Yeast and Fungal Prions: Amyloid-Handling Systems, Amyloid Structure, and Prion Biology.

Yeast prions (infectious proteins) were discovered by their outré genetic properties and have become important models for an array of human prion and amyloid diseases. A single prion protein can become any of many distinct amyloid forms (called prion variants or strains), each of which is self-propagating, but with different biological properties (eg, lethal vs mild). The folded in-register parallel ß sheet architecture of the yeast prion amyloids naturally suggests a mechanism by which prion variant information can be faithfully transmitted for many generations. The yeast prions rely on cellular chaperones for their propagation, but can be cured by various chaperone imbalances. The Btn2/Cur1 system normally cures most variants of the [URE3] prion that arise. Although most variants of the [PSI+] and [URE3] prions are toxic or lethal, some are mild in their effects. Even the most mild forms of these prions are rare in the wild, indicating that they too are detrimental to yeast. The beneficial [Het-s] prion of Podospora anserina poses an important contrast in its structure, biology, and evolution to the yeast prions characterized thus far.

Protein-based inheritance in Saccharomyces cerevisiae: [URE3] as a prion form of the nitrogen regulatory protein Ure2.

The [URE3] element of the yeast Saccharomyces cerevisiae results from the presence of an altered form of the nitrogen regulatory protein Ure2. This altered form acts as an infectious protein (prion). Genes affecting [URE3] initiation and propagation should give valuable information about prion diseases as well as other conformational diseases.

Prion filament networks in [URE3] cells of Saccharomyces cerevisiae.

The [URE3] prion (infectious protein) of yeast is a self-propagating, altered form of Ure2p that cannot carry out its normal function in nitrogen regulation. Previous data have shown that Ure2p can form protease-resistant amyloid filaments in vitro, and that it is aggregated in cells carrying the [URE3] prion. Here we show by electron microscopy that [URE3] cells overexpressing Ure2p contain distinctive, filamentous networks in their cytoplasm, and demonstrate by immunolabeling that these networks contain Ure2p. In contrast, overexpressing wild-type cells show a variety of Ure2p distributions: usually, the protein is dispersed sparsely throughout the cytoplasm, although occasionally it is found in multiple small, focal aggregates. However, these distributions do not resemble the single, large networks seen in [URE3] cells, nor do the control cells exhibit cytoplasmic filaments. In [URE3] cell extracts, Ure2p is present in aggregates that are only partially solubilized by boiling in SDS and urea. In these aggregates, the NH(2)-terminal prion domain is inaccessible to antibodies, whereas the COOH-terminal nitrogen regulation domain is accessible. This finding is consistent with the proposal that the prion domains stack to form the filament backbone, which is surrounded by the COOH-terminal domains. These observations support and further specify the concept of the [URE3] prion as a self-propagating amyloid.

Prions of yeast from cytoplasmic genes to heritable amyloidosis.

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).

[URE3] prion propagation in Saccharomyces cerevisiae: requirement for chaperone Hsp104 and curing by overexpressed chaperone Ydj1p.

The [URE3] nonchromosomal genetic element is an infectious form (prion) of the Ure2 protein, apparently a self-propagating amyloidosis. We find that an insertion mutation or deletion of HSP104 results in inability to propagate the [URE3] prion. Our results indicate that Hsp104 is a common factor in the maintenance of two independent yeast prions. However, overproduction of Hsp104 does not affect the stability of [URE3], in contrast to what is found for the [PSI(+)] prion, which is known to be cured by either overproduction or deficiency of Hsp104. Like Hsp104, the Hsp40 class chaperone Ydj1p, with the Hsp70 class Ssa1p, can renature proteins. We find that overproduction of Ydj1p results in a gradual complete loss of [URE3]. The involvement of protein chaperones in the propagation of [URE3] indicates a role for protein conformation in inheritance.

Prions of yeast as heritable amyloidoses.

Two infectious proteins (prions) of Saccharomyces cerevisiae have been identified by their unusual genetic properties: (1) reversible curability, (2) de novo induction of the infectious prion form by overproduction of the protein, and (3) similar phenotype of the prion and mutation in the chromosomal gene encoding the protein. [URE3] is an altered infectious form of the Ure2 protein, a regulator of nitrogen catabolism, while [PSI] is a prion of the Sup35 protein, a subunit of the translation termination factor. The altered form of each is inactive in its normal function, but is able to convert the corresponding normal protein into the same altered inactive state. The N-terminal parts of Ure2p and Sup35p (the "prion domains") are responsible for prion formation and propagation and are rich in asparagine and glutamine residues. Ure2p and Sup35p are aggregated in vivo in [URE3]- and [PSI]-containing cells, respectively. The prion domains can form amyloid in vitro, suggesting that amyloid formation is the basis of these two prion diseases. Yeast prions can be cured by growth on millimolar concentrations of guanidine. An excess or deficiency of the chaperone Hsp104 cures the [PSI] prion. Overexpression of fragments of Ure2p or certain fusion proteins leads to curing of [URE3].

