Cellular iron utilization is regulated by putative siderophore transporter FgSit1 not by free iron transporter in Fusarium graminearum

This report investigated FgSit1, which encodes a putative ferrichrome transporter of Fusarium graminearum. The identity of the deduced amino acid sequence of FgSit1 with the amino acid sequence of ScArn1p, an FC-Fe(3+) transporter of Saccharomyces cerevisiae, was 51%; both the growth defect related to the Deltafet3Deltaarn1-4 strain of S. cerevisiae in an iron-depleted condition and the FC-Fe(3+) uptake activity were recovered upon the introduction of FgSit1 into the Deltafet3Deltaarn1-4 strain. Although ScArn1p was found in the late endosomal compartment in S. cerevisiae, FgSit1 was found on the plasma membrane in S. cerevisiae; when FgSit1 was expressed exogenously in S. cerevisiae, it showed greater FC-Fe(3+) uptake activity than did ScArn1p. Additionally, in F. graminearum FC-Fe(3+) uptake activity in the Deltafgsit1 strain was found to be one-fourth that of the wild-type. However, Fe(3+) uptake activity in the Deltafgsit1 strain was 5-fold higher than that of wild-type; the gene expression of FgFtr1, a putative iron transporter, was induced by the deletion of FgSit1, but was not induced by the deletion of FgSit2. Taken together, these results strongly suggest that FgSit1 encodes a putative FC-Fe(3+) transporter that mediates FC-Fe(3+) uptake using a different mechanism than ScArn1p and plays an important role in the regulation of cellular iron availability in F. graminearum.     

Regulation of brain anandamide by acute administration of ethanol

FgFtr1 and FgFtr2 are putative iron permeases, and FgFet1 and FgFet2 are putative ferroxidases of Fusarium graminearum. They have high homologies with iron permease ScFtr1 and ferroxidase ScFet3 of Saccharomyces cerevisiae at the amino acid level. The genes encoding iron permease and ferroxidase were localized to the same chromosome in the manner of FgFtr1/FgFet1 and FgFtr2/FgFet2. The GFP (green fluorescent protein)-fused versions of FgFtr1 and FgFtr2 showed normal functions when compared with FgFtr1 and FgFtr2 in an S. cerevisiae system, and the cellular localizations of FgFtr1 and FgFtr2 in S. cerevisiae depended on the expression of their putative ferroxidase partners FgFet1 and FgFet2 respectively. Although FgFtr1 was found on the plasma membrane when FgFet1 and FgFtr1 were co-transformed in S. cerevisiae, most of the FgFtr1 was found in the endoplasmic reticulum compartment when co-expressed with FgFet2. Furthermore, FgFtr2 was found on the vacuolar membrane when FgFet2 was co-expressed. From the two-hybrid analysis, we confirmed the interaction of FgFtr1 and FgFet1, and the same result was found between FgFtr2 and FgFet2. Iron-uptake activity also depended on the existence of the respective partner. Finally, the FgFtr1 and FgFtr2 were found on the plasma and vacuolar membrane respectively, in F. graminearum. Taken together, these results strongly suggest that FgFtr1 and FgFtr2 from F. graminearum encode the iron permeases of the plasma membrane and vacuolar membrane respectively, and require their specific ferroxidases to carry out normal function. Furthermore, the present study suggests that the reductive iron-uptake system is conserved from yeast to filamentous fungi.     

Targeted suppression of the ferroxidase and iron trafficking activities of the multicopper oxidase Fet3p from<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The Fet3 protein in Saccharomyces cerevisiae is a multicopper oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe(2+) to Fe(3+); this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe(2+), one of which is essential also to the coupling of the ferroxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K(M) for Fe(2+) exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferroxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in (59)Fe uptake. For the Fet3p(E185D) mutant, K(M) for iron was 300-fold greater than the wild-type K(M), while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe(2+ )binding to Fet3p and to the subsequent trafficking of the Fe(3+) produced to Ftr1p.     

