Characterization of iron uptake into mitochondria of Saccharomyces cerevisiae
Type of DegreeDissertation
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Iron is abundant in the environment in the oxidized ferric state. However this form is not bioavailable. Cells require soluble ferrous iron and therefore many reduction strategies have evolved. FRE5 encodes a putative iron reductase in Saccharomyces cerevisiae. The gene product is found in mitochondria where iron is converted into the essential cofactors heme and Fe-S. Overall this dissertation investigates the hypothesis that Fre5 reductase activity is required for optimal Fe utilization. I uncover iron related phenotypes in S. cerevisiae that lack the FRE5 gene (referred to throughout as FRE5 null or fre5Δ) including a heme defect compared with the isogenic wild type. This defect could be reversed by adding supplemental iron to the medium, suggesting Fre5 is involved in iron availability in yeast. In addition I found an H2O2 resistance phenotype for fre5Δ. I hypothesize that the Fre5 reductase activity creates redox cycling of iron that increases the potential for Fenton chemistry to explain the H2O2 resistance of this mutant. These phenotypes in fre5Δ are reversed by the introduction of FRE5 gene on a vector. However the overexpression of FRE5 in high copy number also resulted in iron related phenotypes. A reduction in total mitochondrial iron was observed in fre5Δ compared with wild type. FRE5 overexpression increased mitochondrial iron reductase activity compared to fre5∆. Based on statistical analysis of metal profiles in WT, fre5Δ and fre5Δ with FRE5 overexpression strain, I found that fre5Δ has a lower level of mitochondrial iron and magnesium and a higher level of mitochondrial copper and zinc compared to the overexpression strain. Fre reductases are required for both copper and iron transport at the plasma membrane so we compared copper and iron phenotypes in mitochondria. Ferrous iron is transported into mitochondria by Mrs3. Pic2 is a copper transporter in mitochondria but deletion of both PIC2 and MRS3 led to a severe respiratory growth defect independent of iron concentrations therefore we suggest that Mrs3 has a low affinity for copper. To investigate this overlap between copper and iron I studied the affect of copper on heme synthesis and found an overlap that results in a copper-induced heme defect that may be relevant to human health in copper overload disorders.