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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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Analyses of supercomplex formation in the mitochondrial ETC of S. cerevisiae ISC mutants.To analyze ETC supercomplex formation, mitochondrial suspensions isolated from yeast grown on glucose at the late exponential growth phase were solubilized and the proteins separated using blue native polyacrylamide gel electrophoresis (BN-PAGE) as described in the Materials and Methods[27]–[29]. The ETC mitochondrial supercomplexes and their molecular mass in kilodaltons are indicated to the right of the gel (A) as described elsewhere [27], [28], [30]. B) Analysis of band intensities of the supercomplexes observed in (A). C) Immunoblotting of mitochondrial extracts using anti-Rip1 antibody as the first antibody [4] and monoclonal anti-mouse IgG HRP conjugate as the second antibody; the densitometry analysis plot is shown below. Data correspond to three independent assays determining the band intensity by densitometry analysis using Image J software. Values are the mean of three independent mitochondrial isolations. SE values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare yeast strains, and significant differences (p<0.05) with respect to the WT control are indicated by (*).
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pone-0111585-g007: Analyses of supercomplex formation in the mitochondrial ETC of S. cerevisiae ISC mutants.To analyze ETC supercomplex formation, mitochondrial suspensions isolated from yeast grown on glucose at the late exponential growth phase were solubilized and the proteins separated using blue native polyacrylamide gel electrophoresis (BN-PAGE) as described in the Materials and Methods[27]–[29]. The ETC mitochondrial supercomplexes and their molecular mass in kilodaltons are indicated to the right of the gel (A) as described elsewhere [27], [28], [30]. B) Analysis of band intensities of the supercomplexes observed in (A). C) Immunoblotting of mitochondrial extracts using anti-Rip1 antibody as the first antibody [4] and monoclonal anti-mouse IgG HRP conjugate as the second antibody; the densitometry analysis plot is shown below. Data correspond to three independent assays determining the band intensity by densitometry analysis using Image J software. Values are the mean of three independent mitochondrial isolations. SE values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare yeast strains, and significant differences (p<0.05) with respect to the WT control are indicated by (*).

Mentions: Interestingly, the results of the BN-PAGE gels indicated that assembly of the III2IV2 and III2IV1 supercomplexes is dependent on the functionality of the ISC system. The band corresponding to the III2IV2 supercomplex was almost absent in mitochondria from ssq1Δ and isa1Δ mutants, but the band corresponding to the III2IV1 supercomplex was detected at low levels in all ISC mutants (Fig. 7a). Remarkably, in the densitometric analysis of the gels, the intensity of the bands corresponding to the III2IV2 supercomplex was significantly affected in ssq1Δ and isa1Δ mutants; the III2IV1 supercomplex was also diminished in ssq1Δ, isa1Δ, grx5Δ, and mrs4Δ mutants, whereas in atx1Δ mutant, a response similar to the WT was observed (Fig. 7b). In addition, densitometry data indicated that the content of dimeric complex V (i.e. the F1F0 ATPase) and its monomer were significantly increased in grx5Δ mutants, whereas the dimer of complex IV and II remained unaffected in ISC mutants but not in the iron-transporter deficient strains. Immunoblotting assays using anti-Rip1 antibody (Rieske protein) confirmed the supercomplexes formation and Raman spectrometry findings. In western blot analysis of mitochondrial protein extracts from the S. cerevisiae strains, the protein signal corresponding to Rip1p was absent from mitochondria from ssq1Δ mutant, and significantly diminished in the isa1Δ mutant, but in grx5Δ, mrs4Δ, and atx1Δ mutants similar levels to the WT was observed, while mrs4Δ mutant showed a stronger signal (Fig. 7c).


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)

Analyses of supercomplex formation in the mitochondrial ETC of S. cerevisiae ISC mutants.To analyze ETC supercomplex formation, mitochondrial suspensions isolated from yeast grown on glucose at the late exponential growth phase were solubilized and the proteins separated using blue native polyacrylamide gel electrophoresis (BN-PAGE) as described in the Materials and Methods[27]–[29]. The ETC mitochondrial supercomplexes and their molecular mass in kilodaltons are indicated to the right of the gel (A) as described elsewhere [27], [28], [30]. B) Analysis of band intensities of the supercomplexes observed in (A). C) Immunoblotting of mitochondrial extracts using anti-Rip1 antibody as the first antibody [4] and monoclonal anti-mouse IgG HRP conjugate as the second antibody; the densitometry analysis plot is shown below. Data correspond to three independent assays determining the band intensity by densitometry analysis using Image J software. Values are the mean of three independent mitochondrial isolations. SE values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare yeast strains, and significant differences (p<0.05) with respect to the WT control are indicated by (*).
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Related In: Results  -  Collection

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

pone-0111585-g007: Analyses of supercomplex formation in the mitochondrial ETC of S. cerevisiae ISC mutants.To analyze ETC supercomplex formation, mitochondrial suspensions isolated from yeast grown on glucose at the late exponential growth phase were solubilized and the proteins separated using blue native polyacrylamide gel electrophoresis (BN-PAGE) as described in the Materials and Methods[27]–[29]. The ETC mitochondrial supercomplexes and their molecular mass in kilodaltons are indicated to the right of the gel (A) as described elsewhere [27], [28], [30]. B) Analysis of band intensities of the supercomplexes observed in (A). C) Immunoblotting of mitochondrial extracts using anti-Rip1 antibody as the first antibody [4] and monoclonal anti-mouse IgG HRP conjugate as the second antibody; the densitometry analysis plot is shown below. Data correspond to three independent assays determining the band intensity by densitometry analysis using Image J software. Values are the mean of three independent mitochondrial isolations. SE values are indicated as bars (n = 3), one-way ANOVA with Bonferroni's post-hoc test was used to compare yeast strains, and significant differences (p<0.05) with respect to the WT control are indicated by (*).
Mentions: Interestingly, the results of the BN-PAGE gels indicated that assembly of the III2IV2 and III2IV1 supercomplexes is dependent on the functionality of the ISC system. The band corresponding to the III2IV2 supercomplex was almost absent in mitochondria from ssq1Δ and isa1Δ mutants, but the band corresponding to the III2IV1 supercomplex was detected at low levels in all ISC mutants (Fig. 7a). Remarkably, in the densitometric analysis of the gels, the intensity of the bands corresponding to the III2IV2 supercomplex was significantly affected in ssq1Δ and isa1Δ mutants; the III2IV1 supercomplex was also diminished in ssq1Δ, isa1Δ, grx5Δ, and mrs4Δ mutants, whereas in atx1Δ mutant, a response similar to the WT was observed (Fig. 7b). In addition, densitometry data indicated that the content of dimeric complex V (i.e. the F1F0 ATPase) and its monomer were significantly increased in grx5Δ mutants, whereas the dimer of complex IV and II remained unaffected in ISC mutants but not in the iron-transporter deficient strains. Immunoblotting assays using anti-Rip1 antibody (Rieske protein) confirmed the supercomplexes formation and Raman spectrometry findings. In western blot analysis of mitochondrial protein extracts from the S. cerevisiae strains, the protein signal corresponding to Rip1p was absent from mitochondria from ssq1Δ mutant, and significantly diminished in the isa1Δ mutant, but in grx5Δ, mrs4Δ, and atx1Δ mutants similar levels to the WT was observed, while mrs4Δ mutant showed a stronger signal (Fig. 7c).

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