Self-Perpetuating Structural States in Biology, Disease, and Genetics (2002)

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Colloquium Changes in the miciclle region of Sup35 profoundly alter the nature of eDiaenetic inheritance for the yeast prion [PSi+] . _ Jia-Jia Liu*t, Neal Sondheimertt, and Susan L. Lindquist§ Department of Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637 The yeast prion [PSI+] provides an epigenetic mechanism for the inheritance of new phenotypes through self-perpetuating changes in protein conformation. lPSI+] is a nonfunctional, ordered aggre- gate of the translation termination factor Sup35p that influences new Sup35 proteins to adopt the same state. The N-terminal region of Sup35p plays a central role in prion induction and propagation. The C-terminal region provides translation termination activity. The function of the highly charged, conformationally flexible middle region (M) is unknown. An M deletion mutant was capable of existing in either the prion or the nonprion state, but in either case it was mostly insoluble. Substituting a charged synthetic polypeptide for M restored solubility, but the prions formed by this variant were mitotically very unstable. Substituting charged flex- ible regions from two other proteins for M created variants that acquired prion states (defined as self-perpetuating changes in function transferred to them from wild-type [PSI+] elements), but had profoundly different properties. One was soluble in both the prion and the nonprion form, mitotically stable but melotically unstable, and cured by guanidine HCI but not by alterations in heat shock protein 104 (Hsp104p). The other could only maintain the prion state in the presence of wild-type protein, producing Men- delian segregation patterns. The unique character of these M variants, all carrying the same N-terminal prior-determining re- gion, demonstrate the importance of M for [PSI+] and suggest that a much wider range of epigenetic phenomena might be based on self-perpetuating, prior-like changes in protein conformation than suggested by our current methods for defining prion states. Yeast prions represent a fundamentally different mechanism for the transmission of genetic information than DNA based inheritance. With priors, heritable changes in phenotype are produced by self-perpetuating changes in protein conformation rather than by any changes in nucleic acids (1-34. tPSI+] and other genetic elements of this type are called prions because of conceptual similarities between their modes of transmission and that postulated for the infectious agent of the mammalian prion diseases (1, 44. However, yeast prions play a different role in the biology of cells that harbor them. They are not generally pathogenic. Rather, they modify metabolism in an epigenetic manner that can be beneficial to the organism under certain circumstances (S. 6~. The protein determinant of ~PSI+] is Sup35, a subunit of the translation termination factor (7~. In [psi-] cells, which lack the prion, Sup35 protein (Sup35p) is soluble and functional. In [PSI+] cells, most Sup35p is found in self-perpetuating, ordered aggregates. In this state, the protein is nonfunctional. The reduced concentration of functional translation-terminator fac- tor causes ribosomes to occasionally read-through stop codons (2, 3~. Thus, the presence of [PSI+] is routinely monitored by suppression of nonsense-codon mutations in auxotrophic mark- ers (8~. The phenotype is heritable because Sup35p in the [PSI+] 16446-16453 1 PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 state influences newly synthesized Sup35p to adopt the same state, and because the protein is passed from mother cell to daughter during mitosis. When the daughter cell starts to make her own Sup35 proteins, they are influenced by preexisting [PSI+I complexes (inherited from the mother's cytoplasm) to undergo conformational conversion. Thus, the change in Sup35p function is inherited cytoplasmically. Sup35p can be divided into three regions based on sequence analysis and functional investigations. The C-terminal region (C, amino acids 254-685) is responsible for the translation