Prion generation in vitro: amyloid of Ure2p is infectious

[URE3] is a prion (infectious protein) of the Ure2 protein of yeast. In vitro, Ure2p can form amyloid filaments, but direct evidence that these filaments constitute the infectious form is still missing. Here we demonstrate that recombinant Ure2p converted into amyloid can infect yeast cells lacking the prion. Infection produced a variety of [URE3] variants. Extracts of [URE3] strains, as well as amyloid of Ure2p formed in an extract-primed reaction could transmit to uninfected cells the [URE3] variant present in the cells from which the extracts were made. Infectivity and determinant of [URE3] variants resided within the N-terminal 65 amino acids of Ure2p. Notably, we could show that amyloid filaments of recombinant Ure2p are nearly as infectious per mass of Ure2p as extracts of [URE3] strains. Sizing experiments indicated that infectious particles in vitro and in vivo were >20 nm in diameter, suggesting that they were amyloid filaments and not smaller oligomeric structures. Our data indicate that there is no substantial difference between filaments formed in vivo and in vitro.     

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.     

Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p

The [URE3] prion of Saccharomyces cerevisiae shares many features with mammalian prions and poly-glutamine related disorders and has become a model for studying amyloid diseases. The development of the [URE3] phenotype is thought to be caused by a structural switch in the Ure2p protein. In [URE3] cells, Ure2p is found predominantly in an aggregated state, while it is a soluble dimer in wild-type cells. In vitro, Ure2p forms fibrils with amyloid-like properties. Several studies suggest that the N-terminal domain of Ure2p is essential for prion formation. In this work, we investigated the fibril formation of Ure2p by isolating soluble oligomeric species, which are generated during fibrillization, and characterized them with respect to size and structure. Our data support the critical role of the N-terminal domain for fibril formation, as we observed fibrils in the presence of 5 M guanidinium chloride, conditions at which the C-terminal domain is completely unfolded. Based on fluorescence measurements, we conclude that the structure of the C-terminal domain is very similar in dimeric and fibrillar Ure2p. When studying the time course of fibrillization, we detected the formation of small, soluble oligomeric species during the early stages of the process. Their remarkable resistance against denaturants, their increased content of beta-structure, and their ability to 'seed' Ure2p fibrillization suggest that conversion to the amyloid-like conformation has already occurred. Thus, they likely represent critical intermediates in the fibrillization pathway of Ure2p.     

Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.     

The mechanisms of [URE3] prion elimination demonstrate that large aggregates of Ure2p are dead-end products

The yeast prion [URE3] is a self-propagating inactive form (the propagon) of the Ure2 protein. Ure2p is composed of two domains: residues 1-93--the prion-forming domain (PFD)--and the remaining C-terminal part of the protein, which forms the functional domain involved in nitrogen catabolite repression. Guanidine hydrochloride, and the overproduction of Ure2p 1-65 or Ure2-GFP have been shown to induce the elimination of [URE3]. We demonstrate here, two different curing mechanisms: the inhibition of [URE3] replication by guanidine hydrochloride and its destruction by Ure2p aggregation. Such aggregation is observed if PFD or Ure2-GFP are overproduced and in heterozygous URE2/URE2-GFP, [URE3] diploids. We found that the GFP foci associated with the presence of the prion were dead-end products, the propagons remaining soluble. Surprisingly, [URE3] propagated via the Ure2-GFP fusion protein alone is resistant to these two curing mechanisms and cannot promote the formation of foci. The relationship between aggregation, prion and Hsp104 gives rise to a model in which the propagon is in equilibrium with larger aggregates and functional protein.     

