Spatial sequestration and oligomer remodeling during de novo [PSI⁺] formation

Abstract

Prions are misfolded, aggregated, infectious proteins found in a range of organisms from mammals to bacteria. In mammals, prion formation is difficult to study because misfolding and aggregation take place prior to symptom presentation. The study of the yeast prion [PSI+], which is the misfolded infectious form of Sup35p, provides a tractable system to monitor prion formation in real time. Recently, we showed that the de novo formation of prion aggregates begins with the appearance of highly mobile cytoplasmic foci, called early foci, which assemble into larger ring or dot structures. We also observed SDS-resistant oligomers during formation, and lysates containing newly formed oligomers can convert [psi−] cells to the [PSI+] state, suggesting that these oligomers have infectious potential. Here, we further characterize two aspects of prion formation: spatial sequestration of early foci and oligomerization of endogenous Sup35p. Our data provides important insights into the process of prion formation and explores the minimal oligomer requirement for infectivity.

Implications of the Actin Cytoskeleton on the Multi-Step Process of [PSI+] Prion Formation

“…We previously showed that older mothers form multiple aggregates that fail to coalesce compared to their corresponding young daughters [ 42 ]. Furthermore, mature propagating [ PSI + ] aggregates have been shown to use actin/myosin networks to localize to IPOD structures [ 43 ].…”

“…Furthermore, mature propagating [ PSI + ] aggregates have been shown to use actin/myosin networks to localize to IPOD structures [ 43 ]. Based on these results, we previously proposed that rejuvenated daughters have more robust actin networks that drive coalescence [ 42 ]. In this study, we showed that multiple early foci are formed in act1-120 and act1-122 strains ( Figure 1 ), and remain separate for several hours, thus providing evidence that properly formed networks are important to aggregate coalescence.…”

The three faces of Sup35

“…As the oligomers assemble into larger SDS‐stable aggregates over time, small highly mobile fluorescent foci are visually detected. These foci quickly coalesce into a single fluorescent dot before being sequestered near the cell periphery (Lyke & Manogaran, ; Sharma et al, ). Once residing at the periphery, the majority of the diffuse Sup35NM quickly converges into the aggregate within 20 min.…”