Iron Trafficking in the Cytosol and Vacuoles of Saccharomyces cerevisiae

feorganism to study eukaryotic iron metabolism. Much Fe enters the cytosol and shuttles to the mitochondria for heme and iron-sulfur clusters biosynthesis. Little is known about cytosolic iron. Circumstantial evidence suggests that cytosolic Fe is high-spin Fe^II but further evidence is required to validate this. Cytosolic Fe flows not only into mitochondria but also into vacuoles. They are acidic organelles that can store Fe under Fe-overload conditions. The endogenous ligand of vacuolar Fe is proposed to be polyphosphate, but no direct evidence has been shown. The main goal of this dissertation was to investigate the vacuolar and cytosolic Low-Molecular-Mass (LMM) iron complexes in S. cerevisiae. To characterize these complexes, vacuolar and cytosolic fractions were first isolated from whole cells of yeast under low and high iron. LMM complexes were contained in flow through solutions (FTSs) by passing vacuolar and cytosolic fractions through a 10 kDa membrane cut-off. These FTSs were then loaded onto a LC size exclusion column interfaced to an ICP-MS. The results of LMM metal complexes from vacuoles were described in Chapter II. 29 batches of vacuoles were isolated from S.cerevisiae. Since ICP-MS can detect multiple elements simultaneously, I found that nearly all iron, zinc, and manganese ions in the vacuolar FTSs were present as LMM complexes. Phosphorus-detected peaks generally comigrated with metal-detected speaks at 500 – 1700 Da, thus suggesting polyphosphate is the common ligand for metals stored in the vacuoles. The binding of transition metals and vacuolar polyphosphate was further demonstrated by the treatment with phosphatase, which resulted in the loss of LMM metal-bound species. The second aim was to study the speciation of cytosolic iron (Chapter III). Iron with peaks range from 700 – 1300 Da dominated. Cytosol from cells of different genetic knockout strains were isolated and collected the FTSs to study the behavior of these Fe complexes. Interestingly, a pool of copper LMM complexes were detected in the cytosol at 300 – 1300 Da under high copper growth condition and disruption of copper homeostasis