Prions of Yeast: From Cytoplasmic Genes to Heritable Amyloidosis Part 1

Introduction

It was believed that only proteins could carry out enzymatic reactions, and only nucleic acids could mediate inheritance. In recent years, the work of Cech and Altman and others has shown that nucleic acids can catalyze reactions. Now it has been shown that, in yeast, proteins can mediate inheritance.

The infectious protein (prion) concept arose from studies of the transmissible spongiform encephalopathies (TSEs) of mammals (1), and several lines of evidence suggest that TSEs are indeed caused by infectious forms of the PrP protein, but the absence of definitive proof has left substantial doubt and disagreement on this point (2-6). The ease of genetic manipulation of yeast offers experimental possibilities not yet available even in the mouse system. This enabled the discovery of yeast prions (7), and has facilitated the rapid characterization of these systems. The parallels between the yeast and mammalian systems are striking. Moreover, because both of the yeast prion systems appear to involve self-propagating amyloid forms of the respective proteins, these systems may also serve as models for the broader class of diseases for which amyloid accumulation is a central feature. The discovery of the [HET-s] prion of the filamentous fungus Podospora, another genetically manipulable system, adds a new dimension to prion studies (8).

Genetics is not only another useful tool in these studies, but is the essential component for the study of prions. Genetic criteria for prions of yeast, and specific genetic experiments designed to rule out alternative hypotheses, havemade a convincing case that the yeast elements [URE3] and [PSI] are prions. Biochemical studies have now begun to bring evidence for the mechanisms that underlie these prions.

Many reviews of yeast and fungal prions have appeared recently, and the reader may consult these for different emphasis in coverage and different views of the subject (9-13).

Genetic Criteria for Prions

We use the word ‘prion’ to mean a protein that is infectious, by whatever mechanism. Infectious proteins of yeasts and filamentous fungi should therefore be infectious in a similar way to viruses of these organisms. Perhaps because in nature yeast mate frequently, yeast viruses are infectious only via this mating process, and yet they are widely distributed in natural isolates. When an infected cell mates with an uninfected cell, all of the meiotic and mitotic progeny are infected. Thus, the virus appears as a nonchromosomal gene (a non-Mendelian genetic element). Yeast viruses do not pass out of one cell and enter another. The thick wall of yeast cells may contribute to this fact, but the even thicker plant cell walls do not prevent extracellular spread of plant viruses. Viruses of filamentous fungi likewise only spread via cell-to-cell fusion, and are strictly intracellular entities. Infectious proteins (prions) would thus be expected to likewise appear as nonchromosomal genetic elements (7).

Among nonchromosomal genetic elements, three genetic properties were proposed that should distinguish those resulting from nucleic acid replicons, such as viruses or plasmids, from prions (Fig. 1; see ref. 7).

Reversible Curability

If a prion can be cured from a strain, it should be possible to find a rare subclone of the cured strain in which the prion has again arisen spontaneously. The change that makes a protein infectious should occur with some low frequency in any sufficiently large population of the normal form of the molecule. In contrast, curing a plasmid (such as the yeast 2-^ DNA plasmid) or a virus (such as the killer virus) is an irreversible event. Once the virus or plasmid has been cured, it will not arise spontaneously, but can only reappear by being reintroduced from another cell (Fig. 1, top).

Overproduction of Protein Increases the Frequency with Which the Prion Arises

Whatever the mechanism by which prions arise, it is expected that increasing the amount of the normal form should increase the number of molecules that have the potential to undergo the prion change, and thus the frequency with which the prion formation event occurs (Fig. 1, middle). In contrast, there is no chromosome-encoded protein whose overproduction will induce a nucleic acid replicon to arise de novo.

Fig. 1. Genetic criteria for a prion. To distinguish cytoplasmic genetic elements due to a nucleic acid replicon (and a chromosomal gene needed for its replication) from a prion (and the chromosomal gene encoding the protein), three genetic criteria were proposed (7). (Top): Curing [URE3] with guanidine produces cells from which [URE3] can again arise at frequencies similar to that at which it arose from the wild-type ancestor. (Middle) Overproducing Ure2p increases the frequency with which [URE3] arises. (Bottom) The phenotype of ure2 mutants is the same as that of [URE3] strains because both phenotypes arise from deficiency of normal Ure2p; and URE2 is necessary for propagation of [URE3].