Scrambled prion domains form prions and amyloid

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The amino-terminal prion domain of Ure2p is necessary and sufficient for prion formation and has a high glutamine (Q) and asparagine (N) content. Such Q/N-rich domains are found in two other yeast prion proteins, Sup35p and Rnq1p, although none of the many other yeast Q/N-rich domain proteins have yet been found to be prions. To examine the role of amino acid sequence composition in prion formation, we used Ure2p as a model system and generated five Ure2p variants in which the order of the amino acids in the prion domain was randomly shuffled while keeping the amino acid composition and C-terminal domain unchanged. Surprisingly, all five formed prions in vivo, with a range of frequencies and stabilities, and the prion domains of all five readily formed amyloid fibers in vitro. Although it is unclear whether other amyloid-forming proteins would be equally resistant to scrambling, this result demonstrates that [URE3] formation is driven primarily by amino acid composition, largely independent of primary sequence.     

Ure2p function is enhanced by its prion domain in Saccharomyces cerevisiae

The Ure2 protein of Saccharomyces cerevisiae can become a prion (infectious protein). At very low frequencies Ure2p forms an insoluble, infectious amyloid known as [URE3], which is efficiently transmitted to progeny cells or mating partners that consequently lose the normal Ure2p nitrogen regulatory function. The [URE3] prion causes yeast cells to grow slowly, has never been identified in the wild, and confers no obvious phenotypic advantage. An N-terminal asparagine-rich domain determines Ure2p prion-forming ability. Since ure2Delta strains are complemented by plasmids that overexpress truncated forms of Ure2p lacking the prion domain, the existence of the [URE3] prion and the evolutionary conservation of an N-terminal extension have remained mysteries. We find that Ure2p function is actually compromised in vivo by truncation of the prion domain. Moreover, Ure2p stability is diminished without the full-length prion domain. Mca1p, like Ure2p, has an N-terminal Q/N-rich domain whose deletion reduces its steady-state levels. Finally, we demonstrate that the prion domain may affect the interaction of Ure2p with other components of the nitrogen regulation system, specifically the negative regulator of nitrogen catabolic genes, Gzf3p.     

Is the prion domain of soluble Ure2p unstructured?

The [URE3] prion is a self-propagating amyloid form of the Ure2 protein of Saccharomyces cerevisiae. Deletions in the C-terminal nitrogen regulation domain of Ure2p increase the frequency with which the N-terminal prion domain polymerizes into the prion form, suggesting that the C-terminus stabilizes the prion domain or that the structured C-terminal region sterically impairs amyloid formation. We find by in vivo two-hybrid analysis no evidence of interaction of prion domain and C-terminal domain. Furthermore, surface plasmon resonance spectrometry shows no evidence of interaction of prion domain and C-terminal domain, and cleavage at a specific site between the domains frees the two fragments. Our NMR analysis indicates that most residues of the prion domain are in fact disordered in the soluble form of Ure2p. Deleting the tether holding the C-terminal structured region to the amyloid core does not impair prion formation, arguing against steric impairment of amyloid formation. These results suggest that the N-terminal prion domain is unstructured in the soluble protein and does not have a specific interaction with the C-terminus.     

Structure of the prion Ure2p in protein fibrils assembled in vitro

The Ure2 protein from the yeast Saccharomyces cerevisiae has prion properties. In vitro and at neutral pH, soluble Ure2p spontaneously forms long, straight, insoluble protein fibrils. Two models have been proposed to account for the assembly of Ure2p into protein fibrils. The "amyloid backbone" model postulates that a segment ranging from 40 to 70 amino acids in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated beta-structure running along the fibrils. The second model hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and/or intramolecular interactions between Ure2p monomers. Here, we performed a cysteine scan on residues located in the N- and C-terminal parts of Ure2p to determine whether these domains interact. Amino acid sequences centered around residue 6 in the N-terminal domain of Ure2p and residue 137 in the C-terminal moiety interacted at least transiently via intramolecular interactions. We documented the assembly properties of a Ure2p variant in which a disulfide bond was established between the N- and C-terminal domains and showed that it possesses assembly properties indistinguishable from those of wild-type Ure2p. We probed the structure of Ure2pC6C137 within the fibrils and demonstrate that the polypeptide is in a conformation similar to that of its soluble assembly-competent state. Our results constitute the first structural characterization of the N-terminal domain of Ure2p in both its soluble assembly-competent and fibrillar forms. Our data indicate that the flexibility of the N-terminal domain and conformational changes within this domain are essential for fibril formation and provide new insight into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] phenotype in yeast.     

