the purity of the fractions containing the higher-mobility species, 2 �g of the fourth fraction (F4) was analyzedby SDS-PAGE and Coomassie staining (Fig. 8B). Samplebuffer was added with or without SDS, dithiothreitol (DTT),and boiling in order to detect both apo and holo forms of theprotein. As a control, 2 �g of hCp was also analyzed. Thepredominant band in both F4 samples migrates at a molecularmass of �130 kDa, confirming that the fraction contains rela-tively pure rFOX1; however, several less-distinct bands areapparent in the undenatured sample both above and below thepredominant 130-kDa band, making it difficult to determinethe ratio of holo-rFOX1 to apo-rFOX1 according to this anal-ysis.

Fraction F4 also exhibited a slight blue hue. All MCO pro-teins have a characteristic absorbance at 608 nm due to theCu2� at the T1 site(s). This fraction was concentrated 7.5-fold,and an absorbance scan from 300 to 800 nm revealed thisdistinctive absorbance feature (Fig. 8C). If the extinction co-efficient, ε, for holo-rFOX1 is equivalent to that of hCp (10,000M�1 cm�1) (32), then an A608 value of 0.10 would correspondto 10 �M (1.3 mg/ml) holo-rFOX1 in the concentrated F4

sample. This sample contained 24 mg/ml total protein andtherefore �6% holo-rFOX1.

MCO proteins catalyze the oxidation of p-phenylenediamine(PPD) regardless of their substrate specificity (2, 48); thus, thePPD oxidase activity of F4 was measured in comparison tothose of Fet3p and hCp (Fig. 9A). Conditioned medium frommock-transfected HEK293E cells was harvested 5 days post-treatment, concentrated 10-fold, and used as a negative con-trol; various amounts of the latter sample were assayed todetermine if HEK293E cells themselves contributed to oxidaseactivity. The amount of F4 used in the assay corresponded toapproximately 1 �M holo-rFOX1 (20 �M total rFOX1) basedon the 608-nm absorbance and extinction coefficient as out-lined above. The rate of oxidation of PPD by this amount of F4was comparable to that with 0.5 �M Fet3p and hCp, or about50% of the activity of these two ferroxidases used as positivecontrols. The concentrated control medium exhibited no oxi-dase activity, indicating that the secreted rFOX1 contributedall the oxidase activity observed in F4.

A ferrozine-based assay was also used to determine thereactivity of this rFOX1 protein toward ferrous iron. Ferrozineforms a stable complex with Fe2� that absorbs at 550 nm. Inthis endpoint assay, Fe2� is incubated with ferroxidase for 30

FIG. 6. ClustalW alignment of Chlamydomonas FTR1 (CrFtr1) with Saccharomyces cerevisiae Ftr1p (ScFtr1) (30). Stars and dots indicateidentical and similar residues, respectively. The 7 predicted transmembrane domains (boxed in blue) align well with the yeast transmembranedomains. The two REXXE motifs (boxed in yellow) align with their yeast counterparts, placing them in transmembrane domains 1 and 4, whilethe possible iron-channeling EPTD motif in Loop 6 corresponds to the essential DASE motif in ScFtr1p (boxed in yellow). (HA)2 tags were clonedin CrFtr1 in loops 3, 4, and 6 between the residues boxed in purple, and GFP was fused to the carboxy terminus.



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min prior to addition of ferrozine. After 10 min of incubationwith ferrozine, the absorbance at 550 nm is measured. In theabsence of a ferroxidase, a strong absorbance is observed,whereas samples containing ferroxidase have an A550 that isreduced due to the conversion of Fe2� to Fe3�. As shown inFig. 9B, the amount of remaining Fe2� after 30 min wassmaller in the F4 sample than in the samples containing noferroxidase and concentrated conditioned medium, suggestingrFOX1 exhibits Fe2� oxidase activity in addition to activitytoward PPD.

In mammals, the ferroxidase activities of Cp and Hp areproposed to contribute to the Fe3� loading of apotransferrin(Tf) for systemic iron delivery. In a recent study, Griffiths et al.used urea-PAGE to show that purified recombinant hHp cat-alyzed the formation of diferric Tf from Fe2� and apo-Tf (16).We used this assay to examine the ability of rFOX1 (F4) tocatalyze Fe3�-Tf formation. In each sample, apo-Tf was incu-bated with Fe2� in the presence of 0.1 mM ascorbate and thereaction was initiated by the addition of 0.4 �M ferroxidase;yeast Fet3p was used as a positive control. As a function ofincubation time, samples were removed and electrophoreti-cally fractionated on a 6% urea polyacrylamide gel (Fig. 9C).Lanes 1 and 2 show the migration differences between apo-

and diferric Tf, respectively. When apo-Tf was incubated with0.2 mM Fe2� in the presence of ascorbate, very little mono- ordiferric Tf was formed after 3 h (lane 3). As indicated in lanes4 to 6, an increase in monoferric Tf was observed in the F4sample as a function of incubation time. In the negative controlcontaining concentrated conditioned medium from mock-transfected HEK293E cells (lane 7), very little mono- or difer-ric Tf was observed. We conclude that the monoferric Tfformed in the F4 sample can be attributed to rFOX1-depen-dent ferroxidase activity.

