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.     

Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils

Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.     

The core of Ure2p prion fibrils is formed by the N-terminal segment in a parallel cross-β structure: evidence from solid-state NMR

Intracellular fibril formation by Ure2p produces the non-Mendelian genetic element [URE3] in Saccharomyces cerevisiae, making Ure2p a prion protein. We show that solid-state NMR spectra of full-length Ure2p fibrils, seeded with infectious prions from a specific [URE3] strain and labeled with uniformly ¹⁵N-¹³C-enriched Ile, include strong, sharp signals from Ile residues in the globular C-terminal domain (CTD) with both helical and nonhelical ¹³C chemical shifts. Treatment with proteinase K eliminates these CTD signals, leaving only nonhelical signals from the Gln-rich and Asn-rich N-terminal segment, which are also observed in the solid-state NMR spectra of Ile-labeled fibrils formed by residues 1-89 of Ure2p. Thus, the N-terminal segment, or “prion domain” (PD), forms the fibril core, while CTD units are located outside the core. We additionally show that, after proteinase K treatment, Ile-labeled Ure2p fibrils formed without prion seeding exhibit a broader set of solid-state NMR signals than do prion-seeded fibrils, consistent with the idea that structural variations within the PD core account for prion strains. Measurements of ¹³C-¹³C magnetic dipole-dipole couplings among ¹³C-labeled Ile carbonyl sites in full-length Ure2p fibrils support an in-register parallel β-sheet structure for the PD core of Ure2p fibrils. Finally, we show that a model in which CTD units are attached rigidly to the parallel β-sheet core is consistent with steric constraints.     

Flexibility of the Ure2 prion domain is important for amyloid fibril formation

Ure2, the protein determinant of the Saccharomyces cerevisiae prion [URE3], has a natively disordered N-terminal domain that is important for prion formation in vivo and amyloid formation in vitro; the globular C-domain has a glutathione transferase-like fold. In the present study, we swapped the position of the N- and C-terminal regions, with or without an intervening peptide linker, to create the Ure2 variants CLN-Ure2 and CN-Ure2 respectively. The native structural content and stability of the variants were the same as wild-type Ure2, as indicated by enzymatic activity, far-UV CD analysis and equilibrium denaturation. CLN-Ure2 was able to form amyloid-like fibrils, but with a significantly longer lag time than wild-type Ure2; and the two proteins were unable to cross-seed. Under the same conditions, CN-Ure2 showed limited ability to form fibrils, but this was improved after addition of 0.03?M guanidinium chloride. As for wild-type Ure2, allosteric enzyme activity was observed in fibrils of CLN-Ure2 and CN-Ure2, consistent with retention of the native-like dimeric structure of the C-domains within the fibrils. Proteolytically digested fibrils of CLN-Ure2 and CN-Ure2 showed the same residual fibril core morphology as wild-type Ure2. The results suggest that the position of the prion domain affects the ability of Ure2 to form fibrils primarily due to effects on its flexibility.     

The yeast prion protein Ure2: insights into the mechanism of amyloid formation

Ure2, a regulator of nitrogen metabolism, is the protein determinant of the [URE3] prion state in Saccharomyces cerevisiae. Upon conversion into the prion form, Ure2 undergoes a heritable conformational change to an amyloid-like aggregated state and loses its regulatory function. A number of molecular chaperones have been found to affect the prion properties of Ure2. The studies carried out in our laboratory have been aimed at elucidating the structure of Ure2 fibrils, the mechanism of amyloid formation and the effect of chaperones on the fibril formation of Ure2.     

Alcohol oxidase (AOX1) from Pichia pastoris is a novel inhibitor of prion propagation and a potential ATPase

Previous results suggest that methylotrophic yeasts may contain factors that modulate prion stability. Alcohol oxidase (AOX), a key enzyme in methanol metabolism, is an abundant protein that is specific to methylotrophic yeasts. We examined the effect of Pichia pastoris AOX1 on prion phenotypes in Saccharomyces cerevisiae . The S. cerevisiae prion states [ PSI ⁺] and [ URE3 ] arise from aggregation of the proteins Sup35p and Ure2p respectively, and correlate with the ability of Sup35p and Ure2p to form amyloid-like fibrils in vitro . We found that expression of P. pastoris AOX1 in S. cerevisiae had no effect on propagation of the [ PSI ⁺] prion, but inhibited propagation of [ URE3 ]. Addition of AOX1 early in the time-course of fibril formation inhibits Ure2p fibril formation in vitro . AOX1 has not previously been identified as an ATPase. However, we discovered that in addition to its flavin adenine dinucleotide-dependent AOX activity, AOX1 possesses ATPase activity. This study identifies AOX1 as a novel prion inhibitory factor and a potential ATPase.     

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.     

The yeast prion Ure2p retains its native alpha-helical conformation upon assembly into protein fibrils in vitro

The yeast inheritable phenotype [URE3] is thought to result from conformational changes in the normally soluble and highly helical protein Ure2p. In vitro, the protein spontaneously forms long, straight, insoluble protein fibrils at neutral pH. Here we show that fibrils of intact Ure2p assembled in vitro do not possess the cross beta-structure of amyloid, but instead are formed by the polymerization of native-like helical subunits that retain the ability to bind substrate analogues. We further show that dissociation of the normally dimeric protein to its constituent monomers is a prerequisite for assembly into fibrils. By analysing the nature of early assembly intermediates, as well as fully assembled Ure2p fibrils using atomic force microscopy, and combining the results with experiments that probe the fidelity of the native fold in protein fibrils, we present a model for fibril formation, based on assembly of native-like monomers, driven by interactions between the N-terminal glutamine and asparagine-rich region and the C-terminal functional domain. The results provide a rationale for the effect of mutagenesis on prion formation and new insights into the mechanism by which this, and possibly other inheritable factors, can be propagated.     