Internal initiation drives the synthesis of Ure2 protein lacking the prion domain and affects [URE3] propagation in yeast cells

The [URE3] phenotype in Saccharomyces cerevisiae is caused by the inactive, altered (prion) form of the Ure2 protein (Ure2p), a regulator of nitrogen catabolism. Ure2p has two functional domains: an N-terminal domain necessary and sufficient for prion propagation and a C-terminal domain responsible for nitrogen regulation. We show here that the mRNA encoding Ure2p possesses an IRES (internal ribosome entry site). Internal initiation leads to the synthesis of an N-terminally truncated active form of the protein (amino acids 94-354) lacking the prion-forming domain. Expression of the truncated Ure2p form (94-354) mediated by the IRES element cures yeast cells of the [URE3] phenotype. We assume that the balance between the full-length and truncated (94-354) Ure2p forms plays an important role in yeast cell physiology and differentiation.     

In vitro analysis of SpUre2p, a prion-related protein, exemplifies the relationship between amyloid and prion

The yeast Saccharomyces cerevisiae contains in its proteome at least three prion proteins. These proteins (Ure2p, Sup35p, and Rnq1p) share a set of remarkable properties. In vivo, they form aggregates that self-perpetuate their aggregation. This aggregation is controlled by Hsp104, which plays a major role in the growth and severing of these prions. In vitro, these prion proteins form amyloid fibrils spontaneously. The introduction of such fibrils made from Ure2p or Sup35p into yeast cells leads to the prion phenotypes [URE3] and [PSI], respectively. Previous studies on evolutionary biology of yeast prions have clearly established that [URE3] is not well conserved in the hemiascomycetous yeasts and particularly in S. paradoxus. Here we demonstrated that the S. paradoxus Ure2p is able to form infectious amyloid. These fibrils are more resistant than S. cerevisiae Ure2p fibrils to shear force. The observation, in vivo, of a distinct aggregation pattern for GFP fusions confirms the higher propensity of SpUre2p to form fibrillar structures. Our in vitro and in vivo analysis of aggregation propensity of the S. paradoxus Ure2p provides an explanation for its loss of infective properties and suggests that this protein belongs to the non-prion amyloid world.     

The yeast prion [URE3] can be greatly induced by a functional mutated URE2 allele

The non-Mendelian element [URE3] of yeast is considered to be a prion form of the Ure2 protein. The [URE3] phenotype occurs at a frequency of 10(-5) in haploid yeast strains, is reversible, and its frequency is increased by overexpressing the URE2 gene. We created a new mutant of the Ure2 protein, called H2p, which results in a 1000-fold increase in the rate of [URE3] occurrence. To date, only the overexpression of various C-terminal truncated mutants of Ure2p gives rise to a comparable level. The h2 allele is, thus, the first characterized URE2 allele that induces prion formation when expressed at a low level. By shuffling mutated and wild-type domains of URE2, we also created the first mutant Ure2 protein that is functional and induces prion formation. We demonstrate that the domains of URE2 function synergistically in cis to induce [URE3] formation, which highlights the importance of intramolecular interactions in Ure2p folding. Additionally, we show using a green fluorescent protein (GFP) fusion protein that the h2 allele exhibits numerous filiform structures that are not generated by the wild-type protein.     

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].     

