Figure 4. FBP directly regulates formation and disassembly of reversible Cdc19 amyloids.

(A) Illustration of how FBP binding may regulate Cdc19 amyloid formation and disassembly. FBP (1) depletes aggregation-prone monomers by inducing Cdc19 tetramerization [4], and (2) directly promotes aggregate re-solubilization.

(B) – (D) Purified Cdc19^WT (B), Cdc19^ΔFBP (C) or Cdc19^irrev (D) proteins were mixed with the indicated metabolites and incubated at 30 °C for 14 hours. After centrifugation, the supernatant (Sup) and pellet (Pellet) fractions were analysed by SDS-PAGE and quantified relative to buffer controls (shown as mean ± S.E.M, n = 3 independent experiments, two-tailed t-test, P = 0.0342).

(E) – (F) Cdc19^irrev is monomeric in vivo and in vitro. (E) Extracts from cells expressing GFP-tagged Cdc19^WT or Cdc19^irrev were separated by size-exclusion chromatography (SEC) and selected peak fractions were analysed by Western blot with an α-GFP antibody. Elution volume (ml) and size markers (kDa) are indicated. (F) Recombinant Cdc19^WT, Cdc19^irrev and for control tetrameric Cdc19^ΔPEP mutant proteins were purified and analysed by SEC. Normalized absorbance at 215 nm(arbitrary units; a.u.) was plotted as a function of elution volume (ml). n = 3 independent experiments.

(G) Direct FBP binding to Cdc19 is necessary to re-solubilize Cdc19 amyloids in vivo. Wild-type or tps1Δ cells expressing GFP-tagged Cdc19^WT or Cdc19^ΔFBP and over-expressing Pyk2 were heat shocked (42 °C, 30 min) and allowed to recover at 30 °C in the presence of 25 μg/ml cycloheximide. Representative GFP-images (three independent experiments) were taken 60 or 240 min after heat shock. Arrowheads indicate Cdc19-GFP aggregates. Scale bars: 5 μm.

(H) Heat shock-induced (42 °C, 10 min) Cdc19^WT, Cdc19^ΔFBP and Cdc19^irrev amyloids were incubated with FBP (4 or 8 mM) or for control with F6P (4 mM) or buffer. Scale bar: 500 nm. Black arrows mark fibrils that became positively stained due to FBP binding. Representative fibrils (dashed squares) were 1.5 x enlarged to better visualize distinct fibril morphologies (inserts). n = 3 independent experiments. Unprocessed original scans of gels and blots and source data for all graphical representations are shown in Source Data Fig. 4.