FIG. 3.

Interactions of wild-type Hsp104, mutant Hsp104-KT, and Hsp70-Ssa. (A) Antagonistic interaction between mutant and wild-type Hsp104 in [PSI⁺] curing. The [PSI⁺ PIN⁺] strain OT56 was transformed with pH28 (Hsp104), pLA1-HSP104-KT (Hsp104-KT), a combination of both plasmids, or a control matching plasmid. Cultures were induced in liquid −Ura-His/Gal+Raf medium (see Materials and Methods) and plated onto YPD medium at various time points to detect [PSI⁺] curing. Both the wild-type and mutant Hsp104 proteins cure [PSI⁺] almost completely by the fourth generation of growth. Coexpression of wild-type Hsp104 and mutant Hsp104 reduces [PSI⁺] curing efficiency. (B) Antagonistic interactions between mutant and wild-type Hsp104 in thermotolerance. Strain OT60 was transformed with the URA3 plasmid pKT218,620 bearing HSP104-KT under the endogenous HSP104 promoter (Hsp104-KT) or matching control plasmid (Control). Transformants were grown either in the synthetic −Ura medium (which is selective for the plasmid and causes slight induction of Hsp104) or in YPD medium at 25°C. In YPD cultures, HSP104 expression was induced by a 30-min pretreatment at 37°C prior to heat shock (40). Cultures were heat shocked at 50°C, and serial dilutions were spotted onto −Ura medium. Plates were photographed after 3 days of incubation. The plasmid pKT218,620 greatly decreases the thermotolerance of the culture, confirming the dominant-negative effect of Hsp104-KT. The same result was reproduced with strain GT17 (not shown). (C) Effects of Hsp104-Ssa on [PSI⁺] curing: the quantitative assay. Yeast strain, OT56; plasmids, pH28 + pRS316GAL (Hsp104), pH28 + pGAL-SSA1 (Hsp104 + Ssa), pLA1-HSP104-KT + pRS316GAL (Hsp104-KT), and pLA1-HSP104-KT + pGAL-SSA1 (Hsp104-KT + Ssa). Production of the Hsp104, Hsp104-KT, and Hsp70-Ssa proteins was induced in the liquid −Ura-His/Gal+Raf medium. The number of generations after induction is shown. Excess Hsp70-Ssa protects [PSI⁺] from curing by excess Hsp104 but not from curing by excess Hsp104-KT. (D) Effects of Hsp70-Ssa on [PSI⁺] curing: the color assay. Yeast strain, OT56; plasmids, pLA1 (Control), pGAL-SSA1 (↑Ssa), pH28 (↑Hsp104), pH28 + pGAL-SSA1 (↑Hsp104 + ↑Ssa), pLA1-HSP104-KT (↑Hsp104-KT), and pLA1-HSP104-KT + pGAL-SSA1 (↑Hsp104-KT + ↑Ssa). Transformants were grown on solid −Ura-His/Gal medium and were then velveteen replica plated onto YPD medium and analyzed by color phenotype. A lighter color is indicative of more intense suppression, apparently due to a larger proportion of [PSI⁺] cells in the culture. Results confirm that excess Hsp70-Ssa decreases the [PSI⁺] curing effect of ↑Hsp104 but increases the [PSI⁺] curing effect of ↑Hsp104-KT.