Two Prion-Inducing Regions of Ure2p Are Nonoverlapping

Ure2p of Saccharomyces cerevisiae normally functions in blocking utilization of a poor nitrogen source when a good nitrogen source is available. The non-Mendelian genetic element [URE3] is a prion (infectious protein) form of Ure2p, so that overexpression of Ure2p induces the de novo appearance of infectious [URE3]. Earlier studies defined a prion domain comprising Ure2p residues 1 to 64 and a nitrogen regulation domain included in residues 66 to 354. We find that deletion of individual runs of asparagine within the prion domain reduce prion-inducing activity. Although residues 1 to 64 are sufficient for prion induction, the fragment from residues 1 to 80 is a more efficient inducer of [URE3]. In-frame deletion of a region around residue 224 does not affect nitrogen regulation but does eliminate prion induction by the remainder of Ure2p. Larger deletions removing the region around residue 224 and more of the C-terminal part of Ure2p restore prion-inducing ability. A fragment of Ure2p lacking the original prion domain does not induce [URE3], but surprisingly, further deletion of residues 151 to 157 and 348 to 354 leaves a fragment that can do so. The region from 66 to 80 and the region around residue 224 are both necessary for this second prion-inducing activity. Thus, each of two nonoverlapping parts of Ure2p is sufficient to induce the appearance of the [URE3] prion.     

The [URE3] yeast prion: from genetics to biochemistry

[URE3] is a non-Mendelian genetic element of the yeast Saccharomyces cerevisiae, an altered prion form of Ure2 protein. We show that recombinant Ure2p is a soluble protein that can assemble in vitro into dimers, tetramers, and octamers or form insoluble fibrils observed for PrP in its filamentous form or for Sup35p upon self-assembling, suggesting a similar mechanism for all prions. Computational, genetic, biochemical, and structural data allow us to specify a new boundary between the so-called prion-forming and nitrogen regulator (catalytic) domains of the protein and to map this boundary to Met-94. We bring strong evidence that the COOH-terminal (94-354) part of the protein forms a tightly folded domain, while the NH2-terminal (1-94) part is unstructured. These domains (or various parts of these domains) were shown (by means of the two-hybrid system approach and affinity binding experiments) to interact with each other (both in vivo and in vitro). We bring also evidence that the COOH-terminal (94-354) catalytically active part of the protein can be synthesized (both in vitro and in vivo) via an internal ribosome-binding mechanism, independently of the production of the full-length protein. We finally show that Ure2p aggregation in vivo (monitored by fluorescence of Ure2p--GFP fusion) does not necessarily give rise to [URE3] phenotype. The significance of these findings for the appearance and propagation of the yeast prion [URE3] is discussed.     

Structure and Assembly Properties of the N-Terminal Domain of the Prion Ure2p in Isolation and in Its Natural Context

Background

The aggregation of the baker''s yeast prion Ure2p is at the origin of the [URE3] trait. The Q- and N-rich N-terminal part of the protein is believed to drive Ure2p assembly into fibrils of amyloid nature and the fibrillar forms of full-length Ure2p and its N-terminal part generated in vitro have been shown to induce [URE3] occurrence when introduced into yeast cells. This has led to the view that the fibrillar form of the N-terminal part of the protein is sufficient for the recruitment of constitutive Ure2p and that it imprints its amyloid structure to full-length Ure2p.

Results

Here we generate a set of Ure2p N-terminal fragments, document their assembly and structural properties and compare them to that of full-length Ure2p. We identify the minimal region critical for the assembly of Ure2p N-terminal part into amyloids and show that such fibrils are unable to seed the assembly of full length Ure2p unlike fibrils made of intact Ure2p.

Conclusion

Our results clearly indicate that fibrillar Ure2p shares no structural similarities with the amyloid fibrils made of Ure2p N-terminal part. Our results further suggest that the induction of [URE3] by fibrils made of full-length Ure2p is likely the consequence of fibrils growth by depletion of cytosolic Ure2p while it is the consequence of de novo formation of prion particles following, for example, titration within the cells of a specific set of molecular chaperones when fibrils made of Ure2p N-terminal domain are introduced within the cytoplasm.     

