The life of [PSI]
The AAA+ disaggregase Hsp104 is essential for the maintenance and inheritance of nearly all known prions of the yeast Saccharomyces cerevisiae. Uniquely for [PSI+], the prion form of the Sup35 protein, there seem to be two activities, involving differing co-chaperones, by which Hsp104 affects the inheritance of [PSI+], the prion form of the Sup35 protein. Each pathway is also involved in protection against ageing, one through disaggregation of damaged proteins and the other through their retention in the mother cell during budding. Mutations in both Hsp104 and Sup35 affect prion inheritance by one or other of these pathways, as does manipulation of either Hsp104 enzyme activity or expression, in both vegetative (budding) divisions and in sporulation. Based on our recent finding (Ness et al. in Molec Microbiol 104:125–143, 2017) we suggest that the management of the heritable prion forms of Sup35 in [PSI+] cells in sporulation may be a marker for a role for Hsp104 in rejuvenation during sporulation.
KeywordsPrion [PSI] Sup35 Hsp104 Sporulation Yeast
Prions are proteins which can adopt various forms of aggregation and folding which affect the phenotype of the ‘host’ organism and are heritable or infectious. Commonly cited models are the neurodegenerative diseases in mammals linked to the prion forms of the PrP protein expressed in the brain (Prusiner 2013, review), in E. coli, a protein, curli, which is excreted and helps in the formation of biofilms and in the yeast Saccharomyces cerevisiae, the [PSI+] prion, which is an aggregate of the polypeptide chain termination factor Sup35/eRF3 (Tuite et al. 2015, review). In S. cerevisiae, the prion-associated phenotypes are stable over thousands of cell generations, while PrP-based infections are essentially incurable.
Both inheritance and infection of prions are the consequence of the ability of amyloid fibre to template the addition and refolding of monomeric protein to the growing fibres with fragmentation increasing their numbers. The remodeling and addition of molecules to an aggregate can occur spontaneously, but is greatly enhanced by pre-existing remodeled molecules. The new additions adopt the form of the model template, and fragmentation promotes an exponential increase in amount of amyloid from a pool of monomer both in vitro and in vivo.
A number of factors have been found to affect the stability of inheritance of prions. The stability of [PSI+] is affected by environmental stress (e.g. Singh et al. 1979; Tuite et al. 1981; Newnam et al. 2011) or by mutations in either the SUP35 or the HSP104 gene (Young and Cox 1971; Doel et al. 1994; DePace et al. 1998) or by manipulation of gene expression (Chernoff et al. 1995; Glover and Lum 2009; Helsen and Glover 2012a, b; Chernova et al. 2017; Ness et al. 2017). Some of the variant forms of the prion amyloid also show an inherent instability during cell division (Uptain et al. 2001). The degree of instability can range from total loss of the prion (often referred to as ‘curing’) to stochastic random loss characteristically giving rise to red sectors on white [PSI+] colonies. The involvement of Hsp104 is because this chaperone is required for the fragmentation of amyloid fibres of nearly all yeast prions so far identified, including the Sup35p [PSI+] prion.
The Hsp104 ‘curing’ paradox
Recently, we provided evidence that definitively supports one of two theories which have been offered for a long-running paradox surrounding the inheritance of [PSI+] (Ness et al. 2017). The paradox is as follows: the AAA+ disaggregase Hsp104 is essential for [PSI+] inheritance and blocking the ATPase activity of Hsp104 using guanidine hydrochloride (GdnHCl) allows the prion to be diluted out during cell division due to the loss of fragmentation by this disaggregase (Eaglestone et al. 2000; Byrne et al. 2007). But [PSI+] can also be eliminated from cells by elevating the levels of the same enzyme (Chernoff et al. 1995). The most straight-forward explanation for this oddity, first offered by Paushkin et al. (1996), was that over-expression caused an increase in the disaggregase activity of the enzyme such that the amyloid aggregates of the prion form of Sup35 were resolved into its constituent monomers in their native form. This would result in the restoration of normal polypeptide chain termination activity to Sup35 and abolishing both the read-through of nonsense codons characteristic of the presence of [PSI+] and the templating activity of the prion form of the protein on which its inheritance depends. Yet this hypothesis has now been challenged by our new findings (Ness et al. 2017).
