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

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

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

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

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

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

Age- and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease

<p>Although exact causes of Parkinson disease (PD) remain enigmatic, mitochondrial dysfunction is increasingly appreciated as a key determinant of dopaminergic neuron susceptibility in both familial and sporadic PD. Two genes associated with recessive, early-onset PD encode the ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase PRKN/PARK2/Parkin, which together orchestrate a protective mitochondrial quality control (mitoQC) pathway. Upon stress, both enzymes cooperatively identify and decorate damaged mitochondria with phosphorylated poly-Ub (p-S65-Ub) chains. This specific label is subsequently recognized by autophagy receptors that further facilitate mitochondrial degradation in lysosomes (mitophagy). Here, we analyzed human post-mortem brain specimens and identified distinct pools of p-S65-Ub-positive structures that partially colocalized with markers of mitochondria, autophagy, lysosomes and/or granulovacuolar degeneration bodies. We further quantified levels and distribution of the ‘mitophagy tag’ in 2 large cohorts of brain samples from normal aging and Lewy body disease (LBD) cases using unbiased digital pathology. Somatic p-S65-Ub structures independently increased with age and disease in distinct brain regions and enhanced levels in LBD brain were age- and Braak tangle stage-dependent. Additionally, we observed significant correlations of p-S65-Ub with LBs and neurofibrillary tangle levels in disease. The degree of co-existing p-S65-Ub signals and pathological PD hallmarks increased in the pre-mature stage, but decreased in the late stage of LB or tangle aggregation. Altogether, our study provides further evidence for a potential pathogenic overlap among different forms of PD and suggests that p-S65-Ub can serve as a biomarker for mitochondrial damage in aging and disease.</p> <p><b>Abbreviations:</b> BLBD: brainstem predominant Lewy body disease; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DLB: dementia with Lewy bodies; DLBD: diffuse neocortical Lewy body disease; EOPD: early-onset Parkinson disease; GVB: granulovacuolar degeneration body; LB: Lewy body; LBD: Lewy body disease; mitoQC: mitochondrial quality control; nbM: nucleus basalis of Meynert; PD: Parkinson disease; PDD: Parkinson disease with dementia; p-S65-Ub: PINK1-phosphorylated serine 65 ubiquitin; SN: substantia nigra; TLBD: transitional Lewy body disease; Ub: ubiquitin </p