Genome-Wide Screen of Genes Required for Caffeine Tolerance in Fission Yeast

Abstract

BackgroundAn excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.Methodology/Principal FindingsWe searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H2O2-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner.Conclusions/SignificanceWith our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.

Genome-wide Screening of Regulators of Catalase Expression

“…Growth Curves-Yeast cells were grown in YES from an initial absorbance (A 600 ) of 0.1 with or without the addition of 2 mM H 2 O 2 using an assay based on automatic measurements of optical densities as previously described (14). For each strain, we calculated the "arrest after H 2 O 2 " by subtracting the minutes required to reach an A 600 of 0.5 between 2 mM H 2 O 2 -treated and untreated cultures.…”

Cells Lacking Pfh1, a Fission Yeast Homolog of Mammalian Frataxin Protein, Display Constitutive Activation of the Iron Starvation Response

“…Solid Sensitivity Assay-To analyze sensitivity to different agents on plates, S. pombe strains were grown and spotted as described (30). Serially diluted cells were spotted into minimal medium or YE5S plates containing or not the indicated concentrations of hydrogen peroxide (H 2 O 2 ), deferoxamine mesylate (Sigma), or iron (FeCl 3 ⅐6H 2 O, Sigma).…”

“…Gels were transferred to membranes, and they were probed with polyclonal anti-Pap1 antiserum and anti-Sty1 antiserum (as loading control) (36) Growth Curves-Yeast cells were grown in YE5S, and the cultures of NG60, NG147, and NG148 strains were grown anaerobically. The initial A 600 of the growth curves was 0.1, and recording of the growth curves was performed as described (30).…”

Calcineurin-Crz1 Signaling in Lower Eukaryotes

“…1 (30), caffeine (31), temperature (32), pH (33), blue light (34), the nature of the nitrogen source (35), and antifungal drugs (36). All of these signals increase the intracellular Ca 2ϩ concentration.…”

“…Further defects of ⌬crz1 mutants have been found during growth at alkaline pH (16,17) and at elevated temperature (16,32), as well as in resistance to ethanol (30), H 2 O 2 (67), and caffeine (31). It has been shown in C. neoformans that Crz1 is involved in survival under conditions of limited aeration and biofilm formation (68).…”