Prions of Yeast: From Cytoplasmic Genes to Heritable Amyloidosis Part 3

[Het-s], A Prion of Podospora Required for a Normal Function

Sexual Mating vs Hyphal Anastomosis

Most strains of Saccharomyces cerevisiae are diploid, and the starvation conditions that initiate the meiosis-sporulation pathway are commonplace in nature. The haploid mating-competent products of meiosis quickly find a compatible partner, and mating reconstitutes the diploid state. This sexual mating seems designed to generate diversity in the progeny, so that some may survive uncertain future environmental conditions.

Filamentous fungi have two kinds of mating. Sexual mating, like that of S. cerevisiae (and higher organisms), is designed to produce diverse offspring, and so it requires that parents differ at one or more special loci (mating-type loci). Hyphal anastomosis is another form of mating, in which two fungal colonies fuse when they meet, probably in order to share nutrients. In yeast and other fungi, infectious entities, such as viruses, pass from cell to cell exclusively via cell-to-cell contact, such as occurs in mating and hyphal anastomosis. This poses a risk, which is essentially a sexually transmitted disease of fungi. In filamentous fungi, the sexual spores are usually devoid of viruses, perhaps because these cells have little cytoplasm. However, hyphal anastomosis is accompanied by the free flow of cytoplasm, and even nuclei, from one colony to another. Perhaps for this reason, fungi limit hyphal anastomosis to partners that pass a test for genetic identity, the presumption being that genetically identical partners already carry the same viruses. In Podospora anserina, this test is identity at nine chromosomal loci, called het loci (reviewed in ref. 80). Partners with different alleles at even one of the het loci will attempt to fuse hyphae, but quickly recognize that they were not "meant for each other," and form a barrier between the colonies that prevents further fusion attempts.

One of these loci, called het-s, has two alleles, het-s and het-S. The het-s locus is unusual, in that it can have either of two phenotypes, and this difference in phenotypes is controlled by a nonchromosomal genetic element, called [Het-s] (81). Colonies that are het-s and have [Het-s] show the usual reaction of incompatibility when they meet a colony that is het-S. However, het-s colonies that do not have the [Het-s] nonchromosomal genetic element are able to fuse equally with het-s or het-S partners (a neutral phenotype) (Fig. 10).

[Het-s] Has Properties of a Prion of the HET-s Protein

The [Het-s] nonchromosomal genetic element spreads from one colony to another by hyphal anastomosis. However, [Het-s] is apparently not included in sexual spores, and so it is lost from the meiotic offspring of a sexual cross (82). This is a form of natural curing. In a colony arising from a meiotic offspring lacking [Het-s], this element often arises again, and spreads through the colony (82).

Fig. 10. [Het-s] is a prion necessary for heterokaryon incompatibility, a normal fungal function. Two colonies of the filamentous fungus Podospora will fuse to form heterokaryons when their hyphae meet, if they are genetically identical at 9 chromosomal loci, called het loci. Heterokaryon incompatibility results from different alleles at any of these loci, and the colonies do not fuse. One such locus, called het-s, with alleles het-s and het-S, produces heterokaryon incompability, only if the protein product of the het-s allele is in a prion form (8). [Het-s*] denotes the absence of [Het-s].

This is reminiscent of the reversible curing criteria for yeast prions (7). Coustou et al. (8) have found that overproduction of the protein encoded by het-s increases the frequency with which the [Het-s] element arises, and the het-s gene is necessary for the propagation of the [Het-s] non-chromosomal genetic element. They therefore proposed that [Het-s] is a prion form of the HET-s protein, a suggestion that they support by showing, further, that the het-s protein is relatively protease-resistant in extracts of a [Het-s] strain, compared to a strain lacking [Het-s].

The HET-s Minimal Prion Domain Is the N-terminal 26 Amino Acids

Deletion mutants of het-s were tested for their ability to propagate [Het-s], and to show the incompatibility reaction (83). It was found that expression of residues 1-26 was sufficient to allow propagation of the [Het-s] prion; to express the incompatibility reaction required residues 1-112 (Fig. 4). Residues 86-289 were also sufficient to propagate the [Het-s] prion. Thus, just as Ure2p has two nonoverlapping regions, either of which is sufficient, when overexpressed, to induce the de novo appearance of [URE3], the protein encoded by het-s has two nonoverlapping regions, either of which can propagate the [Het-s] prion (83).