Amyloid of the Candida albicans Ure2p prion domain is infectious and has an in-register parallel β-sheet structure

Ure2p of Candida albicans (Ure2(albicans) or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2(glabrata)) cannot, even though the Ure2(glabrata) N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2(cerevisiae)) than Ure2(albicans) is. We show that the N-terminal N/Q-rich prion domain of Ure2(albicans) forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p(1-90), we show that this infectious amyloid has an in-register parallel β-sheet structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2(glabrata) does not readily form amyloid, and that formed upon prolonged incubation is not infectious.     

Reporter assay systems for [URE3] detection and analysis

The Saccharomyces cerevisiae prion [URE3] is the infectious amyloid form of the Ure2p protein. [URE3] provides a useful model system for studying amyloid formation and stability in vivo. When grown in the presence of a good nitrogen source, [URE3] cells are able to take up ureidosuccinate, an intermediate in uracil biosynthesis, while cells lacking the [URE3] prion can not. This ability to take up ureidosuccinate has been commonly used to assay for the presence of [URE3]. However, this assay has a number of practical limitations, affecting the range of experiments that can be performed with [URE3]. Here, we describe recently developed alternative selection methods for the presence or absence of [URE3]. They make use of the Ure2p-regulated DAL5 promoter in conjunction with ADE2, URA3, kanMX, and CAN1 reporter genes, and allow for higher stringency in selection both for and against [URE3], nonselective assay of prion variants, and direct transformation of prion filaments. We discuss advantages and limitations of each of these assays.     

Induction of distinct [URE3] yeast prion strains

[URE3] is a non-Mendelian genetic element in Saccharomyces cerevisiae, which is caused by a prion-like, autocatalytic conversion of the Ure2 protein (Ure2p) into an inactive form. The presence of [URE3] allows yeast cells to take up ureidosuccinic acid in the presence of ammonia. This phenotype can be used to select for the prion state. We have developed a novel reporter, in which the ADE2 gene is controlled by the DAL5 regulatory region, which allows monitoring of Ure2p function by a colony color phenotype. Using this reporter, we observed induction of different [URE3] prion variants ("strains") following overexpression of the N-terminal Ure2p prion domain (UPD) or full-length Ure2p. Full-length Ure2p induced two types of [URE3]: type A corresponds to conventional [URE3], whereas the novel type B variant is characterized by relatively high residual Ure2p activity and efficient curing by coexpression of low amounts of a UPD-green fluorescent protein fusion protein. Overexpression of UPD induced type B [URE3] but not type A. Both type A and B [URE3] strains, as well as weak and strong isolates of type A, were shown to stably maintain different prion strain characteristics. We suggest that these strain variants result from different modes of aggregation of similar Ure2p monomers. We also demonstrate a procedure to counterselect against the [URE3] state.     

Molecular chaperone Hsp104 can promote yeast prion generation

[URE3] is an amyloid-based prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. The Ure2p of the human pathogen Candida albicans can also be a prion in S. cerevisiae. We find that overproduction of the disaggregating chaperone, Hsp104, increases the frequency of de novo [URE3] prion formation by the Ure2p of S. cerevisiae and that of C. albicans. This stimulation is strongly dependent on the presence of the [PIN(+)] prion, known from previous work to enhance [URE3] prion generation. Our data suggest that transient Hsp104 overproduction enhances prion generation through persistent effects on Rnq1 amyloid, as well as during overproduction by disassembly of amorphous Ure2 aggregates (generated during Ure2p overproduction), driving the aggregation toward the amyloid pathway. Overproduction of other major cytosolic chaperones of the Hsp70 and Hsp40 families (Ssa1p, Sse1p, and Ydj1p) inhibit prion formation, whereas another yeast Hsp40, Sis1p, modulates the effects of Hsp104p on both prion induction and prion curing in a prion-specific manner. The same factor may both enhance de novo prion generation and destabilize existing prion variants, suggesting that prion variants may be selected by changes in the chaperone network.