The genetic properties of the [URE3] non-Mendelian element of Saccharomyces cerevisiae suggest that it is a prion (infectious protein) form of Ure2p, a regulator of nitrogen catabolism. In extracts from [URE3] strains, Ure2p was partially resistant to proteinase K compared with Ure2p from wild-type extracts. Overexpression of Ure2p in wild-type strains induced a 20- to 200-fold increase in the frequency with which [URE3] arose. Overexpression of just the amino-terminal 65 residues of Ure2p increased the frequency of [URE3] induction 6000-fold. Without this “prion-inducing domain” the carboxyl-terminal domain performed the nitrogen regulation function of Ure2p, but could not be changed to the [URE3] prion state. Thus, this domain induced the prion state in trans, whereas in cis it conferred susceptibility of the adjoining nitrogen regulatory domain to prion infections.

The [ PSI ⁺ ] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the structure of amyloid fibrils formed in vitro from purified recombinant Sup35 ^1–253 , consisting of the glutamine- and asparagine-rich N-terminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole–dipole couplings among ¹³ C nuclei in a series of Sup35NM fibril samples, ¹³ C-labeled at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular ¹³ C– ¹³ C distances of ≈0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of ≈0.5 nm. These results indicate that an in-register parallel β-sheet structure underlies the [ PSI ⁺ ] prion phenomenon.

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 HSP104results 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.

Viruses, plasmids, and prions can spread in nature despite being a burden to their hosts. Because a prion arises de novo in more than one in 10 ⁶ yeast cells and spreads to all offspring in meiosis, its absence in wild strains would imply that it has a net deleterious effect on its host. Among 70 wild Saccharomyces strains, we found the [ PIN ⁺ ] prion in 11 strains, but the [URE3] and [ PSI ⁺ ] prions were uniformly absent. In contrast, the “selfish” 2μ DNA was in 38 wild strains and the selfish RNA replicons L-BC, 20S, and 23S were found in 8, 14, and 1 strains, respectively. The absence of [URE3] and [ PSI ⁺ ] in wild strains indicates that each prion has a net deleterious effect on its host.

Many proteins can adopt self-propagating β-sheet-rich structures, termed amyloid fibrils. The [URE3] and [PSI ⁺ ] prions of Saccharomyces cerevisiae are infectious amyloid forms of the proteins Ure2p and Sup35p, respectively. Ure2p forms prions primarily as a result of its sequence composition, as versions of Ure2p with the prion domain amino acids shuffled are still able to form prions. Here we show that prion induction by both Ure2p and Ure2-21p, one of the scrambled versions of Ure2p, is clearly dependent on the length of the inducing fragment. For Ure2-21p, no single sequence is found in all of the inducing fragments, highlighting the sequence independence of prion formation. Furthermore, the sequence of the Sup35p prion domain can also be randomized without blocking prion formation. Indeed, a single shuffled sequence could give rise to several prion variants. These results suggest that [PSI ⁺ ] formation is driven primarily by the amino acid composition of the Sup35p prion domain, and that the Sup35p oligopeptide repeats are not required for prion maintenance.