Figure 1.
Scoring for [PSI+] induction in deletion strains.
All deletion library candidates in the BY4741 background were cured on GuHCl and cytoduced using kar1 donor strains containing the Sup35PD-GFP and ura3–14 plasmids and either high [PIN+] (right) or [pin−] (left). Expression of Sup35PD-GFP was induced on plasmid selective media containing 50 µM copper for two days and then spotted either onto -Ura to score for [PSI+] induction or +Ura to assay growth. All cytoductions were performed in duplicate and cytoductants were tested for the ability to induce [PSI+] multiple times. Control strains (top row) show growth on -Ura in [PIN+], but not [pin−], strains after 8 days. Both rnq1Δ (second row) and bem1Δ (third row) strains show no growth on -Ura after 11 days, indicative of no [PSI+] induction. Deletion strains showing very low induction, e.g. arf1Δ, exhibit very few colonies after 11 days of growth on -Ura (fourth row), whereas strains showing low induction (e.g. bug1Δ, last row) show slow growth on -Ura after eight days.
Table 1.
Yeast deletion library strains that show no, very low, or low induction of [PSI+].
Figure 2.
Nine deletions show reduced [PSI+] appearance.
Genes were disrupted in the 74D-694 [PIN+] strain background. Deletion strains, transformed with the Sup35PD-GFP plasmid, were grown in 10 ml of plasmid selective media supplemented with copper for 24 hours at 30°C and plated on SD-Ade in order to determine [PSI+] induction frequency. Induction frequency and standard error were calculated with at least one transformant from each independent knock out (see Table S3) for a total of three transformants per deletion. The exception was pre9Δ which was calculated from three transformants from a single independent knock out. Only deletion strains that showed a significant difference in induction frequency compared to wildtype strains (p<0.05 unpaired t-test) were included in the figure.
Table 2.
Properties of deletions that inhibit de novo induction of [PSI+].
Figure 3.
A. [PIN+] cells containing Sup35PD-GFP were induced in copper containing liquid media for 24 hours. The percentage of cells containing rings was determined by counting more than 300 cells from at least one transformant from each independent knockout line (see Table S3) for a total of three transformants per deletion. Bars indicate standard error and (*) indicates deletion strains that showed a significant difference in the percentage of cells containing rings compared to wildtype (p<0.01 unpaired t-test). B. Ring containing cells (purple bars) or cells with diffuse cytoplasmic fluorescence (green bars) were isolated by micromanipulation, placed on rich media and assayed for growth. At least 20 ring containing cells and 10 diffuse cells, from at least one transformant from each independent knock out line for a total of three transformants, were tested. Bars indicate standard error and (*) indicates the deletion strains that showed a significant difference in viability compared to wildtype strains (p<0.01 unpaired t-test).
Figure 4.
Aggregation and toxicity of 103Q in deletion strains.
A. Log phase wildtype [PIN+] cells containing the 103Q-GFP plasmid were induced in galactose containing media for one to two hours and then visualized. Within the population, most cells exhibited strong fluorescent aggregates on a non-fluorescent background (bottom), some cells had faint fluorescent foci on a cloudy fluorescent background (middle), and a very small population of cells had diffuse cytoplasmic fluorescence (top). B. Deletions containing 103Q-GFP were induced and scored for the different cell types indicated in (A). The percentages of cells containing strong fluorescent aggregates (green bars), faint foci (purple bars), and diffuse cytoplasmic fluorescence (gray bars) were calculated from over 300 cells from at least one transformant from each independent knock out line for a total of three transformants. C. Wildtype [PIN+], wildtype [pin−], rnq1Δ, and the indicated [PIN+] deletion strains were transformed with either the 103Q-GFP or 25Q-GFP plasmid and grown to late log phase in glucose (uninducing) media. Cells were serially diluted 20-fold and plated on media that either induced (right column; galactose containing media) or did not induce (left column; glucose containing media) the overexpression of the 25Q-GFP or 103Q-GFP plasmid. White arrowhead indicates the lowest dilution displaying growth. Deletion strains in (C) are in the 74D-694 background except sac6Δ, which is in the BY4741 [PIN+] background as indicated. Pictures shown are representative of the multiple independent deletion lines tested for 103Q-GFP toxicity.
Figure 5.
Proposed effects of class I and II genes on the multistep pathways of de novo prion and polyglutamine aggregate formation.
A. Induction of [PSI+] by transient overexpression of Sup35PD-GFP in a [psi−] [PIN+] strain with endogenous Sup35 tagged with GFP. As described in Mathur et al., [55] the first stages of [PSI+] induction involve the formation of a peripheral fluorescent ring. Class II deletions appear to inhibit this step. As the ring matures, diffuse Sup35 diminishes in intensity. The peripheral ring then collapses to surround the vacuole. The daughters of the ring containing cell contain multiple Sup35:GFP aggregates characteristic of [PSI+]. Class I deletions interfere with transitioning to [PSI+] after the ring appears. B. Hypothetical model of 103Q aggregation in [PIN+] cells. Class II proteins function in the initial formation of small toxic 103Q oligomers (left), since class II gene deletions result in diffuse fluorescence and suppression of toxicity. Class I proteins most likely facilitate incorporation of these small oligomers into large protective aggregates (right).