A Screen for Nigericin-Resistant Yeast Mutants Revealed Genes Controlling Mitochondrial Volume and Mitochondrial Cation Homeostasis

Little is known about the regulation of ion transport across the inner mitochondrial membrane in Saccharomyces cerevisiae. To approach this problem, we devised a screening procedure for facilitating the identification of proteins involved in mitochondrial ion homeostasis. Taking advantage of the growth inhibition of yeast cells by electroneutral K(+)/H(+) ionophore nigericin, we screened for genetic mutations that would render cells tolerant to this drug when grown on a nonfermentable carbon source and identified several candidate genes including MDM31, MDM32, NDI1, YMR088C (VBA1), CSR2, RSA1, YLR024C, and YNL136W (EAF7). Direct examination of intact cells by electron microscopy indicated that mutants lacking MDM31 and/or MDM32 genes contain dramatically enlarged, spherical mitochondria and that these morphological abnormalities can be alleviated by nigericin. Mitochondria isolated from the Δmdm31 and Δmdm32 mutants exhibited limited swelling in an isotonic solution of potassium acetate even in the presence of an exogenous K(+)/H(+) antiport. In addition, growth of the mutants was inhibited on ethanol-containing media in the presence of high concentrations of salts (KCl, NaCl, or MgSO(4)) and their mitochondria exhibited two- (Δmdm31 and Δmdm32) to threefold (Δmdm31Δmdm32) elevation in magnesium content. Taken together, these data indicate that Mdm31p and Mdm32p control mitochondrial morphology through regulation of mitochondrial cation homeostasis and the maintenance of proper matrix osmolarity

Identification of Yeast Mutants Exhibiting Altered Sensitivity to Valinomycin and Nigericin Demonstrate Pleiotropic Effects of Ionophores on Cellular Processes

<div><p>Ionophores such as valinomycin and nigericin are potent tools for studying the impact of ion perturbance on cellular functions. To obtain a broader picture about molecular components involved in mediating the effects of these drugs on yeast cells under respiratory growth conditions, we performed a screening of the haploid deletion mutant library covering the <i>Saccharomyces cerevisiae</i> nonessential genes. We identified nearly 130 genes whose absence leads either to resistance or to hypersensitivity to valinomycin and/or nigericin. The processes affected by their protein products range from mitochondrial functions through ribosome biogenesis and telomere maintenance to vacuolar biogenesis and stress response. Comparison of the results with independent screenings performed by our and other laboratories demonstrates that although mitochondria might represent the main target for both ionophores, cellular response to the drugs is very complex and involves an intricate network of proteins connecting mitochondria, vacuoles, and other membrane compartments.</p></div

Summary of the numbers of strains categorized based on the localization of the corresponding proteins (see also Fig 3). PM, plasma membrane; ER, endoplasmic reticulum, values in brackets include dual localizations.

<p>Summary of the numbers of strains categorized based on the localization of the corresponding proteins (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164175#pone.0164175.g003" target="_blank">Fig 3</a>). PM, plasma membrane; ER, endoplasmic reticulum, values in brackets include dual localizations.</p

All viable <i>erg</i> mutants exhibit changes in sensitivity to ionophores in both BY4741 and SCY325 genetic backgrounds.

<p>(A) Growth of the mutant strains was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164175#pone.0164175.g001" target="_blank">Fig 1</a> and in Materials and Methods. (B) Scheme of the pathway of conversion of squalene to ergosterol and phenotypes of the mutants in term of their sensitivity to valinomycin and nigericin, respectively. RD, respiratory deficient.</p

Summary of the number of strains in each ionophore-resistance-related category identified in this study.

<p>Summary of the number of strains in each ionophore-resistance-related category identified in this study.</p

Functional groups enriched in the screen for mutants with altered sensitivity to ionophores.

<p>Functional groups enriched in the screen for mutants with altered sensitivity to ionophores.</p

Examples of strains exhibiting different sensitivity to ionophores compared with the wild-type.

<p>Three microliters of the suspensions with the indicated concentrations of cells were spotted on the corresponding sSG media containing ionophores.</p

Organelle morphology in yeast cells treated with ionophores.

<p>The wild-type strain (BY4741) and the corresponding transformants were cultivated in sSG medium. After addition of nigericin (15 mg/l) or valinomycin (15 mg/l) the cells were observed by fluorescence microscopy. Mitochondria were visualized using DiOC₆ or mt-pHluorin (expressed from a plasmid) and vacuoles were stained with FM4-64, neutral red or Vba1-yEGFP3 (expressed from the plasmid pDF01).</p

Distribution of localization of the proteins encoded by the genes whose deletion results in altered sensitivity to valinomycin and/or nigericin (see Table 3 for details).

<p>Proteins with dual localization are counted twice (once for every localization) to reflect here every occurrence of the proteins in the cell.</p