Some proteins can change their fold from normal to a specific alternative form, called prion, which is able to catalyze this change (1). In man and animals such process causes prion diseases like Creutzfeldt-Jacob disease, bovine spongiform encephalopathy, and scrapie of sheep. A similar autocatalytic mechanism is shared by human amyloid diseases, which are noninfectious, in contrast to prion diseases (2). In yeast, there are several proteins, which can undergo prion-like structural conversion. The most studied of them are translation termination factor eRF3, also called Sup35, and Ure2 involved in regulation of nitrogen metabolism (3 was not lost (9). Hsp104 was shown to break large aggregates of denatured protein into smaller pieces (10, 11). We proposed that Hsp104 acts similarly on fiber-shaped prion polymers, thus fragmenting them into shorter polymers and increasing their number (12). This is essential for their inheritance and accelerates the prion conversion by multiplying the ends of prion polymers, where the conversion occurs. The overproduction of Hsp104 should cause excessive fragmentation, increased levels of soluble Sup35, and possibly [PSI ϩ ] loss. An alternative model proposed that Hsp104 is primarily required to facilitate the prion conversion in one or another way (13,14).These two models may be distinguished, since they make different predictions for alteration of the size of prion particles upon inhibition of the Hsp104 function. By the former model, the size should increase due to blocked fragmentation, while by the latter it should stay constant or decrease due to block of polymerization. Recent studies provided some support for the "fragmentation" model. Decrease of the Hsp104 expression caused increase in the size of Sup35 prion aggregates, suggesting decreased disaggregation by Hsp104 (15). The activity of Hsp104 is inhibited by growing yeast cells in the presence of 3-5 mM guanidine HCl (GuHCl) 1 (16). Such treatment cures efficiently [PSI ϩ ] (17) and other known yeast prions. Study of the kinetics of [PSI ϩ ] loss in the presence of GuHCl allowed concluding that it blocks replication of prion "seeds" (18,19). Thus, Hsp104 inhibition correlates with the block of fragmentation (replication) of prion particles (seeds). However, in these experiments the relation of the studied prion entities to the Sup35 polymers considered by the above models was not characterized. The prion seeds were defined genetically, but their physical nature was not studied. In the work (15) the size of

[URE3] is a prion (infectious protein), a self-propagating amyloid form of Ure2p, a regulator of yeast nitrogen catabolism. We find that overproduction of Btn2p, or its homologue Ypr158 (Cur1p), cures [URE3]. Btn2p is reported to be associated with late endosomes and to affect sorting of several proteins. We find that double deletion of BTN2 and CUR1 stabilizes [URE3] against curing by several agents, produces a remarkable increase in the proportion of strong [URE3] variants arising de novo and an increase in the number of [URE3] prion seeds. Thus, normal levels of Btn2p and Cur1p affect prion generation and propagation. Btn2p-green fluorescent protein (GFP) fusion proteins appear as a single dot located close to the nucleus and the vacuole. During the curing process, those cells having both Ure2p-GFP aggregates and Btn2p-RFP dots display striking colocalization. Btn2p curing requires cell division, and our results suggest that Btn2p is part of a system, reminiscent of the mammalian aggresome, that collects aggregates preventing their efficient distribution to progeny cells.

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The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.

Prions are infectious, self-propagating amyloid-like protein aggregates of mammals and fungi. We have studied aggregation propensities of a yeast prion domain in cell culture to gain insights into general mechanisms of prion replication in mammalian cells. Here, we report the artificial transmission of a yeast prion across a phylogenetic kingdom. HA epitope-tagged yeast Sup35p prion domain NM was stably expressed in murine neuroblastoma cells. Although cytosolically expressed NM-HA remained soluble, addition of fibrils of bacterially produced Sup35NM to the medium efficiently induced appearance of phenotypically and biochemically distinct NM-HA aggregates that were inherited by daughter cells. Importantly, NM-HA aggregates also were infectious to recipient mammalian cells expressing soluble NM-HA and, to a lesser extent, to yeast. The fact that the yeast Sup35NM domain can propagate as a prion in neuroblastoma cells strongly argues that cellular mechanisms support prion-like inheritance in the mammalian cytosol.PrP ͉ Sup35 P rions are infectious particles composed exclusively or predominantly of proteins. In mammals, prions are the causative agent of transmissible spongiform encephalopathies or prion diseases that are associated with the conversion of the normal host encoded prion protein, PrP C , to its infectious, aggregated prion isoform, PrP Sc (1). Mammalian prion diseases belong to the group of protein misfolding diseases that are associated with the abnormal aggregation of diverse host proteins into highly ordered, ␤-sheet-rich fibrillar aggregates, the so-called amyloids. A hallmark of prions is the existence of different strains that are associated with characteristic disease phenotypes (2). Remarkably, prions have also been identified in fungi, where they represent epigenetic elements of inheritance that replicate via a similar mechanism of selfpropagating protein conformation. Prions of eukaryotic microorganisms have been invaluable in elucidating basic concepts of prion biology. In fact, discoveries in yeast prion biology allowed formal demonstration of the protein-only hypothesis originally proposed for mammalian prions. Unlike mammalian prions, however, yeast prions are generally not lethal. The Saccharomyces cerevisiae epigenetic element [PSI ϩ ] is a prion of the translation termination factor subunit Sup35p (3, 4) that arises from conversion of soluble active monomers to an inactive amyloid (5-9), leading to a change in the yeast metabolic phenotype. Translation termination activity is conferred by the carboxyl-terminal domain C, whereas the amino-terminal Sup35NM domain is necessary and sufficient for prion-based inheritance (10). The N region comprises a prionforming domain (amino acids 1-97), which is defined as the minimal region essential for induction and propagation of the prion state (10). Acting as a linker between the N and C regions, the highly charged M region increases the solubility of the protein (8) and imparts stability of the prion during mitosis and meiosis (11). Yeast prion b...

