Sex, prions, and plasmids in PNAS PLUS

Amy C. Kellya, Frank P. Shewmakerb, Dmitry Kryndushkinb, and Reed B. Wicknera,1

aLaboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; and bDepartment of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814

Contributed by Reed B. Wickner, August 8, 2012 (sent for review April 22, 2012) Even deadly prions may be widespread in nature if they spread [PSI+] when assayed using a synthetic Sup35p with an elevated by infection faster than they kill off their hosts. The yeast prions prion-forming tendency (25). Here we test these conditions for [PSI+] and [URE3] (amyloids of Sup35p and Ure2p) were not found an effect on prion formation by the wild-type Sup35p. in 70 wild strains, while [PIN+] (amyloid of Rnq1p) was found in Our argument, that the rare occurrence of an infectious agent ∼16% of the same population. Yeast prion infection occurs only in nature implies pathologic effects on the host, relies on outcross by , balancing the detrimental effects of carrying the prion. mating being a fairly frequent event. Yeast prions spread only by We estimated the frequency of outcross mating as about 1% of mating (there is no extracellular prion species in the replication mitotic doublings from the known detriment of carrying the 2-μm cycle), and if outcross mating is extremely rare, then yeast prions DNA plasmid (∼1%) and its frequency in wild populations (38/70). could be rare even if their effects on the host were neutral. Yeast We also estimated the fraction of total that are outcross germinate with mating type either a or α, and a and α matings (∼23–46%) from the fraction of heterozygosity at the spores from the same tetrad may mate (intratetrad or brother– highly polymorphic RNQ1 (∼46%). These results show that sister). The mating-type switching system (homothallism) results the detriment of carrying even the mildest forms of [PSI+], [URE3], in the clone from a single germinated becoming a mixture or [PIN+] is greater than 1%. We find that Rnq1p polymorphisms in of a and α cells which can mate with each other. These two kinds wild strains include several premature stop codon that can- of “selfing” are unlikely to spread any virus, plasmid, or prion, not propagate [PIN+] from the reference and others with because probably both mating partners will lack the element, or several small deletions and point mutations which show a small both will have it. Alternatively, a germinated spore may mate transmission barrier. Wild strains carrying [PIN+] are far more with a haploid not derived from the same parent diploid (out- likely to be heterozygous at RNQ1 and other loci than are [pin−] cross mating), and this type of mating has the potential to spread EVOLUTION strains, probably reflecting its being a sexually transmitted disease. the nonchromosomal genetic element. The frequency of outcross Because sequence differences are known to block prion propagation mating of Saccharomyces paradoxus in the wild has been esti- or ameliorate its pathogenic effects, we hypothesize that polymor- mated at one per 105 mitotic divisions based on studies of 20 wild phism of RNQ1 was selected to protect cells from detrimental strains (26). Another study similarly estimated 0.5 outcross matings effects of the [PIN+] prion. per 105 mitoses for S. cerevisiae (27). Furthermore, outcross mat- ings in S. paradoxus were estimated to comprise only ∼1% of total two-micron DNA | population genetics matings (28). This very rare outcross mating then was used to calculate the detriment of [PSI+] (29) using our finding of [PSI+] rions (infectious ) include agents causing the spongi- being rare in the wild (20). Here, we use two different methods Pform encephalopathies of mammals (1–3) and an array of to measure the frequency of outcross matings, each resulting in a nonchromosomal genetic elements of the yeast Saccharomyces dramatically higher result than previous estimates, and with a cerevisiae and the filamentous fungus Podospora anserina (4–6). corresponding dramatic impact on estimates of the detriment of Most prions are self-propagating amyloids, linear filamentous carrying [PSI+], [URE3], and [PIN+]. polymers of a single . The prions [PSI+], [URE3], and Prion transmission generally is limited by differences in amino [PIN+] of S. cerevisiae are parallel in-register β-sheet amyloids of acid sequence between donor and recipient strains, a phenomenon Sup35p, Ure2p, and Rnq1p, respectively (7–10); the [Het-s] prion first seen in the species barrier of scrapie transmission (reviewed of P. anserina is a β-helical amyloid of the HET-s protein (11). in ref. 30), later recognized as a requirement for homozygosity at Sup35p is a subunit of the translation termination factor (12, 13), PrP residue 129 for Creutzfeldt–Jakob disease (31), and recently Ure2p is a regulator of nitrogen catabolism (14), and Rnq1p has reported in a barrier to the transmission of [PSI+] and of no known function (15). [URE3] between Saccharomyces species (32, 33). Even within S. The mammalian prions are uniformly lethal but nonetheless cerevisiae, there are several groups of polymorphs of Sup35p are found in wild animals. For example, chronic wasting disease of between which prion transmission is inefficient; we suggest the deer and elk is found in ∼10% of wild deer in parts of Wyoming, existence of these polymorphs was selected to protect yeast from Colorado, Illinois, and Wisconsin (16). Evidently, infectious the detrimental effects of [PSI+] (34), just as polymorphisms of spread outpaces the lethal effects on the animals. The [Het-s] human PrP are believed to have been selected to protect against prion of P. anserina is involved in heterokaryon incompatibility, kuru-like epidemics of prion disease (31). As for the interspecies a normal function of that species, and, as a result, ∼80% of wild barrier (35), we found that sequence differences in both the strains with the appropriate chromosomal genotype carry the [Het-s] prion (17, 18). The yeast prions [PSI+] and [URE3] also can be lethal (19), but the variants of [PSI+], [URE3], and [PIN+] used in most experiments are mild in their effects. While the lethal variants Author contributions: A.C.K., F.P.S., D.K., and R.B.W. designed research; A.C.K., F.P.S., D.K., and R.B.W. performed research; A.C.K., F.P.S., D.K., and R.B.W. contributed new of yeast prions doubtless are detrimental to their hosts, even the reagents/analytic tools; A.C.K., F.P.S., D.K., and R.B.W. analyzed data; and A.C.K., F.P.S., mild variants of [PSI+] and [URE3] are rare in the wild (20), D.K., and R.B.W. wrote the paper. a result confirmed by others (21) and which we interpreted as The authors declare no conflict of interest. meaning that they are substantially detrimental. Nonetheless, Data deposition: The sequences reported in this paper have been deposited in the others argue that these prions are beneficial, promoting survival by GenBank database (accession nos. JX468101–JX468345). allowing cells to resist stress (22, 23), although the reported effects 1To whom correspondence should be addressed. E-mail: [email protected]. were not reproducible with the same strains (24). It is reported This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. further that certain stress conditions increase the frequency of 1073/pnas.1213449109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1213449109 PNAS Early Edition | 1of8 Downloaded by guest on October 1, 2021 Sup35p N and M domains are important in the intraspecies- found in >5% of isolates. The frequencies of the most common transmission barriers (34). Rnq1p has been reported to be quite alleles are shown in Fig. 2 and Table S2). polymorphic in clinical isolates of S. cerevisiae (36). Here we ex- Nucleotide diversity (π, the average number of pairwise nu- amined a wider range of wild S. cerevisiae strains and tested the cleotide differences per nucleotide between all possible strain effects of these polymorphisms on transmission of the [PIN+] prion. pairs) at RNQ1 was similar to that in YGL108C and to the average for S. cerevisiae but was slightly higher than in TRP1 (Table 1). Results In contrast, nucleotide polymorphism (θ, the total number of Estimation of Mating Frequency Based on 2-μm DNA Plasmid. Both μ polymorphic sites per nucleotide, standardized by the number of Futcher and Cox (37) and Mead et al. (38) have shown that 2- m strains) for RNQ1 was higher than in the two control or in DNA slows the growth of laboratory haploid or diploid strains the S. cerevisiae genome (39) (Table 1). by 1–3%. We used similar mixed-culture competition methods Nucleotide diversity and nucleotide polymorphism were highly (Materials and Methods), constructing duplicates of each strain, fi variable across the length of RNQ1 (Fig. S2), reaching maximum to con rm this result with the wild strain, Y1089, which shows an π ∼ μ values in the C-terminal prion domain (maximum = 0.021, 16% slowing of growth by 2- mDNA(Fig. S1). Strain Y1089 θ originally lacked 2-μm DNA, perhaps for this reason. For further maximum = 0.019). When analyzed separately, the prion do- calculations,weusetheconservativeestimateof1%growthslowing main showed slightly higher values of nucleotide diversity, nucle- to get a minimum estimate of the frequency of outcross mating. otide polymorphism, and polymorphism frequency (on average, To calculate the frequency of outcross mating, we note that one polymorphism every 14 bp in the C domain vs. every 23 bp in the 1% slowing of growth by 2-μm DNA (and the rare spontaneous the N domain) (Table 1). loss of the plasmid) must be just balanced by the spreading of the Nearly half (49%) of wild isolates were heterozygous at RNQ1 plasmid by outcross matings, resulting in an equilibrium frequency for one or more polymorphisms, whereas 26% were heterozygous of occurrence of the plasmid in 38 of 70 wild strains (Discussion, at TRP1, and only 9% were heterozygous at YGL108C. Observed Outcross Mating Calculated from 2-μmDNAData). This fact heterozygosity was much higher in [PIN+] than in [pin−] strains implies that there is about one outcross mating per 100 mitotic for all three loci analyzed (Table 1). divisions, similar to analogous estimates by Futcher and Cox (37). Tajima’s D is roughly the normalized difference (diversity − polymorphism). Positive values of Tajima’s D suggest balancing Natural Variants of RNQ1 in Wild Strains of S. cerevisiae. To quantify selection (e.g., heterozygote advantage) or diversifying selection intraspecies genetic variation and examine patterns of natural (minor allele advantage). Negative values may suggest purifying selection, we sequenced the RNQ1 locus and, as controls, TRP1, selection (against minor alleles). Values calculated for RNQ1, a critical for growth we expected to be conserved, and TRP1,andYGL108C were slightly negative although not sig- YGL108C, a nonessential gene with no known function that nificantly different from zero. When calculated exclusively for might evolve neutrally. We used the S. cerevisiae population nonsynonymous sites at RNQ1,Tajima’s D was negative (D = genomic structure inferred by Liti et al. (39) as a guide to obtain − fi yeast strains from as many genetic subpopulations as possible. 1.66) and marginally nonsigni cant (P = 0.06), indicative of Eighty-three wild and domesticated yeast isolates originating weak purifying selection typical for S. cerevisiae genomes (39). ’ from a variety of ecological niches and geographic locations were However, Tajima s D values were variable across the RNQ1 locus sampled (Table S1). RNQ1 was more polymorphic than the (Fig. S2) and, when calculated for 100-bp regions of DNA, revealed control genes, with 16 indels, 37 synonymous SNPs, and 22 marginally significant positive values (D =2.2,P < 0.05) for nonsynonymous SNPs, five of which resulted in premature stop nucleotides 1002–1126 within the C-terminal domain of the protein. codons (Fig. 1). Overall we detected 75 polymorphisms resulting The McDonald–Kreitman tests for neutrality (40) were not in 66 distinct nucleotide haplotypes and 44 distinct protein significant for the three loci tested (Table S3), indicating that haplotypes in the sampled population. Most of the polymorphisms adaptive evolution has not acted on these loci since the consisted of low-frequency alleles, although nearly a third were S. cerevisiae and S. paradoxus lineages split.

