title: Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid-induced oxidative stress in saccharomyces cerevisiae.
abstract: Mutations in adenine biosynthesis pathway genes ADE1 and 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 owing to accumulation of a red intermediate compound in vacuoles. In [PSI+] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing 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 colour development in ade1 and ade2 mutant yeast requires reduced-glutathione, which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid-forming human proteins TDP-43, Aβ-42 and Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour 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 emulated in ade2-1, ade2Δ, and ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, SangareddyMedak District, Telanagana, 502285, India.
authors.forename: Vidhya
authors.initials: V
authors.lastname: Bharathi
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, SangareddyMedak District, Telanagana, 502285, India.
authors.forename: Amandeep
authors.initials: A
authors.lastname: Girdhar
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, SangareddyMedak District, Telanagana, 502285, India.
authors.forename: Archana
authors.initials: A
authors.lastname: Prasad
authors.affiliation: Genomics and Molecular Medicine, CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi, 110007, India.
authors.forename: Meenkshi
authors.initials: M
authors.lastname: Verma
authors.affiliation: Department of Research, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi, 110060, India.
authors.forename: Vibha
authors.initials: V
authors.lastname: Taneja
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, SangareddyMedak District, Telanagana, 502285, India.
authors.forename: Basant K
authors.identifier: 0000-0001-9465-4803
authors.initials: BK
authors.lastname: Patel
mesh_terms: D000225:Adenine / Q000096:biosynthesis; D000682:Amyloid / Q000235:genetics* / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D053898:Biosynthetic Pathways / Q000235:genetics; D008856:Microscopy, Fluorescence; D009154:Mutation; D018384:Oxidative Stress; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
chemical_list: D000682:Amyloid; D029701:Saccharomyces cerevisiae Proteins; D000225:Adenine
keywords: Aβ-42; Rnq1; TDP-43; adenine biosynthesis; amyloid; oxidative stress; yeast prion [PSI+]