In 1994 we discovered that yeast can have prions, infectious proteins analogous to the transmissible spongiform encephalopathies of mammals. We showed that the non-mendelian genetic element, URE3, is a prion of the Ure2 protein, and that PSI+ is a prion of Sup35p (1). We found the first biochemical evidence confirming our discovery (2) and defined the prion domain of Ure2p (2). These prions are amyloids of the respective protein (reviewed in 3). Unexpectedly, shuffling the prion domain amino acid sequence of Ure2p or Sup35p did not alter the ability of these domains to support prion formation, suggesting that the amyloid structure is parallel in-register (4). We have shown by solid-state NMR (in collaboration with Rob Tycko of NIDDK) that the amyloids of Ure2p, Sup35p and Rnq1p are indeed in-register parallel beta sheets (5-7). It has not escaped our notice that this in-register parallel beta sheet structure can explain how a given protein sequence can encode any of several biologically distinct prion variants based on biochemically distinct self-propagating amyloid structures (8). We have examined the URE3 prions based on Ure2 proteins from non-cerevisiae species of Saccharomyces, and have demonstrated a similar species barrier to that seen among mammals of different species in their transmission of spongiform encephalopathies (9). We showed that the variant properties, as defined by species barrier, are maintained even during passage through a different species. We also noted that the Ure2p of Saccharomyces castellii cannot become a prion (9). We find that the Candida albicans Ure2p can form a URE3 prion in S. cerevisiae, but that of Candida glabrata cannot, even though the prion domain of glabrata is closer in sequence to that of cerevisiae than is that of abicans (10). Thus the conservation of sequence in the Ure2 prion domains is not for prion-forming ability, but must reflect the function of this domain in protecting the full length protein from degradation in vivo (11). We find that the C. albicans Ure2 protein or its prion domain each readily form amyloid which is highly infectious for yeast, and, like the other yeast prions, has a parallel in-register beta sheet architecture (12). We have shown that PSI+ is rare in wild strains, though it would be common if it were advantageous (13). To further examine the biology of PSI+, we designed a method to screen for a lethal (Suicidal) PSI+ which efficiently incorporated all of the essential Sup35 protein into amyloid, should such a variant exist. We indeed found that lethal variants of PSI+ and those which produce extremely slow growth comprise more than half of total isolates (15). We also found that common variants of the URE3 prions cause extremely slow growth, although deletion of the URE2 gene in these strains did not slow growth (14). This toxic URE3 therefore cannot be due to a simple deficiency of Ure2p, but must be a due to a pathogenic amyloid. Current efforts are directed to understanding the nature of this toxicity. These results confirm the pathologic nature of the yeast prions PSI+ and URE3. Understanding their mechanisms of pathogenesis may be useful in understanding human amyloidoses. We have sequenced the SUP35 genes of 55 wild S. cerevisiae isolates, finding three groups of common polymorphs (15). PSI+ transmission between polymorphs is largely blocked, suggesting that these changes are selected to protect yeast from the detrimental effects of the prion (15). We find that the rare wild PSI+ variants are sensitive to these blocks as well, supporting this interpretation. M domain changes in one polymorph are important in blocking prion spread (15). We used population genetics to quantify the detriment to cells of carrying the yeast prions PSI+, URE3 and PIN+ (16). From the known detriment of carrying the 2 micron DNA plasmid, determined by three different groups, and the incidence of 2 micron DNA in wild strains (13), we could infer the frequency of outcross mating by S. cerevisiae. Using this information, and the rarity of the yeast prions in wild strains (13), we showed that even the mildest variants of each of these prions must confer at least a 1% growth/survival defect (16). Our structural studies of amyloid of transthyretin showed that it is unique among characterized pathologic amyloids in not having the in-register parallel beta sheet structure (17). 