FIG. 6.

Visual detection of the Sup35^PSI+ aggregates. (A and B) Effects of Hsp104 depletion. Yeast strains were GT81-1C (wild type); [PSI⁺] and [psi⁻] derivatives of GT238–1A (hsp104Δ [P_MOD-HSP104]) (low Hsp104); and GT81-1C grown in the presence of 5 mM GuHCl (GuHCl). [PSI⁺] aggregates tagged by GFP were visualized by fluorescence microscopy using the LEU2 plasmid pLSpSUP35NM-GFP (A) or by immunostaining with Sup35-specific primary rabbit antibodies and rhodamine-labeled secondary antibodies (B), as described in Materials and Methods. Exponential cultures were grown in the synthetic complete glucose medium with 5 mM GuHCl (GuHCl) or in the −Leu-His/Glu medium (all other strains). Representative examples are shown. The Sup35NM-GFP aggregates become larger and less numerous in the [PSI⁺] low-Hsp104 strain. (C) Comparison of the frequencies of the cells containing easily detectable medium-size Sup35NM-GFP aggregates or huge Sup35NM-GFP agglomerates in the [PSI⁺] and [psi⁻] deivatives of the low-Hsp104 strain, GT238-1A, and in the isogenic control wild-type [PSI⁺] strain, GT81-1C. From 70 to 325 cells were screened in each case. (D) Comparison of the frequencies of the cells containing huge Sup35NM-GFP agglomerates and frequencies of the [psi⁻] (red) colonies produced by two [PSI⁺] isolates of the low-Hsp104 strain, GT238-1A. Correlation between these numbers suggests that huge Sup35 agglomerates possess reduced ability to transmit the prion state. Limits of variation (95%) are shown. (E and F) Effects of the overexpressed dominant-negative Hsp104-KT protein on Sup35NM-GFP aggregate size. The yeast strain used was GT81-1C; the inducible constructs were URA3-GAL-HSP104-KT (↑Hsp104-KT) and URA3-GAL-HSP104 (↑Hsp104). Media were −Ura-Leu/Glu (before induction) and −Ura-Leu/Gal+Raf after 18 h of incubation (↑Hsp104-KT and ↑Hsp104). After induction, about 91% of 50 cells screened in the ↑Hsp104-KT culture exhibited detectable medium-size Sup35NM-GFP aggregates. No aggregates of this type were observed in the culture overexpressing wild-type Hsp104. In the same conditions, about 85% of cells of the ↑Hsp104-KT culture formed [psi⁻] colonies.