Figure 1.
(A) Collection of [PSI] strains. The genetic background is indicated on the left. [PSI] strains are indicated on top. (B) Strain typing by GFP labeling. The strain type is indicated on top. GFP fusion constructs are indicated on the left. Three types of labeling are observed: the 47k strain exhibits particulate labeling by Sup(1-61)-GFP and Sup(1-61)(G20D)-GFP, but diffused GFP fluorescence with Sup(1-61)(Q23P)-GFP; the 21ℓ strain exhibits particulate labeling by all three GFP fusion constructs; 21s, 21w, 28s, 28w, 47s and 47w are only labeled by Sup(1-61)-GFP. (C) Strain typing by colony color changes. A set of yeast centromere-based plasmids (YCp33) encoding the wild type Sup35 protein and single mutants (indicated on top of each panel) is introduced into yeast bearing different [PSI] strains (indicated on the left). Each [PSI] strain gives a distinctive color pattern.
Table 1.
[PSI] strain distribution of random spores.
Table 2.
21s generates 21w.
Table 3.
Inter-conversion of [PSI] strains by particle infection.
Figure 2.
Inter-conversion of new [PSI] strains.
(A) Infection by prion particles. [PSI] strains are represented by geometric shapes with black frames. Genetic backgrounds are in italic. Arrows indicate the direction of transmission and the outcome. “x” indicates [PSI] curing. (B) Transmission via mating and sporulation. [PSI] strains are transmitted to heterozygous backgrounds (represented by two-colored hexagons) by mating. The heterozygotes are then sporulated. When forming heterozygotes causes [PSI] curing of the diploids, the percentage of cured colonies is indicated. 21 = N21L; 28 = R28P; 47 = Gi47. Prion strains are unambiguously determined by all strain typing methods shown in Figure 1 (see also Table 3 and Table 4).
Table 4.
Inter-conversion of [PSI] strains: transmission by mating and sporulation.
Figure 3.
Bar graphs of prion strain type distribution of yeast spores. Top panel: spores from the wt/R28P background; middle: wt/Gi47 background; bottom panel: wt/N21L background. Genetic cross is indicated under each bar. Prion strain types are color-coded. Each bar represents the average of 4 distributions, each obtained from an independent heterozygote. For each heterozygote, more than 200 random spores are counted (see Methods: spore analysis for experimental procedures, and Table S1 for exact mean values and standard deviations). The heterozygotes formed by VHs and VHw give similar spore distribution, suggesting that VHs = VHw.
Figure 4.
Distinct structures of [PSI] strains.
(A) Yeast harboring a [PSI] strain (labeled on the left) is mated with different genetic backgrounds (labeled on top). Homozygotes always exhibit stronger suppression of the ade2-1 nonsense mutation, resulting in lighter colony color. (B) Nucleated growth of prion fibers monitored by ThT fluorescence in vitro. Top panel: 21w seeds. Bottom: 28w seeds. 21 = 1 µM solution of Sup(1-253)(N21L) (blue); 28 = 1 µM solution of Sup(1-253)(R28P) (red). The 0.5∶0.5 mixture (green) exhibits lower ThT fluorescence at early time points compared with the averaged signal (dotted black line) of the two pure solutions.