1. Wickner RB (1994) URE3 as an altered URE2 protein: evidence for a prion analog in S. cerevisiae. Science 264: 566 - 569. 2. Masison DC &Wickner RB (1995) Prion-inducing domain of yeast Ure2p and protease resistance of Ure2p in prion-containing cells. Science 270: 93 - 95. 3. Wickner RB, Edskes HK, Ross ED, Pierce MM, Baxa U, Brachmann A &Shewmaker F (2004) Prion Genetics: New Rules for a New Kind of Gene. Ann. Rev. Genetics 38: 681-707. 4. Ross ED, Minton AP &Wickner RB (2005) Prion domains: sequences, structures and interactions. Nat. Cell Biol. 7: 1039-1044. 5. Shewmaker F, Wickner RB &Tycko R (2006) Amyloid of the prion domain of Sup35p has an in-register parallel beta-sheet structure. Proc. Natl. Acad. Sci. USA 103: 19754 - 19759. 6. Baxa U, Wickner RB, Steven AC, Anderson D, Marekov L, Yau W-M &Tycko R (2007) Characterization of beta-sheet structure in Ure2p1-89 yeast prion fibrils by solid state nuclear magnetic resonance. Biochemistry 46: 13149 - 13162. 7. Wickner RB, Dyda F &Tycko R (2008) Amyloid of Rnq1p, the basis of the PIN+ prion, has a parallel in-register beta-sheet structure. Proc Natl Acad Sci U S A 105: 2403 - 2408. 8. Wickner RB, Shewmaker F, Kryndushkin D &Edskes HK (2008) Protein inheritance (prions) based on parallel in-register beta-sheet amyloid structures. Bioessays 30: 955 - 964. 9. Edskes HK, McCann LM, Hebert AM &Wickner RB (2009) Prion variants and species barriers among Saccharomyces Ure2 proteins. Genetics 181: 1159 - 1167. 10. Edskes HK, Engel A, McCann LM, Brachmann A, Tsai H-F, Wickner RB. Prion-forming abilityof Ure2 of yeasts is not evolutionarily conserved. Genetics 2011;188:81 - 90. 11. Shewmaker F, Mull L, Nakayashiki T, Masison DC, Wickner RB. Ure2p function is enhanced by its prion domain in Saccharomyces cerevisiae. Genetics 2007;176:1557 - 65. 12. Engel A, Shewmaker F, Edskes HK, Dyda F, Wickner RB. Amyloid of the Candida albicans Ure2p prion domain is infectious and has a parallel in-register b-sheet structure. Biochemistry 2011;50:5971 - 8. 13. Nakayashiki T, Kurtzman CP, Edskes HK, Wickner RB. Yeast prions URE3 and PSI+ are diseases. Proc Natl Acad Sci U S A 2005;102:10575-80. 14. McGlinchey R, Kryndushkin D, Wickner RB. Suicidal PSI+ is a lethal yeast prion. Proc Natl Acad Sci USA 2011;108:5337 - 41. 15. Bateman, DA and Wickner, RB. PSI+ Prion Transmission Barriers Protect Saccharomyces cerevisiae from Infection: Intraspecies 'Species Barriers'. Genetics 2012;190:569-579. 16. Kelly, AC, Kryndushkin, D, Shewmaker, FP, and Wickner, RB. Sex, prions and plasmids in yeast. Proc Natl Acad Sci USA 2012, in press. 17. Bateman, DA, Tycko, R and Wickner, RB. Experimentally derived structural constraints for amyloid fibrils of wild-type transthyretin. Biophys. J. 2011;101:2485-2492.

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Edskes, Herman K; Kryndushkin, Dmitry; Shewmaker, Frank et al. (2017) Prion Transfection of Yeast. Cold Spring Harb Protoc 2017:pdb.prot089037
Gorkovskiy, Anton; Reidy, Michael; Masison, Daniel C et al. (2017) Hsp104 disaggregase at normal levels cures many [PSI(+)] prion variants in a process promoted by Sti1p, Hsp90, and Sis1p. Proc Natl Acad Sci U S A 114:E4193-E4202
Wickner, Reed B; Edskes, Herman K; Kryndushkin, Dmitry et al. (2017) Genetic Methods for Studying Yeast Prions. Cold Spring Harb Protoc 2017:pdb.prot089029
Kryndushkin, Dmitry; Edskes, Herman K; Shewmaker, Frank P et al. (2017) Prions. Cold Spring Harb Protoc 2017:pdb.top077586
Wickner, Reed B; Kelly, Amy C (2016) Prions are affected by evolution at two levels. Cell Mol Life Sci 73:1131-44
Wickner, Reed B (2016) Yeast and Fungal Prions. Cold Spring Harb Perspect Biol 8:
Wickner, R B; Edskes, H K; Gorkovskiy, A et al. (2016) Yeast and Fungal Prions: Amyloid-Handling Systems, Amyloid Structure, and Prion Biology. Adv Genet 93:191-236
Wickner, Reed B; Edskes, Herman K (2015) Yeast killer elements hold their hosts hostage. PLoS Genet 11:e1005139
Wickner, Reed B; Shewmaker, Frank P; Bateman, David A et al. (2015) Yeast prions: structure, biology, and prion-handling systems. Microbiol Mol Biol Rev 79:1-17
Wickner, Reed B; Edskes, Herman K; Bateman, David A et al. (2015) Yeast prions: proteins templating conformation and an anti-prion system. PLoS Pathog 11:e1004584

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