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...
The plasma-membrane of Saccharomyces cerevisiae contains high affinity permeases for Cu(I) and Fe(II). A low affinity Fe(II) permease has also been identified, designated Fet4p. A corresponding low affinity copper permease has not been characterized, although yeast cells that lack high affinity copper uptake do accumulate this metal ion. We demonstrate in the present study that Fet4p can function as a low affinity copper permease. Copper is a non-competitive inhibitor of (55)Fe uptake through Fet4p (K(i)=22 microM). Fet4p-dependent (67)Cu uptake was kinetically characterized, with K(m) and V(max) values of 35 microM and 8 pmol of copper/min per 10(6) cells respectively. A fet4-containing strain exhibited no saturable, low affinity copper uptake indicating that this uptake was attributable to Fet4p. Mutant forms of Fet4p that exhibited decreased efficiency in (55/59)Fe uptake were similarly compromised in (67)Cu uptake, indicating that similar amino acid residues in Fet4p contribute to both uptake processes. The copper taken into the cell by Fet4p was metabolized similarly to the copper taken into the cell by the high affinity permease, Ctr1p. This was shown by the Fet4p-dependence of copper activation of Fet3p, the copper oxidase that supports high affinity iron uptake in yeast. Also, copper-transported by Fet4p down-regulated the copper sensitive transcription factor, Mac1p. Whether supplied by Ctr1p or by Fet4p, an intracellular copper concentration of approx. 10 microM caused a 50% reduction in the transcriptional activity of Mac1p. The data suggest that the initial trafficking of newly arrived copper in the yeast cell is independent of the copper uptake pathway involved, and that this copper may be targeted first to a presumably small 'holding' pool prior to its partitioning within the cell.
The plasma-membrane of Saccharomycescerevisiae contains high affinity permeases for Cu(I) and Fe(II). A low affinity Fe(II) permease has also been identified, designated Fet4p. A corresponding low affinity copper permease has not been characterized, although yeast cells that lack high affinity copper uptake do accumulate this metal ion. We demonstrate in the present study that Fet4p can function as a low affinity copper permease. Copper is a non-competitive inhibitor of 55Fe uptake through Fet4p (Ki = 22µM). Fet4p-dependent 67Cu uptake was kinetically characterized, with Km and Vmax values of 35µM and 8pmol of copper/min per 106 cells respectively. A fet4-containing strain exhibited no saturable, low affinity copper uptake indicating that this uptake was attributable to Fet4p. Mutant forms of Fet4p that exhibited decreased efficiency in 55/59Fe uptake were similarly compromised in 67Cu uptake, indicating that similar amino acid residues in Fet4p contribute to both uptake processes. The copper taken into the cell by Fet4p was metabolized similarly to the copper taken into the cell by the high affinity permease, Ctr1p. This was shown by the Fet4p-dependence of copper activation of Fet3p, the copper oxidase that supports high affinity iron uptake in yeast. Also, copper-transported by Fet4p down-regulated the copper sensitive transcription factor, Mac1p. Whether supplied by Ctr1p or by Fet4p, an intracellular copper concentration of approx. 10µM caused a 50% reduction in the transcriptional activity of Mac1p. The data suggest that the initial trafficking of newly arrived copper in the yeast cell is independent of the copper uptake pathway involved, and that this copper may be targeted first to a presumably small ‘holding’pool prior to its partitioning within the cell.
Mannose 6-phosphate receptors (MPRs) deliver soluble acid hydrolases to the lysosome in higher eukaryotic cells. The two MPRs, the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/cation-independent MPR, carry out this process by binding with high affinity to mannose 6-phosphate residues found on the N-linked oligosaccharides of their ligands. To elucidate the key amino acids involved in conveying this carbohydrate specificity, site-directed mutagenesis studies were conducted on the extracytoplasmic domain of the bovine CD-MPR. Single amino acid substitutions of the residues that form the binding pocket were generated, and the mutant constructs were expressed in transiently transfected COS-1 cells. Following metabolic labeling, mutant CD-MPRs were tested for their ability to bind pentamannosyl phosphate-containing affinity columns. Of the eight amino acids mutated, four (Gln-66, Arg-111, Glu-133, and Tyr-143) were found to be essential for ligand binding. In addition, mutation of the single histidine residue, His-105, within the binding site diminished the binding of the receptor to ligand, but did not eliminate the ability of the CD-MPR to release ligand under acidic conditions.
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