The [PSI + ] prion is a self-propagating amyloid of the Sup35 protein, normally a subunit of the translation termination factor, but impaired in this vital function when in the amyloid form. The Sup35 N, M and C domains are the amino-terminal prion domain, a connecting polar domain and the essential C-terminal domain resembling eukaryotic elongation factor 1alpha, respectively. Different [PSI + ] isolates (prion variants) may have distinct biological properties, associated with different amyloid structures. Here we use solid state NMR to examine the structure of infectious Sup35NM amyloid fibrils of two prion variants. We find that both variants have an in-register parallel β -sheet structure, both in fully hydrated and in lyophilized form. Moreover, we confirm that some leucine residues in the M domain participate in the in-register parallel β-sheet structure. Transmission of the [PSI + ] prion by amyloid fibrils of Sup35NM and of the [URE3] prion by amyloid fibrils of recombinant full length Ure2p are similar whether they have been lyophilized or not (wet or dry).A prion is an infectious protein, able to transmit a disease or trait without any essential nucleic acid. This concept arose from studies of the mammalian transmissible spongiform encephalopathies (TSEs), but there are now six known distinct prions in yeast, [URE3] Ure2p, Sup35p, Rnq1p, Prb1p, Swi1p, Mca1p, and Cyc8p,. Extensive evidence, culminating in transfection by the corresponding amyloid of the recombinant protein, has shown that at least [PSI + ], [URE3], and [PIN + ] are based on self-propagating amyloids (7-10). Amyloid is a fibrillar protein aggregate characterized by partial protease resistance, birefringence on staining with Congo Red and a cross-β-sheet structure (11).The Sup35 protein is a subunit of the translation termination factor that is inactivated by its aggregation as amyloid in cells infected with the [PSI + ] prion. The diminished levels of Sup35p lead to inefficient translation termination and thus more frequent read-through of premature termination codons, for example, suppressing a nonsense mutation in ADE2 and allowing adenine biosynthesis. Sup35p includes an N-terminal 123 residue prion domain (N), whose normal function is in mRNA turnover (12), a middle 130 residue charged domain (M), and the † This work was supported by the Intramural Program of the National Institute of Diabetes Digestive and Kidney Diseases. *For communication: Phone: 301-496-3452, Fax: 301-402-0240, wickner@helix.nih.gov, Bldg. 8, Room 225, NIH, 8 Center Drive MSC 0830, Bethesda, MD 20892-0830. Supporting Information Available: Supplementary Fig. 1 shows the PITHIRDS-CT data uncorrected for signal from natural abundance 13 C. This material is available free of charge via the Internet at http://pubs.acs.org. NMR experiments require relatively large amounts of material packed into a small volume. Lyophilized fibrils are routinely used for this purpose, and previous solid state NMR studies have shown that lyophilization does not perturb the mo...

The Sup35 protein of Saccharomyces cerevisiae belongs to the family of eRF3, one of the two key factors required for the termination of translation in eukaryotes (1). This protein has a three-domain structure, but only its C-terminal part (C domain) of 432 amino acids is responsible for the function in translation termination and conserved (2). The N-terminal (N) Sup35 domain of 123 amino acids allows protein to switch into the aggregated prion-like state. This aggregation reduces the efficiency of translation termination, which manifests as non- ] cells, overproduced Sup35 is present mostly as amyloid aggregates, which differ from the Sup35 prion amyloids by much higher frequency of appearance and lower efficiency of Sup35 polymerization. Due to this, such cells show normal nonsense codon readthrough but easily develop the suppressor phenotype upon selective pressure. Similar properties were observed for the hybrid Sup35, in which the N domain was replaced for a sequence of 66 glutamine residues. Thus, yeast Sup35 can form non-heritable amyloid structures, which may have phenotypic manifestation. ura3-52 leu2-3,112 trp1-289 his3-⌬200 ade1-14) strain (9) and its derivative 74-D694/Q66 (obtained by replacing SUP35 for the chimerical SUP35-Q66 allele encoding glutamines 1-66 instead of amino acids 1-120 of Sup35 under control of MET17 promoter, see below) were used. Since the ade1-14 UGA mutation carried by this strain caused accumulation of red pigment, its suppression (suppressor phenotype, Sup ϩ ) could be easily detected by the appearance of colonies with lighter color (white or pink, depending on the suppression efficiency). The 5V-H19 and 1-5V-H19 (same, but with the SUP35-C allele, which lacks a region encoding the N and M domains of Sup35) strains were also used in some experiments (10). Yeast were grown at 30°C in rich (YPD, 10 g of yeast extract, 20 g of peptone, 20 g of glucose/liter) or synthetic (6.7 g of yeast nitrogen base, 20 g of glucose supplemented with the required amino acids) medium. Selective media lacking leucine, uracil, or adenine are designated as ϪLeu, ϪUra, or ϪAde, respectively. To select cells that have lost URA3 plasmids, corresponding transformants were grown on 5-fluoroorotic acid medium (11). To cure cells of the [PSI ϩ ] determinants, corresponding strains were grown from single cells to colonies on medium containing 3 mM guanidine hydrochloride (12). MATERIALS AND METHODS Strains and Genetic Methods-In most cases, the 74-D694 (MATaPlasmids-YEplac181-SUP35 was constructed by inserting the XhoI-XbaI fragment of SUP35 into the SalI and XbaI sites of YEplac181. The HpaI-XbaI fragment of YEplac181-SUP35 encoding the