DISCUSSION

Our model for high-affinity iron uptake in Chlamydomonasis one in which the extracellular ferroxidase, FOX1, hands offits ferric iron product to the ferric importer, FTR1. In supportof this model, we show that FOX1 directly supports high-affinity iron uptake in Chlamydomonas utilizing a mechanismconsistent with channeling between a ferroxidase and per-mease. Additionally, the data presented here confirm the pre-dicted localization, topology, and orientation of FOX1 andFTR1. Finally, the spectral and catalytic characteristics of pu-rified recombinant FOX1 clearly indicate it is a ferroxidase.

FIG. 7. Topology analysis of FTR1 in HEK293E cells. Indirect immunofluorescence was performed on cells expressing a series of (HA)2- andGFP-tagged versions of FTR1. The (HA)2 tags were cloned into the predicted loops as noted to the left of each set of images, while GFP was at thecarboxy terminus. Permeabilized and unpermeabilized samples were analyzed for each transfection, and the fluorescent and phase-contrast images areshown for each condition. The absence of fluorescence in the unpermeabilized samples (panels on right) indicates that the designated region of theprotein is located inside the cell, while fluorescence under both conditions indicates that the region is outside the cell. Although fluorescence is visualizedin one cell of the unpermeabilized loop 3 (HA)2 sample, this was the only fluorescent cell and was likely due to permeabilization of that cell.



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The iron uptake analyses indicate FOX1 directly supportshigh-affinity iron uptake. Iron uptake was analyzed under threeindependent conditions in which FOX1 protein abundance wasreduced, and in each case a decrease in uptake activity corre-lated with the decrease in FOX1 protein abundance. For ex-ample, the Vmax for Fe uptake in the fox1 knockdown strainkd11 was reduced by about 70% relative to that of the wildtype; perhaps more significant was that the Fe2� Km valueswere the same, indicating that the same kinetic process wasresponsible for Fe uptake in both strains. This result indicatesthat the residual uptake in the knockdown strain was not dueto a secondary, kinetically distinguishable Fe uptake system.Consistent with previous results, we found that Cu-depletedcells have reduced FOX1 protein levels and diminished Feuptake (20, 29). Finally, the crd2 strain also exhibited de-creased iron uptake that correlated with decreased FOX1abundance. In addition to the reduction in the FOX1 protein,Eriksson et al. previously indicated that mutations at this locusresult in copper-conditional iron deficiency, suggesting CRD2may function in copper transport (11). It should be noted,however, that the crd2 strain is not Cu depleted when grownunder Cu-replete conditions; thus, the reduction of FOX1 pro-

tein abundance (and Fe uptake) does not appear to reflect thecellular Cu status solely.

In order to probe the mechanism of iron uptake, we usedFe3�-specific chelators of various strengths as inhibitors of theiron uptake reaction. We found that the inhibitory effectsquantitatively represented only 1.5% of the strength of thechelator. The � value (�0.39) associated with this correlationsuggests that a direct transfer of Fe3� from FOX1 to FTR1 isapproximately 70-fold more likely than dissociation of Fe3�

from FOX1 into bulk solvent. By analogy to the fungal system,a model is proposed in which the ferric iron product of FOX1is handed off to an extracellular loop in FTR1 and passed intothe cytosol via the RExxE motifs residing in the first and fourthmembrane-spanning helices of the permease (cf. Fig. 1). Thischanneling mechanism implies that there are specific residueson each protein responsible for the “hand-off” from FOX1 toFTR1. Our topology analysis places the EPTD motif, similar tothe DASE motif in Ftr1p, outside the cell; the acidic residues

