The prion hypothesis was originally proposed to explain the etiology of the transmissible spongiform encephalopathies, a group of mammalian neurodegenerative diseases that arise and spread through a protein-only mechanism: a self-perpetuated change in the folding of a cellularly encoded protein PrP [1,2]. The idea that a protein conformation could encode genetic information gained additional support when two enigmatic traits in Saccharomyces cerevisiae, [URE3] and [PSI⁺], were identified as prions [3]. The [URE3] and [PSI⁺] phenotypes mimic loss-of-function mutations in the URE2 and SUP35 genes, respectively, but the prion phenotypes are epigenetic and arise when the Ure2 or Sup35 proteins assemble into functionally compromised higher-order complexes that are self-replicating [4–6]. Importantly, specific conformations of the Ure2 or Sup35 proteins confer heritable [URE3] and [PSI⁺] when exogenously supplied to non-prion cells, the ultimate proof of the prion mechanism for these traits [7–9].

The cornerstone of this protein-only mechanism is the ability of the prion form to self-replicate by templating the conversion of other conformers of the same protein to a like state. This process has been modeled in vitro for both Ure2 and Sup35, providing additional support for the prion hypothesis [10–13]. In each case, denatured recombinant protein spontaneously assembles into ordered, β sheet–rich fibers that bind amyloid-specific dyes upon dilution from denaturant. Remarkably, fibrillization of freshly added protein occurs at an accelerated rate in the presence of pre-formed fibers or of lysates from cells propagating the prion form, recapitulating the self-replication process.

Although prion proteins clearly have the capacity to direct the folding of other conformers in a defined purified system, extension of the prion hypothesis to practice becomes a multistep endeavor within the context of a living cell (Figure 1). First, newly synthesized prion protein must acquire the prion form, likely through its incorporation into existing prion complexes. Second, prion complexes must be stably maintained yet continually subdivided to yield additional replication templates known as prion seeds or propagons. Third, propagons must be partitioned to daughter cells in actively dividing cultures, where they continue to direct the conversion of newly made protein to the prion form and ensure inheritance of the associated phenotype. Thus, inhibition of the pathway at any point would lead to the same outcome: prion loss (also known as curing).

Progression through this in vivo prion cycle (Figure 1) is modulated by parallel, and sometimes competing, quality-control pathways [14], and the interplay between these processes has emerged as a powerful tool for dissecting the mechanism of protein-only inheritance in vivo. Not surprisingly, factors involved in the quality control of protein folding are the largest group of characterized prion regulators in yeast [15–22]. Members of both the Hsp70 (Ssa1 and Ssb1) and Hsp40 (Ydj1) families have been implicated in [URE3] [15–17] and [PSI⁺] [18,20–24] inheritance, but the molecular disaggregase Hsp104 has, by far, the most pronounced effects on fungal prion propagation.

Hsp104 is a member of the heat-inducible Clp/Hsp100 family of AAA⁺ ATPases and functions as a hexamer to resolve stress-induced aggregates [25–28]. Hsp104 is also constitutively expressed at low levels in logarithmically growing yeast cultures [28]. Under these normal growth conditions, Hsp104 is required for both the de novo appearance and propagation of all known S. cerevisiae prions [15,20,28–30]. Given its essential role, the mechanism by which Hsp104 mediates protein-only inheritance has been the subject of intense study.

In the case of [PSI⁺], deletion of Hsp104 induces prion loss or curing [20], and strains devoid of Hsp104 function cannot reacquire heritable [PSI⁺] [29], leading to the initial suggestion that Hsp104 activity was required for Sup35 to access and/or maintain a prion-competent intermediate form [5,20]. Contrary to this idea, [PSI⁺] can be induced de novo in the absence of Hsp104 if Sup35 is overexpressed along with a second prion protein such as New1 or Rnq1, suggesting that the Hsp104 requirement for de novo [PSI⁺] formation is actually an indirect effect through the propagation of a co-prion [30,31]. In line with this model, newly synthesized Sup35 can join existing complexes upon Hsp104 inhibition in vivo [32], and full-length Sup35, or a fragment of the protein comprising only the prion determining domain (NM), can assemble into amyloid fibers in the absence of Hsp104 in vitro [10,11]. However, Hsp104 does accelerate the spontaneous assembly of Sup35 fibers in vitro [13,33,34] and, at early time points in fibrillization reactions, promotes the steady-state accumulation of a Sup35 conformer that immunoreacts with an antibody specific for amyloidogenic intermediates [33]. Thus, although Hsp104 may not be absolutely required for conversion of Sup35 to the prion state, its activity may increase the efficiency of this process.

