Energy depletion causes reversible condensation of Sup35 into intracellular puncta in S. cerevisiae cells expressing GFP-fused Sup35 (in vivo). Sup35 particles dissolved within a few minutes of removing energy stress when cells started growing independent of the presence of chaperones (e.g. Hsp104). Stress-induced Sup35 particles also did not have any of the biochemical features of amyloid-like aggregates (morphology). Starved and energy-depleted yeast experience a reduction in cytosolic pH. By manipulating the cytosolic pH it turned out that acidification was sufficient to induce Sup35 condensates. When 2 μM of purified Sup35 was incubated in physiological buffer in vitro, the protein remained diffuse (protein localization), however, when the pH was reduced from 7.5 to 6.0, condensates of Sup35 formed (protein localization, particle size and count). Sup35 condensates adopted spherical shapes in solution, fused when brought together, deformed when contacting the microscope slide and their photobleached regions quickly recovered fluorescence (morphology), suggesting that they are liquid-like. Sup35 was mobile in growing cells in vivo, but it became immobile (protein localization) when sequestered into condensates, upon stress. This behavior was confirmed in vitro, where Sup35 initially phase-separated to form liquid droplets but then solidified into a gel-like state (morphology) as suggested by fusion and photobleaching experiments. Cryo–electron tomography (imaging assay evidence) of Sup35 droplets revealed that gel-like droplets consisted of an amorphous, yet well-defined, meshwork with an average mesh size of ~10 nm (morphology). Gel-like condensates dissolved (particle size and count) when the salt concentration or pH was raised or in the presence of small amounts of detergents, demonstrating reversibility in vitro. A minimal module consisting only of the prion (N) and the M domain (NM) (mutation, truncation) formed droplets in a reversible and pH-dependent manner in vitro. Removing a linear cluster of ionizable glutamic acid residues within the M domain (mutation, Sup35M3 variant) yields a fully functional Sup35 variant but with altered phase behavior, such that protein-rich droplets formed at pH 7.5 and the pH dependence of droplet formation was discernibly reduced in vitro and in vivo (particle size and count, other change in phenotype/functional readout). Deletion studies suggest that the disordered NM domain alters the phase behavior of the C domain by promoting the formation of reversible gels instead of irreversible aggregates (morphology). The fitness of cells expressing similar levels of full-length Sup35 and the C domain alone (in vivo deletion mutants) have been compared: in the absence of stress the C domain was diffuse and soluble (protein localization) and cells grew normally, showing that the C domain is not aggregation-prone in the cellular environment, presumably because of the presence of ligands such as guanosine 5′-triphosphate. After stress (perturbation of the cell environment to induce phenotypic changes), however, the C domain aggregated in a manner that was similar to that of the full-length protein but after removal of stress, dissolution of C domain aggregates could take several hours, while full length Sup35 condensates dissolved within minutes in wild-type cells (other change in phenotype/functional readout, protein localization). Concomitantly, Sup35C cells took longer to restart growth and exhibited reduced fitness when recovering from stationary phase. This suggests that the NM domain determines the material properties (reversible gel versus irreversible aggregate) of Sup35 in vivo (PMID:29301985)