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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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Effect of Fe2+ treatment on S. cerevisiae ISC mutants.YPD-grown yeast cultures were harvested and suspended in YPD at 1×107 cell/mL, loaded with ROS-sensitive probes, and subjected to Fe2+ dose-response assays. Fluorescence intensity was determined by flow cytometry using DHE probe (A), and DHR123 (B). Yeast suspensions were treated with Fe2+ [FeSO4(NH4)] (10 µM) with and without the metal chelator 1,10-phenanthroline (1 mM), and the percentage of fluorescent cells was determined by flow cytometry (C). Values are the mean of three independent experiments with 20,000 cells counted by flow cytometry for each point. (D) Yeast survival using ethanol as a ROS-inducer. Yeast suspensions were treated with and without ethanol (10%) and with 1,10-phenanthroline (20 mM), and the percentage of surviving cells was determined using Trypan Blue staining; yeast counting was performed using a Neubauer chamber [20]. SEM values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants with the control WT strain (A–C), significant differences (p<0.05) are indicated by (*); or Tukey's post-hoc for (D), significant differences (p<0.05) with respect to the WT control are indicated with different lowercase letters.
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pone-0111585-g005: Effect of Fe2+ treatment on S. cerevisiae ISC mutants.YPD-grown yeast cultures were harvested and suspended in YPD at 1×107 cell/mL, loaded with ROS-sensitive probes, and subjected to Fe2+ dose-response assays. Fluorescence intensity was determined by flow cytometry using DHE probe (A), and DHR123 (B). Yeast suspensions were treated with Fe2+ [FeSO4(NH4)] (10 µM) with and without the metal chelator 1,10-phenanthroline (1 mM), and the percentage of fluorescent cells was determined by flow cytometry (C). Values are the mean of three independent experiments with 20,000 cells counted by flow cytometry for each point. (D) Yeast survival using ethanol as a ROS-inducer. Yeast suspensions were treated with and without ethanol (10%) and with 1,10-phenanthroline (20 mM), and the percentage of surviving cells was determined using Trypan Blue staining; yeast counting was performed using a Neubauer chamber [20]. SEM values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants with the control WT strain (A–C), significant differences (p<0.05) are indicated by (*); or Tukey's post-hoc for (D), significant differences (p<0.05) with respect to the WT control are indicated with different lowercase letters.

Mentions: To confirm that enhanced ROS generation was correlated with the free Fe2+ content, real-time quantification of ROS by flow cytometry in a medium containing sufficient iron was performed using Fe2+dose-response tests (Fig. 5). As expected, all yeast strains displayed a significant, dose-dependent increment in levels of fluorescence (indicating O2•− and H2O2 generation) when treated with increased concentrations of Fe2+. ssq1Δ and isa1Δ mutants showed the highest ROS generation; in contrast, grx5Δ mutants showed a moderate increment in ROS compared to the WT strain, but this increment was lower than that observed in ssq1Δ and isa1Δ mutants. For atx1Δ, mrs4Δ, and aft1Δ iron-homeostasis deficient mutants, both the DHE and DHR123 probes showed that the ROS content was similar to those observed in the WT and grx5Δ strains (Fig. 5a–b). These results indicated that at Fe2+concentrations below 10 µM, O2•− was the main species produced, while at concentrations of Fe2+ between10–20 µM, an increment of H2O2 was also observed (Fig. 5a–b). In addition, determination of ROS generation in YPD-grown cultures showed that the ROS increment was significantly decreased in all mutants by addition of the metal chelator phenanthroline, although this effect was not statistically significant in the WT cells (Fig. 5c). These results confirm the notion that free iron is responsible for an important proportion of the ROS generated in both ISC and defective iron-transport mutants.


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)

Effect of Fe2+ treatment on S. cerevisiae ISC mutants.YPD-grown yeast cultures were harvested and suspended in YPD at 1×107 cell/mL, loaded with ROS-sensitive probes, and subjected to Fe2+ dose-response assays. Fluorescence intensity was determined by flow cytometry using DHE probe (A), and DHR123 (B). Yeast suspensions were treated with Fe2+ [FeSO4(NH4)] (10 µM) with and without the metal chelator 1,10-phenanthroline (1 mM), and the percentage of fluorescent cells was determined by flow cytometry (C). Values are the mean of three independent experiments with 20,000 cells counted by flow cytometry for each point. (D) Yeast survival using ethanol as a ROS-inducer. Yeast suspensions were treated with and without ethanol (10%) and with 1,10-phenanthroline (20 mM), and the percentage of surviving cells was determined using Trypan Blue staining; yeast counting was performed using a Neubauer chamber [20]. SEM values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants with the control WT strain (A–C), significant differences (p<0.05) are indicated by (*); or Tukey's post-hoc for (D), significant differences (p<0.05) with respect to the WT control are indicated with different lowercase letters.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111585-g005: Effect of Fe2+ treatment on S. cerevisiae ISC mutants.YPD-grown yeast cultures were harvested and suspended in YPD at 1×107 cell/mL, loaded with ROS-sensitive probes, and subjected to Fe2+ dose-response assays. Fluorescence intensity was determined by flow cytometry using DHE probe (A), and DHR123 (B). Yeast suspensions were treated with Fe2+ [FeSO4(NH4)] (10 µM) with and without the metal chelator 1,10-phenanthroline (1 mM), and the percentage of fluorescent cells was determined by flow cytometry (C). Values are the mean of three independent experiments with 20,000 cells counted by flow cytometry for each point. (D) Yeast survival using ethanol as a ROS-inducer. Yeast suspensions were treated with and without ethanol (10%) and with 1,10-phenanthroline (20 mM), and the percentage of surviving cells was determined using Trypan Blue staining; yeast counting was performed using a Neubauer chamber [20]. SEM values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare mutants with the control WT strain (A–C), significant differences (p<0.05) are indicated by (*); or Tukey's post-hoc for (D), significant differences (p<0.05) with respect to the WT control are indicated with different lowercase letters.
Mentions: To confirm that enhanced ROS generation was correlated with the free Fe2+ content, real-time quantification of ROS by flow cytometry in a medium containing sufficient iron was performed using Fe2+dose-response tests (Fig. 5). As expected, all yeast strains displayed a significant, dose-dependent increment in levels of fluorescence (indicating O2•− and H2O2 generation) when treated with increased concentrations of Fe2+. ssq1Δ and isa1Δ mutants showed the highest ROS generation; in contrast, grx5Δ mutants showed a moderate increment in ROS compared to the WT strain, but this increment was lower than that observed in ssq1Δ and isa1Δ mutants. For atx1Δ, mrs4Δ, and aft1Δ iron-homeostasis deficient mutants, both the DHE and DHR123 probes showed that the ROS content was similar to those observed in the WT and grx5Δ strains (Fig. 5a–b). These results indicated that at Fe2+concentrations below 10 µM, O2•− was the main species produced, while at concentrations of Fe2+ between10–20 µM, an increment of H2O2 was also observed (Fig. 5a–b). In addition, determination of ROS generation in YPD-grown cultures showed that the ROS increment was significantly decreased in all mutants by addition of the metal chelator phenanthroline, although this effect was not statistically significant in the WT cells (Fig. 5c). These results confirm the notion that free iron is responsible for an important proportion of the ROS generated in both ISC and defective iron-transport mutants.

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
Related in: MedlinePlus