Fig. 1. Polymorphisms in RNQ1 (Upper) and Rnq1p (Lower) sequences detected in 83 wild S. cerevisiae strains. Polymorphic sites are indicated by base-pair position in RNQ1 and codon in Rnq1p. Insertions are denoted by “ins” after the nucleotides/amino acid(s) flanking the insertion site. Deletions are indicated by “Δ” followed by the deleted positions. An asterisk indicates a mutation that was found in only one strain. A indicates that the polymorphism resulted in a premature stop codon.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1213449109 Kelly et al. Downloaded by guest on October 1, 2021 rad) or outcross matings, then outcross matings constitute PNAS PLUS >23% of total matings (Discussion, Calculation of the Fraction of Outcross Mating from the Heterozygosity at RNQ1). Our data do not allow us to discern what fraction of non-outcross matings are homothallic and what fraction are intratetrad, but calcula- tion of these two extreme cases indicates that the true fraction of outcross matings must be at least 23–46%.

Estimate of Pathogenicity of [PSI+], [URE3], and [PIN+]. Above, we used the known detriment of carrying 2-μm DNA and its in- cidence in the wild to calculate the frequency of outcross mating, the only way that its incidence can be maintained at the observed levels. Using the same equations (Discussion, What About [PSI+], [URE3], and [PIN+]?), we can use the now known frequency of outcross mating and the observed low frequency of [PSI+], [URE3], and [PIN+] to calculate the detriment to the cell of carrying each prion. Even before calculation, several elementary considerations show that each of these prions must be at least as detrimental for the cell as 2-μm DNA. Each prion arises de novo − at a measurable frequency {[PSI+] arises at about 10 5 in cells − − with [PIN+] (41), and [URE3] at about 10 5 to 10 6 (4, 42)}, but 2-μm DNA does not arise de novo short of geologic time. The Fig. 2. Protein haplotypes for Rnq1p. We used the program DNAsp version −4 fi plasmid is lost at about 10 , but laboratory variants of [PSI+] 5 to reconstruct haplotype phase. Phase reconstruction was veri ed in het- ∼ −5 erozygous individuals using sequences generated from single spores or from are lost at only 10 . Each of these facts should make the prions cloning reactions. A description of the protein polymorphisms encoded by more widespread than 2-μm DNA, but [PSI+], [URE3], and each haplotype and the frequency (%) in the population are shown. [PIN+] are each found less frequently in wild strains than the plasmid, so each must be at least as detrimental as 2-μm DNA. EVOLUTION Our model calculation indicates that [PSI+], [URE3], and [PIN Transmission Barriers Between Rnq1p Polymorphs. Laboratory strains +] each confer detriments of ∼1.0% on their hosts, given the were prepared with RNQ1 from several wild strains (Table S4), ∼1%detrimentof2-μmDNA(Discussion, What About [PSI+], and [PIN+] was introduced by cytoplasmic mixing (cytoduction) [URE3], and [PIN+]?), As is evident in Table 3 (see also- from a strain with the reference RNQ1 sequence (Table 2). Most Discussion, What About [PSI+], [URE3], and [PIN+]?), the recipients could propagate [PIN+], showing no transmission detriment calculated in our model is not very sensitive to prev- barrier. The premature termination polymorphs with the largest alence in the range observed for the prions and 2-μm DNA but is residual fragments each failed to propagate [PIN+], and we pre- critically sensitive to the frequency of outcross mating. sume those making even shorter fragments would fail also. An allele with several point mutations and two small deletions also Stress Does Not Induce [PSI+] Formation. It has been reported that showed a mild transmission barrier (Table 2). under certain stress conditions the frequency with which [PSI+] arises de novo is elevated in cells with a synthetic Sup35p prion Estimate of the Fraction of Outcross Matings Based on RNQ1 domain modified to make it more susceptible to prion formation Heterozygosity. Because RNQ1 is highly polymorphic, the level (25). Although under most such conditions, being [PSI+] made the of heterozygosity is particularly well suited for estimating the cells less able to survive the corresponding stress, it was con- fraction of matings that are outcross matings, as opposed to cluded, paradoxically, that this stress induction of [PSI+] was intratetrad matings or homothallism matings (Introduction). adaptive (25). We tested whether these conditions would elevate If we assume that matings can be only homothallic or outcross [PSI+] formation in cells with a wild-type Sup35p prion domain matings, then outcross matings constitute >46% of total mating to determine if this mechanism might be relevant to the biological (Discussion, Calculation of the Fraction of Outcross Mating from role of this prion. We found that under four of the conditions the Heterozygosity at RNQ1). If we assume that matings can be reported to elevate [PSI+] formation, there was no significant only intratetrad (between germinating spores of the same tet- change after 7 d incubation (Fig. S3). If stress induction of [PSI+]

