Abstract
Previous studies on Fe(II) uptake in Saccharomyces cerevisiae suggested the presence of two uptake systems with different affinities for this substrate. We demonstrate that the FET3 gene is required for high affinity uptake but not for the low affinity system. This requirement has enabled a characterization of the low affinity system. Low affinity uptake is time-, temperature-, and concentration-dependent and prefers Fe(II) over Fe(III) as substrate. We have isolated a new gene, FET4, that is required for low affinity uptake, and our results suggest that FET4 encodes an Fe(II) transporter protein. FET4's predicted amino acid sequence contains six potential transmembrane domains. Overexpressing FET4 increased low affinity uptake, whereas disrupting this gene eliminated that activity. In contrast, overexpressing FET4 decreased high affinity activity, while disrupting FET4 increased that activity. Therefore, the high affinity system may be regulated to compensate for alterations in low affinity activity. These analyses, and the analysis of the iron-dependent regulation of the plasma membrane Fe(III) reductase, demonstrate that the low affinity system is a biologically relevant mechanism of iron uptake in yeast. Furthermore, our results indicate that the high and low affinity systems are separate uptake pathways.
Highlights
Strate that the FET3 gene is required for high affinity Many studies have suggestetdhat a plasma membraneFe(1II)
The high up to 600 p ~ th, e low affinity activity appeared saturable in affinity system was saturated for substrate at a concentration both wild type and fet3 mutant cells and had similar apparent of 1p~ Fe(I1).An increase in uptakerate attributable to the low K, values (-30 PM)in thesetwo strains (Fig. 1B)
The observed affinity system was observed at concentrations greater than 5 plateau could have resulted fromincomplete reduction of p ~ T.hese results suggest that the activity of the low affinity Fe(II1) because of insufficient ascorbate in the assay, but a system can be estimated in wild type cells by subtracting the 10-fold increase in ascorbate concentration (10 mM) had no high affinity system's contribution (i.e.the uptakerate at1 p ~ ) effect on the apparent K, of the low affinity system (data not from the rateobserved at higher concentrations (Fig. l A, open shown).,we conclude that theplateau effect is caused triangles)
Summary
Examine the role of FET3 in low affinity uptake, we assayed mutation They indicate that the activity of the this activity in wild type and fet mutant cells over a broad low affinity system can be measured directly in fet mutant range of Fe(I1) concentrations. The high up to 600 p ~ th, e low affinity activity appeared saturable in affinity system was saturated for substrate at a concentration both wild type and fet mutant cells and had similar apparent of 1p~ Fe(I1).An increase in uptakerate attributable to the low K,,, values (-30 PM)in thesetwo strains (Fig. 1B). The estimated V,, of the low affinity system in the assayed in the fet mutant strainwere similar in magnitude to wild type strain was 143 fmol/min/106 cells, whilethe V, in the low afflnity activity observed in thewild type strain. All values represent the mean of two experiments each performed in duplicate
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