In addition to a potential role in protein-state replication, Hsp104 has been linked to the generation of additional [PSI⁺] propagons, a process most frequently described as fragmentation of prion complexes [6,35]. Such fragmentation would increase the converting capacity of the existing steady-state pool of Sup35 by distributing the protein among a greater number of complexes and would also facilitate the transmission of these prion templates to daughter cells upon division. In support of this model, prion loss upon inhibition of Hsp104 is progressive in dividing cultures, with preferential retention of propagons in mother cells [36,37]. Reactivation of Hsp104 in these cells prior to prion loss results in a cell division– and protein synthesis–independent increase in propagon number, suggesting an Hsp104-mediated change in existing Sup35 [32,37]. Although the biochemical nature of propagons is currently unknown, the steady-state size of Sup35 complexes is larger in cultures upon Hsp104 inhibition, consistent with a block to Hsp104-dependent fragmentation [38,39], and recent in vitro studies report the direct fragmentation of Sup35 or NM fibers by Hsp104 [33,34].

Despite these dynamic changes in Sup35 physical state during the prion cycle, existing prion complexes must remain sufficiently stable at steady state to maintain and transmit the prion phenotype. Recent reports, employing Sup35 or NM fusions to the green fluorescent protein (GFP) have suggested that Sup35 prion complexes are inherently unstable [40] and that Hsp104 is required to maintain the integrity of these complexes [41]. Consistent with this latter point, prion loss upon Hsp104 inhibition was reported in the absence of cell division, suggesting that curing occurs through the disassembly of existing prion oligomers [41]. However, these observations are at odds with previous reports. For example, resolubilized Sup35 is not biochemically detectible upon Hsp104 inhibition in [PSI⁺] cultures [32], and [PSI⁺] curing by loss of Hsp104 does not occur in non-dividing, stationary phase cultures [36,41].

Does Hsp104 actually mediate each step of the [PSI⁺] prion cycle in vivo, or does the interdependency of these events complicate interpretation of the available experimental observations? To resolve this issue, we have systematically probed each individual step in the [PSI⁺] prion cycle in vivo (Figure 1) by monitoring the Hsp104-dependent dynamics of either newly made or existing Sup35 in single live cells and correlating these changes in protein state to transitions in the prion phenotype. Our observations resolve many controversies in the literature and support a model in which Hsp104 acts at a single point in the [PSI⁺] prion cycle to remodel existing prion complexes. In the absence of this event, Sup35 complexes increase in size and decrease in mobility. As a result, these complexes are inefficiently transmitted to daughter cells, and newly synthesized Sup35 accumulates in a soluble form when the number of pre-existing oligomers becomes limiting.

Together, our in vivo studies probe the molecular mechanism by which Hsp104 mediates propagation of the Sup35/[PSI⁺] prion. We suggest that Hsp104 acts at a single point in the prion cycle to remodel existing Sup35^[PSI+] complexes (Figure 1). By tightly regulating expression of Sup35-GFP, we provide direct evidence for this event: the decreased fluorescence of existing Sup35-GFP^[PSI+] in wild-type microcolonies (Figure 4). Despite this change in fluorescence, the protein persists (Figure 5A) and remains assembled in complexes (Figure 5C). Because Sup35-GFP^[PSI+] foci remain uniformly detectable in wild-type [PSI⁺] cells that constitutively express the fusion [43], we interpret our observations as a redistribution of existing Sup35-GFP molecules to new complexes containing an excess of untagged Sup35 (Figure 4D), providing direct experimental support in vivo for fragmentation [6,35].

In the absence of Hsp104-dependent remodeling of Sup35-GFP^[PSI+] complexes, a number of crucial events in the [PSI⁺] prion cycle are compromised. First, Sup35-GFP^[PSI+] complexes increase in size, initially detected as brighter Sup35-GFP fluorescence (Figure 2B). By separately expressing Sup35-GFP in either [PSI⁺] or [psi⁻] cells and inhibiting fusion protein synthesis upon mating, we unambiguously link this change to incorporation of soluble Sup35-GFP^[psi−] into existing complexes (Figure 2). Importantly, when [PSI⁺] complexes are marked with Sup35-GFP prior to mating, no change in fluorescence intensity is detected upon Hsp104 inhibition, indicating that existing complexes do not laterally associate to form larger particles (Figure 2C). Thus, the number of Sup35-GFP^[PSI+] complexes becomes static when Hsp104 activity is lost, as previously predicted in mathematical models of curing [36,37].