Phenotype Relationship of the Prion and a Prion Maintenance Gene

If the prion causes cellular pathology because the normal form is converted to an abnormal nonfunctional form, then the deficiency of the normal form should produce the same phenotype as that produced by mutation of the chromosomal gene for the normal form (Fig. 1, bottom). Furthermore, this chromosomal gene for the normal form should be necessary for the propagation of the prion. Thus, a prion would have, as one of the chromosomal genes needed for its propagation, one whose mutant phenotype was the same as that produced by the presence of the prion. This is not the relationship between a nucleic acid replicon and the chromosomal genes needed for its propagation (Table 1). For example, the mitochondrial DNA encodes proteins necessary for utilization of glycerol, a nonfermentable carbon source. Cells carrying mitDNA can grow on glycerol, but mutants in the chromosomal gene for the DNA polymerase responsible for replicating mitDNA lose mitDNA, and cannot grow on glyc-erol, which is the opposite phenotype.

TSEs Do Not Yet Fulfill Genetic Criteria for a Prion

None of the TSEs can yet be cured, so reversible curing is not even testable. Transgenic mice overproducing PrP have been constructed, and these mice do become sick and die as a result, but their tissues have not been shown to contain infectious material. That is, the overproduction of PrP is not, in these mice, giving rise to an infectious entity (14). PrP is necessary for the propagation of the scrapie agent (15), but the phenotype of deletion of the PRNP gene (16) is not at all like that of scrapie. This result is consistent with scrapie being caused by a virus or other nucleic acid replicon, but it does not rule out scrapie being a prion. The prion form of PrP presumably has a positively harmful effect on cells. Thus, the yeast prions were established, based on evidence of a type not yet available for the mammalian TSEs.

[URE3] Is a Nonchromosomal Genetic Element Affecting Regulation of Nitrogen Catabolism

When presented with a rich nitrogen source, such as ammonia or glutamine, yeast represses the transcription of genes whose products are involved in the utilization of poor nitrogen sources, such as allantoate, a degradation product of purine metabolism (Fig. 2; 17,18). The chance resemblance of allantoate to ureidosuccinate, an intermediate in uracil biosynthesis, results in uptake of ureidosuccinate being controlled by the nitrogen regulation system (19,20).

Lacroute et al. isolated mutants able to take up ureidosuccinate on media containing ammonia (21-25). These mutants defined two chromosomal genes, urel and ure2, and a nonchromosomal genetic element, [URE3]. Although the urel and ure2 mutants were recessive or partially recessive, [URE3] was dominant, and could be transferred from cell to cell by cytoplasmic mixing (25). The molecular basis of [URE3] was unclear, and studies of [URE3] lapsed for many years. However, the role of Ure2p in nitrogen regulation was found to be its negative regulation of the activity of Gln3p, a positive transcription regulator necessary for transcription of many of the genes regulated by this system (Fig. 2; 26-28).

Table 1

Relation of Phenotypes of Nucleic Acid Replicon, Prion and Chromosomal Genes Needed for Their Propagation

		Phenotypes
Non-Mendelian element	Presence of non-Mendelian element	Chromosomal mutant that loses the element		Does replacing the chromosomal mutant gene restore the phenotype?
			Relation
M dsRNA	Killer +	Killer -	Opposite	No
mitDNA	Glycerol +	Glycerol -	Opposite	No
mitDNA-DI	Glycerol -	Glycerol -	Same	No
Prion	Defective	Defective	Same	Yes
[URE3]	USA uptake +	USA uptake +	Same	Yes
[PSI]			Same	Yes

Since either the presence of the prion or mutation in the gene for the protein result in deficiency of the normal protein, any phenotype resulting from the absence of this normal protein will be similar in these two conditions. The chromosomal gene for the protein is also required for propagation of the prion. Usually, the presence of a non-chromosomal genetic element produces the phenotype opposite that of mutation of a chromosomal gene needed for its propagation. One exception is a mutant of the non-chromosomal nucleic acid replicon that makes its presence known by elimination of its normal parent (like a defective – interfering [DI] virus). This can be distinguished from a prion by replacing the chromosomal maintenance gene, which will restore the phenotype to normal, in the case of the prion, but not in the case of the mutant nucleic acid replicon.

Since either the presence of the prion or mutation in the gene for the protein result in deficiency of the normal protein, any phenotype resulting from the absence of this normal protein will be similar in these two conditions. The chromosomal gene for the protein is also required for propagation of the prion. Usually, the presence of a non-chromosomal genetic element produces the phenotype opposite that of mutation of a chromosomal gene needed for its propagation. One exception is a mutant of the non-chromosomal nucleic acid replicon that makes its presence known by elimination of its normal parent (like a defective – interfering [DI] virus). This can be distinguished from a prion by replacing the chromosomal maintenance gene, which will restore the phenotype to normal, in the case of the prion, but not in the case of the mutant nucleic acid replicon.

