SUMMARY A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants.

The [PSI+] prion is a self-propagating amyloid of the translation termination factor, Sup35p, of Saccharomyces cerevisiae. The N-terminal 253 residues (NM) of this 685-residue protein normally function in regulating mRNA turnover but spontaneously form infectious amyloid in vitro. We converted the three Ile residues in Sup35NM to Leu and then replaced 16 single residues with Ile, one by one, and prepared Ile-1-13 C amyloid of each mutant, seeding with amyloid formed by the reference sequence Sup35NM. Using solid-state NMR, we showed that 10 of the residues examined, including six between residues 30 and 90, showed the ∼0.5-nm distance between labels diagnostic of the in-register parallel amyloid architecture. The five scattered N domain residues with wider spacing may be in turns or loops; one is a control at the C terminus of M. All mutants, except Q56I, showed little or no [PSI+] transmission barrier from the reference sequence, suggesting that they could assume a similar amyloid architecture in vitro when seeded with filaments of reference sequence Sup35NM. Infection of yeast cells expressing the reference SUP35 gene sequence with amyloid of several mutants produced [PSI+] transfectants with similar efficiency as did reference sequence Sup35NM amyloid. Our work provides a stringent demonstration that the Sup35 prion domain has the folded inregister parallel β-sheet architecture and suggests common locations of the folds. This architecture naturally suggests a mechanism of inheritance of conformation, the central mystery of prions.[PSI+] prion | solid-state NMR | dipolar recoupling | amyloid | Sup35 P rions are infectious proteins, mostly self-propagating amyloids. Amyloid is a filamentous polymer, rich in β-sheet structure, in which the β-strands run perpendicular to the long axis of the filament and the hydrogen bonds joining β-strands to make a sheet are along the long axis of the filament. In mammals, prions are uniformly lethal diseases, caused by amyloid formation of the PrP protein. In yeast and fungi, prions are not uniformly fatal and have widely varying effects (reviewed in ref. 1). Perhaps the most remarkable feature of prions is that they have strains or variants, distinct self-propagating forms of the same protein, analogous to alleles of a gene, each relatively stably propagated. The existence of different self-propagating prion variants implies an array of self-propagating structures, each based on the same protein sequence. Because each prion variant is self-propagating, and each variant represents a different amyloid structure/ conformation, there must be some mechanism by which the protein can template its conformation. This mechanism must operate for each of the many conformations that are possible for a given prion.An amino acid residue in a β-sheet can have interactions in three dimensions (Fig. 1A): (a) along the peptide chain; (b) with the residues it faces within the β-sheet but perpendicular to the peptide chain, including the residues to which its main chain N-H and >C = O are hydrog...

] prion (10, 11). Amyloid is a filamentous β-sheet-rich protein polymer, and the yeast prion amyloids have a folded, in-register, parallel β-sheet architecture (12-15). This architecture provides a mechanism by which proteins can template their conformation, much as DNA templates its sequence, and explains the rather stable propagation of many different prion variants (called "prion strains" in mammals) based on different conformations of a single prion protein (16, 17).Chernoff's seminal discovery that Hsp104 overproduction or deficiency could cure the [PSI + ] prion (18, 19) led to detailed dissection of the mechanisms of these effects, and discovery of the involvement of many other chaperones and cochaperones. Hsp104 (20) is a disaggregating chaperone, which acts with Hsp70s and Hsp40s to solubilize proteins (21). Monomers are removed from the aggregate and fed through the central cavity of the Hsp104 hexamer, thereby denaturing them and allowing them a chance to properly refold (22)(23)(24). Millimolar guanidine HCl is a surprisingly specific inhibitor of Hsp104 (25-29), and has been used to show that the effect of Hsp104 inactivation on prion propagation is to block the generation of new seeds (also called propagons) (30-32). Hsp104's prion-propagating activity, like its general disaggregating activity, also involves Hsp70s and nucleotide-exchange factors, as well as Hsp40s. Hsp70s, the cytoplasmic Ssas of Saccharomyces cerevisiae, are necessary for stable prion propagation (33-37), and can antagonize the curing of ] remains controversial. One proposal is that overproduced Hsp104 binds to a special site in the middle (M) domain of Sup35p (49) and so prevents Hsp70s from having access to the filaments, which access is believed necessary for the Hsp104-Hsp70-Hsp40 machine to extract a monomer from the filament and thereby cleave it (37). variants arising spontaneously in the absence of this Hsp104 overproduction curing activity are cured when that activity is restored at normal levels. This activity is thus an antiprion system, largely protecting the cells from prion formation by this protein.