termi- nation activity and is essential for viability (9-12~. The N- terminal region (N. amino acids 1-123) is required for the induction and maintenance of tPSI+] (11-13~. Deletion of N eliminates IPSI+ l, whereas even transient over expression of N induces [PSI+] (12~. N is also responsible for the species barrier: in chimeric Sup35 proteins created from different species, the prion state is efficiently transferred only between proteins that share the same critical region of N (14-16~. The role of the region between N and C (M, amino acids 124-253) remains unclear. In inter-specific comparisons of Sup35p amino acid sequences, N and M are less conserved than C (7, 17~. However, general features of these regions have been retained for long periods of evolution (14-16, 18~. N regions from even distantly related Hemiascomycetes are rich in glut amine and asparagine residues (16, 19~. M regions are highly charged, and their sequences are heavily biased toward a subset of charged amino acids (9, 16, 18, 19~. In Saccharomyces cerevisiae, 42% of the residues in M are charged. All positively charged residues are lysines, and these cluster at the N-terminal end of M. The negatively charged residues, mostly glutamates, are concentrated at the C-terminal end. Although tPSI+] is inherited in an orderly way, both mitoti- cally and meiotically, it is metastable. tPSI+] cells occasionally give rise to ~?si-] cells and vice versa as the tPSI+] conformation is lost or gained (20~. The rate at which [PSI+] elements are lost greatly increases during growth on media containing guanidine hydrochloride (Gdn HCl) (21, 22~. The inheritance of [P.~+1 is . ~ _ ~ This paper results from the Arthur M. Sackier Colioqulum of the Nationai Acaclemy of Sciences, "Self-Perpetuating Structurai States in Biology, Disease, ancl Genetics," heicl March 22-24, 2002, at the Nationai Acaclemy of Sciences in Washington, DC. Abbreviations: Gcin.HCi, guanicline hycirochioricle; YPD, yeast extract/peptone/clextrose. *Present aciciress: Department of Neurology ancl Neurologicai Sciences, Stanforcl University Schooi of Medicine, MSES Bulicling, Room P259, 1201 Weich Roacl, MC5489, Stanforcl, CA 94305. t3.-~.L and N.S. contributecl equally to this work. $Present aciciress: Department of Pecliatrics, Chiiciren's Hospitai of Philaclelphia, 34th ancl Civic Center Bouievarcl, Phliacleiphia, PA ~ 9104. §To whom reprint requests shouicl be sent at the present aciciress: Whiteheacl insti- tute of Biomeclicai Research, 9 Cambricige Center, Cambricige, MA 02142. E-maii: iinclquisLacimin~wi.mit.eclu. www.pnas.org/cgi/cloi/1 0.1 073/pnas.252652099

also modulated by several protein chaperones (23-26~. This effect is most striking with heat shock protein 104 (HsplO4p), a protein remodeling factor. Both overexpression and deletion of the HSP104 gene cure cells of tPSI+] (23~. HsplO4p must be present at an intermediate concentration for tPSI+] propagation. The ability of Sup35p to exist in two stable and heritable conformations is fundamental to the conversion of cells from .tpsi-] to tPSI+] and vice versa. It is known that the crucial features of Sup35p required for this transition are confined to N and M: transferring N and M to a heterologous protein is sufficient to confer all of the prion behaviors of tPSI+] to that hybrid protein (27~. Moreover, the transition from the nonprion to the prion state in vivo has been modeled in vitro by using the purified NM fragment of Sup35p. This polypeptide can exist for extended periods in the soluble state with a high degree of random coil and converts to a f-sheet-rich amyloid by seeded polymerization (28-30~. To date, all of the critical elements for prion induction and propagation have been mapped to N. However, the M region (amino acids 124-253) is critical for solubility of NM in vitro. M alone remains soluble for months and cannot be seeded by preformed amyloids. On the other hand, N alone is soluble only in denaturing buffers (29, 31~. This obser- vation would suggest that M might play an important role in prion biology but its function has not been investigated in vivo. We created several alterations in the M region. They produced