A protein required for prion generation: [URE3] induction requires the Ras-regulated Mks1 protein.

Infectious proteins (prions) can arise de novo as well as by transmission from another individual. De novo prion generation is believed responsible for most cases of Creutzfeldt-Jakob disease and for initiating the mad cow disease epidemic. However, the cellular components needed for prion generation have not been identified in any system. The [URE3] prion of Saccharomyces cerevisiae is an infectious form of Ure2p, apparently a self-propagating amyloid. We now demonstrate a protein required for de novo prion generation. Mks1p negatively regulates Ure2p and is itself negatively regulated by the presence of ammonia and by the Ras-cAMP pathway. We find that in mks1Delta strains, de novo generation of the [URE3] prion is blocked, although [URE3] introduced from another strain is expressed and propagates stably. Ras2(Val19) increases cAMP production and also blocks [URE3] generation. These results emphasize the distinction between prion generation and propagation, and they show that cellular regulatory mechanisms can critically affect prion generation.

Prions: Portable prion domains.

Self-propagating abnormal proteins, prions, have been identified in yeast; asparagine/glutamine-rich 'prion domains' within these proteins can inactivate the linked functional domains; new prion domains and reporters have been used to make 'synthetic prions', leading to discoveries of new natural prions.

[URE3] and [PSI] are prions of yeast and evidence for new fungal prions.

[URE3] and [PSI] are two non-Mendelian genetic elements discovered over 25 years ago and never assigned to a nucleic acid replicon. Their genetic properties led us to propose that they are prions, altered self-propagating forms of Ure2p and Sup35p, respectively, that cannot properly carry out the normal functions of these proteins. Ure2p is partially protease-resistant in [URE3] strains and Sup35p is aggregated specifically in [PSI] strains supporting this idea. Overexpression of Hsp104 cures [PSI], as does the absence of this protein, suggesting that the prion change of Sup35p in [PSI] strains is aggregation. Strains of [PSI], analogous to those described for scrapie, have now been described as well as an in vitro system for [PSI] propagation. Recently, two new potential prions have been described, one in yeast and the other in the filamentous fungus, Podospora.

Prions in Saccharomyces and Podospora spp.: protein-based inheritance.

Genetic evidence showed two non-Mendelian genetic elements of Saccharomyces cerevisiae, called [URE3] and [PSI], to be prions of Ure2p and Sup35p, respectively. [URE3] makes cells derepressed for nitrogen catabolism, while [PSI] elevates the efficiency of weak suppressor tRNAs. The same approach led to identification of the non-Mendelian element [Het-s] of the filamentous fungus Podospora anserina, as a prion of the het-s protein. The prion form of the het-s protein is required for heterokaryon incompatibility, a normal fungal function, suggesting that other normal cellular functions may be controlled by prions. [URE3] and [PSI] involve a self-propagating aggregation of Ure2p and Sup35p, respectively. In vitro, Ure2p and Sup35p form amyloid, a filamentous protein structure, high in beta-sheet with a characteristic green birefringent staining by the dye Congo Red. Amyloid deposits are a cardinal feature of Alzheimer's disease, non-insulin-dependent diabetes mellitus, the transmissible spongiform encephalopathies, and many other diseases. The prion domain of Ure2p consists of Asn-rich residues 1 to 80, but two nonoverlapping fragments of the molecule can, when overproduced, induce the de nova appearance of [URE3]. The prion domain of Sup35 consists of residues 1 to 114, also rich in Asn and Gln residues. While runs of Asn and Gln are important for [URE3] and [PSI], no such structures are found in PrP or the Het-s protein. Either elevated or depressed levels of the chaperone Hsp104 interfere with propagation of [PSI]. Both [URE3] and [PSI] are cured by growth of cells in millimolar guanidine HCl. [URE3] is also cured by overexpression of fragments of Ure2p or fusion proteins including parts of Ure2p.

Mks1p is a regulator of nitrogen catabolism upstream of Ure2p in Saccharomyces cerevisiae.

