Background

The yeast Saccharomyces cerevisiae cells mutant for ADE1 or ADE2 genes (e.g., ade1∆, ade2∆, ade1-14, ade2-1), when grown on YPD (Yeast Peptone Dextrose) medium, accumulate red pigment in the vacuole which is an intermediate metabolite of adenine biosynthesis pathway (Sharma et al., 2003). The suppressible allele ade1-14 which contains a premature stop codon, has been widely used to score for [PSI⁺] prion state of the translation termination factor Sup35 protein. In a [psi^-] yeast, the Sup35p remains soluble and functional, therefore translation is terminated efficiently at the premature stop codon of the ade1-14 allele leading to synthesis of truncated and non-functional Ade1 protein. Thus, the adenine biosynthesis cascade remains incomplete leading to accumulation of intermediate metabolite yielding the red phenotype of the [psi^-] yeast. In contrast, in a [PSI⁺] yeast, the Sup35p is aggregated and partially inactivated, thereby causing read-through of the premature non-sense codon of the ade1-14 allele that leads to functional Ade1 protein synthesis. Thus, adenine biosynthesis pathway is completed and no red intermediate metabolite is accumulated, consequently giving the [PSI⁺] cells, a white phenotype (Chernoff et al., 1993). In the view that the red color development from this adenine biosynthesis intermediate metabolite requires presence of reduced glutathione(Sharma et al., 2003), we reasoned that presence of oxidative stress which would oxidize glutathione, would also cause white color conversion alike to the white phenotype of the [PSI⁺] yeast. Additionally, it is known that the aggregations of several amyloid proteins cause cellular oxidative stress, therefore we attempted to develop a red/white reporter color assay for amyloid-induced oxidative stress in the ade1/ade2 mutant yeast background similar to the widely used red/white assay for the [psi^-] to [PSI⁺] conversion (Bharathi et al., 2016). We present here red/white color switch assay using two amyloid-like proteins, TAR DNA binding protein 43 (TDP-43) and Fused in Sarcoma (FUS), both of which are implicated in the pathogenesis of the motor neuron disease, Amyotrophic Lateral Sclerosis (ALS) (Rossi et al., 2016). Such simplistic color assay for amyloid-induced oxidative stress in yeast has never been reported previous to our Bharathi et al., 2016 manuscript and has the potential to be a highly useful methodology to study for amyloid protein-induced toxicity in yeast.