The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein

The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p.     

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.     

Structural characterization of the fibrillar form of the yeast Saccharomyces cerevisiae prion Ure2p

The protein Ure2 from the yeast Saccharomyces cerevisiae has prion properties. It assembles in vitro into long, straight, insoluble fibrils that are similar to amyloids in that they bind Congo Red and show green-yellow birefringence and have an increased resistance to proteolysis. We recently showed that Ure2p fibrils assembled under physiologically relevant conditions are devoid of a cross-beta-core. A model for fibril formation, where assembly is driven by non-native inter- and/or intramolecular interaction between Ure2p monomers following subtle conformational changes was proposed [Bousset et al. (2002) EMBO J. 21, 2903-2911]. An alternative model for the assembly of Ure2p into fibrils where assembly is driven by the stacking of 40-70 N-terminal amino acid residues of Ure2p into a central beta-core running along the fibrils from which the C-terminal domains protrude was proposed [Baxa et al. (2003) J. Biol. Chem. 278, 43717-43727]. We show here that Ure2p fibril congophilia and the associated yellow-green birefringence in polarized light are not indicative that the fibrils are of amyloid nature. We map the structures of the fibrillar and soluble forms of Ure2p using limited proteolysis and identify the reaction products by microsequencing and mass spectrometry. Finally, we demonstrate that the C-terminal domain of Ure2p is tightly involved in the fibrillar scaffold using a sedimentation assay and a variant Ure2p where a highly specific cleavage site between the N- and C-terminal domains of the protein was engineered. Our results are inconsistent with the cross-beta-core model and support the model for Ure2p assembly driven by subtle conformational changes and underline the influence of the natural context of the N-terminal domain on the assembly of Ure2p.     

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.     

The [URE3] yeast prion results from protein aggregates that differ from amyloid filaments formed in vitro

The [URE3] yeast prion is a self-propagating inactive form of the Ure2 protein. Ure2p is composed of two domains, residues 1-93, the prion-forming domain, and the remaining C-terminal part of the protein, which forms the functional domain involved in nitrogen catabolite repression. In vitro, Ure2p forms amyloid filaments that have been proposed to be the aggregated prion form found in vivo. Here we showed that the biochemical characteristics of these two species differ. Protease digestions of Ure2p filaments and soluble Ure2p are comparable when analyzed by Coomassie staining as by Western blot. However, this finding does not explain the pattern specifically observed in [URE3] strains. Antibodies raised against the C-terminal part of Ure2p revealed the existence of proteolysis sites efficiently cleaved when [URE3], but not wild-type crude extracts, were submitted to limited proteolysis. The same antibodies lead to an equivalent digestion pattern when recombinant Ure2p (either soluble or amyloid) was analyzed in the same way. These results strongly suggest that aggregated Ure2p in [URE3] yeast cells is different from the amyloid filaments generated in vitro.     

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.     

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.     

Structure of the globular region of the prion protein Ure2 from the yeast Saccharomyces cerevisiae

BACKGROUND: The [URE3] non-Mendelian element of the yeast S. cerevisiae is due to the propagation of a transmissible form of the protein Ure2. The infectivity of Ure2p is thought to originate from a conformational change of the normal form of the prion protein. This conformational change generates a form of Ure2p that assembles into amyloid fibrils. Hence, knowledge of the three-dimensional structure of prion proteins such as Ure2p should help in understanding the mechanism of amyloid formation associated with a number of neurodegenerative diseases. RESULTS: Here we report the three-dimensional crystal structure of the globular region of Ure2p (residues 95--354), also called the functional region, solved at 2.5 A resolution by the MAD method. The structure of Ure2p 95--354 shows a two-domain protein forming a globular dimer. The N-terminal domain is composed of a central 4 strand beta sheet flanked by four alpha helices, two on each side. In contrast, the C-terminal domain is entirely alpha-helical. The fold of Ure2p 95--354 resembles that of the beta class glutathione S-transferases (GST), in line with a weak similarity in the amino acid sequence that exists between these proteins. Ure2p dimerizes as GST does and possesses a potential ligand binding site, although it lacks GST activity. CONCLUSIONS: The structure of the functional region of Ure2p is the first crystal structure of a prion protein. Structure comparisons between Ure2p 95--354 and GST identified a 32 amino acid residues cap region in Ure2p exposed to the solvent. The cap region is highly flexible and may interact with the N-terminal region of the partner subunit in the dimer. The implication of this interaction in the assembly of Ure2p into amyloid fibrils is discussed.     

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.     

Equilibrium folding properties of the yeast prion protein determinant Ure2.

The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The [URE3] phenotype is equivalent to loss of function of Ure2, a protein involved in regulation of nitrogen metabolism. The prion-like behaviour of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine. This region has been termed the prion-determining domain (PrD). Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the [URE3] phenotype. Removal of the PrD does not affect the regulatory activity of Ure2. The C-terminal portion of the protein has homology to glutathione S -transferases, which are dimeric proteins. We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene. The recombinant purified protein is shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared. The stability of Ure2, DeltaGD-N, H2O, determined by chemical denaturation and monitored by fluorescence, is 12.1(+/-0.4) kcal mol-1at 25 degrees C and pH 8.4. A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concentration. The stability is the same within error for Ure2 variants lacking all or part of the prion-determining domain. The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermolecular interactions at micromolar protein concentrations. This suggests that the C-terminal domain may stabilise the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodynamic stability of the folded protein.     

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.     

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.