Biophysical Investigation of the Iron in Aft1-1^up 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 up-regulated 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 do 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 HS Fe(III) species. These species are in a non-mitochondrial 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.

Mössbauer, EPR, and Modeling Study of Iron Trafficking and Regulation in Δccc1 and CCC1-up Saccharomyces cerevisiae

“…8,25 Thus, we briefly summarize the Fe content of DY150 to provide a baseline against which Δccc1 and CCC1-up strains can be compared.…”

Compartmentalization of iron between mitochondria and the cytosol and its regulation

“…1). At the same time, levels of the mitochondrial iron transporter Mrs4 are increased, which eventually results in a redistribution of the net flux of cytosolic iron from the vacuole into the mitochondria where iron accumulates in form of insoluble ferric phosphate nano-particles (Belli et al, 2004;Foury and Talibi, 2001;Hausmann et al, 2008;Miao et al, 2011;Miao et al, 2009;Miao et al, 2008). This is an un-physiological event as the vacuole serves as the natural iron reservoir in fungi (Blaiseau, 2011 #3149).…”

Cellular Iron Distribution in Bacillus anthracis

“…However, investigations of the subcellular distribution of iron and iron-proteins in the literature are rare (11,12,23), in part due to the technical challenges involved. Knowledge of the identities and quantities of iron-proteins and of the amount of iron with which they are associated under different growth conditions is a key factor in systems analyses of iron homeostasis (20).…”