very different effects, demonstrating that M plays crucial roles in tPSI+] biology. In fact, the self-perpetuating prion states of these altered Sup35 proteins are so strikingly different from those of wild-type (WT) protein, they suggest that a much broader range of behaviors might involve prior-like changes in proteins than has previously been suspected. Materials and Methods Strains, Cultivation Procedures, and Genetic Analysis. The tPSI+] and [psi-] isogenic strain pair used were 74-D694a [MATa, adel- 14(~UGA), trpl-289(<UAG), his3l`-200, ura3-52, leu2-3,112] and 74-D694a~ fMATcx, adel-14(~UGA), trpl-289(~UAG), his32\-200, ura3-52, leu2-3,112~. The HSP104 disruption strain used was 74-D694:hspA-lL fadel-14(~UGA'), trpl-289(<UAG), his3~-200, ura3-52, leu2-3, 112, hspl O4::LEU2~. fETA +] strain: SL1010-lA fETA+] tpsi-] fadel-14 (<UGA), his 5-2, met8-1, ura3-52, leu2- 1(UAA) trpl (~1 or 289~] (The fETA+] strain was a kind gift from S. W. Liebman, University of Illinois, Chicago). Cells were grown at 30°C in rich media tyeast extract/ pep/one/dextrose (YPD)] until mid-logarithmic phase (32~. ~PSI+] was monitored by its ability to suppress the adel-14 (UGA) mutation and allow cells to grow on synthetic medium (SC) lacking adenine (-ADE). The 74-D694 [psi-] cells are red and tPSI+] cells are white or pink on rich medium (13, 23~. tPSI+] curing on medium containing Gdn.HCl, CuS04 induction of tPSI+], and GFP fluorescence analysis of Sup35p aggregates were performed as described (33~. Standard procedures for crossing, sporulation, and tetrad analysis were used (34~. Plasmid Construction. The SUP35 M deletion (amino acids 124- 253) was generated by site-directed mutagenesis using oligonu- cleotide primers and a QuikChange Site-Directed Mutagenesis kit (Stratagene). The primers used were 5'-CAC AGT CTC AAG GTA TGT TTG GTG GTA AAG-3' and 5'-CTT TAC CAC CAA ACA TAC CTT GAG ACT GTG-3'. The template plasmid for mutagenesis was p316CUPSup35GFPSG. The middle region-deleted SUP35 gene fragment was then PCR amplified and inserted at the EcoRI/BamHI sites of the SUP35 integrative construct pJLI-sup35. PCR primers were as follows: primer A 5'-CG GAA TTC ATG TCG GAT TCA AAC CAA GG-3' and primer B 5'-CGC GGA TCC TTA CTC GGC AAT TTT AAC-3'. Thus, in the resultant construct pJLI-sup352\M, amino acid 123 was fused directly to amino acid 254. Liu eta/. The KDG tripeptide repeats were inserted between SUP35 N and C by site-directed mutagenesis. Primers were as follows: 5'-CCA CAG TCT CAA GGT AAG GAT GGT AAG GAT GGT AAG GAT GGT AAG GAT GGT AAG GAT GGT AAG GAT GGT TTT GGT GGT AAA OAT-3' and 5'-ATC TTT ACC ACC AAA ACC ATC CTT ACC ATC CTT ACC ATC CTT ACC ATC CTT ACC ATC CTT ACC ATC CTT ACC TTG AGA CTG TGG-3'. The SUP35 N-KDG6-C fragment was then PCR amplified and inserted into the SUP35 integrative construct pJLI-SUP35 at the EcoRI/BamHI sites. The N9C substitution was created by amplifying the region of Hsp90p coding for amino acids 210-262 from genomic 74D-694 DNA by using the primers 5P-CAT CTA GAA AGG AAG TTG AAA AGG AAG-3' and 5'-CTC CGC GGC TTG TTT AGT TCT TCT ATC TC-3'. The resulting fragment was inserted into pN164 by using the SacII and XbaI restriction sites. The NTC substitution was created by using the same strategy after ampli- fication of amino acids 635-713 of human topoisomerase I from plasmid pKM10 (a kind gift from J. Champoux, University of Washington, Seattle). The primers used were 5'-GAT CTA GAG CAC CAC CAA AAA CTT TTG AG-3' and 5'-CAC CGC GGC ACT GTG ATC CTA GGG TCC AG-3'. Fidelity of all constructs was confirmed by sequencing. Yeast Transformation. One milliliter of overnight culture was pelleted and resuspended in 0.1 ml LiAc-PEG-TE solution (40% wt/vol PEG 4000/100 mM LiAc/10 mM Tris HCl, pH 7.5/1 mM EDTA). Cells were mixed with 10 ,ug/,ul carrier DNA (salmon testis DNA, boiled for 10 min and rapidly chilled on ice) and 0.5 ,ug/,ul transforming DNA and incubated overnight at room temperature. Cells were heat shocked at 42°C for 15 min, rapidly chilled on ice, spread directly onto synthetic defined medium selective for transformation, and incubated at 30°C. Gene Replacement. The two-step gene integration and replace- ment