Recently, we have found evidence that the ammonia signal is transmitted to Ure2p by Mks1p (Fig. 2; 29). Ure2p is present in the cytoplasm of cells grown on either a rich or poor nitrogen source (29). Ure2p and Gln3p form a cytoplasmic complex (30). There is also evidence that the Ure2p-Gln3p pathway is regulated by the target of rapamycin (TOR) kinases, two protein kinases whose activity is inhibited by the antineoplastic drug, rapamycin (30-32). Rapamycin treatment, by affecting the TOR kinases, affects the phosphorylation of Gln3p, thereby dissociating the Ure2p-Gln3p complex, and allowing Gln3p to enter the nucleus where it can promote transcription of many genes for using poor nitrogen sources (30). Ure2p itself is phosphorylated in a TOR-dependent reaction in response to rapamycin treatment (31,32). It remains unclear whether the TOR pathway mediates the turn-on of nitrogen assimilation genes induced by poor nitrogen sources, or if this is a different signal transduction pathway that feeds into Ure2p and Gln3p.

Fig. 2. Ure2p and regulation of nitrogen catabolism. Ure2p receives a signal of abundant ammonia supplies through Mks1p, and passes this signal to Gln3p, preventing it from activating transcription of genes encoding proteins for utilization of poor nitrogen sources.

[PSI], a Nonchromosomal Genetic Element Affecting Translation Termination

In 1965, Cox discovered a nonchromosomal genetic element that increased the efficiency with which a weak tRNA suppressor, called SUQ5, allowed readthrough of a nonsense mutation (UAA, the "ochre" termination codon) in the ade2 gene (33). He named this nonchromosomal element [PSI], perhaps in preparation for naming mutants unable to propagate (PSI) as PNM for "PSI no more" (34). Efforts to identify [PSI] with any of the known nonchromosomal replicons were unsuccessful (35,36); reviewed in ref. 37, and the molecular nature of both [PSI] and [URE3] were long-standing mysteries in the yeast world.

In apparently unrelated work, omnipotent suppressor mutations, recessive chromosomal mutations resulting in elevated readthrough of nonsense mutations, were found to define two genes, SUP35 and SUP45 (38,39). Recent studies have shown that Sup35p and Sup45p are the subunits of the translation release factor that recognizes the termination codon on the mRNA, and releases the completed peptide from the last tRNA (Fig. 3; 40,41). Suppressor tRNAs have a mutated anticodon that recognizes a termination codon and results in insertion of an amino acid, instead of chain termination. This type of suppres-sion event is always in competition with the translation release factor, so sup35 or sup45 mutations are expected to increase the frequency of suppression by suppressor tRNAs.

Fig. 3. Sup35p is a subunit of the translation termination factor. Mutant tRNAs that read termination codons (suppressor tRNAs) compete with the normal translation termination factor. Therefore, mutants in sup35, or an abnormal (prion) form of Sup35p, should allow suppressor tRNAs to be more efficient.

[URE3] Satisfies the Genetic Criteria for a Prion of Ure2p

Growing cells in the presence of millimolar concentrations of guanidine results in efficient curing of [URE3] (7,37). When cells are cured in this manner, one may isolated derivatives that have again become [URE3] (7). Thus, [URE3] is reversibly curable. Overproduction of Ure2p increases by 20-200-fold, the frequency with which [URE3] arises de novo (7). Finally, the ure2 mutants and [URE3] strains have the same phenotype, and ure2 mutants are unable to propagate the [URE3] nonchromosomal genetic element (7,25). Thus, [URE3] satisfies all three genetic criteria as a prion of Ure2p.

[PSI] Satisifies the Genetic Criteria for a Prion of Sup35p

Growth of [PSI]-containing cells in high-osmotic-strength media results in efficient curing (42). From cells cured in this manner can again be isolated cells that have acquired [PSI] (43). The frequency of de novo appearance of [PSI] is increased 100-fold by the overproduction of Sup35p (44). Finally, the phenotype of sup35 mutants and PSI strains is essentially the same, and sup35 mutants are unable to propagate PSI (45,46). Thus, PSI satisfies the three genetic criteria as a prion of Sup35p. The clear parallel between the evidence in these two systems suggested that they were both prions particularly convincingly (7). These three characteristics are each evidence not only against a nucleic acid replicon, but also evidence specifically for a prion. Various other epigenetic phenomena are known to show the equivalent of reversible curability, but none of those are nonchromosomal genetic elements.

