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Malfunctioning of the iron-sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes.

Gomez M, Pérez-Gallardo RV, Sánchez LA, Díaz-Pérez AL, Cortés-Rojo C, Meza Carmen V, Saavedra-Molina A, Lara-Romero J, Jiménez-Sandoval S, Rodríguez F, Rodríguez-Zavala JS, Campos-García J - PLoS ONE (2014)

Bottom Line: Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected.Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants.These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

View Article: PubMed Central - PubMed

Affiliation: Lab. Biotecnología Microbiana, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México.

ABSTRACT
Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

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Determination of Fe2+ release in S. cerevisiae ISC mutants.Yeast cultures were grown in liquid YPD medium, harvested and suspended in YPD at 1×107 cells/mL and charged with the fluorescent probe PGFL and incubated for 2 h at 30°C with light shaking in darkness. Then, yeast suspensions were treated with and without a stressor and incubated at 30°C with light shaking. Samples (100 µL) were collected, suspended in PBS buffer, and the fluorescence intensity in the cells was evaluated by real-time flow cytometry within 6 h. Free Fe2+ determination in yeast suspensions without a stressor (dashed lines) and with stressor treatment (continuous lines). A) H2O2 12 mM, B) menadione 80 µM, C) ethanol 10% v/v, D) Free Fe2+ determination at 6 h of treatment with ethanol (10%). Results represent the fluorescence intensity of yeast cells. Values are the mean of three independent experiments with 20,000 cells counted by flow cytometry per each point. SEM values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants to controls. Significant differences (p<0.05) are indicated with (*) for (A–C). Tukey's post-hoc test was used for (D), and significant differences (p<0.05) with respect to the WT control are indicated with different letters for the treatments; lowercase and uppercase letters indicate without and with ethanol treatment, respectively.
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pone-0111585-g003: Determination of Fe2+ release in S. cerevisiae ISC mutants.Yeast cultures were grown in liquid YPD medium, harvested and suspended in YPD at 1×107 cells/mL and charged with the fluorescent probe PGFL and incubated for 2 h at 30°C with light shaking in darkness. Then, yeast suspensions were treated with and without a stressor and incubated at 30°C with light shaking. Samples (100 µL) were collected, suspended in PBS buffer, and the fluorescence intensity in the cells was evaluated by real-time flow cytometry within 6 h. Free Fe2+ determination in yeast suspensions without a stressor (dashed lines) and with stressor treatment (continuous lines). A) H2O2 12 mM, B) menadione 80 µM, C) ethanol 10% v/v, D) Free Fe2+ determination at 6 h of treatment with ethanol (10%). Results represent the fluorescence intensity of yeast cells. Values are the mean of three independent experiments with 20,000 cells counted by flow cytometry per each point. SEM values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants to controls. Significant differences (p<0.05) are indicated with (*) for (A–C). Tukey's post-hoc test was used for (D), and significant differences (p<0.05) with respect to the WT control are indicated with different letters for the treatments; lowercase and uppercase letters indicate without and with ethanol treatment, respectively.

Mentions: To evaluate whether the increment in ROS following treatment with oxidant agents was correlated with the functionality of ISC assembly system and the level of the free iron pool, in vivo real-time free Fe2+ quantification was performed using flow cytometry. Cell suspensions of ssq1Δ, grx5Δ, and isa1Δ mutants showed a significant, time-dependent increment in levels of free Fe2+ compared to the WT strain, independent of the presence or absence of oxidants (Fig. 3). As expected, yeast suspensions treated with toxic concentrations of H2O2 (12.5 mM), menadione (80 µM), or ethanol (10%) exhibited higher iron- and time-dependent fluorescence increments than untreated yeast strains, leading to a 2–5 fold augmentation of the quantity of free Fe2+ in ISC mutants after 6 h of treatment. Notably, ssq1Δ and isa1Δ mutants showed higher levels of Fe2+ than grx5Δ mutants. In addition, Fe2+ release was higher in atx1Δ mutants than in aft1Δ and mrs4Δ mutants, and in these mutants, free Fe2+ was also increased when they were treated with oxidant agents (Fig. 3). However, aft1Δ and mrs4Δ mutants showed behavior intermediate between the WT strain and ISC mutants; these cells showed higher free Fe2+ than WT, but lower free Fe2+ than ISC and atx1Δ mutants (Fig. 3b–c). When the Fe2+ release data were analyzed following 6 h of treatment with ethanol, a significant increment in fluorescence values was observed for ssq1Δ, grx5Δ, and isa1Δ strains, but not for the iron-transport defective strains atx1Δ, mrs4Δ, and aft1Δ. However, in the absence of ethanol, only ssq1Δ and isa1Δ mutants showed significant differences in free Fe2+ release values (Fig. 3d). As mentioned above, with ethanol treatment, the free Fe2+ value was significantly increased in all strains, compared to untreated yeast cultures.


