Generating new prions by targeted mutation or segment duplication

Abstract

Yeasts contain various protein-based genetic elements, termed prions, that result from the structural conversion of proteins into self-propagating amyloid forms. Most yeast prion proteins contain glutamine/asparagine (Q/N)-rich prion domains that drive prion activity. Here, we explore two mechanisms by which new prion domains could evolve. First, it has been proposed that mutation and natural selection will tend to result in proteins with aggregation propensities just low enough to function under physiological conditions and thus that a small number of mutations are often sufficient to cause aggregation. We hypothesized that if the ability to form prion aggregates was a sufficiently generic feature of Q/N-rich domains, many nonprion Q/N-rich domains might similarly have aggregation propensities on the edge of prion formation. Indeed, we tested four yeast Q/N-rich domains that had no detectable aggregation activity; in each case, a small number of rationally designed mutations were sufficient to cause the proteins to aggregate and, for two of the domains, to create prion activity. Second, oligopeptide repeats are found in multiple prion proteins, and expansion of these repeats increases prion activity. However, it is unclear whether the effects of repeat expansion are unique to these specific sequences or are a generic result of adding additional aggregation-prone segments into a protein domain. We found that within nonprion Q/N-rich domains, repeating aggregation-prone segments in tandem was sufficient to create prion activity. Duplication of DNA elements is a common source of genetic variation and may provide a simple mechanism to rapidly evolve prion activity.

Keywords: Sup35; amyloid; prion; yeast.

Fig. 1.

Design of prion-promoting mutations. (A) Sequences of the wild-type and mutant Puf4, YLR177W, Yck1, and Pdc2 PrLDs. Strongly prion-promoting amino acids (W, Y, F, V, I, and M) are indicated in green, whereas strongly prion-inhibiting amino acids (P, K, R, D, and E) are in red. Positions that were mutated are underlined. (B) PrLDs that do not show detectable prion activity tend to lack extended peptide stretches without prion-inhibiting residues. Alberti et al. (14) identified 100 yeast fragments with prion-like composition and tested each in four assays for prion-like activity. Shown are box-and-whiskers plots of the longest stretch without any prion-inhibiting residues for each of the proteins that showed prion-like activity in all four assays (Prion) and each of the proteins that failed all four assays (Nonprion). (C) Box-and-whiskers plot of the longest segments with no more than one prion-inhibiting residue.

Fig. 2.

Mutations in the PrLDs cause foci formation. Each of the wild-type and mutant PrLDs was fused to GFP and expressed from the GAL1 promoter. Cells were grown in galactose/raffinose dropout medium for 24 h and then visualized by confocal microscopy. The percentage of fluorescing cells with GFP foci (either rings or dots) is indicated; a minimum of 50 fluorescing cells were counted per construct.

Fig. 3.

Mutations in the PrLDs cause prion formation. (A–D) The wild-type and mutant PrLDs from Puf4 (A), YLR177W (B), Yck1 (C), and Pdc2 (D) were fused to the Sup35MC domain and expressed from the SUP35 promoter as the sole copy of Sup35 in the cell. Strains were transformed with either an empty vector (−) or a plasmid expressing the matching PrLD under control of the GAL1 promoter (+). Cells were grown in galactose/raffinose dropout medium for 3 d and then plated onto dextrose medium lacking adenine to select for [PSI⁺] cells. (E) For each mutant PrLD, to test for stability of the Ade⁺ phenotype, Ade⁺ colonies were streaked onto synthetic complete medium (−GdHCl) or synthetic complete medium supplemented with 4 mM guanidine HCl (+GdHCl). Cells were then restreaked onto YPD to test for loss of the Ade⁺ phenotype. Two prion isolates are shown for Puf4^mut and YLR177W^mut; two representative isolates are shown for Pdc2^mut and Yck1^mut.

Fig. 4.

Prion activity in the mutant PrLDs is sensitive to the number of prion-promoting amino acids. The mutated positions in Puf4^mut and YLR177W^mut were either deleted (Puf4^∆inhib and YLR177W^∆inhib) or replaced with an increased ratio of prion-promoting to neutral amino acids (Puf4^6PP,1N and YLR177W^4PP,1N). (A) Sequences of the mutants. Positions mutated are underlined. Blank spaces indicate deletions. (B and C) Ade⁺ colony formation assay for each of the mutant PrLDs fused to Sup35MC.

Fig. 5.

Prion formation can be observed with as few as two mutations. (A) Sequences of mutants designed to test the minimal number of mutations required to create prion activity for the Puf4 and YLR177W PrLDs. (B and C) Ade⁺ colony formation assay for the Puf4 (B) and YLR177W (C) mutant PrLDs fused to Sup35MC.

Fig. S1.

Prion activity can be created by mutations at other positions. (A) Sequences of mutants targeting other regions of the Puf4 and YLR177W. (B and C) Ade⁺ colony formation assay for the Puf4 (B) and YLR177W (C) mutant PrLDs fused to Sup35MC.

Fig. 6.

Repeat expansions can create prion activity. (A) Short segments lacking any prion-inhibiting amino acids were identified in the Puf4 PrLD (indicated as α, β, γ, and δ in the PrLD sequence). (B) Versions of the Puf4 PrLD containing varying numbers of repeats of each segment were made. These repeat expansion mutant PrLDs were fused to Sup35MC and tested for prion activity. For each construct, the region duplicated and the copy number of the repeats are indicated. PAPA scores of each PrLD-Sup35MC fusion are indicated in parentheses. (C) For each of the repeat expansions of the α and β segments of Puf4, two scrambled versions were made in which the primary sequence of the full repeat region was randomized while keeping amino acid composition unchanged. Each PrLD containing scrambled repeats was fused to Sup35MC and tested for prion activity.

Fig. S2.

Repeat expansions in the YLR177W PrLD. (A) Short segments lacking any prion-inhibiting amino acids were identified in the YLR177W PrLD (indicated as α, β, γ, and δ in the PrLD sequence). (B) Versions of the YLR177W PrLD containing varying numbers of repeats of each segment were made. These repeat expansion mutant PrLDs were fused to Sup35MC and tested for prion activity. For each construct, the region replicated and the copy number of the repeats are indicated. PAPA scores of each PrLD-Sup35MC fusion are indicated in parentheses. For the γ segment, four and five repeat versions were designed; however, cells expressing these fusion proteins as the sole copy of Sup35 were inviable. For the δ4 segment, it is unclear why fewer Ade⁺ colonies were seen upon PrLD overexpression. This may reflect toxicity of this overexpressed PrLD, particularly in prion-positive cells. Alternatively, the overexpressed PrLD may preferentially form structures that are poorly propagated.