Proteasome Control of [URE3] Prion Propagation by Degradation of Anti-Prion Proteins Cur1 and Btn2 in Saccharomyces cerevisiae

[URE3] is a prion of the nitrogen catabolism controller, Ure2p, and [PSI+] is a prion of the translation termination factor Sup35p in S. cerevisiae. Btn2p cures [URE3] by sequestration of Ure2p amyloid filaments. Cur1p, paralogous to Btn2p, also cures [URE3], but by a different (unknown) mechanism. We find that an array of mutations impairing proteasome assembly or MG132 inhibition of proteasome activity result in loss of [URE3]. In proportion to their prion—curing effects, each mutation affecting proteasomes elevates the cellular concentration of the anti-prion proteins Btn2 and Cur1. Of >4,600 proteins detected by SILAC, Btn2p was easily the most overexpressed in a pre9Δ (α3 core subunit) strain. Indeed, deletion of BTN2 and CUR1 prevents the prion—curing effects of proteasome impairment. Surprisingly, the 15 most unstable yeast proteins are not increased in pre9Δ cells suggesting altered proteasome specificity rather than simple inactivation. Hsp42, a chaperone that cooperates with Btn2 and Cur1 in curing [URE3], is also necessary for the curing produced by proteasome defects, although Hsp42p levels are not substantially altered by a proteasome defect. We find that pre9Δ and proteasome chaperone mutants that most efficiently lose [URE3], do not destabilize [PSI+] or alter cellular levels of Sup35p. A tof2 mutation or deletion likewise destabilizes [URE3], and elevates Btn2p, suggesting that Tof2p deficiency inactivates proteasomes. We suggest that when proteasomes are saturated with denatured/misfolded proteins, their reduced degradation of Btn2p and Cur1p automatically upregulates these aggregate-handling systems to assist in the clean-up.     

Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity

[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.     

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.     

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].     

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.     

Prion-forming ability of Ure2 of yeasts is not evolutionarily conserved

[URE3] is a prion (infectious protein) of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. We show that wild S. paradoxus can be infected with a [URE3] prion, supporting the use of S. cerevisiae as a prion test bed. We find that the Ure2p of Candida albicans and C. glabrata also regulate nitrogen catabolism. Conservation of amino acid sequence within the prion domain of Ure2p has been proposed as evidence that the [URE3] prion helps its host. We show that the C. albicans Ure2p, which does not conserve this sequence, can nonetheless form a [URE3] prion in S. cerevisiae, but the C. glabrata Ure2p, which does have the conserved sequence, cannot form [URE3] as judged by its performance in S. cerevisiae. These results suggest that the sequence is not conserved to preserve prion forming ability.     

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.     

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.     

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.     

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.     

Antagonistic interactions between yeast [PSI(+)] and [URE3] prions and curing of [URE3] by Hsp70 protein chaperone Ssa1p but not by Ssa2p

The yeast [PSI(+)], [URE3], and [PIN(+)] genetic elements are prion forms of Sup35p, Ure2p, and Rnq1p, respectively. Overexpression of Sup35p, Ure2p, or Rnq1p leads to increased de novo appearance of [PSI(+)], [URE3], and [PIN(+)], respectively. This inducible appearance of [PSI(+)] was shown to be dependent on the presence of [PIN(+)] or [URE3] or overexpression of other yeast proteins that have stretches of polar residues similar to the prion-determining domains of the known prion proteins. In a similar manner, [PSI(+)] and [URE3] facilitate the appearance of [PIN(+)]. In contrast to these positive interactions, here we find that in the presence of [PIN(+)], [PSI(+)] and [URE3] repressed each other's propagation and de novo appearance. Elevated expression of Hsp104 and Hsp70 (Ssa2p) had little effect on these interactions, ruling out competition between the two prions for limiting amounts of these protein chaperones. In contrast, we find that constitutive overexpression of SSA1 but not SSA2 cured cells of [URE3], uncovering a specific interaction between Ssa1p and [URE3] and a functional distinction between these nearly identical Hsp70 isoforms. We also find that Hsp104 abundance, which critically affects [PSI(+)] propagation, is elevated when [URE3] is present. Our results are consistent with the notion that proteins that have a propensity to form prions may interact with heterologous prions but, as we now show, in a negative manner. Our data also suggest that differences in how [PSI(+)] and [URE3] interact with Hsp104 and Hsp70 may contribute to their antagonistic interactions.     