Malfunctioning of the iron-sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes.

Gomez M, Pérez-Gallardo RV, Sánchez LA, Díaz-Pérez AL, Cortés-Rojo C, Meza Carmen V, Saavedra-Molina A, Lara-Romero J, Jiménez-Sandoval S, Rodríguez F, Rodríguez-Zavala JS, Campos-García J - PLoS ONE (2014)

Determination of Fe2+ release in S. cerevisiae ISC mutants.Yeast cultures were grown in liquid YPD medium, harvested and suspended in YPD at 1×107 cells/mL and charged with the fluorescent probe PGFL and incubated for 2 h at 30°C with light shaking in darkness. Then, yeast suspensions were treated with and without a stressor and incubated at 30°C with light shaking. Samples (100 µL) were collected, suspended in PBS buffer, and the fluorescence intensity in the cells was evaluated by real-time flow cytometry within 6 h. Free Fe2+ determination in yeast suspensions without a stressor (dashed lines) and with stressor treatment (continuous lines). A) H2O2 12 mM, B) menadione 80 µM, C) ethanol 10% v/v, D) Free Fe2+ determination at 6 h of treatment with ethanol (10%). Results represent the fluorescence intensity of yeast cells. Values are the mean of three independent experiments with 20,000 cells counted by flow cytometry per each point. SEM values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants to controls. Significant differences (p<0.05) are indicated with (*) for (A–C). Tukey's post-hoc test was used for (D), and significant differences (p<0.05) with respect to the WT control are indicated with different letters for the treatments; lowercase and uppercase letters indicate without and with ethanol treatment, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214746&req=5

pone-0111585-g003: Determination of Fe2+ release in S. cerevisiae ISC mutants.Yeast cultures were grown in liquid YPD medium, harvested and suspended in YPD at 1×107 cells/mL and charged with the fluorescent probe PGFL and incubated for 2 h at 30°C with light shaking in darkness. Then, yeast suspensions were treated with and without a stressor and incubated at 30°C with light shaking. Samples (100 µL) were collected, suspended in PBS buffer, and the fluorescence intensity in the cells was evaluated by real-time flow cytometry within 6 h. Free Fe2+ determination in yeast suspensions without a stressor (dashed lines) and with stressor treatment (continuous lines). A) H2O2 12 mM, B) menadione 80 µM, C) ethanol 10% v/v, D) Free Fe2+ determination at 6 h of treatment with ethanol (10%). Results represent the fluorescence intensity of yeast cells. Values are the mean of three independent experiments with 20,000 cells counted by flow cytometry per each point. SEM values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants to controls. Significant differences (p<0.05) are indicated with (*) for (A–C). Tukey's post-hoc test was used for (D), and significant differences (p<0.05) with respect to the WT control are indicated with different letters for the treatments; lowercase and uppercase letters indicate without and with ethanol treatment, respectively.
Mentions: To evaluate whether the increment in ROS following treatment with oxidant agents was correlated with the functionality of ISC assembly system and the level of the free iron pool, in vivo real-time free Fe2+ quantification was performed using flow cytometry. Cell suspensions of ssq1Δ, grx5Δ, and isa1Δ mutants showed a significant, time-dependent increment in levels of free Fe2+ compared to the WT strain, independent of the presence or absence of oxidants (Fig. 3). As expected, yeast suspensions treated with toxic concentrations of H2O2 (12.5 mM), menadione (80 µM), or ethanol (10%) exhibited higher iron- and time-dependent fluorescence increments than untreated yeast strains, leading to a 2–5 fold augmentation of the quantity of free Fe2+ in ISC mutants after 6 h of treatment. Notably, ssq1Δ and isa1Δ mutants showed higher levels of Fe2+ than grx5Δ mutants. In addition, Fe2+ release was higher in atx1Δ mutants than in aft1Δ and mrs4Δ mutants, and in these mutants, free Fe2+ was also increased when they were treated with oxidant agents (Fig. 3). However, aft1Δ and mrs4Δ mutants showed behavior intermediate between the WT strain and ISC mutants; these cells showed higher free Fe2+ than WT, but lower free Fe2+ than ISC and atx1Δ mutants (Fig. 3b–c). When the Fe2+ release data were analyzed following 6 h of treatment with ethanol, a significant increment in fluorescence values was observed for ssq1Δ, grx5Δ, and isa1Δ strains, but not for the iron-transport defective strains atx1Δ, mrs4Δ, and aft1Δ. However, in the absence of ethanol, only ssq1Δ and isa1Δ mutants showed significant differences in free Fe2+ release values (Fig. 3d). As mentioned above, with ethanol treatment, the free Fe2+ value was significantly increased in all strains, compared to untreated yeast cultures.

Bottom Line: Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected.Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants.These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

View Article: PubMed Central - PubMed

Affiliation: Lab. Biotecnología Microbiana, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México.

ABSTRACT
Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

Show MeSH