Table 1. Genetic variability and natural selection metrics for 83 wild isolates of S. cerevisiae Length Nucleotide Nucleotide Nonsynonymous Synonymous [pin−] [PIN+] † ‡ § ¶ Locus (bp) Polymorphisms* diversity π polymorph. θ SNPs SNPs Ho Ho

RNQ1 1,218 75 0.005 0.009 22 37 0.38 0.80 RNQ1N 458 20 0.004 0.008 6 14 0.26 0.67 RNQ1C (prion domain) 760 55 0.006 0.010 16 17 0.26 0.73 TRP1 (essential) 675 18 0.002 0.005 8 10 0.15 0.73 YGL108C 423 13 0.004 0.006 4 9 0.03 0.13 (nonessential) S. cerevisiae genome|| 0.006 0.006

*Includes all SNPs, insertions, and deletions. † π, nucleotide diversity; the average number of nucleotide differences per base between two strains. ‡ θ, nucleotide polymorphism; the total number of nucleotide differences per gene, standardized by the number of samples. §The proportion of the 68 [pin-] strains that were heterozygous. Only polymorphisms observed in >1 strain were included in the calculation. ¶The proportion of the 15 [PIN+] strains that were heterozygous. Only polymorphisms observed in >1 strain were included in the calculation. ||From Supplementary Information in ref. 39.

Kelly et al. PNAS Early Edition | 3of8 Downloaded by guest on October 1, 2021 Table 2. Transmission of [PIN+] between alleles Donor (reference allele) Recipient (wild mutant) Wild allele [PIN+] cytoductant [pin−] cytoductant

4974 5000 G59R 12 0 4974 4999 G59R 12 0 4974 5006 Q235ter 0 12 4974 5005 Q235ter 0 14 4962 5014 Y249ter 0 8 4962 5016 Y249ter 0 9 4962 5110 Q193ter, Δ156–161 0 12 4962 5111 Q193ter, Δ156–161 0 11 4974 4995 GQ@166, Δ297–307 10 0 4974 4993 GQ@166, Δ297–307 12 0 4962 4996 GQ@166, Δ297–307 12 0 4962 5009 N377K 30 0 4974 5054–12B GQ@166 4 0 4962 5054–7C GQ@166 17 0 4962 5054–9D GQ@166 13 0 4962 5052–2B Δ165–166, Q360H 4 0 4962 5052–1A Δ165–166, Q360H 9 0 4962 5052–10B Δ165–166, Q360H 7 0 4974 5061–13C G158S, Δ167–170, N238S, 67 4 5061–19D Δ286–296, Q360H, Q383H 5061–21A 5061–28B 4962 5061–25C G158S, Δ167–170, N238S, 26 1 Δ286–296, Q360H, Q383H

Recipient strains were each cured of [PIN+] (if any) by growth three times to single colonies on YPAD with 3 mM guanidine. The [PIN+] strains 4962 and 4974 with the reference allele of RNQ1 were used as cytoplasm donors to strains carrying the indicated alleles from wild strains in cytoduction experiments. Cytoductants were scored for [PIN+]/[pin−] as described in Materials and Methods. GQ@166, insertion of GQ before residue 166.

were a conserved mechanism, we would expect reproducible and rate of slowing of growth plus spontaneous loss must be balanced substantial induction, much like the induction that is achieved by spread by outcross mating to maintain the incidence of 2-μm easily by NM overexpression (Fig. S3). DNA in 38 of 70 wild strains (20). We calculate that, assuming a conservative 1% slowing of growth, outcross mating of yeast Discussion must occur about once per 100 mitotic divisions (see calculations Yeast prions, like yeast viruses, never propagate via the extra- for outcross mating below). cellular space, so infectivity is limited by the frequency of out- Conservative assumptions in our calculation of mating fre- cross matings. Naturally occurring viruses and plasmids that are quency include (i) taking 1% as the detriment of 2-μm DNA, (ii) only slightly detrimental provide a yardstick for comparison with assuming random mating, (iii) ignoring mitotic recombination, μ prions. Previous work from two groups showed that the 2- m (iv) assuming and sporulation are completed as quickly as DNA plasmid slows the growth of haploid or diploid laboratory , and (v) neglecting plasmid loss in meiosis. Nonrandom S. cerevisiae by ∼1–3% (37, 38), and our data with a wild strain distribution of cells with and without 2-μm DNA leads to non- confirm this picture. Although past attempts to find an advantage random mating and a slower spread of the plasmid. The actual of carrying 2-μm DNA have failed, it remains possible that 2-μm DNA is advantageous under some circumstances, and such an outcross mating must be greater than our estimate to give the advantage would weigh against the detriment shown here and observed incidence of the plasmid. Cluster analysis of our sequence by others. data suggests that our wild strains are grouped into several μ Because 2-μm DNA does not infect detectably by an extra- subpopulations, but strains with and without 2- mDNAare cellular route (37) or arise de novo short of geologic time, the found in each subpopulation. The assumed even probability of meiosis and sporulation in each generation certainly is unrealistic but again is a conservative assumption, because a series of meioses Table 3. Prion detriment localized in space and time could saturate the population with 2-μm Detriment of [PSI+] (1 − R) DNA or waste meioses if none of the local population had the plasmid. Because outcross diploids could be either more or less fit P = prevalence of [PSI+] if M = 0.1 if M = 0.01 if M = 0.00001 than intratetrad or intraspore diploids, we have assumed that there fi 0.1 0.092 0.0099 0.00001 is no difference. If outcross diploids were generally more t, we 0.01 0.092 0.0100 0.0001 would get a falsely high outcross mating estimate, but selective 0.001 0.092 0.0109 0.001 survival of outcross diploids also would favor spread of plasmid- or 0.0001 0.098 0.0197 0.01 prion-containing cells. The conclusion that the prions are more 0.00001 0.173 0.095 0.099 detrimental than 2-μm DNA is independent of this issue. We examined wild alleles of the RNQ1 gene and found that − Prion detriment = 1 R, where R = growth/survival rate of the prion RNQ1 is somewhat more variable than the control genes TRP1 -arrying strain compared with the strain lacking the prion) as a function of θ prion prevalence found in the population (p) and the outcross mating fre- and YGL108C, having both a larger number of polymorphs ( = quency per mitosis of S. cerevisiae (M). This calculation was done by iterative nucleotide polymorphism) and a slightly higher average diversity application of Eqs. A and B using Dataset S1. (π = nucleotide diversity). We recognize that our widely dis-