procedure was used to create M mutant strains as described (34, 35~. Genotypes were confirmed by genomic PCR, and protein expression was confirmed by immunoblotting. To disrupt HSP104 in tPSI+~^M and tPSI+]NKC strains, an hsplO4::URA3 disruption plasmid (pYSU2) was inserted in the genome. The plasmid was digested with EcoRI/HindIII to release the hsplO4::URA3 fragment, and the restriction digest was used to transform yeast cells. Transformants were selected on SC-URA medium, and the HSP104 disruption was confirmed by immunoblotting with anti-HsplO4p antibodies (mix of mono- clonal 8-1 and 2-3; ref. 36~. HSP104 was disrupted in tPSI+iNTC and SUP35N9C backgrounds by the short flanking homology method as described (37~. Sup35p Solubility Assay. Cells grown to mid-logarithmic phase in liquid YPD medium were washed with 0.1 M EDTA (pH 8.0), resuspended in spheroplasting buffer (1.0 M sorbitol/0.1 M EDTA, pH 7.5/50 mM DTT/0.166 mg/ml zymolyase 100 T) and incubated at 30°C for 1 h. Cells were collected, washed with the spheroplasting buffer without zymolyase, and lysed by resuspen- sion in lysis buffer (50 mM Tris HCl, pH 7.2/10 mM KCl/100 mM EDTA/1 mM DTT/0.2% wt/vol SDS/1% vol/vol Triton X-100/1 mM benzamidine/2 mM phenylmethylsulfonyl fluo- ride/10 ,ug/ml leupeptin/2 ~g/ml pepstatin A) with incubation on ice for 5 min. Lysates were subjected to centrifugation at 100,000 x g (48,000 rpm in a TLA-100 rotor, Beckman Coulter) for 20 min at 4°C. Supernatant and pellet fractions were analyzed by SDS/10% PAGE (NOVEX, San Diego) and transferred to a poly~vinylidene difluoride) membranes (Millipore). Membranes were incubated with primary antibody (antiSup35p- rabbit poly- clonal antipeptide against amino acids 55-68) 1:1,000 in PBS buffer with 0.1% Tween 20 for 1 h, washed with PBS with 0.1% Tween 20, and incubated with goat anti-rabbit antibody conju- PNAS | December 10, 2002 | vol. 99 | supp~. 4 | 16447

A cost Asia] SUP35^M B C APSIS Nisi-] Sup35^M S P S P S P _— ~ C! d~#~ .,, ._ ~ ~ ~ ~~ ~ ~ Pup— NM-GFP GFP NM-GFP SC-ADE SC-ADE ~ ~~ ~ ~ ~: ~~ ~__- Sup35AM :: :: :: i: SUP35 SUP35^M Fig. 1. Sup357\Mp can convert to [PSI+]. (A) Five-fold serial dilution and growth of SUP35AM cells compared with ~ [PSI+] and [psi-] cells on SC-ADE plates (30°C, 5 days). (B) Sup35p solubility assay of [PSI+], [psi-], and Sup35AMp. After cell Iysis, high speed centrifugation and separation by SDS/10% PAGE Sup35AMp was detected by immunoblotting with anti- Sup35p antibody. S. supernatant fraction; P. pelleted fraction. (C) Induction of heritable [PSI+] factors in WT (SUP35) [psi-] and SUP35AM [psi-] strains. Five-fold serial dilutions of cells after 24-h induction of NM-GFP or GFP alone, plated on SC-ADE (30°C, 5 days). gated to horseradish peroxidase (1:5,000), and immune com- plexes were visualized with enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia). Results Sup35AMp Is Functional and Mostly Insoluble but Can Exist in Both Prion and Nonprion States. Starting with tpsi-] cells, the WT copy of SUP35 was replaced with a gene carrying a deletion of the middle region (SUP35~. The strain contained a tPSI+~- suppressible nonsense mutation in the ADE1 gene. In this background, tPSI+] cells grow on synthetic media deficient in adenine (SC-ADE) and are white on rich media. [psi-] cells do not grow on SC-ADE and produce red colonies on rich media because of the buildup of a colored byproduct of adenine biosynthesis. Recombinant strains containing the SUP35AM gene (n = 16) at the SUP35 locus were red on rich medium but showed a faint background of growth (dark red in color) on SC-ADE. Thus, at least some Sup35AM protein is functional in translation termi- nation, but the protein is not as active as WT Sun35D (Fig. 1 A and C). r r ~ c7 Differential centrifugation of cell lysates revealed that a much smaller fraction of Sup357\Mp was present in the supernatant after a 100,000 x g spin, compared with WT Sup35p in [psi-] strains (Fig. 1B). Coomassie blue staining demonstrated equal loading of total proteins in these fractions and revealed no detectable changes in the solubility of other proteins (data not shown). The partial insolubility of Sup357\Mp