Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI ⁺ ] by Causing Dissolution of the Prion Seeds

Abstract

S accharomyces cerevisiae [PSI ϩ ], the infectious misfolded amyloid form of the protein Sup35, is one of the better-characterized yeast prions (1)(2)(3)(4)(5). Sup35 is an essential protein with three domains, an N-terminal Asn/Gln-rich aggregation-prone domain, a middle domain, and a C-terminal domain that mediates translation termination. In [psi Ϫ ] yeast cells, Sup35 is properly folded, whereas in [PSI ϩ ] yeast cells, most of the Sup35 is misfolded into amyloid seeds, resulting in significant readthrough at stop codons because of the absence of functional Sup35. The conversion of properly folded Sup35 into its misfolded amyloid conformation can occur either spontaneously or by seeding with misfolded Sup35. Once conversion occurs, the [PSI ϩ ] prion conformation continues to pass from generation to generation, with new seeds constantly being generated to maintain a steady-state level of seeds in the yeast population.New seeds are produced when preexisting seeds are severed by Hsp104, a member of the triple-A ATPase family (6). Hsp104 is composed of six identical subunits that form a hexameric ring. Threading of Sup35 molecules through the pore of the Hsp104 hexamer severs the prion seeds in an ATP-dependent reaction (7). Inactivation of the Hsp104 ATPase activity inhibits its severing activity, which in turn cures [PSI ϩ ] and other prions by causing the seeds to dilute out as the cells divide (3,(8)(9)(10)(11)(12)(13)(14).Surprisingly, unlike other yeast prions, the [PSI ϩ ] prion is also cured by overexpression of Hsp104 (3), and even though this was shown more than a decade ago, the mechanism of this curing is still not understood. However, it does appear that the mechanism of [PSI ϩ ] curing by Hsp104 overexpression is very different from the mechanism of curing by Hsp104 inactivation. First, the kinetics of [PSI ϩ ] curing when Hsp104 is overexpressed are very different from the kinetics of curing when Hsp104 is inactivated (8,15,16 One model that has been proposed for the curing of [PSI ϩ ] by the overexpression of Hsp104 is that it involves disaggregation or dissolution of the prion seeds (3,4,8). The role of Hsp104 in this dissolution process could be due to its severing activity, which reduces the size of the prion seeds and concomitantly increases the number of seeds. . This observation was supported by measuring the mobility of GFP-labeled Sup35 in the cytosol and also by measuring the soluble fraction of GFP-labeled Sup35 in cell lysates; both measurements showed an increase in the level of free Sup35. Therefore, in contrast to severing activity, trimming activity reduces the size of the prion seeds without increasing their number.Recently, two other models have been proposed for the curing of [PSI ϩ ] by Hsp104 overexpression. One model proposes that the overexpression of Hsp104 blocks fragmentation of the amyloid fiber (21). This model was based on the observation that when Hsp104 was overexpressed, rather than being recruited to the NMG fibrils by Ssa1, it bound directly to the amyloid ...

Heat shock protein 104 (Hsp104)-mediated curing of [PSI+] yeast prions depends on both [PSI+] conformation and the properties of the Hsp104 homologs

“…We previously showed that overexpression of Hsp104 cures [PSI ϩ ] by a dissolution mechanism involving the trimming of the prion seeds by Hsp104 (14). Because our data support a mechanism in which trimming is necessary for curing by Hsp104 overexpression, a change in trimming should be accompanied by a change in curing.…”

“…The question then arises as to whether a lower level of Sc-Hsp104 would act like overexpression of Sp-Hsp104 and Ca-Hsp104 in that it would neither trim nor cure the strong [PSI ϩ ] variants. To achieve low levels of Hsp104 overexpression, we used the TET-ON promoter, which induces about 3-fold overexpression of Sc-Hsp104, compared with the GAL1 promoter, which induces 10 -20-fold overexpression (14). This relatively low level of Hsp104 overexpression was previously shown to trim the prion seeds and cures the relatively weak 1074 [PSI ϩ ] variant (14), as well as the weak L2888[PSI ϩ ] variant (Fig.…”

“…The dissolution of the prion seeds caused by overexpression of Sc-Hsp104 was shown to require the trimming activity of Hsp104. Mutants of Hsp104 that cannot trim also cannot cure [PSI ϩ ] prion by overexpression (14). For example, overexpression of the T160M point mutant of Sc-Hsp104 that lacks trimming activity but has severing activity comparable with wildtype Sc-Hsp104 does not cure [PSI ϩ ].…”

Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

“…One of the distinguishing features of yeast prions is that they can pass from one cell to another cell by cytoplasmic mixing. This can be achieved by cytoduction, which involves mating of yeast strains involving a kar1 mutation that hinders nuclear fusion (27,28). Recipient haploid cells (kar1 [rho Ϫ ]) with functional p53 and reporter (ADE2 or HIS3) were mated with donor haploid cells (KAR1 [rho ϩ ]) with functional (minus seed) or nonfunctional (plus seed) p53.…”

“…Further, curing of yeast prion [PSI ϩ ] by overexpression of chaperone, Hsp104, due to solubilization of the aggregated form to a soluble one is well known in S. cerevisiae (28). However, recent reports have suggested that overexpression of Hsp104 results in malpartition of [PSI ϩ ] propagons (29).…”