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Iron content of Saccharomyces cerevisiae cells grown under iron-deficient and iron-overload conditions.

ABSTRACT: Fermenting cells were grown under Fe-deficient and Fe-overload conditions, and their Fe contents were examined using biophysical spectroscopies. The high-affinity Fe import pathway was active only in Fe-deficient cells. Such cells contained ~150 ?M Fe, distributed primarily into nonheme high-spin (NHHS) Fe(II) species and mitochondrial Fe. Most NHHS Fe(II) was not located in mitochondria, and its function is unknown. Mitochondria isolated from Fe-deficient cells contained [Fe(4)S(4)](2+) clusters, low- and high-spin hemes, S = (1)/(2) [Fe(2)S(2)](+) clusters, NHHS Fe(II) species, and [Fe(2)S(2)](2+) clusters. The presence of [Fe(2)S(2)](2+) clusters was unprecedented; their presence in previous samples was obscured by the spectroscopic signature of Fe(III) nanoparticles, which are absent in Fe-deficient cells. Whether Fe-deficient cells were grown under fermenting or respirofermenting conditions had no effect on Fe content; such cells prioritized their use of Fe to essential forms devoid of nanoparticles and vacuolar Fe. The majority of Mn ions in wild-type yeast cells was electron paramagnetic resonance-active Mn(II) and not located in mitochondria or vacuoles. Fermenting cells grown on Fe-sufficient and Fe-overloaded medium contained 400-450 ?M Fe. In these cells, the concentration of nonmitochondrial NHHS Fe(II) declined 3-fold, relative to that in Fe-deficient cells, whereas the concentration of vacuolar NHHS Fe(III) increased to a limiting cellular concentration of ~300 ?M. Isolated mitochondria contained more NHHS Fe(II) ions and substantial amounts of Fe(III) nanoparticles. The Fe contents of cells grown with excessive Fe in the medium were similar over a 250-fold change in nutrient Fe levels. The ability to limit Fe import prevents cells from becoming overloaded with Fe.

SUBMITTER: Holmes-Hampton GP

PROVIDER: S-EPMC3914732 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Iron content of Saccharomyces cerevisiae cells grown under iron-deficient and iron-overload conditions.

Holmes-Hampton Gregory P GP Jhurry Nema D ND McCormick Sean P SP Lindahl Paul A PA

Biochemistry 20121219 1

Fermenting cells were grown under Fe-deficient and Fe-overload conditions, and their Fe contents were examined using biophysical spectroscopies. The high-affinity Fe import pathway was active only in Fe-deficient cells. Such cells contained ~150 μM Fe, distributed primarily into nonheme high-spin (NHHS) Fe(II) species and mitochondrial Fe. Most NHHS Fe(II) was not located in mitochondria, and its function is unknown. Mitochondria isolated from Fe-deficient cells contained [Fe(4)S(4)](2+) cluster ...[more]

PMID: 23253189

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Project description:Fermenting cells growing exponentially on rich (YPAD) medium underwent a transition to a slow-growing state as glucose levels declined and their metabolism shifted to respiration. During exponential growth, Fe import and cell-growth rates were matched, affording an approximately invariant cellular Fe concentration. During the transition period, the high-affinity Fe import rate declined slower than the cell-growth rate declined, causing Fe to accumulate, initially as Fe(III) oxyhydroxide nanoparticles but eventually as mitochondrial and vacuolar Fe. Once the cells had reached slow-growth mode, Fe import and cell-growth rates were again matched, and the cellular Fe concentration was again approximately invariant. Fermenting cells grown on minimal medium (MM) grew more slowly during the exponential phase and underwent a transition to a true stationary state as glucose levels declined. The Fe concentration of MM cells that just entered the stationary state was similar to that of YPAD cells, but MM cells continued to accumulate Fe in the stationary state. Fe initially accumulated as nanoparticles and high-spin Fe(II) species, but vacuolar Fe(III) also eventually accumulated. Surprisingly, Fe-packed 5-day-old MM cells suffered no more reactive oxygen species (ROS) damage than younger cells, suggesting that the Fe concentration alone does not accurately predict the extent of ROS damage. The mode and rate of growth at the time of harvesting dramatically affected cellular Fe content. A mathematical model of Fe metabolism in a growing cell was developed. The model included the import of Fe via a regulated high-affinity pathway and an unregulated low-affinity pathway. The import of Fe from the cytosol to vacuoles and mitochondria and nanoparticle formation were also included. The model captured essential trafficking behavior, demonstrating that cells regulate Fe import in accordance with their overall growth rate and that they misregulate Fe import when nanoparticles accumulate. The lack of regulation of Fe in yeast is perhaps unique compared to the tight regulation of other cellular metabolites. This phenomenon likely derives from the unique chemistry associated with Fe nanoparticle formation.

Project description:Thioredoxins are small proteins that regulate the cellular redox state, prevent oxidative damage, and play an active role in cell repair. Oxidative stress has proven to be of much relevance in biotechnological processes when the metabolism of Saccharomyces cerevisiae is mainly respiratory. During wine yeast starter production, active dry yeast cytosolic thioredoxin Trx2p is a key player in protecting metabolic enzymes from being oxidized by carbonylation. Less is known about the role of redox control during grape juice fermentation. A mutant strain that lacked both cytosolic thioredoxins, Trx1p and Trx2p, was tested for grape juice fermentation. Its growth and sugar consumption were greatly impaired, which indicates the system's relevance under fermentative conditions. A proteomic analysis indicated that deletion of the genes TRX1 and TRX2 caused a reduction in the ribosomal proteins and factors involved in translation elongation in addition to enzymes for glycolysis and amino acid biosynthesis. A metabolomic analysis of the trx1Δ trx2Δ mutant showed an increase in most proteogenic amino acids, phospholipids, and sphingolipids and higher fatty acid desaturase Ole1p content. Low glycolytic activity was behind the reduced growth and fermentative capacity of the thioredoxin deletion strain. All three hexokinases were downregulated in the mutant strain, but total hexokinase activity remained, probably due to posttranslational regulation. Pyruvate kinase Cdc19p presented an early level of aggregation in the trx1Δ trx2Δ mutant, which may contribute to a diminished hexose metabolism and trigger regulatory mechanisms that could influence the level of glycolytic enzymes.IMPORTANCE Oxidative stress is a common hazardous condition that cells have to face in their lifetime. Oxidative damage may diminish cell vitality and viability by reducing metabolism and eventually leading to aging and ultimate death. Wine yeast Saccharomyces cerevisiae also faces oxidative attack during its biotechnological uses. One of the main yeast antioxidant systems involves two small proteins called thioredoxins. When these two proteins are removed, wine yeast shows diminished growth, protein synthesis, and sugar metabolism under wine-making conditions, and amino acid and lipid metabolism are also affected. Altogether, our results indicate that proper redox regulation is a key factor for metabolic adaptations during grape juice fermentation.

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Iron content of Saccharomyces cerevisiae cells grown under iron-deficient and iron-overload conditions.

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Iron content of Saccharomyces cerevisiae cells grown under iron-deficient and iron-overload conditions.

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