©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The FET3 Gene Product Required for High Affinity Iron Transport in Yeast Is a Cell Surface Ferroxidase (*)

(Received for publication, September 30, 1994; and in revised form, November 9, 1994)

Deepika M. De Silva (§) Candice C. Askwith David Eide (1) Jerry Kaplan (¶)

From the Division of Immunology and Cell Biology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132 Department of Biochemistry and Molecular Biology, University of Minnesota College of Medicine at Duluth, Duluth, Minnesota 55812

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The yeast FET3 gene is required for high affinity iron transport (Askwith, C., Eide, D., Ho, A. V., Bernard, P. S., Li, L., Davis-Kaplan, S., Sipe, D. M., and Kaplan, J. (1994) Cell 76, 403-410). The gene has extensive sequence homology to the family of multi-copper oxidases. In this communication, we demonstrate that the gene product is a cell surface ferroxidase involved in iron transport. Cells that contain a functional FET3 gene product exhibited an iron-dependent non-mitochondrial increase in oxygen consumption. Comparison of the rate of iron oxidation to O(2) consumption yielded an approximate value of 4:1, as predicted for a ferroxidase. Spheroplasts obtained from cells grown under low iron conditions also displayed an iron-dependent increase in O(2) consumption. Treatment of spheroplasts with trypsin or affinity-purified antibodies directed against the putative external ferroxidase domain of Fet3 had no effect on basal O(2) consumption but inhibited the iron-dependent increase in O(2) consumption. Anti-peptide antibodies directed against the cytosolic domain of Fet3 had no effect on O(2) consumption. These studies indicate that Fet3 is a plasma membrane ferroxidase required for high affinity iron uptake, in which the ferroxidase-containing domain is localized on the external cell surface.


INTRODUCTION

Saccharomyces cerevisiae have (at least) two distinct transport systems that mediate the uptake of elemental iron, a high affinity and a low affinity system(1) . Both systems recognize Fe(II), generated by surface reductases(2) , which are the products of the FRE1 and FRE2 genes(3, 4) . The high affinity transport system is specific for iron(1) . High affinity Fe(II) transport is regulated inversely by cellular iron content(1) , and iron transport is reduced upon copper depletion(5, 6) .

Analysis of mutants unable to grow in low iron media revealed a gene that was required for high affinity iron transport. Mutations in FET3 abrogate high affinity iron transport, and transcription of the gene is inversely regulated by cellular iron content(6) . The translated sequence indicates that the protein has only one potential membrane-spanning domain and is therefore probably not the transmembrane iron transporter. Further sequence analysis indicated that the gene had signature motifs and homology to the multi-copper oxidase family(6) . Members of this family are defined by their mechanism of action; 4 moles of substrate are oxidized with the concomitant reduction of 1 mole of molecular oxygen(7, 8) .

Ceruloplasmin is a member of the multi-copper oxidase family(7) . Copper-deficient animals have extremely low levels of ceruloplasmin and are unable to transport iron from intestinal mucosal cells and macrophages into plasma(9) . Injection of ceruloplasmin into copper-deficient animals resulted in the immediate appearance of plasma iron. These and other studies suggested that ceruloplasmin functioned as an Fe(II)-Fe(III) ferroxidase and that the ferroxidase activity was essential to remove iron from cell surface transport elements(9, 10) . Because of the homology between Fet3 and ceruloplasmin, we suggested that Fet3 functioned as a ferroxidase. We present data here that indicate that Fet3 is a ferroxidase and that the ferroxidase activity is required for high affinity iron transport. We also demonstrate that Fet3 is a type 1 membrane protein in which the ferroxidase-containing domain is located on the extracellular surface.


EXPERIMENTAL PROCEDURES

Yeast Strains and Culture Conditions

The stains employed in this study were derived from W303, DY150, and DY1457 as previously described (6) and the BJ5460-derived strain DY1632 (MAT A, pep4::HIS3, prb1Delta1.6R, His3Delta200, Leu2Delta1, Lys2-801, Trp1, Ura3-52, Can1). The cells were grown in YPD (1.0% yeast extract, 0.2% peptone, 2.0% glucose) or CM (2.0% glucose, 0.69% yeast nitrogen base, and 0.13% amino acid supplement). Either media was supplemented with 80 or 20 µM bathophenanthroline sulfonate, respectively, to make the media iron deficient(6) . Protease deficient cells were converted to spheroplasts by a 10-min incubation in 1.0 M Tris, pH 9.3, 10 mM dithiothreitol followed by the addition of oxalyticase (10 µg/ml, Enzogenetics, Corvallis, OR) in 1.2 M sorbitol, 20 mM potassium phosphate, pH 7.4, at 30 °C for 20 min. Spheroplasts were washed three times in 1.2 M sorbitol prior to use.

