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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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Respiration test of cell suspensions from S. cerevisiae ISC mutants.Mitochondrial functionality was evaluated in yeast suspensions obtained from cultures grown in liquid YPD medium, cells were harvested in the late exponential growth phase and re-suspended in MES-TEA buffer with glucose or with ethanol and incubated at 30°C with light shaking. Cells were used for oxygen consumption rate (OCR) measurements with a Clark-type oxygen electrode coupled to a biological oxygen monitor as described in the Materials and Methods. A–C) Basal OCR with glucose as substrate, D–F) with ethanol treatment. A and D) OCR under coupled state conditions, B and E) OCR under uncoupled state conditions using CCCP for uncoupling, C and F) OCR under complex III blocking conditions using antimycin A as an inhibitor. Values are the mean of three independent experiments. SE values are indicated as bars (n = 3), one-way ANOVA with Tukey's post-hoc test was used to compare yeast strains, significant differences (p<0.05) are indicated with different lowercase letters.
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pone-0111585-g008: Respiration test of cell suspensions from S. cerevisiae ISC mutants.Mitochondrial functionality was evaluated in yeast suspensions obtained from cultures grown in liquid YPD medium, cells were harvested in the late exponential growth phase and re-suspended in MES-TEA buffer with glucose or with ethanol and incubated at 30°C with light shaking. Cells were used for oxygen consumption rate (OCR) measurements with a Clark-type oxygen electrode coupled to a biological oxygen monitor as described in the Materials and Methods. A–C) Basal OCR with glucose as substrate, D–F) with ethanol treatment. A and D) OCR under coupled state conditions, B and E) OCR under uncoupled state conditions using CCCP for uncoupling, C and F) OCR under complex III blocking conditions using antimycin A as an inhibitor. Values are the mean of three independent experiments. SE values are indicated as bars (n = 3), one-way ANOVA with Tukey's post-hoc test was used to compare yeast strains, significant differences (p<0.05) are indicated with different lowercase letters.

Mentions: Raman spectroscopy observations of mitochondria isolated from S. cerevisiae under iron sufficiency indicated that the [Fe–S] cluster content was diminished in ssq1Δ and isa1Δ mutants, and native gels showed a clear alteration in the amount of the III2IV2 and III2IV1 supercomplexes. We therefore evaluated the in situ mitochondrial oxygen consumption rates (OCR) to determinate the functionality of the ETC in ISC mutants. In this case, atx1Δ and mrs4Δ mutants were included as controls, because aft1Δ cells showed decreased mitochondrial content and severely impaired function following isolation (data not shown). Respiration was completely abolished in both the coupled and uncoupled states in ssq1Δ and isa1Δ mutants, whereas in grx5Δ, atx1Δ, and mrs4Δ mutants, the OCR was partially decreased in comparison with the WT strain (Fig. 8a–b). Remarkably, ethanol treatment caused oxygen release in the assay chamber (i.e. negative values for OCR, Fig. 8d–f) instead of oxygen consumption in ISC mutants, except for grx5Δ and atx1Δ. This is suggestive of ROS production, since superoxide dismutase catalyzes the conversion of O2•− to O2 and H2O2, while catalase converts the latter species into H2O and O2. In S. cerevisiae mitochondria, complex III is the only site of ROS generation in the ETC, since it lacks a rotenone-sensitive complex I, the other site of ROS production in the ETC of superior eukaryotes [35]. We exposed the cells to antimycin A, an inhibitor of complex III, to further explore the possible role of complex III in ROS generation. In the absence of ethanol, a small amount of O2 generation was detected in all ISC mutants except in grx5Δ, whereas in the presence of ethanol, higher rates of O2 generation were detected in these mutants, and O2 consumption was fully inhibited in grx5Δ cells (Fig. 8f). Conversely, antimycin A-insensitive oxygen consumption was observed in WT, grx5Δ, and atx1Δ cells in the absence of ethanol, and only in WT and atx1Δ cells in the presence of ethanol. In contrast to the behavior of ISC mutants, mrs4Δ mutant, which shows affected iron homeostasis, displayed an OCR similar to that of the WT strain in the presence of glucose in the coupled state (Fig. 8a). To further corroborate the degree of mitochondrial dysfunction in the various strains, we evaluated mitochondrial membrane potential (Δp). In concordance with their inability to respire, ssq1Δ and isa1Δ mutants did not exhibit Δp (Fig. 9a). Furthermore, OCR correlated with the magnitude of Δp in WT, grx5Δ, atx1Δ, and mrs4Δ cells, since membrane potential was 2–4-fold higher in WT than in grx5Δ or atx1Δ and mrs4Δ mutants. The OCR in a coupled state followed a similar pattern (Fig. 8a and 9a), which in turn may be related to the decreased complex II signal observed for these mutants (Fig. 7a and 7b).