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1213449109 Kelly et al. Downloaded by guest on October 1, 2021 tributed sample may include subpopulations with properties not resulted from underestimating the population from which those PNAS PLUS detected in our analysis that assumes random mating. clones arose. We find that these stress conditions do not elevate We tested the effects of RNQ1 polymorphisms on transmission [PSI+] formation in a strain with a normal Sup35 prion domain, of [PIN+] from cells with the reference RNQ1 sequence. The again indicating that [PSI+] formation is not adaptive. More- polymorphs we tested with premature termination codons could over, amyloid formation by prion proteins is a random process, is not be infected with this [PIN+], consistent with previous data not easily regulated, and the character of prions arising de novo delimiting the Rnq1p prion domain (15, 43). Strains with such is very heterogeneous, ranging from mild forms to toxic or even alleles also would have much lower frequencies of [PSI+] or lethal variants. At this time, no nonartificial condition reliably [URE3] generation. An allele with two small deletions and a few activates known prions. It is argued that an unstable [PSI+] could missense mutations (found, for example, in CBS405) produced a help yeast overcome stress transiently (46), but if there is no small transmission barrier for [PIN+], but other alleles appar- reproducible stress resistance imparted by [PSI+], the prion ently were compatible with free transmission of [PIN+]. Poly- is detrimental, whether stable or not. Thus, prions would appear morphisms of the Sup35p prion domain among wild S. cerevisiae to be an unsuitable means of regulating cellular activities. In produce strong barriers to [PSI+] transmission between any pair contrast, the [Het-s] prion is present in most wild strains, has of the three sequence groups observed (34). The greater barriers only one known variant, and appears to be functional (17, 18). for [PSI+] than for [PIN+] transmission may reflect the fact that Other lines of evidence indicate that yeast prions [URE3] and Sup35p is an essential protein (44) and that [PSI+] often is lethal [PSI+] are diseases. The presence of either prion induces the (or near-lethal) (19), whereas RNQ1 is nonessential (15), and the expression of Hsp104 and Hsp70, suggesting that the cells con- only documented danger of [PIN+] in a wild-type strain is the sider these prions a stress (47, 48). Frequent variants of [URE3] very rare priming of the formation of other prions (45). and [PSI+] can be toxic or lethal, and the variety of such harmful This polymorphism also provides an independent measure of variants has only begun to be examined (19). Extensive barriers what fraction of matings are outcrosses. Although the S. para- to transmission of [PSI+] between wild S. cerevisiae have been doxus study found only one of 28 diploids to be heterozygous and found, suggesting that this protection from “catching” a prion has estimated outcross matings as representing only 1% of total been selected in evolution (34). The notion that prion-forming matings (28), we found nearly 50% of wild S. cerevisiae to be ability is generally conserved (46) is not true for Ure2p, as the heterozygous for RNQ1, indicating a much higher fraction of Saccharomyces castellii, Kluyveromyces lactis,andCandida glabrata matings being outcrosses (compared with homothallic matings Ure2ps cannot become [URE3] even though they are closely re- and intratetrad matings). Our result indicates that a large frac- latedtothatofS. cerevisiae Ure2p, whereas the more distantly EVOLUTION tion of matings are outcross matings, consistent with our esti- related Ure2p can form a prion (33, 49, 50). mate of outcross mating based on the 2-μm plasmid. Our data do Prion-forming ability appears to be sporadic rather than conserved. not allow us to determine independently what fraction of matings The continued presence of prion domains reflects their nonprion were homothallic and what fraction were intratetrad. If we assume functions. The prion domain of Sup35p is important for the gen- that matings were either outcross or intratetrad, then outcross eral mRNA turnover process (51, 52), and that of Ure2p is im- matings were >23% of the total. If we assume matings were either portant in stabilizing the protein against degradation in vivo (53). outcross or homothallic, then outcross matings were >46% of all Previous theoretical analysis (29) of the detriment to cells matings. Because mitotic recombination produces homozygosity carrying [PSI+] necessarily used the values then current for and we cannot correct for such events, these are minimum esti- various parameters. The very low outcross mating frequency of S. − mates. Similar calculations using heterozygosity at TRP1 and paradoxus,10 5 per mitosis, was assumed to apply to S. cerevisiae. YGL108C give estimates of 13–35% for outcross mating. Our results indicate that this assumption results in a 1,000-fold It also was striking that 80% of the 15 [PIN+] strains were het- underestimate of the rate of spread of [PSI+] and a comparable erozygotes and thus were the products of outcross matings, whereas 100-fold underestimate of the degree of detriment it must impart only 45% of [pin−] strains were heterozygotes, a difference signif- to remain so rare. In addition, the assumption that [PSI+] arises − icantattheP = 0.015 level (Fisher’s exact test). The high hetero- at a frequency of 10 5 is based on determinations that ignore the zygosity of RNQ1 in [PIN+] strains may reflect an amelioration of greater number of very sick or suicidal [PSI+] that now are the toxicity of [PIN+] in a heterozygote or the recent acquisition known (19) and the likelihood that other lethal forms of the prion of [PIN+] (a sexually transmitted disease) in an outcross. exist. It was argued (29) that the Sup35p prion domain has no Using our revised value of outcross mating frequency, we can important function that would preclude its loss of [PSI+]-forming estimate the growth/survival defect that must occur in the wild ability by mutation, because a 19-residue deletion (Δ19) in the to explain the rarity of the yeast prions [PSI+], [URE3], and repeat part of Sup35N could not form [PSI+] (36). In fact, we [PIN+] in wild strains (Discussion, What About [PSI+], [URE3] have shown that the Δ19 Sup35p can form [PSI+], although there and [PIN+]?). Assuming that [PSI+], [URE3], and [PIN+] are is a transmission barrier from the reference Sup35p (34). The present in ∼1%, 1%, and 16% of wild strains, respectively, they argument is paradoxical, because if Resende et al. (36) had shown must each confer a detriment of about 1%. It is evident that in that the Δ19 Sup35p (a wild polymorph) cannot become [PSI+], our model the frequency of outcross mating drives the calculated it would negate the assertion that [PSI+]-forming ability is con- detriment (Table 3). served. Of course, the role of the prion domain in general mRNA A report that [PSI+] makes cells resistant to heat or to ele- turnover via interaction with the polyA binding protein (51, 52, vated ethanol concentration (22) was not confirmed by a second 54), and probably in other important functions (55), can explain report (23), and the benefits of [PSI+] reported in this second why this part of the molecule is preserved. Legs are conserved report again were not confirmed by a third report using the same among mammals to facilitate locomotion, not to be broken, but strains (24). If yeast prions do provide a benefit, then yeast a broken leg could aid the survival of a young man in a 1916 should be able to induce their appearance under suitable con- Russian village when the army draft board called. Likewise, a wild ditions. The frequency of [PSI+] arising de novo is reported to yeast that just happens to have a nonsense mutation in a vital be elevated under certain stress conditions in a strain with an gene might survive if it is [PSI+]. At present, however, there is expanded Sup35 prion domain that converts to the prion form no evidence that [PSI+], [URE3], or [PIN+] is beneficial. more often, even without stress (25). The extensive cell death In sexual organisms, the mutation rate appears to be lowered found under the stress conditions may have supplied adenine to to a level limited by the cost of further fidelity [reviewed in ref. the remaining cells, allowing residual growth, and thus the ap- 56] or by the power of selection for an even lower mutation rate parent slight elevation of [PSI+] clones recovered may have (57), not by a need for beneficial mutations. If the mating fre-