explains the weak suppressor phenotype of SUP35AM cells because less Sup35p is available for translational termination than in WT cells. Clearly, the highly charged M region provides a solubilizing function for 16448 1 www.pnas.org/cgi/doi/10.1073/pnas.252652099 the Sup35 protein as a whole in vivo, as it does for the NM fragment in vitro. Insolubility is a characteristic of Sup35p in the tPSI+] state (2) (Fig. 1B). The weak suppressor phenotype of SUP35AM could be due to a weak tPSI+] variant (see below). However the pheno- type was unaffected by growth on Gdn HCl, which efficiently cures tPSI+] (data not shown), suggesting that the insolubility of Sup357\Mp might not be caused by prion formation. Also suggesting that the aggregates were not prior-like, the suppres- sor phenotype was recessive when SUP35AM cells were mated with WT Nisi-] cells (data not shown). Moreover, differential centrifugation showed that aggregates of Sup352\Mp in diploid cells did not cause WT Sup35p to fractionate into the pellet. That is, the Sup35AM protein in these cells could not recruit WT Sup35p to take on the tPSI+] state (data not shown). The aggregates had none of the characteristics of a prion and were more likely simply caused by loss of the solubilizing activity normally conferred by the highly charged M region. Sup357\Mp was, however, capable of acquiring a heritable tPSI+~-like state when SUP35AM cells were mated to WT tPSI+] cells. After mating, the strong suppressor phenotype of the tPSI+] parent was invariably dominant (data not shown), indi- cating that WT protein had converted Sup35AMp into a form that reduced its ability to function in translation. Moreover, the strong suppressor phenotype of the SUP35AM x tPSI+] diploid was cured by growth on medium containing Gdn HCl (data not shown), indicating that Sup352\Mp had acquired the tPSI+] state from WT protein, and could be cured of this state with Gdn HCl, as is the WT protein. Next, we asked whether Sup35l`Mp could acquire the prion state through another common mechanism, by transient over expression of NM. In previous studies, we used NM fused to GFP to monitor the formation and propagation of tPSI+] in living cells (2~. Sup35p in the tPSI+] state has the capacity to capture NM-GFP and induce it to adopt the same state, forming GFP aggregates visible by fluorescence microscopy. Furthermore, overexpression of NM-GFP induces new tPSI+] element forma- tion in [psi-] cells (2~. This state is retained even when the NM-GFP plasmid is lost. SUP35AM cells were transformed with expression plasmids for GFP alone or NM-GFP. After 4 h of induction, intense coales- cent foci were observed in many cells expressing NM-GFP, but never in cells expressing GFP alone (ref. 2 and data not shown). When plated to copper-free medium without selection for the plasmid, cells that had expressed NM-GFP produced colonies with a tPSI+] phenotype at a much higher frequency than those expressing GFP alone (Fig. 1C), suggesting that transient over expression of NM had converted the Sup35l\Mp to the prion state. This was confirmed by 4:0 segregation of the suppressor phenotype in crosses to WT tpsi-] cells and curing by growth on medium containing Gdn HCl (data not shown). Therefore, the Sup352\Mp can exist in two different states (we designate them PSI+ and Ski- that are genetically analogous to the tPSI+] and [psi-] states of the WT Sup35 protein. Unlike WT Sup35p, however, the protein is largely insoluble in both cases (Figs. 1B and 2B). Sup35AMp Can Maintain Different Prion Variants. Although haploid PSI+ strains were readily obtained by overexpressing NM- GFP, they were not readily obtained by other standard methods. For example, in one case SUP35AMwas integrated at the site of WT SUP35 (in tandem with it) in a tPSI+] strain (i.e., both SUP35AM and SUP35 were present). These transformants re- tained the tPSI+] phenotype, as expected because WT Sup35p in the prion state converts Sup352\Mp to that state. However, when one of the two genes was excised by selection against the URA3 marker that had been used for transformation, clones carrying only SUP35AM did not survive. SUP35AM derivatives Liu et a/.

