(Received for publication, September 30, 1994; and in revised form, November 9, 1994)
From the
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 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
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
consumption but inhibited the iron-dependent increase in O
consumption. Anti-peptide antibodies directed against the
cytosolic domain of Fet3 had no effect on O
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.
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.
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 g for 30 min to remove
debris. Membrane fractions were obtained by centrifuging the
supernatant at 72,000
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.
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.
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 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
consumption
upon addition of ascorbate-reduced iron (Fig. 2). The increase
in O
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
consumption required the expression and activity of Fet3.
Iron-dependent O
consumption was not observed in either the fet3 disruption mutant (
FET3) or the original fet3 mutant. Transfection of these mutants with FET3 plasmids
restored both the iron-dependent O
consumption and high
affinity iron transport (data not shown). The rate of iron-dependent
O
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
consumption by 85 and 33%, respectively. This reduction in
ferroxidase activity closely parallels the reduction in high affinity
iron transport activity(6) . (
)
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
10
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
consumption was measured using an oxygen electrode. Reactions
were carried out at 25 °C. Basal rates of O
consumption
were measured, and iron-dependent O
consumption was
determined by the addition of 100 µM FeCl
. The slope of the line indicates loss of O
from the medium. Thus, the more negative the slope the higher the
rate of O
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 consumption with no effect on
iron-induced O
consumption, indicating that the
iron-induced increase in O
consumption cannot be mediated
by an iron-regulated mitochondrial signal (Fig. 2B). Furthermore, mitochondrial
respiration-deficient rho cells had little basal O
consumption but still showed an iron-induced increase in O
consumption.
Simultaneous with measurements of O consumption, we measured rates of Fe(II) oxidation. The
relationship of O
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
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
consumption was reduced to approximately 2:1. This low background
rate of O
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 consumption (Fig. 3), but treatment of spheroplasts with
trypsin resulted in a decrease in iron-induced O
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
10
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 -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
consumption (Fig. 5).
Antibodies to peptides A and B but not C resulted in a decrease in
iron-induced O
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
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 consumption in
spheroplasts. Effect of anti-peptide antibodies on
iron-dependent O
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
consumption. The effect of
antibody P16B was competed by the addition of 15 µg/ml P16B. For
comparison, the rate of iron-dependent O
consumption is
listed below each trace. Results are representative of at least two
independent experiments.
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 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 (
FET3, fet3), regulation (reduction
of mRNA by iron regulation or removal of glucose) or depletion of
copper) resulted in a decrease in iron-dependent O
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 consumption strongly
reduced iron-dependent O
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L25090[GenBank].