Academic journal article Genetics

Hermes Transposon Mutagenesis Shows [URE3] Prion Pathology Prevented by a Ubiquitin-Targeting Protein: Evidence for Carbon/Nitrogen Assimilation Cross Talk and a Second Function for Ure2p in Saccharomyces Cerevisiae

Academic journal article Genetics

Hermes Transposon Mutagenesis Shows [URE3] Prion Pathology Prevented by a Ubiquitin-Targeting Protein: Evidence for Carbon/Nitrogen Assimilation Cross Talk and a Second Function for Ure2p in Saccharomyces Cerevisiae

Article excerpt

THE prions (infectious proteins) [URE3] and [PSI+] are amyloidoses of Ure2p and Sup35p, respectively, in Saccharomyces cerevisiae [reviewed in Liebman and Chernoff (2012), Wickner et al. (2015), and Saupe et al. (2016)], and are important models for the human prion and amyloid diseases (Kraus et al. 2013; Prusiner 2017). Ure2p is necessary for nitrogen catabolite repression (NCR), the shutoff of transcription of genes for the utilization of poor nitrogen sources when a good nitrogen source is available (Cooper 2002). In a [URE3] strain, most of the Ure2p is sequestered in amyloid filaments and so genes for assimilation of poor nitrogen sources (such as DAL5, encoding the allantoin transporter) are inappropriately derepressed, a result detected as an Ade+ phenotype in a strain with a DAL5 promoter driving transcription of the ADE2 gene. Sup35p is a subunit of the translation termination factor that is essential for growth (Frolova etal. 1994; Stansfield and Tuite 1994). [PSI+] cells have most (but not all) of their Sup35p tied up in the amyloid filaments, and therefore frequently read through termination codons.

A single prion protein with a single amino acid sequence can form any of a large number of prion variants or strains, with different biological properties due to different conformations of the protein in the different variants (Derkatch et al. 1996; Collinge and Clarke 2007). Each prion variant is rather stably propagated, implying that a mechanism exists for templating of protein conformation. The parallel in-register folded ß-sheet architecture known for several infectious yeast prion amyloids (Shewmaker et al. 2006; Baxa et al. 2007; Wickner et al. 2008; Gorkovskiy et al. 2014) naturally suggests a mechanism of conformational templating based on the favorable interactions of aligned identical polar or hydrophobic amino acid side chains (Wickner et al. 2007, 2015).

While a majority ofvariants of [URE3] or [PSI+] are highly toxic, or even lethal (McGlinchey et al. 2011), there are mild variants of each prion that are also in fact detrimental (Nakayashiki et al. 2005), but not severely so. Several antiprion systems have now been described in yeast, either preventing prions from arising (Chernoff et al. 1999) or curing most prions as they arise (Wickner et al. 2014, 2017; Gorkovskiy et al. 2017; Son and Wickner 2018). In a study of the role of the essential Hsp40 chaperone Sis1p in [PSI+] propagation, Kirkland, Reidy and Masison found that deletion of the C-terminal domain did not impair cell growth in the absence of [PSI+], but resulted in a severe growth defect on the introduction of an otherwise mild [PSI+] (Kirkland et al 2011). Sis1p is necessary for [PSI+] propagation (Higurashi et al. 2008) and the C-terminal deletion mutants were not losing [PSI + ], but were no longer protecting the cells from [PSI+] toxicity (Kirkland et al. 2011). We carried out a general screen for such genes that normally protect the cell from adverse effects of a prion. We used a transposon mutagenesis method based on the Hermes transposon originally from the house fly (Gangadharan et al. 2010; Guo et al. 2013).

Materials and Methods

The supplemental material has a detailed description of the culture conditions, induction of Hermes transposition, selection of colonies carrying a transposition, extraction of cellular DNA, PCR amplification and isolation of the junction points between transposon and chromosomal insertion site, nextgeneration sequencing of these sites, and analysis of the data by visual display and by counting insertions per open reading frame (ORF). The supplemental material also includes: "Exon Intron Counts.xlsx," giving the insertions in every yeast ORF, distinguishing exons from introns where appropriate; "Sorted Hits.xlsx," giving prominent hits sorted by functional group; "TY gag-pol Counts.xlsx," comparing insertions in the Ty retrotransposons at different locations in the genome; "Count Insertions in ORFs and Introns. …

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