A B C DETACH AM DETACH Sip ~ ~ SC-ADE T 1 23 4 5 Cal [PSI~ [PSI~ ~ _,, T S P T S P SC-ADE _ HI - c. _ Su p35 ~ ~ Sup35/\M FETAL. T 1 2 3 Cal ~ YPD second round mating PETALS [psi l '1 [pX~ _ ~ _ . _ ~ ~ _ lit ~ _ i. *O SC-ADE - Fig. 2. Sup35AMp can maintain different [PSI+] variants. (A) Most SUP35AM spores from SUP35/`M x WT [PSI+] crosses were not viable. (Left) Five repre- sentative tetrads from spores of a [PSI+] diploid from a SUP35AM [psi-] x SUP35 [PSI+] cross, dissected on YPD medium. (Right) Growth of one set of tetrads with one surviving SUP35AM spore on YPD and SC-ADE. T. tetrad; S. spore. (B) Sup35p solubility properties of the WT spore and the SUP35AM spore (methods as described in Fig. 1). T. total protein; S. supernatant; P. pellet. (C) Sup35~Mp can form and propagate [ETA+]. (Upper Left) Growth of SUP35AM [psi-] haploid strain ([pSi-]~M), ~ [ETA+] haploid strain ([ETA+]~, and the diploid from the cross of these two strains on SC-ADE medium. (Upper Right) Three representative tetrads dissected from the diploid SUP35/`M[psi-] x SUP35[ETA+] on YPD medium. (Lower Right) Grovvth on SC-ADE of haploid parental strains and two diploid strains from a cross of a SUP35AM spore from SUP35l`M [psi~] ([psi-]~M)/SUP35 [ETA+] ([ETA+]WT) diploid to WT [psi-]. were readily obtained when the initial insertion had been in a tpsi-] background and other SUP35 [PSI+] variants are readily obtained by this method (35~. Similarly, sporulating SUP35/ SUP35AM ~psi-] diploids yielded the expected number of viable SUP35AM spores, but sporulating SUP35/SUP35AM tPSI+] diploids yielded very few (Fig. 2A). Those that were recovered grew very slowly, even on rich media (Fig. 2A), and by differ- ential centrifugation, virtually all of their Sup351\M protein was found in the pellet (Fig. 2B). Genetic crosses between these slow-growing strains and the WT tpsi-] strain generated tPSI+] diploids (data not shown). Thus, Sup357\Mp in the prion state can transmit that state to WT protein. But [PSI+] cells in which the only copy of Sup35p is Sup357\Mp can have unexpected problems with viability. One explanation is that Sup352\Mp, like WT Sup35p, can form prion variants with different "strengths." The phenomenon of different prion states (called prion "strains" or variants) that are strong, moderate, weak, and very weak is well characterized (13, Liu et a/. 38-40~. These variants are not caused by genetic differences, but are caused by epigenetic differences in the rates that prion variants capture and convert new Sup35p to the prion state. They have different levels of soluble Sup35p and different rates of translation termination (39-41~. Because Sup35AMp is inher- ently less soluble than WT Sup35p, if it acquired a strong prion state there might be too little translation termination activity to keep cells viable. The haploid tPSI+~^M strains induced by transient overexpression of NM-GFP might represent weak variants, viable because a greater fraction of the Sup35AM protein remains soluble and active. To test this possibility, we mated sit- cells to the weak tPSI+] variant fETA+~. Conversion of Sup351\Mp by this weak variant should leave a greater fraction of Sup35AMp in solution and produce more viable haploid SUP35AM PSI+ spores. The diploid showed the same suppression of the adel-14 marker as the fETA+] haploid parent (Fig. 2C Left), suggesting that the Sup357\M protein had converted to a weak prion state. In contrast to the poor viability of SUP35AM spores after mating to strong tPSI+] strains (Fig. 2A), nearly all SUP35AM progeny from the fETA+] were viable (Fig. 2C UpperRight). When these progeny were mated to WT [psi-] tester strains, the diploids exhibited the suppressor phenotype characteristic of fETA+] strains (Fig. 2C Lower Right). Thus, Sup35~\Mp could acquire, maintain, and transmit the fETA+] state to WT protein. The N region is sufficient to form prion variants of different strengths. The M Region Promotes Mitotic Stability of the [PSI+] State. On rich media, tPSI+~-mediated nonsense suppression is not required for growth, yet WT cells retain tPSI+] with high fidelity. In contrast tPSI+~^M strains lost the prion at a high rate (Fig. 3A). We asked whether we could restore stability simply by restoring solubility to the protein. To do this, a DNA fragment encoding a highly charged polypeptide