Kelly et al. PNAS Early Edition | 5of8 Downloaded by guest on October 1, 2021 quency of S. cerevisiae were once per 105 mitoses, as estimated Cells that go through mating-meiosis (fraction M of the total) for S. paradoxus (26), one might argue that this yeast is basically will have the plasmid if both mating partners have the plasmid 2 asexual, and selection for diversity can operate in such circum- (probability pna ) or if the MATa partner has the plasmid and the stances (58). Our evidence that S. cerevisiae mates ∼1,000-fold MATα partner lacks it [pna(1 − pna)] or the reverse [(1 − pna)pna]. 2 − more frequently indicates that it is largely sexual and argues that So the part of pnb for mating cells is M[pna +2pna(1 pna)]. ¼ ¼ð − Þ½ð Þð − Þþð − Þð Þþ selection for diversity is not feasible. Cells have a wide array Therefore, pnb pnþ1 1 M pna 1 L 1 pna G ð 2 þ ð − ÞÞ: of systems to prevent and repair DNA damage, suggesting that M pna 2 pna 1 pna thereisnotashortageofdiversity. At steady state, pn+1 = pn; i.e., the fraction of cells with The evidence presented here indicates that S. cerevisiae mates plasmid does not change. μ far more often than had been thought, that a large proportion of Substituting values for 2- m DNA: these matings are outcross matings, and thus that any stable À Á À Á ¼ ¼ ¼ : ¼ − : : nonchromosomal genetic element that is not found in most cells pnb pnþ1 pn 0 54 1 ÀM 0 5375 À0 9999 ÁÀ ÁÁ þ : þ : : must be detrimental. M 0 2889 2 0 5375 0 4625 ¼ 0:5374 − 0:5374M þ 0:7861M Outcross Mating Calculated from 2-μm DNA Data. We present an ¼ 0:5374 þ 0:2486M elementary treatment, following Hickey (59), of a subject that M ¼ 0:0026=0:2486 ¼ 0:01046: has been dealt with in much greater depth and sophistication by others (29, 59–62). We divide the calculation into phase “a,” the effect of the Note that we have assumed in the phase-b analysis that the average time taken for mating-meiosis is the same as for mitotic growth defect (or advantage) of cells with the plasmid, and phase growth. Actually, mating-meiosis, particularly the meiosis-spor- “b,” which accounts for the loss or gain of the plasmid. We as- ulation part, generally takes more than 10 times as long as one sume that these effects are independent. Let the fraction of cycle of mitotic growth. This approximation again is a conserva- n p plasmid-positive cells at the start of generation be n; after tive assumption that results in an underestimate of the frequency phase a it is pna; after phase b it is pnb = pn+1. The fraction of − − of mating and meiosis. plasmid-negative cells at generation n is 1 pn; similarly for 1 pna, ≥ − Thus, mating-meiosis occurs 1% as often as mitotic division. and 1 pnb. This conclusion is reasonable, because we assumed the growth Growth defect evaluation (phase a). R = growth rate of plasmid- – – defect is 1% (it has been measured at 1 3%, and even higher in positive cells compared with plasmid-negative cells (0.97 0.99 a wild strain), and the mating must compensate for that deficit. The μ − for 2- m DNA): rate of plasmid loss is only 10 4 andsoisamuchsmallerfactorthan ¼ = ¼ð Þð Þ=½ð Þð Þþð − Þ: the growth defect caused by the plasmid. Futcher and Cox (37) pna plasmid-pos cells total cells R pn R pn 1 pn made a similar estimate on similar grounds (once per 200–400 divisions), but Futcher et al. (64) apparently made a calculation

For pn = 38/70 = 0.54, R = 0.99, we get pna = 0.99(0.54)/[0.99 error, reaching a value of 0.02 matings per 100 mitotic divisions, (0.54) + 0.46] = 0.5375. assuming 99% of strains carry the plasmid, instead of the ∼2 Note that at equilibrium, the fraction of plasmid-positive matings per 100 mitotic divisions that our calculation implies cells at generation n must equal that at generation n+1, so the with their assumptions. Calculated fractions of outcross mat- decrease in phase a must be just balanced by an increase in ings are tabulated for various possible observed detriment phase b. values in Table S5. Loss or acquisition of plasmid (phase b). Hickey (59) assumes all cells mate every generation and sets the fraction with plasmid after Calculation of the Fraction of Outcross Mating from the Heterozygosity RNQ1 mating at p2 +2p(1 − p). However, in our case only a fraction of at . We observed 38/83 (46%) heterozygosity in wild strains at RNQ1 cells mate in each generation, and matings can be (i) within , counting only alleles observed in more than one strain so that none are caused by mutations arising in the particular wild a spore clone, (ii) between different cells from the same tetrad, strains studied and thus all heterozygosity must originate from or (iii) between cells from different tetrads (an outcross). Only mating (a conservative approximation). A yeast spore originating outcrosses have the possibility to spread the plasmid and are from a heterozygote (at RNQ1) can mate with (i)anothermember assumed to be random in the population. The probability of such of the same tetrad, with a one-in-three chance of becoming ho- outcross per generation is denoted M. μ mozygous (actually a 0.3223 chance, because RNQ1 is loosely As shown by Futcher and Cox (37), 2- m DNA does not linked to the mating type on III); (ii)withanother spread except by mating; there is no extracellular mode of member of the same spore clone whose mating type was switched μ spread. In meiosis, 2- m DNA segregates 4+:0 (63). by homothallism, resulting in complete homozygosis at all loci; or ∼ −4 L = probability of plasmid loss in one generation [ 10 for (iii) with a haploid not from the same tetrad (outcross mating). μ 2- m DNA (37)]. If we ignore intratetrad (type i) mating, then >46% of all mat- G = probability of spontaneous plasmid gain in one genera- ings are outcross matings because homothallic (type ii)matings μ tion (essentially 0 for 2- m DNA but nonzero for prions). make the cells homozygous. The actual percentage is greater, L, G, and M are small, so we assume LG, GM, and LM are because this analysis ignores mitotic recombination and the oc- each negligible. LG and GM are essentially zero. LM must be casional outcross mating with the same allele, each of which can very small, and neglecting it only decreases the estimate of mating produce homozygosity at RNQ1 even in a diploid that came from frequency, a conservative assumption. Unlike [PSI+], some outcross mating. variants of [URE3] are often lost in meiosis, so this analysis only If we ignore homothallic (type ii) matings, then matings will be applies to those variants that segregate 4+:0. either outcross (type iii) or intratetrad (type i). We consider Cells not mating (1 − M of the total) will have the plasmid spores from the 54% homozygous and 46% heterozygous dip- after phase b if they had it at the end of phase a (pna) and did not loids separately. Spores from a heterozygous diploid that mate lose it (probability 1 − L) or if they lacked it at the end of phase within the tetrad (frequency s) will become homozygous with a(1− pna) and gained it (G). a probability of 0.3223 (see above) and will remain heterozygous So the nonmating part of pnb is (1 − M)[(pna)(1 − L)+(1− pna) with a probability of 0.6777. Those that have an outcross mating (G)]. (frequency 1 − s) will become heterozygous with a probability of