Tah1, A Key Component of R2TP Complex that Regulates Assembly of snoRNP,is Involved in De Novo Generation and Maintenance of Yeast Prion [URE3]

The cellular chaperone machinery plays key role in the de novo formation and propagation of yeast prions (infectious protein). Though the role of Hsp70s in the prion maintenance is well studied, how Hsp90 chaperone machinery affects yeast prions remains unclear. In the current study, we examined the role of Hsp90 and its co-chaperones on yeast prions [PSI⁺] and [URE3]. We show that the overproduction of Hsp90 co-chaperone Tah1, cures [URE3] which is a prion form of native protein Ure2 in yeast. The Hsp90 co-chaperone Tah1 is involved in the assembly of small nucleolar ribonucleoproteins (snoRNP) and chromatin remodelling complexes. We found that Tah1 deletion improves the frequency of de novo appearance of [URE3]. The Tah1 was found to interact with Hsp70. The lack of Tah1 not only represses antagonizing effect of Ssa1 Hsp70 on [URE3] but also improves the prion strength suggesting role of Tah1 in both fibril growth and replication. We show that the N-terminal tetratricopeptide repeat domain of Tah1 is indispensable for [URE3] curing. Tah1 interacts with Ure2, improves its solubility in [URE3] strains, and affects the kinetics of Ure2 fibrillation in vitro. Its inhibitory role on Ure2 fibrillation is proposed to influence [URE3] propagation. The present study shows a novel role of Tah1 in yeast prion propagation, and that Hsp90 not only promotes its role in ribosomal RNA processing but also in the prion maintenance.SummaryPrions are self-perpetuating infectious proteins. What initiates the misfolding of a protein into its prion form is still not clear. The understanding of cellular factors that facilitate or antagonize prions is crucial to gain insight into the mechanism of prion formation and propagation. In the current study, we reveal that Tah1 is a novel modulator of yeast prion [URE3]. The Hsp90 co-chaperone Tah1, is required for the formation of small nucleolar ribonucleoprotein complex. We show that the absence of Tah1 improves the induction of [URE3] prion. The overexpressed Tah1 cures [URE3], and this function is promoted by Hsp90 chaperones. The current study thus provides a novel cellular factor and the underlying mechanism, involved in the prion formation and propagation     

The Cellular Concentration of the Yeast Ure2p Prion Protein Affects Its Propagation as a Prion

The [URE3] yeast prion is a self-propagating inactive form of the Ure2p protein. We show here that Ure2p from the species Saccharomyces paradoxus (Ure2p_Sp) can be efficiently converted into a prion form and propagate [URE3] when expressed in Saccharomyces cerevisiae at physiological level. We found however that Ure2p_Sp overexpression prevents efficient prion propagation. We have compared the aggregation rate and propagon numbers of Ure2p_Sp and of S. cerevisiae Ure2p (Ure2p_Sc) in [URE3] cells both at different expression levels. Overexpression of both Ure2p orthologues accelerates formation of large aggregates but Ure2p_Sp aggregates faster than Ure2p_Sc. Although the yeast cells that contain these large Ure2p aggregates do not transmit [URE3] to daughter cells, the corresponding crude extract retains the ability to induce [URE3] in wild-type [ure3-0] cells. At low expression level, propagon numbers are higher with Ure2p_Sc than with Ure2p_Sp. Overexpression of Ure2p decreases the number of [URE3] propagons with Ure2p_Sc. Together, our results demonstrate that the concentration of a prion protein is a key factor for prion propagation. We propose a model to explain how prion protein overexpression can produce a detrimental effect on prion propagation and why Ure2p_Sp might be more sensitive to such effects than Ure2p_Sc.     

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.     

Btn3 is a negative regulator of Btn2-mediated endosomal protein trafficking and prion curing in yeast

Yeast Btn2 facilitates the retrieval of specific proteins from late endosomes (LEs) to the Golgi, a process that may be adversely affected in Batten disease patients. We isolated the putative yeast orthologue of a human complex I deficiency gene, designated here as BTN3, as encoding a Btn2-interacting protein and negative regulator. First, yeast overexpressing BTN3 phenocopy the deletion of BTN2 and mislocalize certain trans-Golgi proteins, like Kex2 and Yif1, to the LE and vacuole, respectively. In contrast, the deletion of BTN3 results in a tighter pattern of protein localization to the Golgi. Second, BTN3 overexpression alters Btn2 localization from the IPOD compartment, which correlates with a sharp reduction in Btn2-mediated [URE3] prion curing. Third, Btn3 and the Snc1 v-SNARE compete for the same binding domain on Btn2, and this competition controls Btn2 localization and function. The inhibitory effects upon protein retrieval and prion curing suggest that Btn3 sequesters Btn2 away from its substrates, thus down-regulating protein trafficking and aggregation. Therefore Btn3 is a novel negative regulator of intracellular protein sorting, which may be of importance in the onset of complex I deficiency and Batten disease in humans.     

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.     

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.     

The [URE3] prion is not conserved among Saccharomyces species

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating inactive form of the nitrogen catabolism regulator Ure2p. To determine whether the [URE3] prion is conserved in S. cerevisiae-related yeast species, we have developed genetic tools allowing the detection of [URE3] in Saccharomyces paradoxus and Saccharomyces uvarum. We found that [URE3] is conserved in S. uvarum. In contrast, [URE3] was not detected in S. paradoxus. The inability of S. paradoxus Ure2p to switch to a prion isoform results from the primary sequence of the protein and not from the lack of cellular cofactors as heterologous Ure2p can propagate [URE3] in this species. Our data therefore demonstrate that [URE3] is conserved only in a subset of Saccharomyces species. Implications of our finding on the physiological and evolutionary meaning of the yeast [URE3] prion are discussed.     

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.