Characterization of the FET4 Protein of Yeast

Abstract

The low affinity Fe 2؉ uptake system of Saccharomyces cerevisiae requires the FET4 gene. In this report, we present evidence that FET4 encodes the Fe 2؉ transporter protein of this system. Antibodies prepared against FET4 detected two distinct proteins with molecular masses of 63 and 68 kDa. In vitro synthesis of FET4 suggested that the 68-kDa form is the primary translation product, and the 63-kDa form may be generated by proteolytic cleavage of the full-length protein. Consistent with its role as an Fe 2؉ transporter, FET4 is an integral membrane protein present in the plasma membrane. The level of FET4 closely correlated with uptake activity over a broad range of expression levels and is itself regulated by iron. Furthermore, mutations in FET4 can alter the kinetic properties of the low affinity uptake system, suggesting a direct interaction between FET4 and its Fe 2؉ substrate. Mutations affecting potential Fe 2؉ ligands located in the predicted transmembrane domains of FET4 significantly altered the apparent K m and/or V max of the low affinity system. These mutations may identify residues involved in Fe 2؉ binding during transport.In many organisms, iron uptake is a two-step process in which extracellular Fe 3ϩ is reduced to the more soluble Fe 2ϩ form by plasma membrane Fe 3ϩ reductases. The Fe 2ϩ product is then taken up by Fe 2ϩ -specific transport systems. This strategy of iron uptake is found in the yeast Saccharomyces cerevisiae (1-3), some bacteria (4, 5), other fungi (6, 7), and many plant species (8,9). Mammalian cells may use a similar mechanism for uptake of iron across the mucosal membrane of the intestine (10 -12) and for the uptake of free iron in blood plasma (13,14). Mammalian cells acquire most of their iron from transferrin. Fe 3ϩ -transferrin complexes bind to transferrin receptors on the cell surface. These receptor-ligand complexes are endocytosed to an endosomal compartment; the iron is dissociated and then transported across the endosomal membrane. Some studies have suggested that transferrin-delivered FRE2 genes (18,19). The Fe 2ϩ product is then taken up by either of two transport systems. One system has a high affinity for iron (apparent K m of 0.15 M), is necessary for iron-limited growth, and requires the products of the FET3 and FTR1 genes for activity (20 -23). The high affinity system is induced in iron-limited cells, and its components are transcriptionally regulated by the product of the AFT1 gene (24). AFT1 is an iron-responsive DNA binding protein that activates transcription of the target promoters to which it binds (25).Iron-replete yeast cells obtain iron through a second, low affinity uptake system with an apparent K m of 30 M. This system requires the FET4 gene for activity. Our previous results suggested that FET4 is the low affinity Fe 2ϩ transporter (26). First, overexpression of the FET4 gene increased activity of an iron uptake system that was indistinguishable from the low affinity system. Second, disruption of the FET4 gene eliminated low affinity uptake acti...

Combinatorial Control of Yeast FET4 Gene Expression by Iron, Zinc, and Oxygen

“…All mutations were confirmed by DNA sequencing. Strain BMW1 (zrt1::LEU2 zrt2::HIS3 fet4:TRP1) was constructed by ␥-deletion of the FET4 locus (19) in strain CM34 (32). The FET4 overexpression plasmid was pCB1 (19) and the corresponding empty vector was pRS316-GAL1 (33).…”

“…This is evident because mutational inactivation of the high affinity systems does not result in nonviable cells. Previous work identified a low affinity iron transporter encoded by the FET4 gene (19,20). The Fet4 protein has six predicted membrane spanning regions and is localized to the plasma membrane.…”

“…First, levels of Fet4 were ϳ4-fold higher in iron-deficient cells than in iron-replete cells (20). A recent microarray study (24) from our laboratory revealed that FET4 transcription was increased under zinc deficiency and that FET4 may be a Zap1 target gene.…”

CCC1 Is a Transporter That Mediates Vacuolar Iron Storage in Yeast

“…Overexpression of CCC1 was accomplished using a multicopy plasmid that contained CCC1 under its own native promoter as described in (12). Overexpression of FET4 was accomplished using a plasmid (pCB1) in which FET4 was placed under the control of the GAL1 promoter (13). DNA transformations of S. cerevisiae and Escherichia coli were performed using standard procedures (14,15).…”

A multicopper oxidase gene from Candida albicans: cloning, characterization and disruption b bThe EMBL accession number for the sequence reported in this paper is Y09329.

“…The two steps are the reduction of Fe(III) to Fe(II) by the surface ferric reductases Fre1p and Fre2p (Dancis et al, 1994 ;Georgatsou & Alexandraki, 1994), and the transport (and oxidation ?) by a low-or highaffinity Fe(II) transport system (Dix et al, 1997 ;Askwith et al, 1994). The high-affinity system has a K m for iron of 0n15 µM .…”