Crude membrane fractions were obtained from spheroplasts disrupted at 4 °C by Dounce homogenization in 0.6 M mannitol, 20 mM Hepes-KOH, pH 7.4. The homogenate was centrifuged at 3,000 times g for 30 min to remove debris. Membrane fractions were obtained by centrifuging the supernatant at 72,000 times g for 30 min at 4 °C. Pellets were resuspended in 20 mM Tris-HCl, pH 7.4, 1.0% Nonidet P-40.

Wild type cells were made rho by treatment with ethidium bromide according to standard procedures(11) . All chemicals were obtained from Sigma unless otherwise indicated.

Oxygen Measurements

Measurements of O(2) consumption were carried out in a 3.0-ml final volume at room temperature using a Clark-type electrode connected to a YSL oxygen monitor. Reaction mixtures contained 5 times 10^8 cells in LIM assay buffer containing 1.0 mM ascorbate but without phosphate or EDTA(12) . When spheroplasts were assayed, the buffer contained 1.2 M sorbitol to maintain osmolarity. Reactions were initiated by the addition of 100 µM FeCl(3) through the capillary bore of the vessel, and the O(2) consumed was continuously monitored. To measure iron oxidation rate, 20 µM ferrous ammonium sulfate was added to media in the presence and absence of cells. The disappearance of Fe(II) was monitored by removing samples at 1-min intervals and quenching the reaction by the addition of 1.0 mM ferrozine. The cells were centrifuged, and the absorbance of the supernatant at 564 nm ( = 27.9 mM cm) was determined. The rate of Fe(II) oxidation in the absence of cells was subtracted to give the final value.

Antibodies and Peptides

Peptides were synthesized based on the deduced amino acid sequence of Fet3 using methods previously described(13) . Cysteine was added to the carboxyl terminus of each peptide (see Fig. 1for amino acid sequence). Rabbit antibodies were raised against the peptides using the published methods (13) . The antibodies were affinity purified using peptides coupled to the Sulflo Link system after purification of IgG using immobilized protein G (Pierce). Horseradish peroxidase-conjugated goat anti-rabbit IgG was obtained from Cappel Laboratories (West Chester, PA). Dot blots were developed using 3,3`-diaminobenzedine(14) ; protein determinations were performed using the BCA reagent (Pierce) with bovine serum albumin as a standard.


Figure 1: The revised predicted sequence of the FET3 protein. Areas in bold indicate the putative signal sequence and membrane-spanning domains. The revised Fet3 protein has a predicted size of 72.3 kDa. Residues 121-141 and 483-494 represent the two multi-copper oxidase motifs. There are 13 potential N-linked glycosylation sites, all of which are located in the predicted extracellular region of the protein. Underlined regions correspond to the sequences of the three synthetic peptides used to generate antibodies. Peptide P16A spans residues 164-178, P16B spans residues 307-321, and P16C spans residues 590-604.




RESULTS

Our initial analysis of the deduced amino acid sequence of Fet3 suggested that it might be a type 2 membrane protein(6) . We subsequently determined that a sequencing error resulted in a predicted sequence with a truncation of the amino terminus of the protein. Reevaluation of the nucleotide sequence revealed that the reading frame extended 72 amino acids upstream and contained a putative signal sequence (15) (Fig. 1). The presence of this signal sequence suggests that the protein is a type 1 membrane protein with the potential ferroxidase domain located on the exterior cell surface.

The hypothesis that Fet3 is a ferroxidase led to the prediction that cells expressing Fet3 would demonstrate an iron dependence in oxygen consumption, with a stoichiometry of iron oxidation to O(2) consumption characteristic of the multi-copper oxidases. To test this hypothesis, wild type cells were grown on low iron media to induce the high affinity iron transport system. These cells had an increased rate of O(2) consumption upon addition of ascorbate-reduced iron (Fig. 2). The increase in O(2) consumption was not observed when iron was added to cell-free media, either fresh media or media conditioned by cell growth. Control experiments indicated that the iron-dependent O(2) consumption required the expression and activity of Fet3. Iron-dependent O(2) consumption was not observed in either the fet3 disruption mutant (DeltaFET3) or the original fet3 mutant. Transfection of these mutants with FET3 plasmids restored both the iron-dependent O(2) consumption and high affinity iron transport (data not shown). The rate of iron-dependent O(2) consumption was reduced in cells grown in high iron media in which FET3 mRNA concentration was dramatically lowered(6) . In high iron media, however, there is some residual high affinity iron transport and ferroxidase activity. Incubation of cells in glucose-free or copper-free media resulted in a reduction in iron-induced O(2) consumption by 85 and 33%, respectively. This reduction in ferroxidase activity closely parallels the reduction in high affinity iron transport activity(6) . (^1)


