The National Institute of Diabetes & Digestive & Kidney Diseases

Prions in Yeast are Protein Genes: Inherited Amyloidosis

We discovered two prions (infectious proteins) in S. cerevisiae, called [URE3] and [PSI]. In doing so, we proved that a protein can be a gene. We find that one of these prions, [URE3], is based on amyloid formation by Ure2p, a protein normally involved in regulating nitrogen metabolism.

The prion concept has a long history, originating with studies of scrapie.

Definition of "prion": A prion is an infectious protein, without any need for an accompanying nucleic acid.

Three Genetic Criteria for a prion.
Are there any prions in nature, and how can they be recognized? We proposed three genetic criteria that allow one to recognize a prion ¹, distinguishing it from a nucleic acid replicon, like a virus or a plasmid:

1. Reversible curability. If a prion can be cured, it can arise again spontaneously in the cured strain. Once cured, a virus or plasmid will not arise de novo or reappear unless re-introduced from outside the cell.
2. Overproduction of the protein should increase the frequency with which the prion form arises.
3. The phenotype produced by the presence of the prion is the same as the phenotype produced by mutation of the chromosomal gene encoding the protein, because in both cases the normal form is missing. It has either been converted into the (inactive) prion form, or is not made at all.

[URE3] and [PSI] have the genetic properties expected of prions. [URE3] is a non-Mendelian genetic element of S. cerevisiae that makes cells able to take up ureidosuccinate when ammonia is the nitrogen source ². [PSI] is a non-Mendelian genetic element that increases the efficiency with which weak suppressor tRNAs allow read-through of translation termination codons ³. We showed that [URE3] had all three properties expected of a prion of Ure2p, a repressor of many genes involved in nitrogen catabolism ¹. We also pointed out that the work of Cox, Sherman, Tuite, Ter-Avanesyan and Chernoff implies that [PSI] is a prion form of Sup35p, a subunit of the translation termination factor ¹.

Confirmation that [URE3] is a prion. We found that Ure2p is protease resistant, specifically in [URE3] strains, indicating, as predicted by the prion model for [URE3], that Ure2p is changed in [URE3] strains ⁴. We showed that this change is not a consequence of derepressed nitrogen regulation, but rather a cause of it ⁵. Further, the nitrogen source does not influence the frequency of [URE3] arising ⁵. [URE3] really does arise de novo when Ure2p is overexpressed, not as a mutant of some putative wild-type element ⁵. Moreover, it is the Ure2 protein whose overexpression induces [URE3] to arise, not the gene in high copy number or the RNA transcript ⁵. These results ruled out several alternate models and confirmed predictions of the prion model. As reviewed elsewhere, the work of Liebman, Ter-Avanesyan, Lindquist, Chernoff and Tuite has confirmed that [PSI] is a prion form of Sup35p.



Link to Science Magazine abstract

The prion domain of Ure2p. Deletion mutants of URE2 were examined for ability to induce de novo [URE3] formation and for ability to carry out nitrogen regulation. The data define a prion domain (residues 1-65) and a nitrogen regulation domain (residues 66-354) ⁴. The prion domain can propagate [URE3] in the absence of the nitrogen regulation domain, and the nitrogen regulation domain can regulate nitrogen catabolism without the prion domain ⁵. In fact, both segments can act independently if they are on separate molecules without mutual interference ⁵. Only if it has a prion domain attached, is the nitrogen regulation domain inactivated by the presence of [URE3] ⁵.

Link to Proceedings of the National Academy of Sciences Online

Ure2p is aggregated in vivo in [URE3] strains. Fusions of Ure2p and the Green Fluorescent Protein (GFP) were expressed in yeast cells in order to study the state of Ure2p in wild-type and [URE3] strains. The full-length Ure2p-GFP fusion protein was active in nitrogen regulation and could participate in the [URE3] change ⁶. We found that in wild-type strains, Ure2p-GFP was evenly distributed throughout the cytoplasm, but in [URE3] strains it showed marked aggregation. The aggregates were apparently randomly distributed in the cell.

Link to Science Magazine article

Prion Domain Initiation of Amyloid Formation in vitro from Native Ure2p. The Ure2 prion domain, Ure2p^1-65, was made as a synthetic peptide. Diluting the peptide from 6M guanidine into buffer resulted in the rapid formation of filaments 40-45 angstroms in diameter ⁷ (panel a). These filaments have all the characteristics of amyloid. In addition to their filamentous structure, they are essentially entirely beta-sheet in structure, are resistant to proteinase K treatment and show typical birefringence on staining with Congo Red. In parallel with the ability of the prion domain to induce de novo [URE3] formation in vivo with high efficiency, the prion domain peptide induces cofilament formation with native Ure2p, purified from normal yeast cells ⁷. These cofilaments (panel b) are also amyloid, and protease digestion leaves only the prion domain of Ure2p intact (panel c). The cofilaments can seed further amyloid formation by native Ure2p, forming thick filaments (panel d) ⁷.

There are now four candidate prions and the evidence that they are such varies:

1. Mammalian spongiform encephalopathies: PrP is necessary for scrapie and is the major component of purified infectious material. Is it sufficient?
2. [URE3]: Clearly a prion of Ure2p.
3. [PSI]: Clearly a prion of Sup35p.
4. [Het-s]: A new putative prion in Podospora anserina⁸.

References:

1. Wickner, R. B. (1994) Evidence for a prion analog in S. cerevisiae: the [URE3] non-Mendelian genetic element as an altered URE2 protein. Science 264, 566-569.
2. Lacroute, F. (1971) Non-Mendelian mutation allowing ureidosuccinic acid uptake in yeast. J. Bacteriol. 106, 519-522.
3. Cox, B. S. (1965) PSI, a cytoplasmic suppressor of super-suppressor in yeast. Heredity 20, 505-521.
4. Masison, D. C. & Wickner, R. B. (1995) Prion-inducing domain of yeast Ure2p and protease resistance of Ure2p in prion-containing cells. Science 270, 93-95.
5. Masison, D. C., Maddelein, M.-L., & Wickner, R. B. (1997) The prion model for [URE3] of yeast: spontaneous generation and requirements for propagation. Proc. Natl. Acad. Sci. USA 94, 12503-12508.
6. Edskes, H. K., Gray, V. T. & Wickner, R. B. (1999) The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments. Proc. Natl. Acad. Sci. USA 96, 1498-1503.
7. Taylor, K. L., Cheng, N., Williams, R. W., Steven, A. C. & Wickner, R. B. (1999) Prion domain initiation of amyloid formation in vitro from native Ure2p. Science 283, 1339-1343.
8. Coustou, V., Deleu, C., Saupe, S. & Begueret, J. (1997) The protein product of the het-s heterokaryon incopatibility gene of the fungus Podospora anserina behaves as a prion analog. Proc. Natl. Acad. Sci. USA 94, 9773 - 9778.