FIGURE 4:

Induction of [PSI⁺] prion formation in autophagy mutants. (A) Fluorescence micrographs for [PIN⁺] [psi⁻] versions of the wild-type and atg1 mutant strains containing the Sup35NM-GFP plasmid induced with copper for the indicated times. Top rows, representative images in which puncta formation was first detected in the wild-type (18 h) and atg1 (1 h) strains. Bottom rows, representative images in which rod- and ribbon-like aggregates, indicative of de novo prion formation, were detected in the wild-type (18 h) and atg1 (12 h) strains. The percentage of cells containing visible puncta or rod- and ribbon-like aggregates is shown for each strain from an average of 300 cells counted. (B) Western blot analysis of the wild-type and atg1 mutant strains after induction of Sup35NM-GFP for 1, 8, 18, or 24 h. Blots were probed with αSup35 or α-Pgk1 as a loading control. (C) Fluorescence micrographs for cured versions of the wild-type and atg1 mutant strains after induction of Sup35NM-GFP for 2 or 24 h. Representative images in which puncta were only detected in atg1 mutant cells (3.6%) after 24-h induction. (D) [PSI⁺] prion formation was quantified in the wild-type and atg1 mutant strains containing the Sup35NM-GFP plasmid after 0 , 1, and 24 h of copper induction. [PSI⁺] formation was quantified using the ade1-14 mutant allele by growth on medium lacking adenine and differentiated from nuclear SUPX gene mutations by their irreversible elimination in GdnHC1. Data shown are the means of at least three independent biological repeat experiments expressed as the number of colonies per 10⁴ viable cells. Error bars denote the SD. (E) Sup35 toxicity was examined in [psi⁻] or [PSI⁺] versions of the indicated strains containing SUP35 under the control of a GAL inducible promoter. Strains were initially grown overnight in raffinose-containing medium before dilution (A₆₀₀ = 1, 0.1, 0.01, 0.001) and spotting onto agar plates containing galactose or glucose. Overexpression of Sup35 inhibits growth in the [PSI⁺] versions of autophagy mutants compared with [psi⁻] versions.