rich in lysine and glut amic acid (6xKDG) was inserted in place of M creating the replacement SUP35NKC. As expected, when the WT SUP35 gene was replaced by SUP35NKC in [psi-] cells, they retained a tpsi-] phenotype. The solubility of the Sup35NKC protein in this state was comparable to that of WT Sup35p (Fig. 3B). To determine whether this protein could acquire the prion state, SUP135NKC mutants were mated to a typical strong prion strain (Fig. 3C) and the diploid strain showed the suppressor phenotype. Sporulation of this diploid (data not shown) produced haploid SUP35NKC tPSI+] cells (tPSI+]NKC). Most Sup35NKCp was soluble in ~si-]NKC strains, and most of the protein became insoluble when it adopted the tPSI+]NKC state (Fig. 3B). Unlike PSI+ cells, tPSI+]NKC cells exhibited no growth defect when streaked on rich medium (data not shown). However, even though Sup35NKCp appeared to be as soluble as WT protein and produced no general growth disadvantage, the tPSI+]NKC phenotype was highly unstable (Fig. 3D). Thus, replacement of the M region with a charged polypeptide that increases its inherent solubility in vivo is not sufficient to restore stability to the prion state. M provides more than a simple solubilizing function to Sup35p. It also promotes the mitotic stability of tPSI+~. The M region is highly charged and, in the soluble state, circular dichromism spectroscopy shows it to have a highly flexible structure (~60% random coil; A. Cashikar and T. Scheibel, personal communication). Our next alterations were to replace the M region with two naturally occurring polypeptides that, like WT M, are highly charged and are known to have conformational flexibility. The Human Topoisomerase Linker Restores Mitotic Stability but Causes Meiotic Instability. The human topoisomerase linker (T) has a percentage of charged residues similar to Sup35Mp. The linker has been characterized by x-ray crystallography and contains both structured and unstructured regions that link PNAS | December 10, 2002 | volt. 99 | supple. 4 | 16449

A [PSI~M APSIS B CD [PSI~ 1psi ] T S P T S P S u p 3 5 F ~ ~ ., D, [no;-lN K\; [PSI~NKC ~Sj-lNKC T S P T S P _ _ _ Sup35NKC [PS/~ ~ .2N [PSI~NKC a_ it_ - - - - - - Fig. 3. The M region promotes mitotic stability of strong [PSI+]. (A) Appear- ance of red sectors ([psi-]) out of white colonies when [pS/+]^M cells were streaked onto YPD and incubated for several days (Left). Growth of [PSI+] cel Is on YPD shown for comparison (Right). (B) Sup35p solubility assay of the NKC mutant protein compared with that of V\/T Sup35p (methods as described in Fig. 1). (Left) WT [PSI+] and [psi~] strains. (Right) SUP35NKC [PSI+] and [psi~] ([PS/+]NKCand [pSi ]NKC) haploid strains. (C) Mating SUP35NKC[psi ] ([psi ]NKC) to [PSI+] generated [PSI+] diploid. Growth of the two parental strains and a diploid (2N) progeny on SC-ADE is shown. (D) [PSI+]NKC cells were mitotically unstable. Cells were grown in liquid YPD for 16 h and plated onto YPD plates. Most colonies were red/white sectored (Let). A close-up image of one of the sectored colonies is shown (Right). other functional domains of the protein (42~. When an M to T replacement (SUP35NTC) was inserted in tandem with SUP35 in [psi-] cells, strains retaining only SUP35NTC were obtained at an equal frequency to WT. SUP35NTC strains were phenotyp- ically identical to WT tpsi-] cells with respect to growth on rich media and SC-ADE. Sup35NTCp could readily be converted to the prion state, tPSI+]NTC, by matings to WT tPSI+] strains. The haploid progeny of sporulation showed normal viability. tPSI+iNTC strains were also obtained after transient overexpres- sion of Sup35NTCp from an inducible plasmid in the SUP35NTC background. tPSI+]NTC was tested for other common prion properties including curability, mitotic stability, and non-Mendelian inher- itance during meiosis (see Table 1~. It was mitotically stable (Fig. 16450 1 www.pnas.org/cgi/doi/10.1073/pnas.252652099 4A), capable of growth on SC-ADE (Fig. 4B) and was cured by growth on media containing Gdn HCl, but was not cured by either the overexpression or the deletion of HSP104 (Table 1 Fig. 4C). To further characterize tPSI+]NTC and ~si-]NTC states, we analyzed the solubility of the Sup35NTC protein. In both tPSI+iNTC and [psi-]NTC cells, most Sup35NTCp was soluble after a 100,000 x g spin (Fig. 