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)

Respiration test of cell suspensions from S. cerevisiae ISC mutants.Mitochondrial functionality was evaluated in yeast suspensions obtained from cultures grown in liquid YPD medium, cells were harvested in the late exponential growth phase and re-suspended in MES-TEA buffer with glucose or with ethanol and incubated at 30°C with light shaking. Cells were used for oxygen consumption rate (OCR) measurements with a Clark-type oxygen electrode coupled to a biological oxygen monitor as described in the Materials and Methods. A–C) Basal OCR with glucose as substrate, D–F) with ethanol treatment. A and D) OCR under coupled state conditions, B and E) OCR under uncoupled state conditions using CCCP for uncoupling, C and F) OCR under complex III blocking conditions using antimycin A as an inhibitor. Values are the mean of three independent experiments. SE values are indicated as bars (n = 3), one-way ANOVA with Tukey's post-hoc test was used to compare yeast strains, significant differences (p<0.05) are indicated with different lowercase letters.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111585-g008: Respiration test of cell suspensions from S. cerevisiae ISC mutants.Mitochondrial functionality was evaluated in yeast suspensions obtained from cultures grown in liquid YPD medium, cells were harvested in the late exponential growth phase and re-suspended in MES-TEA buffer with glucose or with ethanol and incubated at 30°C with light shaking. Cells were used for oxygen consumption rate (OCR) measurements with a Clark-type oxygen electrode coupled to a biological oxygen monitor as described in the Materials and Methods. A–C) Basal OCR with glucose as substrate, D–F) with ethanol treatment. A and D) OCR under coupled state conditions, B and E) OCR under uncoupled state conditions using CCCP for uncoupling, C and F) OCR under complex III blocking conditions using antimycin A as an inhibitor. Values are the mean of three independent experiments. SE values are indicated as bars (n = 3), one-way ANOVA with Tukey's post-hoc test was used to compare yeast strains, significant differences (p<0.05) are indicated with different lowercase letters.
Mentions: Raman spectroscopy observations of mitochondria isolated from S. cerevisiae under iron sufficiency indicated that the [Fe–S] cluster content was diminished in ssq1Δ and isa1Δ mutants, and native gels showed a clear alteration in the amount of the III2IV2 and III2IV1 supercomplexes. We therefore evaluated the in situ mitochondrial oxygen consumption rates (OCR) to determinate the functionality of the ETC in ISC mutants. In this case, atx1Δ and mrs4Δ mutants were included as controls, because aft1Δ cells showed decreased mitochondrial content and severely impaired function following isolation (data not shown). Respiration was completely abolished in both the coupled and uncoupled states in ssq1Δ and isa1Δ mutants, whereas in grx5Δ, atx1Δ, and mrs4Δ mutants, the OCR was partially decreased in comparison with the WT strain (Fig. 8a–b). Remarkably, ethanol treatment caused oxygen release in the assay chamber (i.e. negative values for OCR, Fig. 8d–f) instead of oxygen consumption in ISC mutants, except for grx5Δ and atx1Δ. This is suggestive of ROS production, since superoxide dismutase catalyzes the conversion of O2•− to O2 and H2O2, while catalase converts the latter species into H2O and O2. In S. cerevisiae mitochondria, complex III is the only site of ROS generation in the ETC, since it lacks a rotenone-sensitive complex I, the other site of ROS production in the ETC of superior eukaryotes [35]. We exposed the cells to antimycin A, an inhibitor of complex III, to further explore the possible role of complex III in ROS generation. In the absence of ethanol, a small amount of O2 generation was detected in all ISC mutants except in grx5Δ, whereas in the presence of ethanol, higher rates of O2 generation were detected in these mutants, and O2 consumption was fully inhibited in grx5Δ cells (Fig. 8f). Conversely, antimycin A-insensitive oxygen consumption was observed in WT, grx5Δ, and atx1Δ cells in the absence of ethanol, and only in WT and atx1Δ cells in the presence of ethanol. In contrast to the behavior of ISC mutants, mrs4Δ mutant, which shows affected iron homeostasis, displayed an OCR similar to that of the WT strain in the presence of glucose in the coupled state (Fig. 8a). To further corroborate the degree of mitochondrial dysfunction in the various strains, we evaluated mitochondrial membrane potential (Δp). In concordance with their inability to respire, ssq1Δ and isa1Δ mutants did not exhibit Δp (Fig. 9a). Furthermore, OCR correlated with the magnitude of Δp in WT, grx5Δ, atx1Δ, and mrs4Δ cells, since membrane potential was 2–4-fold higher in WT than in grx5Δ or atx1Δ and mrs4Δ mutants. The OCR in a coupled state followed a similar pattern (Fig. 8a and 9a), which in turn may be related to the decreased complex II signal observed for these mutants (Fig. 7a and 7b).

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