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1213449109 Kelly et al. Downloaded by guest on October 1, 2021 2 PNAS PLUS 95% (= 1 − Σalleles pallele ) based on the distribution of alleles we treatments tested by Tyedmers (25) that resulted in increased [PSI+] gener- find in the population (Table S2). All spores from a homozygous ation killed most cells. If the majority of plated cells (for the prion induction assay) were dead, they would provide enough nutrients for the live cells to diploid will remain homozygous if they have an intratetrad mating fi and will remain homozygous with average probability 0.05 if they replicate without selection, thus causing an arti cial increase in the number of [PSI+] colonies. Therefore, we could test the ability to induce prion gen- have an outcross mating. We assume that the observed fractions eration in only the four least toxic conditions (below). Strain FPS372 was of homozygotes and heterozygotes are equilibrium values. grown overnight in synthetic complete (SC) medium. Cells (108−109) were Thus, the fraction of homozygotes = p(homo) = 0.54. washed and transferred to SC medium supplemented with water or with ¼ 0:46 ðsð0:3223Þþð1 − sÞð0:05ÞÞ þ 0:54ðs þ 0:05ð1 − sÞÞ each of the following: 2M KCl, 1M MgAc, 1M MnCl2, and 2M NaCl. Cells : ¼ ð : − : þ : − : Þþ : were incubated at 30 °C for 24 h and then were washed and plated as serial 0 54 s 0 1483 0 023 0 54 0 027 0 05 dilutions on solid SC medium and on solid SC medium without adenine. ¼ sð0:6383Þþ0:05 After 7 d the total numbers of adenine-positive cells were calculated as s ¼ 0:49 = 0:6383 ¼ 0:768 fraction of matings within a percentage of the total live cells plated. Selected adenine-positive isolates the tetrad; 1 − s ¼ outcross frequency > 23:2% subsequently were grown on 3 mM guanidine medium to distinguish be- tween [PSI+] and chromosomal mutants. About 20% of the selected ade- nine-positive isolates were confirmed to be [PSI+]. This value is a minimum estimate of the fraction of outcross matings because mitotic recombination also can result in homo- Two-Micron Plasmid Growth Competition Experiment. Our experimental de- zygosis at RNQ1, although RNQ1 is relatively close to its centro- sign, modeled on that of Futcher and Cox (37), used the red ade2 phenotype mere (43 kb), so this effect will not be a big effect (mitotic to mark [cir+] and [cir-o] strains in cis and trans. We created four isogenic recombination homozygosis only affects markers centromere-distal derivatives of the [cir-o] wild fermentation strain, Y1089: (i) ADE+ [cir+], (ii) to the site of the crossover). It was probably a much larger factor in ade2 [cir-o], (iii) ADE+ [cir-o], and (iv) ade2 [cir+]. The naturally heterothallic the study of S. paradoxus (26) because the loci used included three Y1089 was grown in sporulation medium (0.3% potassium acetate, 0.02% that were 326, 362, and 935 kb from their respective centromeres. raffinose) at 30 °C for 10 d. Following tetrad dissection, a single MATα spore was isolated and streaked to single colonies. In two of these colonies, the What About [PSI+], [URE3], and [PIN+]? The stable [PSI+] prion ADE2 gene was replaced by a PCR-generated kanMX4 cassette. To ensure that the kanMX4 cassette integrated properly into the ADE2 locus, we used variants used in most experiments are lost less often than 2-μm −5 PCR to amplify deletion junctions from genomic DNA of G418-resistant DNA and arise de novo at about 10 in strains with the [PIN+] colonies. Two colonies streaked from the original spore clone and two ade2:: prion (41); 11/70 wild strains do have [PIN+]. Like 2-μm DNA, kanMX4 colonies were transformed with overlapping fragments of 2-μm EVOLUTION [PSI+] is uniformly present in all meiotic segregants if either plasmid DNA amplified from genomic extracts of a wild [cir+] strain. We parent is [PSI+]. It was not found in the 70 wild strains exam- confirmed in vivo ligation of fragments in the transformants by PCR using ined. Each of these facts argues that [PSI+] must be more of primers that overlapped the ligation junctions within the 2-μm plasmid. The a detriment than 2-μm DNA. We can estimate this detriment primers used for confirming ADE2 gene replacement, amplifying 2-μm − fi (1 – R, when R = growth rate of [PSI+] relative to [psi ]) as fragments, and con rming in vivo ligation are shown in Table S6. Two different isolates of each of the four cell types were isolated so that a function of its actual abundance in nature using the value of + μ competition experiments could be done in replicate. ADE [cir-o] cells were outcross frequency obtained from the 2- m DNA data. This mixed with ade2 [cir+] cells and ADE+ [cir+] were mixed with ade2 [cir-o] in a 1:1 calculation is the inverse of that done in calculating outcross ratiosothatthefinal concentration of cells was 5 × 103 cells/mL in 50 mL of mating from the 2-μm DNA data, where we knew the detriment yeast extract peptone dextrose plus adenine (YPAD) medium. Replicate mixed and calculated the outcross mating frequency. cultures were grown at 30 °C with shaking and were maintained in log phase where cells were diluted to 5 × 103 cells/mL in 50 mL of fresh YPAD every 24 h. − − L ¼ 10 5; G ¼ 1:6 × 10 6; M ¼ 0:01 Every 48 h ∼1,000 cells from each culture were plated on 1/2YPD medium. Plates weregrownfor48hat30°C,andtheratioofwhitetoredcellswasrecorded.

