Mitochondrial Iron-Sulfur Cluster Activity and Cytosolic Iron Regulate Iron Traffic in Saccharomyces cerevisiae

Abstract

An ordinary differential equation-based mathematical model was developed to describe trafficking and regulation of iron in growing fermenting budding yeast. Accordingly, environmental iron enters the cytosol and moves into mitochondria and vacuoles. Dilution caused by increasing cell volume is included. Four sites are regulated, including those in which iron is imported into the cytosol, mitochondria, and vacuoles, and the site at which vacuolar Fe(II) is oxidized to Fe(III). The objective of this study was to determine whether cytosolic iron (Fecyt) and/or a putative sulfur-based product of iron-sulfur cluster (ISC) activity was/were being sensed in regulation. The model assumes that the matrix of healthy mitochondria is anaerobic, and that in ISC mutants, O2 diffuses into the matrix where it reacts with nonheme high spin Fe(II) ions, oxidizing them to nanoparticles and generating reactive oxygen species. This reactivity causes a further decline in ISC/heme biosynthesis, which ultimately gives rise to the diseased state. The ordinary differential equations that define this model were numerically integrated, and concentrations of each component were plotted versus the concentration of iron in the growth medium and versus the rate of ISC/heme biosynthesis. Model parameters were optimized by fitting simulations to literature data. The model variant that assumed that both Fecyt and ISC biosynthesis activity were sensed in regulation mimicked observed behavior best. Such "dual sensing" probably arises in real cells because regulation involves assembly of an ISC on a cytosolic protein using Fecyt and a sulfur species generated in mitochondria during ISC biosynthesis and exported into the cytosol.

Keywords: Friedreich ataxia; Mossbauer spectroscopy; cellular regulation; mathematical modeling; metal homeostasis.

FIGURE 1.

Chemical model of iron trafficking and regulation in S. cerevisiae. Nutrient Fe^III citrate (N) becomes cytosolic Fe^II (C) as it enters the cell. C moves into the vacuole forming F2 (Fe^II), which oxidizes to Fe^III (F3) and converts into nanoparticles (VP). C also moves into mitochondria, forming FM (Fe^II), which is used to generate FS. This component symbolizes ISCs and heme centers. FS is inserted into respiratory complexes, which function to maintain an O₂-free environment in healthy mitochondria. Some O₂ that diffuses into the matrix reacts with FM to generate mitochondrial nanoparticles and ROS. Red dots indicate the four regulated sites.

FIGURE 2.

Iron regulation pathways in S. cerevisiae. ISC assembly in mitochondria is thought to generate a sulfur-based species called X-S that is exported from the organelle, possibly through Atm1. X-S and Fe_cyt combine in the cytosol to generate a Fe₂S₂ cluster bridged between two glutaredoxin monomers (red star). This reaction is proposed to be the origin of Dual regulation. In the Aft1/2 signaling pathway (purple symbols) and under iron-sufficient conditions, the cluster is passed to Aft1/2 (via Fra2), which prevents activation of the iron regulon in the nucleus. Under iron-deficient conditions, cluster-free monomeric Aft1/2 activates the iron regulon including the Fet3/Ftr1 importer on the plasma membrane. Cth2, Fet5, and Smf3 are also regulated to control vacuole iron levels. Less is known about Yap signaling pathway (red symbols). An ISC is likely built on an unknown protein and transferred eventually to Yap5. Cluster-bound Yap5 activates Ccc1, which imports cytosolic iron into the vacuoles.

FIGURE 3.

Simulated concentrations of iron components in S. cerevisiae at increasing concentrations of iron in the growth medium. The Dual-Reg variant was assumed. The trace of [C] has been multiplied by 5 for ease of viewing.

FIGURE 4.

Simulated concentrations of iron components in S. cerevisiae at increasing rates by which iron-sulfur clusters and heme centers are synthesized. The Dual-Reg model variant is assumed along with [N] = 40 μm. The diseased state is on the left; the healthy state is on the right.

FIGURE 5.

Selected plots from simulations. A, concentrations of mitochondrial iron species under anaerobic growth conditions assuming the Dual-Reg variant. In the diseased state, [FM] is ∼20-fold higher than [MP]. B, [F3] (dashed purple line, right axis) simulated by the C-Reg variant does not decline in the diseased state. [C] (green line, left axis) simulated by the FS-Reg variant increases to unrealistically high concentrations in the diseased state. C, import rates simulated by the C-Reg variant are invarient in the diseased state: no accumulation of iron in mitochondria. D, simulation of [F2] and [F3] by the Dual-Reg variant in CCC1-UP cells that are adenine-deficient. [F2] is subtantially higher than [F3] as observed.