Analysis of [SWI+] formation and propagation events
The budding yeast, Saccharomyces cerevisiae, harbors several prions that are transmitted as altered, heritable protein conformations. [SWI+] is one such prion whose determinant is Swi1, a subunit of the evolutionarily conserved chromatin-remodeling complex SWI/SNF. Despite the importance of Swi1, the molecular events that lead to [SWI+] prionogenesis remain poorly understood. In this study, we have constructed floccullin-promoter-based URA3 reporters for [SWI+] identification. Using these reporters, we show that the spontaneous formation frequency of [SWI+] is significantly higher than that of [PSI+] (prion form of Sup35). We also show that preexisting [PSI+] or [PIN+] (prion form of Rnq1), or overproduction of Swi1 prion-domain (PrD) can considerably promote Swi1 prionogenesis. Moreover, our data suggest a strain-specific effect of overproduction of Sse1 – a nucleotide exchange factor of the molecular chaperone Hsp70, and its interaction with another molecular chaperone Hsp104 on [SWI+] maintenance. Additionally, we show that Swi1 aggregates are initially ring/ribbon-like then become dot-like in mature [SWI+] cells. In the presence of [PSI+] or [PIN+], Swi1 ring/ribbon-like aggregates predominantly colocalize with the Sup35 or Rnq1 aggregates; without a preexisting prion, however, such colocalizations are rarely seen during Swi1-PrD overproduction-promoted Swi1 prionogenesis. We have thus demonstrated a complex interacting mechanism of yeast prionogenesis.
We have dissected the yeast FLO11 promoter and constructed several truncated FLO11-promoter-based reporters for studying the events of [SWI+] de novo formation and propagation. We show that in the presence of [PSI+] or [PIN+], cross-seeding with Sup35 or Rnq1 is a prevalent mechanism promoting Swi1 aggregation and prionogenesis, but not when [SWI+] formation is merely promoted by Swi1 prion-domain overproduction. We also show a strain-specific sensitivity of [SWI+] to overproduction of the molecular chaperone Sse1.