Extended Data Fig. 4. The chaperones Hsp104 and Ssa2 co-operate with FBP to efficiently disassemble Cdc19 amyloids in vivo .

(A) Co-overexpression of Hsp104 and Ssa2 partially restores growth of cdc19^irrev cells after heat stress. cdc19^WT and cdc19^irrev cells overexpressing as indicated Hsp104 or Ssa2, or both together by addition of 10 mM estradiol for 3 hours were treated for 30 min at 42 °C. Serial dilutions were spotted on agar plates and grown at 30 °C for 3 days. The image is representative of three independent experiments.

(B) Hsp104 protein levels were quantified in the indicated strains by Western blot either in the absence (-) or presence (+) of 10 mM estradiol for 3 hours. The image is representative of three independent experiments. Unprocessed original scans of blots are shown in Source data Extended data fig. 4.

(C) Increased FBP levels and Hsp104 co-operate to efficiently restart growth after stress in cdc19^irrev cells. Exponentially growing wild-type or tps1Δ cells expressing either Cdc19^WT or Cdc19^irrev were subjected to a 30 min heat shock at 42 °C. Where indicated, overexpression of Hsp104 was induced by treating cells with 10 mM estradiol for 3 hours. Growth restart after stress release of the indicated strains was quantified by measuring the cell density (OD₆₀₀) over time after inoculation of equal cell numbers at 30 °C. Mean cell density 22 hours after stress release is shown, with error bars representing S.E.M. of three independent experiments. Note that Hsp104 overexpression and increased FBP levels co-operate to rescue cdc19^irrev cells after heat shock.

(D) Wild-type or tps1Δ cells co-expressing mCherry-tagged Hsp104 and either GFP-tagged Cdc19^WT or Cdc19^irrev mutant were heat stressed for 30 min at 42 °C and imaged by fluorescence microscopy at the times indicated (minutes). Representative GFP- (left row) and mCherry-images (middle row) are shown, together with the merged image (bottom row) to visualize co-localization of Cdc19 aggregates and Hsp104. Images are representative of three independent experiments. Scale bar: 5 μm. Source data for the graphical representation and unprocessed Western blots can be found in Source Data Extended Data Fig. 4.