Prion Domains of Ure2p AND Sup35p Are Rich in Asparagine and Glutamine

Deletion analysis of the URE2 gene showed that the N-terminal 65 amino acid residues were sufficient to induce [URE3] formation (47). The same region could stably propagate [URE3] in the complete absence of the C-terminal part of the molecule (48). More detailed examination showed that extending this region to residues 1-80 increased the efficiency of prion induction (49). This region is rich in asparagine residues, with several runs, and is relatively rich in serine and threonine as well (Fig. 4). Deletion of any of the asparagine-rich regions dramatically reduced the prion-inducing activity of the remaining protein (49).

The C-terminal part of Ure2p is sufficient to perform its nitrogen regulation function (28,47), although this function is mildly impaired unless the protein is either overproduced or includes the N-terminal prion domain. The C-terminal nitrogen regulation domain is not functionally affected by [URE3], unless the prion domain is covalently attached (48). Deletions in parts of the C-terminal domain result in a dramatic 100-fold increase in the efficiency with which the remainder of the molecule induces [URE3] (47), suggesting that the C-terminal part of Ure2p stabilizes the prion domain, preventing it from undergoing the prion change.

Although the predominant role in [URE3] prion generation and propagation is played by the N-terminal 80 residues of Ure2p, deletion of residues 221-227 within the C-terminal region did not affect nitrogen regulation activity, but did eliminate prion-inducing activity of the otherwise intact overproduced Ure2p(49). This region has no Asn or Gln residues, and is, of course, dispensable for prion-inducing activity by Ure2 fragments with further deletions.

A further indication of the complexity of prion-inducing activity is the finding that two nonoverlapping fragments of Ure2p are each capable of inducing the high-frequency appearance of [URE3] (49). Although a fragment lacking the first defined prion domain (residues 1-65) is inactive in prion induction, further deletion of the prion-inhibiting regions 151-157 and 348-354 produces a fragment that is now able to induce [URE3] (49). If either of the positive segments, 66-80 or 221-227, are deleted from this fragment, this activity is again lost (49). Clearly, prion-inducing activity is a complex process involving both intramolecular and intermolecular interactions. The prion-promoting and prion-inhibiting activities of regions of Ure2p are summarized in Figure 4.

Deletion analysis of Sup35p showed that its N-terminal 123 amino acid residues (the N domain) are critical for prion propagation and generation (46,50,51). The C-terminal residues, 254-685, are essential for cell growth (50) and is the region with homology to EF-1 a (52-54). Deletion of this region makes a strain unable to propagate [PSI] (46), and overproduction of this region induces the de novo appearance of [PSI] (51). Indeed, the frequency of [PSI] prion generation by this prion domain alone is 66-fold greater than that by similar overproduction of the full-length protein, suggesting, as for Ure2p and [URE3] generation, that the C-terminal domain stabilizes the N-terminal prion domain in the nonprion form (55).

Fig. 4. Prion domains of Ure2p, Sup35p, and HET-s. Prion-promoting and -inhibiting domains are indicated. Two nonoverlapping fragments of either Ure2p or HET-s can induce or propagate the prion forms (49,83). The prion domains of Sup35p were determined by TerAvanesyan et al. (46).

The prion domain of Sup35p, like that of Ure2p, is rich in glutamine and asparagine residues. It also has seven copies of an imperfect repeat sequence, QGGYQQQYNP (Fig. 4). Mutagenesis of this region has shown that the glutamines are important for prion generation and propagation, and that the N-terminal part of the prion domain may be replaced with poly(glutamine), without loss of prion activity (34,45,46,56), suggesting a relation with the Gln repeats of some triplet-repeat diseases. In addition, the repeat region contributes to prion activity, with deletions reducing activity, and increasing the number of repeats, increasing prion activity (46,57).

The significance of the repeats of Sup35p, and their parallel to similar repeats in the N-terminal region of PrP, is somewhat uncertain. It is clear that the Sup35p repeat region is involved in prion generation, because it is both necessary for, and its expansion can increase, prion effects. Human mutants, with increased numbers of repeats in PrP, are among those with hereditary Creutzfeldt-Jakob disease (58), but deletion of the repeat region does not interfere with scrapie propagation in mice (59).