There are genetic differences as well as differences in molecular events between the two [PSI+] ‘curing’ regimes mediated by wild type and mutant Hsp104, respectively. These are first, that the N-terminal domain of Hsp104 is dispensable for the propagation of all yeast prions, but is required for curing by over-expression (Hung and Masison 2006). Second, the loss of the Hsp90 co-chaperones Sti1 and Cpr7 interferes neither with the propagation of [PSI+] nor with the curing of [PSI+] by growth in GdnHCl, but loss of either or both does almost abolish the curing by wild type Hsp104 over-expression (Moosavi et al. 2010; Reidy and Masison 2010). Third, an Hsp104 binding site in the M-region of Sup35 allows binding without the cooperation of Hsp70 or Sis1 (Helsen and Glover 2012a, b, Winkler et al. 2012a; Frederick et al. 2014) and deletion of residues 131–140 in the M region of Sup35 eliminates curing by over-expression, but has no other effect on [PSI+] propagation (Helsen and Glover 2012a, b) and curing by inhibition of Hsp104 ATPase proceeds normally.
These genetic differences correlate with different binding regimes of Hsp104 with the Sup35 amyloid substrate: “productive” but labile, leading to prion aggregate fragmentation, presumably common to all Hsp104-dependent yeast prions and “non-productive” and stable, dependent, in Sup35, on Sup35M-domain residues 131–140.
The productive interaction involves Hsp104 being recruited to the substrate by Hsp40 (Sis1)/Hsp70 (Ssa1) chaperones (Tipton et al. 2008; Winkler et al. 2012b; Lee et al. 2013) leading to polymer fragmentation. The second, non-productive binding regime in the M-region of Sup35 allows binding without the cooperation of Hsp70 or Hsp40 but does involve the two Hsp90s, Cpr7 and Sti1 (Helsen and Glover 2012a, b; Winkler et al. 2012a; Frederick et al. 2014). Saarikangas and Barral 2015, have described replicative age-related mother cell retention of, among other proteins, Sup35 [PSI+] aggregates. The aggregates involve Hsp104 associated with Hsp70s and are observed in the absence of over-expression of Hsp104. Instability of [PSI+] is not observed in these conditions, nor is malpartition. It may be that the association of Sup35 with these objects is the labile one described by Frederick et al. (2014), allowing normal partition and becomes stable with over-expression when the binding involves Hsp90s and not Hsp70s.
Hsp104 has also, apparently two different roles: disaggregation of toxic and misfolded proteins, particularly those resulting from heat-shock, and anchoring misfolded proteins to the actin cytoskeleton. The latter role is associated with cell ageing and involves lantrunculin-sensitive anchoring to the actin cytoskeleton of ageing mother cells as part of the rejuvenation of daughter cells (Tessarz et al. 2009; Helsen and Glover 2012a, b). Ness et al. (2017) propose this latter activity is also likely to be responsible for [PSI+] loss induced by over-expression of Hsp104, an activity which is unique to this one prion out of the half-dozen which have been checked.
Inheritance of [PSI+] propagons in zygotes and asci
The effect on the inheritance of [PSI+] propagons in zygotes and asci from matings of (a), MATa, [PSI+] with (b and c) MATα [psi−] or (d) with PNM2(Sup35G58R) [psi−] haploids
(a) ψ+ strong parent
74, 422, 185, 486, 504, 205, 670, 215, 440, 158, 284, 317, 408, 275, 390, 265, 331, 413, 393, 239, 370, 315, 370, 783
Ave.: 382.5; median: 324
(b) 4-h Zygotes (ψ+ × ψ−)
1316, 2600, 612, 798, 529, 642, 504, 1414, 464, 780, 953, 248, 652, 1081, 178, 698, 682, 1749, 2176, 736, 299
Ave.: 848; median: 698
(c) ψ- x ψ+, Propagons in five tetrads from 4-h-old zygotes
Total overall: 4790; Ave./spore 252.1
(d) G58R x ψ+, Propagons in five tetrads from 4-h-old zygotes
Total overall: 191; Ave./spore: 9.05 (19.7 in WT spores)
Hsp104 and transmission of [PSI+] propagons in sporulation
The cross discussed above (Table 1) involved a PNM allele of SUP35 (i.e. PNM2G58R) not HSP104 which leaves open the question whether the elimination of [PSI+] in this cross is due to an unusual sensitivity of the mutant protein to Hsp104 disaggregase. However, we find that dominant PNM1 mutants of Hsp104 totally lacking ATPase activity give results either quantitatively indistinguishable from those in Table 1, or are even more severe in terms of reduction of propagon numbers (Cox, BS unpublished data: see below).