The PSI+ fraction at equilibrium (pn) is assumed to be 1%. Characterization of RNQ1 in Wild Isolates of S, cerevisiae. From 83 wild isolates To calculate R as a function of pn: we amplified and sequenced the RNQ1, TRP1, and YGL108C loci. When necessary, PCR products were cloned to resolve ambiguous sequencing reads pna ¼ðRÞðpnÞ = ½ðRÞðpnÞþ1 − pn [A] that resulted from heterozygous genotypes at one or more loci. A few of our wild isolates were polyploid; at least two spore clones from these strains were sequenced to resolve haploid genotypes. Using CodonCode Aligner p ¼ p þ ¼ð1 − MÞ p ð1 − LÞþð1 − p Þ G nb n 1  na na (version 3.7.1; CodonCode Corporation) we aligned nucleotide sequences of þ ð Þ2þ ð − Þ [B] the three loci to corresponding reference sequences from strain S288C and M pna 2pna 1 pna documented the location and frequency of SNPs, insertions, and deletions. The total number of polymorphisms per locus was determined considering Using the values of L, G, and M above, we find the following each SNP or insertion/deletion event as one site. We calculated observed relation between the equilibrium value of p and the growth de- heterozygosity per locus as the proportion of heterozygous individuals in the population. fect, R, shown in Table 3. The calculation may be done using fi When a gene sequence showed more than one heterozygous site, we used the .xls le, Dataset S1. DNAsp version 5 (66) to reconstruct haplotypes and verified the conclusion by sequencing from single-spore clones or individual DNA clones. We also Materials and Methods used this program to calculate nucleotide diversity (π, the average number Strains, Media, and Genetic Methods. Media were as described in ref. 65, wild of pairwise differences per nucleotide site), nucleotide polymorphism (θ, strains are listed in Table S1, and laboratory strains are listed in Table S4.For the number of polymorphic sites per nucleotide, standardized by the + cytoduction (cytoplasmic mixing) experiments, an excess of [PIN+] ρ donors number of strains), and the number of synonymous and nonsynonymous ο over [pin−] ρ recipient strains were mixed in water, and mating on a rich- polymorphisms for RNQ1, TRP1, and YGL108C. We tested loci for selective medium plate was allowed for ∼7 h. Recipient cells were selected by neutrality using Tajima’s D (67), calculated for all segregating sites and in- streaking on an appropriate plate. The ρ+ colonies with recipient nuclear cluding gaps at each locus. The statistical significance of Tajima’s D values genotype were mated with the [pin−] rnq1Δ p1332 (CEN URA3 RNQ11-375- was determined per Tajima (67). To investigate changes in genetic variation GFP) strains 5074 or 5076, and diploids were examined for aggregates of and selection across the length of each gene, we also conducted a sliding- Rnq11-375-GFP by fluorescence microscopy. window analysis with nucleotide diversity, nucleotide polymorphism, and Tajima’s D values calculated every 100 bp with a 25-bp step-size. [PSI+] Induction Assay. Several treatments were reported to induce the ap- To investigate selective neutrality further, we used the McDonald–Kreit- pearance of [PSI+] in cells with an artificially modified Sup35 prion domain man (40) test to compare synonymous and nonsynonymous polymorphisms that makes the Sup35p more prone to the prion state formation (25). All within wild isolates and between S. cerevisiae and S. paradoxus. All available

Kelly et al. PNAS Early Edition | 7of8 Downloaded by guest on October 1, 2021 RNQ1, TRP1, YGL108C sequences for S. paradoxus were downloaded from were checked by sequencing and were used as cytoduction recipients from publically accessible databases and compiled for use as the outgroup for a [PIN+] strain with the reference RNQ1 sequence. McDonald–Kreitman tests. Nucleotide sequences of S. paradoxus were pro- vided by the Saccharomyces Genome Database and the Genome Informatics ACKNOWLEDGMENTS. This work was supported in part by the Intramural group at the Wellcome Trust Sanger Institute. Program of the National Institute of Diabetes and Digestive and Kidney To test for barriers to [PIN+] transmission, sporulated wild strains were Diseases and in part by the Uniformed Services University of the Health crossed with haploid his4 ho::kanMX ura3 laboratory strains. His+ Ura− Sciences (USUHS). We thank the USUHS Biomedical Instrumentation Center G418-resistant segregants, presumed to have the wild strain’s RNQ1 gene, facilities for providing assistance with microscopy.

