Figure 2: Loss of N-terminal acetylation induces an Hsf-1-mediated stress response, which impacts prion propagation.

(a) Stress response element (pSTRE) activity was analysed using a β-galactosidase reporter (SB757) expressed in ΔNatA [PSI⁺] (SY319) or [psi⁻] (SY978) strains relative to a WT [PSI⁺] strain (SLL2606). (n=6, error bars represent s.d.). (b) Heat-shock element (pHSE) activity was analysed using a β-galactosidase reporter (SB753) expressed in ΔNatA [psi⁻] (SY978) and [PSI⁺] (SY319) strain, and in a ΔNatA [PSI⁺] (SY319) strain also expressing the carboxy-terminal domain of Sup35 (+C; SLL6676) relative to a WT [PSI⁺] strain (SLL2606). (n=6, *P<0.00001 by unpaired Student’s t-test compared with WT [PSI⁺], error bars represent s.d.). (c) Expression of Hsp104, Ssa1/2, Sis1 and Ssb1/2 was determined by analysing lysates of a [PSI⁺] ΔNatA strain (SY319) by SDS–PAGE followed by quantitative western blotting using specific antisera relative to WT [PSI⁺] (SLL2606) lysates. (n=6, P<0.005 by unpaired Student’s t-test compared with WT [PSI⁺], error bars represent s.d.). (d) Lysates from WT (SLL2606), ΔNatA (SY319), SUP35(S2P) (SY1209), HSF1^ON (SY2130) and SUP35(S2P) HSF1^ON (SY2124) [PSI⁺] strains were analysed by SDD-AGE followed by immunoblotting for Sup35. (e) Lysates from [PSI⁺] WT (SLL2606) and ΔNatA strains (SY319) expressing a dominant-negative Hsf1 mutant (Hsf1^DN; SB778) or an empty vector (−) were analysed by SDD-AGE, followed by immunoblotting for Sup35. (f) Cultures of WT (SLL2606) and ΔNatA [PSI⁺] (SY319) strains expressing Hsf1^DN (+) or an empty vector (−) were spotted on complete medium (+ade) and medium lacking adenine (−ade) before incubation at 30 °C. (g) Tenfold serial dilutions of WT [PSI⁺] (SLL2606) and [psi⁻] (SLL2119), [PSI⁺] ΔNatA (SY319), [PSI⁺] SUP35(S2P) (SY1209), [PSI⁺] HSF1^ON (SY2130) and [PSI⁺] SUP35(S2P) HSF1^ON (SY2129) strains were grown on rich medium (YPD) and medium lacking adenine (−ade) at 30 °C.