Figure 2. Curing of the [PSI⁺] prion as a result of titration of chaperone proteins by Pin4C aggregates. (A) Cartoon showing normal propagation of [PSI⁺] in the absence of overexpressed Pin4C (left), and rapid loss of [PSI⁺] when Pin4C is overexpressed (right). Normally Hsp104 and Sis1 bind to [PSI⁺] aggregates, and Hsp104 shears the aggregates producing small [PSI⁺] seeds that are easily transmitted to daughter cells. When Pin4C is overexpressed, it forms aggregates that titrate Hsp104 and Sis1 away from the [PSI⁺] aggregates preventing their shearing. The resulting large [PSI⁺] aggregates remain in the mother cell, so the daughter cells do not inherit the prion. (B) Nonsense suppression color test showing rapid loss of [PSI⁺] when Pin4C is overexpressed. The nonsense reporter is the chromosomal ade1–14 nonsense mutation, which causes cells to be Ade^-, and colonies to be red on YPD medium. In [PSI⁺] cells, the inactivation of the translational release factor causes nonsense suppression and, depending upon the level of the restoration of the Ade⁺ phenotype, the colonies become white or pink. [PSI⁺] propagates stably, as shown by pink (Ade⁺) [PSI⁺] colonies (left). Upon Pin4C overexpression, red (Ade^-) [psi^-] colonies frequently appear (right). (C) Enlargement of visible [PSI⁺] aggregates upon Pin4C overexpression. Sup35 coalesces to small dots in [PSI⁺] cells expressing GFP-tagged Sup35 under the control of the native SUP35 promoter (left). These dots enlarge upon overexpression of Pin4C (right; Sup35). Appearance of enlarged Sup35 foci coincides with the formation of large Pin4C dots, which do not co-localize with Sup35 aggregates (right; Pin4C; the same cell cluster is shown for Sup35 and Pin4C panels). Also, SDS resistant oligomers formed by Sup35 in [PSI⁺] cells (middle, left lane) enlarge upon overexpression of Pin4C (middle, right lane). (D) A shift of Hsp104 into Pin4C aggregates. Hsp104-GFP is diffuse or in tiny particles in the cytoplasm in [PSI⁺] cells (left). Upon Pin4C-dsRED overexpression, Hsp104-GFP co-aggregates with Pin4C-dsRED leaving less Hsp104-GFP in the cytoplasm (right). (E) Overexpression of Hsp104 or Sis1 rescues the curing caused by overexpression of Pin4C. The Hsp104^T160M allele used for this experiment is functional but, unlike wild-type Hsp104, it does not cure [PSI⁺] when overexpressed.³⁵ While overexpression of this Hsp104 allele did not alter the aggregation of Pin4C, overexpression of Sis1 prevented Pin4C from forming large aggregates (data not shown). Parts B-E of this figure were adapted with permission from figures in Yang et al.²⁷