Use of ade1 & ade2 mutations for development of a versatile red/white color assay of amyloid ‐induced oxidative stress in Saccharomyces cerevisiae

Abstract Mutations in adenine biosynthesis pathway genes ADE1 & ADE2 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 vacuoles. In [PSI+] yeast, partial inactivation of the translation termination factor, Sup35 protein, due to its amyloid aggregation allows for read‐through of the ade1‐14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red color development in ade1 & ade2 mutant yeast requires reduced‐glutathione which helps in transport of the intermediate metabolite P‐ribosylaminoimidazole carboxylate (CAIR) into vacuoles that develops the red color. Here, we hypothesized that amyloid‐induced oxidative stress would deplete reduced‐glutathione levels and thus thwart the development of red color in ade1 or ade2 yeast. Indeed, when we over‐expressed amyloid forming human proteins TDP‐43, Aβ‐42 & Poly‐Gln‐103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast, yielded some white color colonies. Further, the white color eventually reverted back to red upon turning‐off the amyloid protein's expression. Also, the aggregate‐bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emula...
Source: Yeast - Category: Molecular Biology Authors: Tags: Research Article Source Type: research