Fre1p Cu2+ reduction and Fet3p Cu1+ oxidation modulate copper toxicity in Saccharomyces cerevisiae

Fre1p is a metalloreductase in the yeast plasma membrane that is essential to uptake of environmental Cu2+ and Fe3+. Fet3p is a multicopper oxidase in this membrane essential for high affinity iron uptake. In the uptake of Fe3+, Fre1p produces Fe2+ that is a substrate for Fet3p; the Fe3+ produced by Fet3p is a ligand for the iron permease, Ftr1p. Deletion of FET3 leads to iron deficiency; this deletion also causes a copper sensitivity not seen in wild type. Deletion of FTR1 leads to copper sensitivity also. Production in the ftr1delta strain of an iron-uptake negative Ftr1p mutant, Ftr1p(RAGLA), suppressed this copper sensitivity. This Ftr1p mutant supported the plasma membrane targeting of active Fet3p that is blocked in the parental ftr1delta strain. A ferroxidase-negative Fet3p did not suppress the copper sensitivity in a fet3delta strain, although it supported the plasma membrane localization of the Fet3p.Ftr1p complex. Thus, loss of membrane-associated Fet3p oxidase activity correlated with copper sensitivity. Furthermore, in vitro Cu1+ was shown to be an excellent substrate for Fet3p. Last, the copper sensitivity of the fet3delta strain was suppressed by co-deletion of FRE1, suggesting that the cytotoxic species was Cu1+. In contrast, deletion of CTR1 or of FET4 did not suppress the copper sensitivity in the fet3delta strain; these genes encode the two major copper transporters in laboratory yeast strains. This result indicated that the apparent cuprous ion toxicity was not due to excess intracellular copper. These biochemical and physiologic results indicate that at least with respect to cuprous and ferrous ions, Fet3p can be considered a metallo-oxidase and appears to play an essential role in both iron and copper homeostasis in yeast. Its functional homologs, e.g. ceruloplasmin and hephaestin, could play a similar role in mammals.     

The yeast FET5 gene encodes a FET3 -related multicopper oxidase implicated in iron transport

The yeast FET3 gene encodes an integral membrane multicopper oxidase required for high-affinity iron uptake. The FET4 gene encodes an Fe(II) transporter required for low-affinity uptake. To identify other yeast genes involved in iron uptake, we isolated genes that could, when overexpressed, suppress the iron-limited growth defect of a fet3 fet4 mutant. The FET5 gene was isolated in this screen and it encodes a multicopper oxidase closely related to Fet3p. Several observations indicate that Fet5p plays a role analogous to Fet3p in iron transport. Suppression of the fet3 fet4 mutant phenotype by FET5 overexpression required the putative FTR1 transporter subunit of the high-affinity system. Fet5p is an integral membrane protein whose oxidase domain is located on the cell surface or within an intracellular compartment. Oxidase activity measured in cells with altered levels of FET5 expression suggested that Fet5p is a functional oxidase. FET5 overexpression increased the rate of iron uptake by a novel uptake system. Finally, FET5 mRNA levels are regulated by iron and are increased in cells grown in iron-limited media. These results suggest that Fet5p normally plays a role in the transport of iron. Received: 12 May 1997 / Accepted: 4 July 1997     

The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake

S. cerevisiae accumulate iron by a process requiring a ferrireductase and a ferrous transporter. We have isolated a mutant, fet3, defective for high affinity Fe(II) uptake. The wild-type FET3 gene was isolated by complementation of the mutant defect. Sequence analysis of the gene revealed the presence of an open reading frame coding for a protein with strong similarity to the family of blue multicopper oxidoreductases. Consistent with the role of copper in iron transport, growth of wild-type cells in copper-deficient media resulted in decreased ferrous iron transport. Addition of copper, but not other transition metals (manganese or zinc), to the assay media resulted in the recovery of Fe(II) transporter activity. We suggest that the catalytic activity of the Fet3 protein is required for cellular iron accumulation.     

