Unlabelled: The yeast prion protein Sup35 is aggregation-prone at high concentrations. De novo Sup35 prion formation occurs at a significantly increased rate after transient overexpression of Sup35 in the presence of another prion, [PIN⁺], but it is still a rare event. Recent studies uncovered an additional and seemingly more prevalent role of Sup35: at its physiological level, it undergoes phase separation to form reversible condensates in response to transient stress. Stress-induced reversible Sup35 condensation in the [psi^-] strain enhances cellular fitness after stress ceases, whereas irreversible Sup35 aggregates in the [PSI⁺] strain do not confer this advantage. However, how Sup35 overexpression, which could potentially lead to irreversible aggregation, affects its condensation under stress conditions remains unclear. In this study, we used a combinatorial method to examine how different levels of Sup35 overproduction and cellular conditions affect the nature, formation, and physical properties of Sup35 assemblies in yeast cells, as well as their impacts on cellular growth. We observed notable morphological distinctions between irreversible Sup35 aggregates and reversible Sup35 condensates, possibly indicating different formation mechanisms. In addition, Sup35 aggregation caused by a very high overexpression level can strongly inhibit cell growth, diminish the formation of stress-induced condensates when Sup35 is completely aggregated, and impair cellular recovery from stress. Together, this study advances our fundamental understanding of the physical properties and formation mechanism of different Sup35 assemblies and their impacts on cellular growth. We conclude that in vivo studies are sensitive to overexpression and can lead to assembly routes that strongly affect functions.

Importance: The role of condensates in living cells is often studied by overexpression. For understanding their physiological role, this can be problematic. Overexpression can shift cellular functions, thereby changing the system under study, and overexpression can also affect the phase behavior of condensates by shifting the position of the system in the underlying phase diagram. Our detailed study of overexpression of Sup35 in S. cerevisiae shows the interplay between these factors and highlights basic features of intracellular condensation such as the balance between condensation and aggregation as well as how cellular localization and responsiveness depend on protein levels. We also apply super-resolution microscopy to highlight details within the cells.

• PMID: 39912644
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Journal Article

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Feng J, Osmekhina E, Timonen JVI, Linder MB

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