1. Caughey B, Baron GS, Chesebro B, Jeffrey M (2009) Getting a grip on prions: 34. Bateman DA, Wickner RB (2012) [PSI+] Prion transmission barriers protect Saccharomyces Oligomers, amyloids, and pathological membrane interactions. Annu Rev Biochem 78: cerevisiae from infection: Intraspecies ‘species barriers’. Genetics 190:569–579. 177–204. 35. Chen B, et al. (2010) Genetic and epigenetic control of the efficiency and fidelity of 2. Aguzzi A, Baumann F, Bremer J (2008) The prion’s elusive reason for being. Annu Rev cross-species prion transmission. Mol Microbiol 76:1483–1499. Neurosci 31:439–477. 36. Resende CG, Outeiro TF, Sands L, Lindquist S, Tuite MF (2003) Prion protein gene 3. Collinge J, Clarke AR (2007) A general model of prion strains and their pathogenicity. polymorphisms in . Mol Microbiol 49:1005–1017. Science 318:930–936. 37. Futcher AB, Cox BS (1983) Maintenance of the 2 microns circle plasmid in populations 4. Wickner RB (1994) [URE3] as an altered URE2 protein: Evidence for a prion analog in of Saccharomyces cerevisiae. J Bacteriol 154:612–622. Saccharomyces cerevisiae. Science 264:566–569. 38. Mead DJ, Gardner DCJ, Oliver SG (1986) The yeast 2 micron plasmid: Strategies for the 5. Wickner RB, Edskes HK, Shewmaker F, Nakayashiki T (2007) Prions of fungi: Inherited survival of a selfish DNA. Mol Gen Genet 205:417–421. structures and biological roles. Nat Rev Microbiol 5:611–618. 39. Liti G, et al. (2009) Population genomics of domestic and wild . Nature 458:337–341. 6. Saupe SJ (2011) The [Het-s] prion of Podospora anserina and its role in heterokaryon 40. McDonald JH, Kreitman M (1991) Adaptive protein evolution at the Adh locus in incompatibility. Semin Cell Dev Biol 22:460–468. Drosophila. Nature 351:652–654. 7. Shewmaker F, Wickner RB, Tycko R (2006) Amyloid of the prion domain of Sup35p has 41. Liebman SW, Bagriantsev SN, Derkatch IL (2006) Biochemical and genetic methods for an in-register parallel β-sheet structure. Proc Natl Acad Sci USA 103:19754–19759. characterization of [PIN+] prions in yeast. Methods 39:23–34. 8. Baxa U, et al. (2007) Characterization of β-sheet structure in Ure2p1-89 yeast prion 42. Bradley ME, Edskes HK, Hong JY, Wickner RB, Liebman SW (2002) Interactions among “ ” – fibrils by solid-state nuclear magnetic resonance. Biochemistry 46:13149–13162. prions and prion strains in yeast. Proc Natl Acad Sci USA 99(Suppl 4):16392 16399. 9. Wickner RB, Dyda F, Tycko R (2008) Amyloid of Rnq1p, the basis of the [PIN+] prion, 43. Kadnar ML, Articov G, Derkatch IL (2010) Distinct type of transmission barrier has a parallel in-register β-sheet structure. Proc Natl Acad Sci USA 105:2403–2408. revealed by study of multiple prion determinants of Rnq1. PLoS Genet 6:e1000824. 10. Shewmaker F, McGlinchey RP, Wickner RB (2011) Structural insights into functional 44. Dagkesamanskaya AR, Ter-Avanesyan MD (1991) Interaction of the yeast omnipotent and pathological amyloid. J Biol Chem 286:16533–16540. suppressors SUP1(SUP45) and SUP2(SUP35) with non-mendelian factors. Genetics 128: – 11. Wasmer C, et al. (2008) Amyloid fibrils of the HET-s(218-289) prion form a beta 513 520. solenoid with a triangular hydrophobic core. Science 319:1523–1526. 45. Derkatch IL, Bradley ME, Hong JY, Liebman SW (2001) Prions affect the appearance of – 12. Frolova L, et al. (1994) A highly conserved eukaryotic protein family possessing other prions: The story of [PIN(+)]. Cell 106:171 182. properties of polypeptide chain release factor. Nature 372:701–703. 46. Halfmann R, Alberti S, Lindquist S (2010) Prions, protein homeostasis, and phenotypic – 13. Stansfield I, Tuite MF (1994) Polypeptide chain termination in Saccharomyces diversity. Trends Cell Biol 20:125 133. cerevisiae. Curr Genet 25:385–395. 47. Jung G, Jones G, Wegrzyn RD, Masison DC (2000) A role for cytosolic hsp70 in yeast – 14. Cooper TG (2002) Transmitting the signal of excess nitrogen in Saccharomyces [PSI(+)] prion propagation and [PSI(+)] as a cellular stress. Genetics 156:559 570. 48. Schwimmer C, Masison DC (2002) Antagonistic interactions between yeast [PSI(+)] and cerevisiae from the Tor proteins to the GATA factors: Connecting the dots. FEMS [URE3] prions and curing of [URE3] by Hsp70 protein chaperone Ssa1p but not by Microbiol Rev 26:223–238. Ssa2p. Mol Cell Biol 22:3590–3598. 15. Sondheimer N, Lindquist S (2000) Rnq1: An epigenetic modifier of protein function in 49. Safadi RA, Talarek N, Jacques N, Aigle M (2011) Yeast prions: Could they be exaptations? yeast. Mol Cell 5:163–172. The URE2/[URE3] system in Kluyveromyces lactis. FEMS Yeast Res 11:151–153. 16. Saunders SE, Bartelt-Hunt SL, Bartz JC (2012) Occurrence, transmission, and zoonotic 50. Edskes HK, et al. (2011) Prion-forming ability of Ure2 of yeasts is not evolutionarily potential of chronic wasting disease. Emerg Infect Dis 18:369–376. conserved. Genetics 188:81–90. 17. Coustou V, Deleu C, Saupe S, Begueret J (1997) The protein product of the het-s 51. Hosoda N, et al. (2003) Translation termination factor eRF3 mediates mRNA decay heterokaryon incompatibility gene of the fungus Podospora anserina behaves as through the regulation of deadenylation. J Biol Chem 278:38287–38291. a prion analog. Proc Natl Acad Sci USA 94:9773–9778. 52. Funakoshi Y, et al. (2007) Mechanism of mRNA deadenylation: Evidence for 18. Dalstra HJP, Swart K, Debets AJM, Saupe SJ, Hoekstra RF (2003) Sexual transmission of a molecular interplay between translation termination factor eRF3 and mRNA the [Het-S] prion leads to meiotic drive in Podospora anserina. Proc Natl Acad Sci USA deadenylases. Genes Dev 21:3135–3148. 100:6616–6621. 53. Shewmaker F, Mull L, Nakayashiki T, Masison DC, Wickner RB (2007) Ure2p function is 19. McGlinchey RP, Kryndushkin D, Wickner RB (2011) Suicidal [PSI+] is a lethal yeast enhanced by its prion domain in Saccharomyces cerevisiae. Genetics 176:1557–1565. prion. Proc Natl Acad Sci USA 108:5337–5341. 54. Cosson B, et al. (2002) Poly(A)-binding protein acts in translation termination via 20. Nakayashiki T, Kurtzman CP, Edskes HK, Wickner RB (2005) Yeast prions [URE3] and eukaryotic release factor 3 interaction and does not influence [PSI(+)] propagation. [PSI+] are diseases. Proc Natl Acad Sci USA 102:10575–10580. Mol Cell Biol 22:3301–3315. 21. Halfmann R, et al. (2012) Prions are a common mechanism for phenotypic inheritance 55. Urakov VN, et al. (2006) N-terminal region of Saccharomyces cerevisiae eRF3 is in wild yeasts. Nature 482:363–368. essential for the functioning of the eRF1/eRF3 complex beyond translation fi 22. Eaglestone SS, Cox BS, Tuite MF (1999) Translation termination ef ciency can be termination. BMC Mol Biol 7:34–46. regulated in Saccharomyces cerevisiae by environmental stress through a prion- 56. Sniegowski PD, Gerrish PJ, Johnson T, Shaver A (2000) The evolution of mutation – mediated mechanism. EMBO J 18:1974 1981. rates: Separating causes from consequences. Bioessays 22:1057–1066. 23. True HL, Lindquist SL (2000) A yeast prion provides a mechanism for genetic variation 57. Lynch M (2011) The lower bound to the evolution of mutation rates. Genome Biol – and phenotypic diversity. Nature 407:477 483. Evol 3:1107–1118. 24. Namy O, et al. (2008) Epigenetic control of polyamines by the prion [PSI+]. Nat Cell 58. Raynes Y, Gazzara MR, Sniegowski PD (2011) Mutator dynamics in sexual and asexual – Biol 10:1069 1075. experimental populations of yeast. BMC Evol Biol 11:158. 25. Tyedmers J, Madariaga ML, Lindquist S (2008) Prion switching in response to 59. Hickey DA (1982) Selfish DNA: A sexually-transmitted nuclear parasite. Genetics 101: environmental stress. PLoS Biol 6:e294. 519–531. 26. Tsai IJ, Bensasson D, Burt A, Koufopanou V (2008) Population genomics of the wild 60. Anderson TF, Lustbader E (1975) Inheritability of plasmids and population dynamics yeast Saccharomyces paradoxus: Quantifying the life cycle. Proc Natl Acad Sci USA of cultured cells. Proc Natl Acad Sci USA 72:4085–4089. 105:4957–4962. 61. Stewart FM, Levin BR (1977) The population biology of bacterial plasmids: Apriori 27. Ruderfer DM, Pratt SC, Seidel HS, Kruglyak L (2006) Population genomic analysis of conditions for the existence of conjugationally transmitted factors. Genetics 87:209–228. outcrossing and recombination in yeast. Nat Genet 38:1077–1081. 62. Bergstrom CT, Lipsitch M, Levin BR (2000) Natural selection, infectious transfer and 28. Johnson LJ, et al. (2004) Population genetics of the wild yeast Saccharomyces the existence conditions for bacterial plasmids. Genetics 155:1505–1519. paradoxus. Genetics 166:43–52. 63. Livingston DM (1977) Inheritance of the 2 micrometer m DNA plasmid from 29. Masel J, Griswold CK (2009) The strength of selection against the yeast prion [PSI+]. Saccharomyces. Genetics 86:73–84. Genetics 181:1057–1063. 64. Futcher B, Reid E, Hickey DA (1988) Maintenance of the 2 micron circle plasmid of 30. Moore RA, Vorberg I, Priola SA (2005) Species barriers in prion diseases—brief review. Saccharomyces cerevisiae by sexual transmission: An example of a selfish DNA. Arch Virol Suppl 19):187–202. Genetics 118:411–415. 31. Mead S, et al. (2003) Balancing selection at the prion protein gene consistent with 65. Sherman F (1991) Getting Started with Yeast. Guide to Yeast Genetics and Molecular prehistoric kurulike epidemics. Science 300:640–643. Biology, Methods in Enzymology, eds Guthrie C, Fink GR (Academic, San Diego), Vol 32. Chen B, Newnam GP, Chernoff YO (2007) Prion species barrier between the closely 194, pp 3–21. related yeast proteins is detected despite coaggregation. Proc Natl Acad Sci USA 104: 66. Librado P, Rozas J (2009) DnaSP v5: A software for comprehensive analysis of DNA 2791–2796. polymorphism data. Bioinformatics 25:1451–1452. 33. Edskes HK, McCann LM, Hebert AM, Wickner RB (2009) Prion variants and species 67. Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by barriers among Saccharomyces Ure2 proteins. Genetics 181:1159–1167. DNA polymorphism. Genetics 123:585–595.

8of8 | www.pnas.org/cgi/doi/10.1073/pnas.1213449109 Kelly et al. Downloaded by guest on October 1, 2021