The yeast FET5 gene encodes a FET3 -related multicopper oxidase implicated in iron transport

The yeast FET3 gene encodes an integral membrane multicopper oxidase required for high-affinity iron uptake. The FET4 gene encodes an Fe(II) transporter required for low-affinity uptake. To identify other yeast genes involved in iron uptake, we isolated genes that could, when overexpressed, suppress the iron-limited growth defect of a fet3 fet4 mutant. The FET5 gene was isolated in this screen and it encodes a multicopper oxidase closely related to Fet3p. Several observations indicate that Fet5p plays a role analogous to Fet3p in iron transport. Suppression of the fet3 fet4 mutant phenotype by FET5 overexpression required the putative FTR1 transporter subunit of the high-affinity system. Fet5p is an integral membrane protein whose oxidase domain is located on the cell surface or within an intracellular compartment. Oxidase activity measured in cells with altered levels of FET5 expression suggested that Fet5p is a functional oxidase. FET5 overexpression increased the rate of iron uptake by a novel uptake system. Finally, FET5 mRNA levels are regulated by iron and are increased in cells grown in iron-limited media. These results suggest that Fet5p normally plays a role in the transport of iron.     

An engineered bifunctional high affinity iron uptake protein in the yeast plasma membrane

High affinity iron uptake in fungi is supported by a plasma membrane protein complex that includes a multicopper ferroxidase enzyme and a ferric iron permease. In Saccharomyces cerevisiae, this complex is composed of the ferroxidase Fet3p and the permease Ftr1p. Fe(II) serves as substrate for Fe-uptake by being substrate for Fet3p; the resulting Fet3p-produced Fe(III) is then transported across the membrane via Ftr1p. A model of metabolite channeling of this Fe(III) is tested here by first constructing and kinetically characterizing in Fe-uptake two Fet3p-Ftr1p chimeras in which the multicopper oxidase/ferroxidase domain of Fet3p has been fused to the Ftr1p iron permease. Although the bifunctional chimeras are as kinetically efficient in Fe-uptake as is the wild type two-component system, they lack the adaptability and fidelity in Fe-uptake of the wild type. Specifically, Fe-uptake through the Fet3p, Ftr1p complex is insensitive to a potential Fe(III) trapping agent - citrate - whereas Fe-uptake via the chimeric proteins is competitively inhibited by this Fe(III) chelator. This inhibition does not appear to be due to scavenging Fet3p-produced Fe(III) that is in equilibrium with bulk solvent but could be due to leakiness to citrate found in the bifunctional but not the two-component system. The data are consistent with a channeling model of Fe-trafficking in the Fet3p, Ftr1p complex and suggest that in this system, Fet3p serves as a redox sieve that presents Fe(III) specifically for permeation through Ftr1p.     

The yeast multicopper oxidase Fet3p and the iron permease Ftr1p physically interact

High affinity iron uptake in yeast is carried out by a multicomponent system formed by the ferroxidase Fet3p and the iron permease Ftr1p. The currently accepted model predicts that Fet3p and Ftr1p are functionally associated, however, a structural interaction between these two proteins has not been proven yet. The methylotrophic yeast Pichia pastoris has been used to perform cross-linking studies aimed to demonstrate the existence of a Fet3p-Ftr1p complex. Cross-linking of membrane suspensions with the membrane-impermeable reagents DTSSP and BS(3) has evidenced the presence of a high molecular weight band with Fet3p oxidase activity. This band has been purified and subjected to N-terminal sequence analysis. Two sequences were found in the cross-linked species, one of which could be assigned to Fet3p and the other to Ftr1p. This is the first experimental demonstration that Fet3p and Ftr1p are physically associated.     

Evidence for iron channeling in the Fet3p-Ftr1p high-affinity iron uptake complex in the yeast plasma membrane

In high-affinity iron uptake in the yeast Saccharomyces cerevisiae, Fe(II) is oxidized to Fe(III) by the multicopper oxidase, Fet3p, and the Fe(III) produced is transported into the cell via the iron permease, Ftr1p. These two proteins are likely part of a heterodimeric or higher order complex in the yeast plasma membrane. We provide kinetic evidence that the Fet3p-produced Fe(III) is trafficked to Ftr1p for permeation by a classic metabolite channeling mechanism. We examine the (59)Fe uptake kinetics for a number of complexes containing mutant forms of both Fet3p and Ftr1p and demonstrate that a residue in one protein interacts with one in the other protein along the iron trafficking pathway as would be expected in a channeling process. We show that, as a result of some of these mutations, iron trafficking becomes sensitive to an added Fe(III) chelator that inhibits uptake in a strictly competitive manner. This inhibition is not strongly dependent on the chelator strength, however, suggesting that Fe(III) dissociation from the iron uptake complex, if it occurs, is kinetically slow relative to iron permeation. Metabolite channeling is a common feature of multifunctional enzymes. We constructed the analogous ferroxidase, permease chimera and demonstrate that it supports iron uptake with a kinetic pattern consistent with a channeling mechanism. By analogy to the Fe(III) trafficking that leads to the mineralization of the ferritin core, we propose that ferric iron channeling is a conserved feature of iron homeostasis in aerobic organisms.     