The increase in fluorescence intensity of Sup35-GFP^[PSI+] complexes also correlates with a decrease in their mobility (Figure 3). Although this relative immobility is immediately apparent upon zygote formation when a large bolus of soluble Sup35 incorporates (Figure 3), the decreased mobility arises over time in GdnHCl-treated haploids as newly synthesized Sup35 continues to join complexes (Video S1 and unpublished data). In either case, this immobility provides a possible molecular explanation for the diminished transmission of propagons to daughter cells (Figure 4) [37,39] and the consequential decrease in the efficiency of incorporation of soluble Sup35^[psi−] (Figure 6) [32].

Can our observations of Sup35-GFP^[PSI+] dynamics in vivo explain the behavior of heritable prion particles? Several lines of evidence suggest that Sup35-GFP^[PSI+] foci are a robust proxy for propagons. First, Sup35-GFP is fully functional in translation termination, accumulates in cytoplasmic foci only in [PSI⁺] cells, supports [PSI⁺] propagation as the only copy of Sup35, and is regulated by Hsp104 levels in a manner that is indistinguishable from wild-type Sup35 [43]. Second, mathematical models of propagon dynamics upon Hsp104 inhibition predict a block in replication of these heritable units and their subsequent dilution by cell division [36]. The changes in Sup35-GFP foci that we observed upon Hsp104 inhibition (Figures 2–4 and 7) provide direct experimental support at the single-cell level for these predictions: Sup35-GFP^[PSI+] foci become static upon Hsp104 inhibition, and their reduced mobility correlates with an inefficient transfer to daughter cells (Figures 3 and 4). Third, genetic analyses of propagon transmission revealed a segregation bias in which mothers are more likely to retain prion seeds than their daughters [37]. The preferential accumulation of existing Sup35-GFP^[PSI+] in zygotes and their immediate progeny mirrors propagon behavior upon division (Figure 4). Fourth, propagon numbers increase independently of cell division and protein synthesis upon reactivation of Hsp104 [32,37]. In line with this observation, static Sup35-GFP^[PSI+] complexes are rapidly resolved coincidently with the bulk inactivation of soluble Sup35 upon removal of GdnHCl from the medium (Figure 7). The close alignment of our observations with the previous genetic characterizations of heritable prion units suggests that propagons are equivalent to or derived from Sup35-GFP^[PSI+] foci. If this relationship is indeed direct, counting propagons by dispersing colonies formed in the presence of GdnHCl is likely to yield an underestimate given the clear segregation bias that we observe for Sup35-GFP^[PSI+] foci (Figure 4), as previously suggested [37].

Our work also resolves some of the seemingly conflicting data present in the literature, which necessitated formation of multiple models for the role of Hsp104 in prion propagation. One of these controversies is the fate of existing Sup35-GFP^[PSI+] upon Hsp104 inhibition. Ness et al. observe persistent aggregation of existing Sup35 in GdnHCl-treated [PSI⁺] cultures using a biochemical assay [32]. In contrast, Wu et al. report disassembly of Sup35-GFP^[PSI+] complexes upon Hsp104 inhibition with the same treatment, as assessed by the accumulation of soluble Sup35-GFP^[PSI+] using microscopic approaches [41]. A key consideration in the interpretation of these studies is the status of Sup35 expression. Wu et al. monitored the behavior of bulk Sup35-GFP under conditions of on-going synthesis, whereas Ness et al. focused their studies on the dynamics of existing Sup35 only [32,41]. By regulating expression of Sup35-GFP, we resolve these apparent conflicts. Accumulation of soluble Sup35-GFP upon Hsp104 inhibition only occurs with continued synthesis of the fusion (Figures 4 and 6). Thus, prion complexes are not destabilized upon Hsp104 loss, but rather become more stable (Figure 4) and less efficient at incorporating newly synthesized Sup35 (Figure 6). Nonetheless, these static complexes remain biologically active substrates that are rapidly remodeled to efficient templates upon Hsp104 reactivation (Figure 7).

