VTC4 Polyphosphate Polymerase Knockout Increases Stress Resistance of Saccharomyces cerevisiae Cells.
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ABSTRACT:
SUBMITTER: Tomashevsky A
PROVIDER: S-EPMC8227513 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Vacuoles play major roles in the trafficking, storage, and homeostasis of metal ions in fungi and plants. In this study, 29 batches of vacuoles were isolated from Saccharomyces cerevisiae. Flow-through solutions (FTS) obtained by passing vacuolar extracts through a 10 kDa cut-off membrane were characterized for metal content using an anaerobic liquid chromatography system interfaced to an online ICP-MS. Nearly all iron, zinc, and manganese ions in these solutions were present as low-molecular-mass (LMM) complexes. Metal-detected peaks with masses between 500-1700 Da dominated; phosphorus-detected peaks generally comigrated. The distribution of metal:polyphosphate complexes was dominated by particular chain-lengths rather than a broad binomial distribution. Similarly treated synthetic FeIII polyphosphate complexes showed similar peaks. Treatment with a phosphatase disrupted the LMM metal-bound species in vacuolar FTSs. These results indicated metal:polyphosphate complexes 6-20 phosphate units in length and coordinated by 1-3 metals on average per chain. The speciation of iron in FTSs from iron-deficient cells was qualitatively similar, but intensities were lower. Under healthy conditions, nearly all copper ions in vacuolar FTSs were present as 1-2 species with masses between 4800-7800 Da. The absence of these high-mass peaks in vacuolar FTS from cup1Δ cells suggests that they were due to metallothionein, Cup1. Disrupting copper homeostasis increased the amount of LMM copper:polyphosphate complexes in vacuoles (masses between 1500-1700 Da). Potentially dangerous LMM copper species in the cytosol of metallothionein-deficient cells may traffic into vacuoles for sequestration and detoxification.
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Project description:Despite major progress in defining the functional roles of genes, a complete understanding of their influences is far from being realized, even in relatively simple organisms. A major milestone in this direction arose via the completion of the yeast Saccharomyces cerevisiae gene-knockout collection (YKOC), which has enabled high-throughput reverse genetics, phenotypic screenings and analyses of synthetic-genetic interactions1-3. Ensuing experimental work has also highlighted some inconsistencies and mistakes in the YKOC, or genome instability events that rebalance the effects of specific knockouts4-6, but a complete overview of these is lacking. The identification and analysis of genes that are required for maintaining genomic stability have traditionally relied on reporter assays and on the study of deletions of individual genes, but whole-genome-sequencing technologies now enable-in principle-the direct observation of genome instability globally and at scale. To exploit this opportunity, we sequenced the whole genomes of nearly all of the 4,732 strains comprising the homozygous diploid YKOC. Here, by extracting information on copy-number variation of tandem and interspersed repetitive DNA elements, we describe-for almost every single non-essential gene-the genomic alterations that are induced by its loss. Analysis of this dataset reveals genes that affect the maintenance of various genomic elements, highlights cross-talks between nuclear and mitochondrial genome stability, and shows how strains have genetically adapted to life in the absence of individual non-essential genes.
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