Expressing both the HET-s and HET-S alleles in the same cell results in a sublethal phenotype, presumably because of the formation of toxic complexes of the two proteins (83). This allowed selection of mutants of the het-s or het-S genes, which could no longer give this phenotype, or which could not propagate [Het-s]. The same selection also produced mutants in chromosomal genes affecting prion propagation, whose analysis will doubtless be of considerable interest.

Curing of Yeast Prions and Blocking Yeast Prion Generation: Can These Methods Be Used in Mammals?

The first curing method for any prion was discovered by Singh et al. in 1979, who found that growth, or just incubation, of cells in hypertonic medium resulted in high frequency loss of [PSI+] (42). Curing was found using either 2.5 M KCl or 1.75 M ethylene glycol. A survey of other chemicals for curing activity showed that growth in the presence of millimolar concentrations of guanidine HCl are efficient cures of [PSI+] (84). The mechanism of curing by guanidine is as yet unknown, but some parameters of its action have been determined. Exposure of [PSI+] cells to curing doses of guanidine do not result in immediate loss of the prion. Four generations of growth are required before cured cells begin to appear (85). Incubation of cells, in stationary phase, in the presence of guanidine does not cure, nor does heat or ethanol stress enhance curing.

Similar concentrations of guanidine also cure [URE3] (7,37). [URE3] can also be cured by overexpression of Ure2p-GFP fusion proteins, or by overexpression of certain fragments of Ure2p (65). It has been suggested that these fusion proteins or fragments bind to the amyloid growing filaments in such a way that they block further propagation of the filament (Fig. 11).

Notes

[URE3] requires Hsp104p for propagation and is cured by overexpressed

Ydjlp. Deletion or mutation of HSP104 results in loss of [URE3] (93), as was previously shown for [PSI] (67,68). However, while [PSI] is also cured by overexpression of Hsp104p, [URE3] is not. Overexpression of the Hsp40 family chaperone Ydj1p cures [URE3] (93), but does not cure [PSI] (Dan Masison, personal communication). Differences in response to the state of cellular chaper-ones may reflect differences in the structure of the Ure2p and Sup35p aggregates.

Evidence for Ure2p amyloid in [URE3] cells. While both Sup35p and Ure2p can form amyloid in vitro, the state of neither protein in prion-contain-ing cells is known. Recently, electron microscopic examination of thin sections of [URE3] cells overproducing Ure2p has revealed the presence of networks of filaments in the cytoplasm (96). These filaments were shown by immuno electron microscopy to be composed of Ure2p.

Fig. 11. Curing of the [URE3] prion by fusion proteins or fragments of Ure2p (65).

Table 2

Comparison of Evidence for the Prion Etiology of Scrapie, [URE3], [PSI], and [Het-s]

Evidence	Scrapie	[URE3]	[PSI]	[Het-s]
Reversible curing	No	Yes	Yes	Yes
	No	Yes	Yes	Yes
Phenotype relation	No	Yes	Yes	No
Spontaneous generation caused by protein	No	Yes	Yes	No
In vitro amyloid propagation	Yes	Yes	Yes	No
Altered protein in infected cells	Yes	Yes	Yes	Yes
UV resistance of infectious agent	Yes	No	No	No
Purification of infectious agent	Yes	No	No	No

No filaments were seen in cells lacking [URE3]. Antibody to the C-terminal domain of Ure2p readily detected the filaments while antibodies specific to the N-terminal were less able to do so. Examination of extracts of these cells suggests an explanation for this result. A substantial fraction of the Ure2p in extracts of [URE3] cells, perhaps 3/4 of the total, is insoluble in boiling 3M urea-20% SDS, a property typical of many amyloids. The portion that can be solubilized under these conditions is protease-resistant, as shown previously. The urea-SDS insoluble material reacts with antibody to the C-terminal domain of Ure2p, but not to the N-terminal domain, suggesting a structure in which the prion domain  forms a compact inaccessable structure of stacked beta sheets to which is attached the peripheral C-terminal domain (96).

Ure2C structure closely resembles glutathione-S-transferases. Two groups have recently reported the structure of the C-terminal domain of Ure2p and found that, consistent with the known amino acid sequence homology with glutathione – S – transferases, the nitrogen regulation domain shows close structural similarity to these enzymes (86,97). However, structural differences at the active site – homologous region (86,97) explain why Ure2p has not been found to have GST activity (88). Two parts of nitrogen regulation domain that stabilize the prion domain (47) are present in the structure near the presumed location of the N-terminal domain (97).

