Biophysical Investigation of the Iron in Aft1-1up and Gal-YAH1 Saccharomyces cerevisiae
Abstract

Aft1p is a major iron regulator in budding yeast Saccharomyces cerevisiae. It indirectly senses cytosolic Fe status and responds by activating or repressing iron regulon genes. Aft1p within the Aft1-1up strain has a single amino acid mutation which causes it to constitutively activate iron regulon genes regardless of cellular Fe status. This leads to elevated Fe uptake under both low and high Fe growth conditions. Ferredoxin Yah1p is involved in Fe/S cluster assembly, and Aft1p-targeted iron regulon genes are also upregulated in Yah1p-depleted cells. In this study Mössbauer, EPR, and UV−vis spectroscopies were used to characterize the Fe distribution in Aft1-1up and Yah1p-depleted cells. Aft1-1up cells grown in low Fe medium contained more Fe than did WT cells. A basal level of Fe in both WT and Aft1-1up cells was located in mitochondria, primarily in the form of Fe/S clusters and heme centers. The additional Fe in Aft1-1up cells was present as mononuclear HS Fe(III) species. These species are in a nonmitochondrial location, assumed here to be vacuolar. Aft1-1up cells grown in high Fe medium contained far more Fe than found in WT cells. The extra Fe was present as HS Fe(III) ions, probably stored in vacuoles, and as Fe(III) phosphate nanoparticles, located in mitochondria. Yah1p-deficent cells also accumulated nanoparticles in their mitochondria, but they did not contain HS Fe(III) species. Results are interpreted by a proposed model involving three homeostatic regulatory systems, including the Aft1 system, a vacuolar iron regulatory system, and a mitochondrial Fe regulatory system.
Throughout this paper, we place “phosphate” in parentheses when referring to Fe(III) (phosphate) nanoparticles in Aft1-1up cells because we presume, but have not demonstrated, that they contain phosphate groups. Phosphate is associated with nanoparticles in Atm1p-deficient and Yfh1-deficient mitochondria. (10, 11, 13) Mössbauer spectra of Aft1-1up nanoparticles are nearly indistinguishable from those of Atm1p-deficient cells.
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