In WT cells grown in low [Femedium], the iron regulon is partially activated. Imported Fe is delivered primarily to the mitochondria, where much of it is assembled into Fe/S clusters and heme centers. Some Fe is sent to vacuoles, where it resides in the HS Fe(III) state. When WT cells are grown in high [Femedium], Aft1 is down-regulated, shifting the burden of importing Fe to Aft1-independent pathways (curved blue arrows). In Aft1-1up cells, Aft1 is constitutively activating the iron regulon regardless of the concentration of Femedium. When grown in low [Femedium], more Fe is imported relative to WT cells. The majority is sent to vacuoles while much of the remainder is sent to mitochondria for heme and ISC biosynthesis. When Aft1-1up cells are grown in high [Femedium], the rate of import is increased, but the imported Fe is distributed similarly. The flow into mitochondria is favored because nanoparticle formation depresses the concentration of the precursor Fe species in the matrix that is both sensed for regulation and converted into nanoparticles. Mitochondria “feel” Fe-deficient, accelerating Fe import. In Yah1p-depleted cells grown at either low or high medium-Fe, nearly all imported Fe is delivered to mitochondria. This massive influx of Fe into the mitochondria depresses the concentration of cytosolic Fe that is sensed by the Aft1 regulatory system and by the vacuolar iron regulatory system (Fecyt). Due to this regulatory structure, these cells “feels” Fe deficient, leading to an increased import of Femedium and the efflux of Fe from the vacuole, as the regulatory system attempts to increase Fecyt.