Data on the role of iba57p in free Fe2+ release and O2∙− generation in Saccharomyces cerevisiae

The related study has confirmed that in Saccharomyces cerevisiae, iba57 protein participates in maturation of the [2Fe–2S] cluster into the Rieske protein, which plays important roles in the conformation and functionality of mitochondrial supercomplexes III/IV in the electron transport chain (Sánchez et al., 2018) [1]. We determined in S. cerevisiae the effects of mutation in the IBA57 gene on reactive oxygen species (ROS) and iron homeostasis. Flow cytometry and confocal microscopy analyses showed an increased generation of ROS, correlated with free Fe2+ release in the IBA57 mutant yeast. Data obtained support that a dysfunction in the Rieske protein has close relationship between ROS generation and free Fe2+ content, and which is possible that free Fe2+ release mainly proceeds from [Fe–S] cluster-containing proteins.


Subject area
Biology More specific subject area

Cell biology
Type of data Graphs, figures How data was acquired ROS and Fe 2 þ determination by flow cytometry using a BD Accuri C6 Flow Cytometer (BD Biosciences) and observation by using a confocal microscope (Olympus FV1000).

Data format
Analyzed and images Experimental factors ROS and Fe 2 þ determination in S. cerevisiae cells using fluorescent probes.

Experimental features
Real-time quantification of ROS and Fe 2 þ in S. cerevisiae cells suspensions were determined by flow cytometry and cellular structures were co-localized by confocal microscopy.

Data accessibility
Data are provided with this article.

Value of the data
There is an established relation between IBA57 mutation and the Rieske protein maturation in S.
cerevisiae, which affects the electron transport chain functionality.
IBA57 mutation in S. cerevisiae is correlated with ROS generation and loss of iron homeostasis. This dataset provides new insights into the mechanism of ROS generation in S. cerevisiae, dependent of the ETC functionality.

Data
Treatments with 80 µM menadione in the Saccharomyces cerevisiae iba57Δ mutant caused significant impairment in its growth rate ( Fig. 1a-b). The levels of free Fe 2 þ even without oxidant were significantly incremented in a time-dependent fashion in cell suspensions of the iba57Δ mutant yeast (Fig. 1c). The iba57Δ mutant displayed a significant increment of superoxide radical (O 2 − ) generation with a dose-dependent of Fe 2 þ , determined by flow cytometry (Fig. 1d). The western blot assays showed that the Rieske protein (Rip1p) was absent in the rip1Δ mutant, and decreased expression level was found in the iba57Δ mutant (Fig. 1e). When extracts from cultures grown on YPD plus high Fe 2 þ concentration (20 µM) or menadione as ROS-inducer were used, the Rip1p expression increased significantly in the WT, but not in the iba57Δ mutant.
Microscopy analysis shows an increment in ROS generation, associated with release of free Fe 2 þ in the iba57Δ mutant (Fig. 2). Interestingly, the high-intensity fluorescence observed in the iba57Δ mutant, which exhibited a full dissipation of mitochondrial membrane potential was associated with loss of iron homeostasis in the yeast cells.

Yeast strains and growth conditions
Mutant strains iba57Δ, rip1Δ, and grx5Δ correspond to the haploid S. cerevisiae BY4741 (Mat a, his3Δ, leu2Δ0, met15Δ0, ura3Δ0) and its KanMX4 interruption gene (Open Biosystems). Growth tests were carried out as described [1].   generation areas are shown as red granules within the cells, and mitochondrial structures (Δp) are shown as cyan granules within the cells, using the Rho123 probe. Images of the cells were taken at 10× to 60× magnifications using a confocal microscope (Olympus FV1000).

Determination of Rip1p expression by Western blot in S. cerevisiae
Mitochondrial protein extracts 50 μg were separated by electrophoresis on SDS-PAGE gels, membranes for Western blot procedure were treated as described [1][2][3]. Bands intensity in films were quantified using the Image J software and data graphed as Rip1p expression intensity.