Genetic and biochemical analysis of functional interactions between proteins involved in iron trafficking and its regulation in Saccharomyces cerevisiae
Singh, Arvinder Jit
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The high affinity iron uptake in Saccharomces cerevisiae involves reduction of Fe 3+ to Fe 2+ , a bioavailable form. Fre1p and Fre2p present in the plasma membrane are responsible for 99% of the plasma membrane ferric reductase activity. The expression of FRE1 is regulated by the iron and copper status of the cell. The iron regulation is mediated by the iron responsive transcription factor Aft1p and its paralog Aft2p while the copper regulation is mediated by Mac1p. This study demonstrates that Aft1p and Aft2p bind to the same cis-element on the FRE1 promoter. Aft1p dependent activation of FRE1 promoter activity requires Mac1p. Aft2p regulates FRE1 promoter independent of Mac1p. Aft1p interacts with Mac1p through its N-terminal domain. The iron reduced by the plasma membrane reductases is oxidized to Fe 3+ by ferroxidase, Fet3p before entering the cell via iron permease, Ftr1p. We have demonstrated an interaction between Fet3p and Ftr1p, the components of high affinity iron uptake in yeast. The carboxyl-terminal domains of these proteins interact and are required for trafficking of the complex to the plasma membrane. Chimeras of Fet3p and Fet5p demonstrate the requirement of specific multicopper-oxidase domain for the complex to be stable in the plasma membrane. Finally, we have demonstrated that Fre6p, a reductase homolog, is required for iron efflux from the vacuole. Smf3p mediated iron efflux from the vacuole is disrupted in the absence of Fre6p. Deletion of FRE6 results in increase in vacuolar iron content. Fre6p tagged with GFP is localized to the vacuolar membrane.