M J, Vidhya Bharathi
(2016)
Use of ade1 mutation for development of a versatile ed/white color assay of amyloid-induced oxidative stress in Saccharomyces cerevisiae.
Masters thesis, Indian Institute of Technology Hyderabad.
Abstract
Mutations in adenine biosynthesis pathway gene ADE1 have been conventionally used to score for prion [PSI+] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [psi-] yeast appear red on YPD medium due to accumulation of a red intermediate compound in the vacuoles. In [PSI+] yeast, partial inactivation of the translation termination factor, Sup35 protein, due to its amyloid aggregation allows for read-through of the stop codon in the ade1-14 allele and yeast appears white as no red intermediate pigment is accumulated. It is known that the red color development in ade1 mutant yeast requires cytoplasmic levels of reduced glutathione which helps in transport of the intermediate metaboliteP ribosylaminoimidazole carboxylate (CAIR) into vacuoles that develops red color. Here, we hypothesized that amyloid-induced oxidative stress in yeast would deplete reduced glutathione levels and thus thwart the development of red color in the ade1yeast. Indeed, when we over-expressed amyloid forming human proteins TDP-43, Amyloid β-42 &Poly-Gln-103, the otherwise red ade1mutant yeast, turned to white color. Further, the color reverted back to red upon turning off the amyloid expression or by growth on media containing reducing agent thereby corroborating that the oxidative stress caused the white color. This red/white assay could also be emulated in an ade1yeast having erg6 gene deletion that increases the cell wall permeability. Thus, this model would be useful tool for drug-screening against general amyloid-induced oxidative stress & toxicity in yeast.
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