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

Abstract

Strains lacking and overexpressing the vacuolar iron (Fe) importer CCC1 were characterized using Mössbauer and EPR spectroscopies. Vacuolar Fe import is impeded in Δccc1 cells and enhanced in CCC1-up cells, causing vacuolar Fe in these strains to decline and accumulate, respectively, relative to WT cells. Cytosolic Fe levels should behave oppositely. The Fe content of Δccc1 cells grown under low-Fe conditions was similar to that in WT cells. Most Fe was mitochondrial with some nonheme high spin (NHHS) FeII present. Δccc1 cells grown with increasing Fe concentration in the medium contained less total Fe, less vacuolar HS FeIII, and more NHHS FeII than in comparable WT cells. As the Fe concentration in the growth medium increased, the concentration of HS FeIII in Δccc1 cells increased to just 60% of WT levels, while NHHS FeII increased to twice WT levels, suggesting that the NHHS FeII was cytosolic. Δccc1 cells suffered more oxidative damage than WT cells, suggesting that the accumulated NHHS FeII promoted Fenton chemistry. The Fe concentration in CCC1-up cells was higher than in WT cells; the extra Fe was present as NHHS FeII and FeIII and as FeIII oxyhydroxide nanoparticles. These cells contained less mitochondrial Fe and exhibited less ROS damage than Δccc1 cells. CCC1-up cells were adenine-deficient on minimal medium; supplementing with adenine caused a decline of NHHS FeII suggesting that some of the NHHS FeII that accumulated in these cells was associated with adenine deficiency rather than the overexpression of CCC1. A mathematical model was developed that simulated changes in Fe distributions. Simulations suggested that only a modest proportion of the observed NHHS FeII in both strains was the cytosolic form of Fe that is sensed by the Fe import regulatory system. The remainder is probably generated by the reduction of the vacuolar NHHS FeIII species.

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

“…Our goal was merely to explore essential aspects of the regulation at four key traffic sites. To do this, we used surrogate mathematical expressions-called "Reg functions"-to mimic regulatory behavior (6,37). Reg functions can be viewed as valves that dynamically adjust between closed and fully opened to smoothly regulate traffic flow through a site.…”

“…A NHHS Fe II species evident in Mössbauer spectra of adenine-deficient whole cells may also be located in vacuoles (7). Under adeninesufficient conditions in which the vacuolar iron importer Ccc1 is either absent or overproduced, high levels of Fe II are present (6).…”

“…They are devoid of iron under iron-deficient conditions. Vacuoles from cells grown on iron-sufficient medium contain high concentrations of a NHHS Fe III complex in which the coordinating ligands are closely related to polyphosphate (6). A NHHS Fe II species evident in Mössbauer spectra of adenine-deficient whole cells may also be located in vacuoles (7).…”

Responses of Saccharomyces cerevisiae Strains from Different Origins to Elevated Iron Concentrations

“…The utilization of biophysical spectroscopies has recently allowed the detailed examination of yeast iron contents in terms of growth phase, chemical form, and distribution within organelles (25,26,29). In the exponential growth phase, the intracellular yeast iron level remains roughly constant due to a balance between iron import and cellular division (26).…”

Iron toxicity in yeast: transcriptional regulation of the vacuolar iron importer Ccc1

“…When mitochondrial Fe–S cluster synthesis is diminished, cytosolic Aft1 and Aft2 are translocated to the nucleus and bind to target DNA resulting in transcription of the “low iron regulon”. While Aft1 and Aft2 are the major low-iron sensing transcription factors, mathematical modeling of yeast iron acquisition suggests that there may be other transcription factors that respond to changes in cytosolic iron levels (Cockrell et al 2014). …”