Figure 2: Iron-dependent O consumption in yeast correlates with Fet3 activity. A, cells were grown to exponential phase in either YPD bathophenanthroline sulfonate (low iron medium) or YPD (high iron medium). The cells were harvested, washed, and resuspended in 1/10 volume assay buffer. Reaction mixtures contained 5 times 10^8 cells and 1 mM ascorbate in LIM assay buffer containing 2% glucose and 0.25 µM copper (unless otherwise indicated). For this figure and all subsequent figures, O(2) consumption was measured using an oxygen electrode. Reactions were carried out at 25 °C. Basal rates of O(2) consumption were measured, and iron-dependent O(2) consumption was determined by the addition of 100 µM FeCl(3). The slope of the line indicates loss of O(2) from the medium. Thus, the more negative the slope the higher the rate of O(2) consumption by the cells. B, sodium cyanide was added to wild type induced cells to give a final concentration of 300 µM. Results are representative of at least three independent experiments.



The addition of cyanide blocked respiratory O(2) consumption with no effect on iron-induced O(2) consumption, indicating that the iron-induced increase in O(2) consumption cannot be mediated by an iron-regulated mitochondrial signal (Fig. 2B). Furthermore, mitochondrial respiration-deficient rho cells had little basal O(2) consumption but still showed an iron-induced increase in O(2) consumption.

Simultaneous with measurements of O(2) consumption, we measured rates of Fe(II) oxidation. The relationship of O(2) consumption to the rate of iron oxidation is shown in Table 1. In every instance in which iron uptake activity was expressed, the ratio of Fe(II) oxidation:O(2) consumption approached 4:1. Under conditions in which high affinity iron transport activity was decreased by removal of glucose or removal of copper, the stoichiometry of iron oxidation to O(2) consumption was reduced to approximately 2:1. This low background rate of O(2) consumption was inhibited by superoxide dismutase (data not shown), suggesting the formation of reduced oxygen species in the absence of Fet3 activity.



To determine if the ferroxidase domain of Fet3 is on the extracellular or intracellular surface of the membrane, experiments were preformed with spheroplasts. Removal of the yeast cell wall did not affect the iron-induced increase in O(2) consumption (Fig. 3), but treatment of spheroplasts with trypsin resulted in a decrease in iron-induced O(2) consumption. The effect of trypsin was abolished if trypsin was pre-incubated with soybean trypsin inhibitor prior to addition to spheroplasts. These results suggest an extracellular location of the ferroxidase domain.


Figure 3: Trypsin abolishes iron-dependent O consumption in spheroplasts. The protease-deficient strain DY1632 was grown in CM bathophenanthroline sulfonate to exponential phase and harvested, and spheroplasts were generated using oxalyticase as described under ``Experimental Procedures.'' Spheroplasts (5 times 10^8 cells) were preincubated with trypsin for 15 min at 25 °C, and residual trypsin was removed by washing spheroplasts in 1.2 M sorbitol prior to assaying. Concentrations of trypsin used in µg/ml are listed below each trace. Soybean trypsin inhibitor (50 µg/ml) was incubated with trypsin for 15 min at 25 °C before addition to spheroplasts. Results are representative of at least two independent experiments.



Further evidence that the ferroxidase domain is extracellular resulted from examination of the effect of anti-peptide antibodies on spheroplast ferroxidase activity. Peptides were synthesized corresponding to three different regions of Fet3. The regions were chosen based on the prediction of alpha-helical structure, in the expectation that they were on the exterior surface of the protein. Two of the peptides (P16A and P16B) are part of the putative ferroxidase domain, and the third (P16C) is on the cytosolic domain (see Fig. 1). Rabbit antibodies raised against the peptides were affinity purified using the specific peptide immobilized on columns. These antibodies did not detect the Fet3 protein if it was treated with SDS but did detect it after extraction of the native protein by non-ionic detergents. On a qualitative level using dot blots, each of the antisera has comparable reactivity (Fig. 4A). Treatment of spheroplasts with trypsin prior to detergent extraction and dot blot analysis resulted in the disappearance of reactivity to antibodies P16A and P16B but not P16C (Fig. 4B). Incubation of spheroplasts with anti-peptide antibodies A, B, or C had no effect on basal O(2) consumption (Fig. 5). Antibodies to peptides A and B but not C resulted in a decrease in iron-induced O(2) consumption by 20 and 64%, respectively. This decrease was prevented if the spheroplasts were simultaneously incubated with the antibody and the antigenic peptide. Addition of antibody P16C had no effect on iron-induced O(2) consumption.


