Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

The intrinsic ability of cells to adapt to a wide range of environmental conditions is a fundamental process required for survival. Potassium is the most abundant cation in living cells and is required for essential cellular processes, including the regulation of cell volume, pH and protein synthesis. Yeast cells can grow from low micromolar to molar potassium concentrations and utilize sophisticated control mechanisms to keep the internal potassium concentration in a viable range. We developed a mathematical model for Saccharomyces cerevisiae to explore the complex interplay between biophysical forces and molecular regulation facilitating potassium homeostasis. By using a novel inference method (“the reverse tracking algorithm”) we predicted and then verified experimentally that the main regulators under conditions of potassium starvation are proton fluxes responding to changes of potassium concentrations. In contrast to the prevailing view, we show that regulation of the main potassium transport systems (Trk1,2 and Nha1) in the plasma membrane is not sufficient to achieve homeostasis. Without potassium, all living cells will die; it has to be present in sufficient amounts for the proper function of most cell types. Disturbances in potassium levels in animal cells result in potentially fatal conditions and it is also an essential nutrient for plants and fungi. Cells have developed effective mechanisms for surviving under adverse environmental conditions of low external potassium. The question is how. Using the eukaryotic model organism, baker's yeast (Saccharomyces cerevisiae), we modeled how potassium homeostasis takes place. This is because, through mathematical modeling and experimentation, we found that the electro-chemical forces regulating potassium concentrations are coupled to proton fluxes, which respond to external conditions in order to maintain a viable potassium level within the cells. Our results challenge the current understanding of potassium homeostasis in baker's yeast, and could potentially be extended to other microorganisms, including non-conventional yeasts such as the pathogenic Candida albicans, and plant cells. In the future, the fundamental bases for this descriptive and predictive model might contribute to the development of new treatments for fungal infections, or developments in crop sciences.

A new YNB medium containing very low concentrations of alkali metal cations has been developed to carry out experiments to study potassium homoeostas

Potassium homeostasis is crucial for living cells. In the yeast Saccharomyces cerevisiae , the uptake of potassium is driven by the electrochemical gradient generated by the Pma1 H ⁺ -ATPase, and this process represents a major consumer of the gradient. We considered that any mutation resulting in an alteration of the electrochemical gradient could give rise to anomalous sensitivity to any cationic drug independently of its toxicity mechanism. Here, we describe a genomewide screen for mutants that present altered tolerance to hygromycin B, spermine, and tetramethylammonium. Two hundred twenty-six mutant strains displayed altered tolerance to all three drugs (202 hypersensitive and 24 hypertolerant), and more than 50% presented a strong or moderate growth defect at a limiting potassium concentration (1 mM). Functional groups such as protein kinases and phosphatases, intracellular trafficking, transcription, or cell cycle and DNA processing were enriched. Essentially, our screen has identified a substantial number of genes that were not previously described to play a direct or indirect role in potassium homeostasis. A subset of 27 representative mutants were selected and subjected to diverse biochemical tests that, in some cases, allowed us to postulate the basis for the observed phenotypes.

Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication. Human mitochondria contain a small double-stranded DNA genome (mtDNA) of only 16,569 base pairs (bp) that encodes 13 essential subunits of the oxidative phosphorylation system. Depletion of mtDNA and different types of mtDNA mutations cause mitochondrial disease, and are also implicated in biological ageing. For almost half a century it has been known that mtDNA contains ribonucleotides, but their identity and precise location are not known. The source of these ribonucleotides and their relevance for mitochondrial genome stability in healthy individuals and in patients with mitochondrial defects has not been addressed. We have used a combination of next-generation sequencing, and in vivo and in vitro biochemistry to address some of these questions. Our findings demonstrate that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of ribonucleotide excision repair pathways in human mitochondria. Our data also reveal that when dNTP pools are limiting, ribonucleotides serves as a second line of building blocks for DNA synthesis. We also demonstrate increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools, which may constitute a new pathogenic mechanism that affects mtDNA stability and impairs later rounds of mtDNA replication.

Summary: Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low‐molecular‐weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)₃ was also detected and direct transport assays demonstrate that As(GS)₃ does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.