FIG. 8. Purified rFOX1 exhibits electrophoretic and spectral prop-erties characteristic of an MCO. rFOX1 was purified from conditionedmedium of HEK293E cells transfected with a plasmid containing theMCO coding sequence of FOX1 fused to the ER targeting signalcoding sequence of hCp. (A) Fractions eluted from Mono-Q resinwere diluted 10-fold, and 5 �l of each was analyzed by immunoblottingfor rFOX1. The samples were left unheated prior to electrophoresis inorder to detect both apo and holo forms of FOX1. The prominentband in all fractions migrates according to the molecular mass ofrFOX1, �120 kDa; fractions 4 and 5 exhibit a species migratingaround 60 kDa as well, which could correspond to holo-rFOX1.(B) Fraction 4 from the Mono-Q column was analyzed for purity by aCoomassie-stained SDS polyacrylamide gel. Protein (3 �g) was incu-bated in sample buffer with or without SDS, DTT, and boiling prior toelectrophoresis (2nd and 3rd lanes, respectively). Although the frac-tion contains one prominent band corresponding to the size of rFOX1,a discrete band corresponding to the higher-mobility band in the im-munoblot was not visualized in the undenatured sample, indicating themajority of the protein is apo-rFOX1. The 4th and 5th lanes show themobility of 2.5 �g denatured and undenatured native hCp, respec-tively. (C) A UV-visible spectrum of fraction 4 concentrated 7.5-foldwas obtained using a BMG Labtech FLUOstar Omega microplatereader. The spectrum exhibits an absorbance peak at 608 nm, which ischaracteristic of all MCO proteins.

FIG. 9. rFOX1 exhibits PPD oxidase and Fe2� oxidase activity andcatalyzes Fe3� loading of Tf. (A) PPD oxidase activity was assayed bymeasuring the absorbance at 530 nm over time. The activities of equiv-alent amounts of Fet3p and hCp (0.5 �M) are shown by the first 2 bars.Conditioned medium from mock-transfected cells was concentrated10-fold and exhibits no PPD oxidase activity (third bar). Pure rFOX1(4th Mono-Q fraction containing mostly apo-rFOX1) was used at 20�M total rFOX1 and exhibits activity within the same range as that ofFet3p and hCp. (B) A ferrozine-based assay was used to measureferroxidase activity. After a 30-min incubation of enzyme with Fe2�,ferrozine was added to chelate the remaining Fe2�, and the absor-bance at 550 nm was measured. The sample in which buffer wasincubated with Fe2� prior to ferrozine addition was used to represent100% Fe2� remaining after 30 min (first bar). As shown by the secondand third bars, 0.5 �M hCp and Fet3p completely oxidize the Fe2�,while 10-fold-concentrated conditioned medium from mock-trans-fected cells oxidized �25% of the Fe2�. Pure rFOX1 (20 �M total)almost completely oxidized the Fe2� within 30 min. (C) Urea-PAGEwas used to assay the catalysis of Fe3� loading of Tf. Pure rFOX1 wasincubated in the presence of Fe2�, ascorbate, and apo-Tf, and sampleswere taken at the times indicated and subjected to separation on aurea-polyacrylamide gel. The migration of the various forms of Tf isindicated in the first two lanes. After 3 h, rFOX1 converts all apo-Tf tomonoferric-Tf (lane 6). Very little mono- or diferric Tf is formed in thebuffer control or 10� mock-transfected conditioned medium samples(lanes 3 and 7, respectively).



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in this motif could potentially serve as “hands” in Fe3� chan-neling. Certainly, mutational analysis of FTR1 is required toassess the role of the EPTD motif in uptake. Furthermore, aniron-channeling mechanism implies that the two proteins are ina complex or at least close enough to one another for such a“hand-off” to occur. Evidence of any interaction betweenFOX1 and FTR1 has yet to be obtained, however.

Iron uptake supported by FOX1 and FTR1 requires theirexpression at the plasma membrane. FOX1 was previouslydetected in the plasma membrane fraction from Chlamydomo-nas by immunoblotting (21); we used the FOX1 antibody foranalysis of the orientation of FOX1 in intact cells. The indirectimmunofluorescence data confirmed that FOX1 localizes atthe plasma membrane while demonstrating that the MCO do-main is exocytoplasmic. Identical results were obtained withrecombinant FOX1 expressed in HEK293E cells. FTR1 local-ization, topology, and orientation were also determined usinga series of (HA)2- and GFP-tagged versions of FTR1 heter-ologously expressed in these cells. The direct fluorescencefrom FTR1::GFP and the indirect immunofluorescence resultsdemonstrate FTR1 plasma membrane localization. Further-more, the topology of FTR1 illustrated in our model (Fig. 1) isconsistent with the indirect immunofluorescence patterns ob-served in unpermeabilized versus permeabilized cells, patternswhich matched those reported for Ftr1p (45); these patternsplace loops 4 and 6 outside and loop 3 and the carboxy termi-nus of both proteins in the cytoplasm. Although the Fe-traf-ficking DASE motif in loop 6 of Ftr1p is not strictly conservedin loop 6 of FTR1, we predict a comparable Fe3�-traffickingrole for the EPTD motif in this loop in the algal permease.Overall, the topologic analyses for FOX1 and FTR1 provide apicture of these two proteins in the algal plasma membranethat is consistent with the hypothesis that they function as aferroxidase and a permease in Fe uptake in this organism.