The supply of nitrogen regulates yeast genes affecting nitrogen catabolism, pseudohyphal growth, and meiotic sporulation. Ure2p of Saccharomyces cerevisiae is a negative regulator of nitrogen catabolism that inhibits Gln3p, a positive regulator of DAL5, and other genes of nitrogen assimilation. Dal5p, the allantoate permease, allows ureidosuccinate uptake (Usa(+)) when cells grow on a poor nitrogen source such as proline. We find that overproduction of Mks1p allows uptake of ureidosuccinate on ammonia and lack of Mks1p prevents uptake of ureidosuccinate or Dal5p expression on proline. Overexpression of Mks1p does not affect cellular levels of Ure2p. An mks1 ure2 double mutant can take up ureidosuccinate on either ammonia or proline. Moreover, overexpression of Ure2p suppresses the ability of Mks1p overexpression to allow ureidosuccinate uptake on ammonia. These results suggest that Mks1p is involved in nitrogen control upstream of Ure2p as follows: NH(3) dash, vertical Mks1p dash, vertical Ure2p dash, vertical Gln3p --> DAL5. Either overproduction of Mks1p or deletion of MKS1 interferes with pseudohyphal growth.

The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments.

The [URE3] nonchromosomal genetic element is a prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. Ure2p1-65 is the prion domain of Ure2p, sufficient to propagate [URE3] in vivo. We show that full length Ure2p-green fluorescent protein (GFP) or a Ure2p1-65-GFP fusion protein is aggregated in cells carrying [URE3] but is evenly distributed in cells lacking the [URE3] prion. This indicates that [URE3] involves a self-propagating aggregation of Ure2p. Overexpression of Ure2p1-65 induces the de novo appearance of [URE3] by 1,000-fold in a strain initially [ure-o], but cures [URE3] from a strain initially carrying the [URE3] prion. Overexpression of several other fragments of Ure2p or Ure2-GFP fusion proteins also efficiently cures the prion. We suggest that incorporation of fragments or fusion proteins into a putative [URE3] "crystal" of Ure2p poisons its propagation.

Mak21p of Saccharomyces cerevisiae, a homolog of human CAATT-binding protein, is essential for 60 S ribosomal subunit biogenesis.

Mak21-1 mutants are unable to propagate M1 double-stranded RNA, a satellite of the L-A double-stranded RNA virus, encoding a secreted protein toxin lethal to yeast strains that do not carry M1. We cloned MAK21 using its map location and found that Mak21p is homologous to a human and mouse CAATT-binding protein and open reading frames in Schizosaccharomyces pombe and Caenorhabditis elegans. Although the human protein regulates Hsp70 production, Mak21p is essential for growth and necessary for 60 S ribosomal subunit biogenesis. mak21-1 mutants have decreased levels of L-A coat protein and L-A double-stranded RNA. Electroporation with reporter mRNAs shows that mak21-1 cells cannot optimally express mRNAs which, like L-A viral mRNA, lack 3'-poly(A) or 5'-cap structures but can normally express mRNA with both cap and poly(A). The virus propagation phenotype of mak21-1 is suppressed by ski2 or ski6 mutations, each of which derepresses translation of non-poly(A) mRNA.

Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus.

The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA. Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression. A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron. Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA. Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I. The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present. This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism. Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV.

Efficient translation of distal cistrons of a polycistronic mRNA of a plant pararetrovirus requires a compatible interaction between the mRNA 3' end and the proteinaceous trans-activator.

Caulimoviruses, a type of plant pararetrovirus, employ a highly unusual mechanism to express the multiple cistrons of their pregenomic RNA. It involves translation of a polycistronic mRNA utilizing cis-acting viral RNA sequences and a transacting virus-encoded protein (P6). In addition to its role in polycistronic translation, the translational trans-activator protein P6 also activates its own expression from a monocistronic subgenomic RNA. Using Nicotiana Edwardsonii cell suspension protoplasts, we analyzed the ability of P6 proteins from three different caulimoviruses to activate viral RNA-based reporter constructs. Cis-acting elements present in figwort mosaic caulimovirus (FMV) are functional not only in the presence of the cognate P6 activator protein, but also in the presence of the heterologous activators from cauliflower mosaic caulimovirus (CaMV) and peanut chlorotic streak caulimovirus (PCISV). However, when 3' cis-acting elements essential for efficient polycistronic expression of FMV are replaced by their counterparts from PCISV, reporter gene expression is only observed in the presence of PCISV P6. Derepression of monocistronic reporter constructs tailed with FMV or CaMV 3' proximal sequences is less efficient in the presence of PCISV P6 than with either FMV or CaMV P6, but more efficient when the constructs contain a cognate PCISV 3' cis-element. Efficient expression of polycistronic and monocistronic caulimovirus mRNAs in plant cells thus requires compatible interactions between P6, a translational trans-activator, and its cognate cis-element at the 3' end of the mRNA.