Loss of [PSI+] in the PNM2G58R [psi−] × [PSI+] would not appear to be an amyloid disaggregation problem, but a propagon segregation problem. Nevertheless, in the absence of any perturbation of Hsp104 function by mutation or environment, all the empirical evidence is consistent with a random distribution of [PSI+] propagons in both vegetative (mitotic) and sporulation (meiotic) divisions (Table 1; Byrne et al. 2009). Mutations or environmental disturbance may nevertheless affect inheritance in either.
The role of Hsp104 in sporulation has not been explored very extensively although sporulation occurs normally in diploids homozygous for a ∆hsp104 deletion (e.g. Ünal et al. 2011). The possibility remains that, without being essential Hsp104 nevertheless has a role which is important for the distribution of [PSI+] propagons and perhaps other organelles to spores (see Suda et al. 2007). The possibility that a link between these observations and the rejuvenation which accompanies spore formation (Ünal and Amon 2011; Ünal et al. 2011) is tantalizing. To explore this we followed the fate of GFP-decorated propagons in sporulation in PNM1-1/+ [psi−] diploids. This diploid segregated an average of seven propagons to spores in a total of 10 tetrads analysed. In spite of a starting cytoplasm replete with diffuse GFP and GFP-decorated punctate spots before the arrest of vegetative division and also for 24 h after the induction of meiosis (Fig. 5a, b), by the time sporulation was complete after four days, asci showed no fluorescence within the four spores and only weak diffuse fluorescence outside them (Fig. 5c). Once returned to growth medium, GFP fluorescence reappeared in the two spores in every ascus to which the Sup35::GFP fusion gene segregated, mostly without the evidence of Sup35::GFP-decorated aggregates (Fig. 5d) and there was minimal evidence of GFP-decorated aggregates inherited from the zygotes in which these ascospores formed. In the presence of the dominant PNM1-1 mutation in these sporulating zygotes we estimate that at most only 10% of the Sup35, whether aggregated or not, was dispersed to the spores. What we see is Mendelian inheritance of autonomous new synthesis of this GFP-linked essential protein in the germinating spores, very little of which decorates any Sup35 aggregates.
Degradation, disaggregation or malpartition?
Another question we raise in our new study (Ness et al. 2017) is that of the source of instability of the [PSI+] prion that is commonly observed in [PSI+] variants, PNM2(SUP35) mutants and heterozygotes formed between them and with strains expressing wild type Sup35, and also in partially dominant PNM1(HSP104) mutants. Such prion instability characteristically appears as sectoring in colonies growing on normal growth medium (Fig. 1c) and the implication is that the instability is associated with malpartition rather than degradation. No doubt having very low numbers of large propagons would contribute to the spectrum of instability, but interaction with Hsp104 in rejuvenation mode may be a common feature (Verges et al. 2011).
What is not certain is that any form of intrinsic or induced instability of Sup35/[PSI+] prions is due to significant degradation or disaggregation of the amyloid form. Disaggregation can be observed in vitro (Shorter and Lindquist 2004) albeit with molar ratios of Hsp104:Sup35 polymer that are far in excess of what may be observed in vivo, but until now (i.e. Ness et al. 2017) has not been directly assayed in vivo.
One of the epidemiological myths about the mammalian PrPSc prion is that it is indestructible by normal physical or chemical methods. For example, sterilizing surgical instruments is not sufficient to quell iatrogenic prion infection, nor is cooking; resistance to proteases has been used as a means of identifying amyloid and Alper et al. (1967) could not kill the infectious scrapie agent by ionizing radiation or UV. The thermodynamic facts are that an amyloid fibre exists in an entropy pit and relatively large amounts of energy input are required to get it out (Eichner and Radford 2011). Fragmentation by Hsp104 with the 12 ATPase sites in the hexamolecular collar is achieved by merely extracting a single molecule from within the fibre (Glover and Lum 2009). In spite of several published claims to the contrary, there has been no direct demonstration of in vivo degradation of [PSI+] Sup35 amyloid aggregates. This leaves us with the need to find other explanations for the appearance of prion-free cells.
BSC wishes to acknowledge the expert instruction, help, advice and insights, free use of their facilities and generous hospitality received from Dr. Tricia Serio, Dr. Prasana Satputa-Krishnan and other members of the Serio group when he was a guest in their laboratory at Brown University, Providence, R. I. This work was done while BSC held an Emeritus Fellowship funded by the Leverhulme Trust, 2012–2014. The research was also supported by a project grant from the BBSRC to MFT (Ref no. BB/H012982/1).
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