The in vitro spectral and kinetic analyses of rFOX1 clearlydefine this protein as an MCO with specificity toward Fe2�.The PPD and ferroxidase activities of FOX1 as determinedhere are comparable to those exhibited by Fet3p and hCp. Thekinetic data were limited, however, by the fact that 10% ofthe purified rFOX1 was isolated in a fully Cu-replete state.This limited Cu activation could be due to an imbalance be-tween the efficiency of rFOX1 protein production and ofrFOX1 Cu-loading in HEK293E cells; these cells do not nor-mally express hCp or hHp. Nonetheless, the data here clearlyshow that rFOX1 catalyzes Fe3� loading of Tf. After 3 h,FOX1 catalyzed the complete formation of monoferric Tf fromapo-Tf. Diferric Tf was not formed as might have been ex-pected; however, the assay was limited to the amount ofrFOX1 that could be used before interfering with the migra-tion of Tf on the urea gel, and thus, an increased concentrationof holo-rFOX1 may have given different results.

While the data presented here indicate the similarity be-tween the components of iron uptake in Chlamydomonas andS. cerevisiae, structural differences between FOX1 and Fet3pindicate that the iron-trafficking contributions of each ferroxi-dase to iron uptake may be different. A homology-based struc-tural model of FOX1 suggests that, like hCp, FOX1 is com-prised of six cupredoxin domains containing T1 Cu sites indomains 2, 4, and 6 and a trinuclear copper cluster betweendomains 1 and 6 (51). In all ferroxidases, at least one acidic

residue within hydrogen bonding distance of a His ligand at theT1 Cu site is required for the efficient outer-sphere electrontransfer that occurs from Fe2� to the T1 Cu(II) atom; theseacidic residues adjacent to T1 Cu sites confer Fe2� specificityto a ferroxidase (41–42, 47). In FOX1, E349, E727, and D1077reside close to the T1 Cu sites in domains 2, 4, and 6, respec-tively, suggesting that Fe2� binding and oxidation occur inde-pendently at each site (51). In contrast, Fet3p has a singlesubstrate oxidation site where E185 not only supports Fe2�

specificity and electron transfer to the T1 Cu atom but alsocontributes to Fe3� channeling to the DASE motif in Ftr1p(27, 45, 47). In the crystal structure of hCp, Lindley et al.observed that acidic residues close to each of the T1 Cu sitescollectively contribute to a negative charge distribution that isformed at the top surface of the molecule; these were sug-gested to be involved in Fe2� binding and oxidation (31). Thisnegative charge distribution is conserved in the FOX1 homol-ogy model and may provide a holding site for Fe3� that awaitsdissociation into solution or trafficking to FTR1 (46). Thus, thethree negatively charged cation binding sites apparent in theFOX1 model may cooperatively form a pool of Fe3� to whichFTR1 has access, rather than the permease obtaining Fe3�

from each site independently. Further analysis of FOX1 andFTR1 is certainly required to determine the precise mecha-nism of high-affinity iron uptake in this alga.

Nonetheless, the similarities between FOX1 and hCp makeChlamydomonas an excellent model for understanding iron-trafficking mechanisms that are applicable to higher eu-karyotes. If the charge distribution on FOX1 described aboveprovides an iron binding platform accessible to FTR1, it isappealing to infer from this how proteins in other organismsmight interact with a ferroxidase in order to traffic iron. Forinstance, in humans, both Fpn and Tf may extend motifs intothe negatively charged surface of Cp, thus coupling this en-zyme’s ferroxidase activity to the release of Fe2� from Fpn tothe binding of Fe3� by Tf. The halotolerant alga D. salinacould serve as a particularly useful iron-trafficking model sinceit expresses an iron uptake system involving a ferroxidase (D-Fox) and a transferrin (TTf). TTf is known to bind Fe3� and isfound in complex with D-Fox, but there has been no demon-stration that D-Fox is essential to Fe3� loading of TTf (13, 37).Clearly, mechanistic analyses of high-affinity Fe uptake in bothalgal systems will undoubtedly benefit our understanding ofiron-trafficking mechanisms in higher eukaryotes.

ACKNOWLEDGMENTS

We thank Sabeeha Merchant and Janette Kropat, University of Cali-fornia, Los Angeles, for providing reagents. We also acknowledge theassistance of the Confocal Microscope and Flow Cytometry Facility in theSchool of Medicine and Biomedical Sciences, University at Buffalo.

This research was supported by an award from the National Instituteof Diabetes and Digestive and Kidney Diseases, DK053820 (to D.J.K.).

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Analysis of the High-Afﬁnity Iron Uptake System at the … · located in the extracellular space. FTR1 contains 7 membrane-span-ning helices with loops 4 and 6 outside and loop