The formation of dynamic, membraneless compartments using intracellular phase transitions such as phase separation and gelation provides an efficient way for cells to respond to environmental changes. Recent work has identified a special class of intrinsically disordered domains enriched for polar amino acids such as glycine, glutamine, serine, or tyrosine as potential drivers of phase separation in cells. However, more traditional work has highlighted the ability of these domains to drive the formation of fibrillar aggregates. Such domains are also known as prion domains. They have first been identified in budding yeast proteins that form amyloid-like aggregates. Because these aggregates are heritable and change the activity of the prion-domain–containing protein, they are thought to be a common mechanism for phenotypic inheritance in fungi and other organisms. However, the aggregation of prion domains has also been associated with neurodegenerative diseases in mammals. Therefore, the relationship between the role of these domains as drivers of phase separation and their ability to form prion-like aggregates is unknown.

The budding yeast translation termination factor Sup35 is an archetypal prion-domain–containing protein. Sup35 forms irreversible heritable aggregates, and these aggregates have been proposed to be either a disease or an adaptation that generates heritable phenotypic variation in populations of budding yeast. Despite having been described almost 25 years ago, the physiological functions of the Sup35 prion domain and other prion-like domains remain unclear. Uncovering these functions is a prerequisite for understanding the evolutionary pressures shaping prion-like sequences and how their physiological and pathological transitions affect cellular fitness.

Here, we show that the prion domain of Sup35 drives the reversible phase separation of the translation termination factor into biomolecular condensates. These condensates are distinct and different from fibrillar amyloid-like prion particles. Combining genetic analysis in cells with in vitro reconstitution protein biochemistry and quantitative biophysical methods, we demonstrate that Sup35 condensates form by pH-induced liquid-like phase separation as a response to sudden stress. The condensates are liquid-like initially but subsequently solidify to form protective protein gels. Cryo–electron tomography demonstrates that these gel-like condensates consist of cross-linked Sup35 molecules forming a porous meshwork. A cluster of negatively charged amino acids functions as a pH sensor and regulates condensate formation. The ability to form biomolecular condensates is shared among distantly related budding yeast and fission yeast. This suggests that condensate formation is a conserved and ancestral function of the prion domain of Sup35. In agreement with an important physiological function of the prion domain, the catalytic guanosine triphosphatase (GTPase) domain of the translation termination factor Sup35 readily forms irreversible aggregates in the absence of the prion domain. Consequently, cells lacking the prion domain exhibit impaired translational activity and a growth defect when recovering from stress. These data demonstrate that the prion domain rescues the essential GTPase domain of Sup35 from irreversible aggregation, thus ensuring that the translation termination factor remains functional during harsh environmental conditions.

The prion domain of Sup35 is a highly regulated molecular device that has the ability to sense and respond to physiochemical changes within cells. The N …