Given these observations, [psi⁻] cells must only appear when existing Sup35^[PSI+] complexes fail to segregate during cell division, a prediction at odds with a previous study, which reported prion curing in arrested cultures [41]. However, recent studies by Byrne, Cox, and Tuite have uncovered a high degree of lethality in the strain employed by Wu et al. upon release from α-factor arrest (L. Byrne, B. S. Cox, and M. F. Tuite, personal communication). Thus, the reported curing actually occurred in dividing cultures, but this division was overlooked due to the extensive, and counterbalancing, cell death.

Another point of controversy is the role of Hsp104 in the efficiency of Sup35^[psi−] conversion to the prion state. We observe quantitative inactivation of Sup35^[psi−] upon introduction of Sup35^[PSI+] in the absence of Hsp104 function in some cells, as monitored by the appearance of a translation termination defect (Figure 2B) [43], suggesting that Hsp104 is not directly required for efficient incorporation. However, the percentage of cells displaying the prion phenotype is diminished in Hsp104-inactivated zygotes. This observation is consistent with a primary role for Hsp104 in the generation of additional prion templates [6,35,36]. Specifically, the converting capacity of some cells falls below the threshold needed to inactivate the vast majority of soluble Sup35^[psi−] contributed by the [psi⁻] mating partner when Hsp104 is inhibited. Since Hsp104 is not available to generate additional prion seeds, this converting potential is dictated by the number of propagons present in the cell at the time of Hsp104 inhibition, a factor that varies widely in individual cells of a homogenous culture [37]. Consistent with this interpretation, newly synthesized Sup35-GFP accumulates in a soluble form soon after Hsp104 inhibition (Figure 6) [32], coincident with the loss of Sup35-GFP^[PSI+] remodeling (Figure 4), and this soluble pool is rapidly inactivated upon restoration of Hsp104 function and remodeling of existing Sup35-GFP^[PSI+] complexes (Figure 7).

An exclusive role for Hsp104 in the generation of new prion seeds is also compatible with observations in vitro. Sup35 self-assembly into amyloid fibers is accelerated by the addition of Hsp104 [13,33], and this effect has been linked to a steady-state increase in the abundance of an amyloidogenic intermediate [33,34]. One interpretation of this observation is that Hsp104 promotes the de novo formation of this species [33], but an alternate explanation is the Hsp104-dependent fragmentation of spontaneously arising intermediates. The incorporation of soluble protein into the smaller complexes predicted in this alternative scenario would also result in an apparent steady-state increase in the abundance of this form. Consistent with this interpretation, Hsp104 preferentially interacts with low molecular weight oligomers of Sup35 [52], which are intermediates in fiber assembly and disassembly reactions in vitro [53], and specifically alters the biophysical properties of Sup35-GFP^[PSI+] complexes in vivo (Figure 3). Thus, the enhanced conversion efficiency in vivo and in vitro is likely to be a secondary consequence of prion-complex remodeling by Hsp104 (Figure 4) [32].

Loss of Hsp104-dependent remodeling also uncovers biological consequences for single-cell differences in a superficially homogenous culture. In addition to the variation in translation termination efficiency, [PSI⁺] cells lacking Hsp104 activity display a broader range of doubling times than their untreated counterparts (Table 1). Again, these distinctions likely arise from cell-to-cell variation in the number of prion particles [37] at the time of Hsp104 inhibition, with cells containing a larger number of prion particles being the most efficient at converting soluble Sup35, but also the most detrimental to cellular physiology (Figure 8).

How is remodeling mechanistically accomplished? Hsp104 forms a hexamer with a central pore [26], and previous studies suggest that Hsp104 functions by threading substrates through this channel [54,55]. How, then, might this extrusion lead to Sup35^[PSI+] remodeling and production of new prion complexes? Other members of the Clp/Hsp100 family may provide some insight. For example, ClpX extracts a single MuA monomer from a DNA-bound tetramer, an event that destabilizes the remainder of the complex and promotes dissociation [56–58]. Hsp104 could act similarly on Sup35^[PSI+] complexes by unfolding constituent monomers and thereby fragmenting the now destabilized oligomer. A future challenge, then, is to determine the contributions of each of these species, the unfolded monomer and the fragmented oligomer, to prion propagation and the viability of [PSI⁺] cells.