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

Abstract

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.