Figure 4: Immunodetection of Fet3 in membrane fractions. 5 µg of a crude membrane fraction obtained from wild type cells grown in low iron medium were applied to nitrocellulose membranes. The individual dots were incubated with each of the indicated antibody. Preimmune serum was used at 1:100 dilution and affinity purified antibody at 1:1,000. For detection, blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG and developed using diaminobenzedine as a substrate.




Figure 5: Antibodies to the predicted extracellular region of Fet3 block iron-dependent O(2) consumption in spheroplasts. Effect of anti-peptide antibodies on iron-dependent O(2) consumption in spheroplasts is shown. Reaction conditions were exactly as described in Fig. 3except that 20 µg/ml antibody was added to spheroplasts where indicated, the mixtures were incubated at 25 °C for 15 min, and the spheroplasts were assayed for O(2) consumption. The effect of antibody P16B was competed by the addition of 15 µg/ml P16B. For comparison, the rate of iron-dependent O(2) consumption is listed below each trace. Results are representative of at least two independent experiments.




DISCUSSION

Our original description of the FET3 gene, required for high affinity iron transport, included a sequence analysis suggesting the presence of only one transmembrane domain. We suggested that the gene product was a type 2 membrane protein and was not likely to represent a transmembrane tranporter. A revised sequence analysis and the data presented here indicate that Fet3 is a type 1 membrane protein in which the putative ferroxidase-containing domain is extracellular. This conclusion is consistent with the findings in other members of the multi-copper oxidase family. All members of the family that have been localized are extracellular proteins, and the cDNA of not yet localized members contain leader sequences(16, 17) .

The multi-copper oxidases have two substrates, oxygen and a reducing substrate(7, 8) , and are the only enzymes known to reduce both atoms of molecular oxygen to water. This is accomplished by sequential oxidation of four molecules of the reduced substrates and simultaneous transfer of all four electrons to molecular oxygen. We reasoned that a study of iron-dependent oxygen consumption in induced yeast cells would yield information about the enzymatic activity of Fet3. Comparison of the rate of Fe(II) oxidation to O(2) consumption in induced cells gave a stoichiometry of approximately 4:1, consistent with the reduction of molecular oxygen to water. Conditions that reduced the level of high affinity iron transport (mutation (DeltaFET3, fet3), regulation (reduction of mRNA by iron regulation or removal of glucose) or depletion of copper) resulted in a decrease in iron-dependent O(2) consumption. The efficiency of Fet3 catalyzed Fe(II) oxidation, and the induction of this activity under conditions that induce the high affinity iron transport system lead us to conclude that the ferroxidase activity of Fet3 is directly associated with the high affinity uptake system.

The accessibility of proteases and antibodies to domains of Fet3 in the periplasmic space was used to determine the orientation of the protein in the plasma membrane. These agents that did not affect basal O(2) consumption strongly reduced iron-dependent O(2) consumption. The inability of the P16C antibody to inhibit the ferroxidase activity of spheroplasts indicates that this portion of Fet3 resides on the inaccessible cytoplasmic side of the membrane.

Taken together, these results demonstrate that Fet3 is a cell surface ferroxidase required for high affinity iron transport. Previously, we speculated that Fe(III) is reduced to Fe(II) by the surface ferrireductases and passes through the transmembrane transporter as Fe(II). On the cytoplasmic surface, Fe(II) is oxidized to Fe(III) by the ferroxidase activity of Fet3(5, 6) . Our current data, however, suggest that the Fet3 ferroxidase domain of the molecule is exposed on the cell surface. If Fet3 converts Fe(II) to Fe(III), this must occur either close to or in the transmembrane channel. Our working hypothesis is that Fet3 is part of the transmembrane transporter and that the transporter is a heteromultimer.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant NIDDK-5-30534. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L25090[GenBank].

§
Supported by National Institutes of Health Training Grant NIDDK-T32 DK07115.

To whom all correspondence should be addressed: Division of Immunology and Cell Biology, Dept. of Pathology, University of Utah, College of Medicine, Salt Lake City, UT 84132. Tel.: 801-581-7427; Fax: 801-581-4517.

(^1)
D. M. De Silva, C. C. Askwith, D. Eide, and J. Kaplan, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Dan Knauer for peptide synthesis.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.