Figure 2. Hsp104 accelerates or abolishes de novo Sup35 and Ure2 prion formation.

(A, B) Sup35 (2.5μM) was incubated with rotation (80rpm) for either 0h, 2h, or 6h without His₆-Hsp104, 2h or 6h with His₆-Hsp104 (1μM), or 2h or 6h with His₆-Hsp104 (0.03μM). Reactions were depleted of His₆-Hsp104 using magnetic Ni-NTA, and the extent of depletion was determined by immunoblot (A). Following His₆-Hsp104 depletion, reactions were sonicated, and used to seed (2% wt/wt) unpolymerized Sup35 (2.5μM) for 4h in unrotated reactions (B). Values represent means±SD (n=3). (C, D) Ure2 (2.5μM) was incubated with rotation (80rpm) without or with His₆-Hsp104 (0.03μM or 1μM) for 0h, 6h or 14h. Reactions were then depleted of His₆-Hsp104 using magnetic Ni-NTA, and the extent of depletion was determined by immunoblot (C). Following His₆-Hsp104 depletion, reactions were sonicated, and used to seed (2% wt/wt) unpolymerized Ure2 (2.5μM) for 4h in unrotated reactions (D). Values represent means±SD (n=3).

(E) Reactions were performed as in (A, B) and reaction products were concentrated, sonicated, and transformed into [psi⁻] cells. The number of [PSI⁺] colonies relative to total transformants was then determined. Values represent means±SD (n=3). (F) Reactions were performed as in (C, D) and reaction products were concentrated, sonicated, and transformed into [ure-o] cells. The number of [URE3] colonies relative to total transformants was then determined. Values represent means±SD (n=3).