Sup35p N-termini from other species can be prion domains. The C-terminal domain of Sup35p from the yeasts Pichia methanolica, Pichia pastoris, Candida albicans, Kluyveromyces lactis, Saccharomycodes ludwigii and Zygosaccharomyces rouxii are closely homologous to that of S. cerevisiae. The N-termini are less homologous, but retain the octapeptide repeat pattern and are all rich in asparagine, glutamine and glycine (92,94). Fusions of the N-ter-mini of these various yeasts to the cerevisiae C-terminus were used to test prion domain activity. In one study, the Pichia pastoris N-terminal domain was shown to have all of the expected genetic and biochemical properties of a prion (91). Properties of other fusions are consistent with prion activity, but less completely documented (87,94). Little or no transmission of the prion state was observed between fusion proteins with N-termini from different species, a result interpreted as the analog of the species barrier seen in the mammalian transmissible spongiform encephalopathies (87,91,94).

Rnq1p can be a prion. Since the prion domains of Ure2p and Sup35p are both rich in asparagine and glutamine, and these residues are important for prion activity (see Subheading 8.) candidates for new yeast prions were sought among asparagine-glutamine rich protein sequences. One protein, whose only known feature is that it is Rich in N and Q (Rnq1p) was found aggregated in some strains and soluble in others (95). Moreover, this aggregation was transmissible by cytoplasmic transfer, curable by guanidine and required Hsp104 for its propagation. Although the presence of aggregation (or indeed deletion of the gene) did not produce a recognized phenotype, this is apparently a prion.

Hsp70s are essential for propagation of [PSI+]. Jung et al. have shown that Hsp70 proteins of the Ssa family are necessary for the propagation of [PSI] (89). In addition, this group showed that [PSI+] cells have elevated levels of Hsp104, indicating that the presence of [PSI+] is perceived by the cell as a stressful situation (89). A hint to the mechanism of curing by millimolar concentrations of guanidine comes from the finding that this compound inhibits the in vivo activities of Hsp104 (90).

Conclusions

Yeast and fungal prions have already had an enormous impact on the prion field. The evidence that [URE3] and [PSI+] are indeed prions is, in several ways, stronger than that available for the mammalian systems, thus finally proving the existence of prions (Table 2). At the same time, this shows that proteins can be genes. The discovery that chaperones are critical in propagation of (PSI+) has obvious implications beyond yeast. Proteins interacting with Sup35p and Ure2p each determine susceptibility to de novo prion generation. The finding that a fungal prion is responsible for a normal cellular function shows that prions need not be a pathological phenomenon, but may in some cases prove to have physiological functions.

We speculate that studies of the cellular factors involved in propagation of yeast prions, and of chemicals or other means capable of curing yeast prions, will suggest treatments useful in human prion or amyloid diseases. Moreover, the use of yeast molecular genetics may also facilitate the discovery of new prions.

Summary

We identified the nonchromosomal genes [URE3] and [PSI] of S. cerevisiae as prions, based on their special genetic properties, which indicated they were not viruses or plasmids, but were infectious protein forms of Ure2p and Sup35p, respectively. These properties are not yet demonstrated for the TSEs. The [URE3] and [PSI] prions arise de novo: they are not mutants of some nucleic acid replicon. The overproduction of the Ure2 and Sup35 proteins, not the mRNAs or the genes, results in ‘spontaneous generation’ of these prion diseases. The prion domains of Ure2p and Sup35p are rich in Asn and Gln and these residues are critical for prion generation and propagation. The C-terminal domains of Ure2p and Sup35p carry out their cellular functions and stabilize the N-terminal prion domains of each molecule in the normal form.

In [URE3] and [PSI] strains, Ure2p and Sup35p are aggregated, respectively, and Ure2p is protease resistant. Ure2p and Sup35p can form self-propagating amyloid in vitro, and the properties of this amyloid formation indicate that it is the basis of these prion phenomena. The chaperone, Hsp104, is critical in [PSI] propagation, and the Hsp70s are minor modifiers of this effect. Both [PSI] and [URE3] are cured by millimolar concentrations of guanidine in the medium, and [URE3] is also cured by overexpression of various Ure2p fragments, and by fusions of Ure2p with green fluorescent protein. The [Het-s] nonchromo-somal gene of the filamentous fungus P. anserina is the first example of a prion necessary for a normal cellular function, the heterokaryon incompatibility reaction used by most fungi to avoid the spread of viruses.