Cloning and functional characterization of the Rhizopus oryzae high affinity iron permease (rFTR1) gene

Rhizopus oryzae is the most common etiologic agent of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. Therefore, the high affinity iron permease (rFTR1) which encodes a protein required to scavenge iron from the environment, is highly likely to be a critical determinant of virulence for R. oryzae. We have cloned rFTR1 by using a PCR approach relying on degenerate primers designed from the conserved regions of Saccharomyces cerevisiae high affinity iron permease. Sequence analysis of a 2.0 kb EcoRI genomic clone revealed a single open reading frame of 1107 bp that lacked introns. The putative rFtr1p had significant homology to known fungal high affinity iron permeases from Candida albicans (46% identity) and S. cerevisiae (44% identity). In R. oryzae, rFTR1 was expressed in iron-depleted and not in iron-rich media. Finally, rFTR1 restored the ability of an ftr1 null mutant of S. cerevisiae to grow on iron-limited medium and to take up radiolabeled iron, whereas S. cerevisiae transformed with the empty vector did not. These data demonstrate that we have cloned the gene encoding a R. oryzae high affinity iron permease and the putative rFtr1p is involved in assimilation of iron from iron-depleted environments.     

The fission yeast ferric reductase gene frp1+ is required for ferric iron uptake and encodes a protein that is homologous to the gp91-phox subunit of the human NADPH phagocyte oxidoreductase.

We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have cloned frp1+, the wild-type allele of the mutant gene. frp1+ mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frp1+ promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FRE1 gene product and the gp91-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease.     

A new ferrous iron-uptake transporter, EfeU (YcdN), from Escherichia coli

Escherichia coli possesses multiple routes for iron uptake. Here we present EfeU (YcdN), a novel iron acquisition system of E. coli strain Nissle 1917. Laboratory strains of E. coli such as K12 lack a functional (efeU) ycdN gene caused by a frameshift mutation. EfeU, a member of the oxidase-dependent iron transporters (OFeT), is a homologue of the iron permease Ftr1p from yeast. The ycdN gene is part of the ycdNOB tricistronic operon which is expressed in response to iron deprivation in a Fur-dependent manner. Expression of efeU resulted in improved growth of an E. coli mutant lacking all known iron-uptake systems and mediated increased iron uptake into cells. Furthermore, the presence of other divalent metal cations did not impair growth of strains expressing efeU. The EfeU protein functioned as ferrous iron permease in proteoliposomes in vitro. Topology analysis indicated that EfeU is an integral cytoplasmic membrane protein exhibiting seven transmembrane helices. Two REXXE motifs within transmembrane helices of OFeT family members are implicated in iron translocation. Site-directed mutagenesis of each REGLE motif of EfeU diminished iron uptake in vivo and growth yield. In vitro the EfeU variant protein with an altered first REGLE motif was impaired in iron permeation, whereas activity of the EfeU variant with a mutation in the second motif was similar to the wild-type protein.     

Cloning of Pichia pastoris Fet3: insights into the high affinity iron uptake system

High-affinity iron uptake by yeast cells appears to require the presence of a complex formed on the plasma membrane by the multicopper oxidase Fet3 and the permease Ftr1 which work together to allow iron to enter safely inside the cell. The Pichia pastoris ferroxidase Fet3 has been cloned and it has been found to display high sequence similarity to other yeast multicopper oxidases, including all the predicted ligands for the catalytic copper atoms and for the iron substrate. P. pastoris appears to possess a high-affinity iron uptake system similar to that of S. cerevisiae, as far as regulation of expression is concerned. However, the P. pastoris high-affinity iron uptake system presents a K(m) value for iron almost ten times higher than that of S. cerevisiae, possibly to control iron fluxes over a wider range of concentrations of this metal, in order to avoid toxic iron overloading.