4D). To test the aggregation of Sup35p by using GFP, we expressed a plasmid with a fusion of the N and T regions to the GFP marker (NT-GFP) in [PSI+ jNTC and LDsi-iNTC cells. NT-GFP showed a diffuse fluorescence pattern in both strain types confirming that the protein does not form large aggregates (Fig. 4E). Therefore this protein can exist in states that are genetically analogous to the prion states of Sup35p, but in both states most of the protein remains soluble after centrifugation at 100,000 x g for 20 min. The tPSI+]NTC state was dominant in crosses to [psi-iNTC cells, indicating that it readily converted soluble Sup35NTCp to the prion state. When tPSI+]NTC homozygous diploids were sporu- lated, the frequency of meiotic transmission of the suppressor phenotype to offspring was not always 4:0, the ratio typical for WT tPSI+] diploids, but it was clearly non-Mendelian (Fig. 4F and Table 1~. This segregation pattern was similar to that of another yeast prion, tURE3] (43~. These findings suggest that the highly charged M region also influences the accurate propaga- tion of tPSI+] elements through meiosis. The different effects of M substitutions on mitotic and meiotic stability suggest that the mechanisms for maintaining meiotic and mitotic stability are, at least in part, distinct. Substitution of the Hsp90p Linker for M Causes Another Distinct Genetic Behavior. The other M substitution we tested was derived from S. cerevisiae Hsp90 protein. This highly charged region (amino acids 210-262 of the polypeptide sequence) connects the two stably folded domains of Hsp90p and is degraded by even very short treatments with proteases, suggesting it is not inher- ently a tightly folded polypeptide (444. As with SUP35NTC, when M was replaced by this portion of the HSP90 coding sequence (~SUP35N9C), [psi-] cells retained a nonsuppressor state (data not shown). In contrast to SUP35NTC, SUP35AM, and SUP35NKC cells, a suppressor state could not be induced in haploid SUP35N9C cells by overexpression of polypeptides containing the N region (data not shown). The protein could, however, acquire a tPSI+~-like state when SUP35N9C cells were mated to WT tPSI+] cells (Fig. 5A). The diploid strain had many other characteristics of [PSI+] strains, including a suppressor phenotype that was eliminated by plating to media containing Gdn HCl (see Table 1~. It also showed strong mitotic stability. But surprisingly, sporulation of this diploid always produced two tPSI+] colonies with a SUP35 genotype and two SUP35N9C with the tpsi-] phenotype (Fig. SA). These observations suggested that Sup35N9Cp could enter a tPSI+~-like state, but could only acquire that state from pre- formed ~PSI+] elements and could not thereafter retain it on its own (when separated by sporulation from the WT protein). To more fully characterize these transitions we examined the solu- bility of the Sup35N9C protein in the haploid SUP35N9C strain, the diploid SUP35/SUP35N9C tPSI+] strain and the haploid progeny of sporulation. The Sup35N9C protein was almost entirely soluble in the SUP35N9C parent, but was insoluble (as was WT Sup35p) in the heterozygous tPSI+] diploid (Fig. SD). After sporulation, Sup35N9Cp became soluble once again in the SUP35N9C haploid progeny, whereas the insoluble prion state was maintained in SUP35 progeny (Fig. SD, right lanes). This result was confirmed by the presence of small foci in the tPSI+] diploid after 2 h of expression of an N9-GFP fusion protein (Fig. SB). In contrast, N9-GFP fluorescence in the nonsuppressed haploid SUP35N9C remained diffuse (Fig. SC). Thus, ~-- ~ r-~ ~ r ciu et a/. i

Table 1. M substitution mutants and their properties Protein [PSI+] inducibility . solubility Hsp104 curability Region length; % charge; Segregation no. of positive amino acid Sup35 N terminus Mate to pattern Stable in Gdn.HCI Hsp104 over residues that are Iysine over express [PSI+] [PSl+]:[psi-] mitosis curable [PSI+] [psi-] Z\HSP104 express WT; 130 aa; 42%; 24 of 24 AM KDG6(NKC); 18 aa; 67%; 6 of 6 HuTop I (NTC); 79 aa; 44%; 17 of 21 + + + + Hsp90 (N9C); 53 aa; 77%; 17 of 17 ~ - + N.T., not tested. *Tested in heterozygous [PSI+] diploid (WT/N9C). Sup35N9Cp can readily enter a tPSI+~-like state under the influence of WT protein in that state, but it cannot maintain that state on its own. Discussion We have demonstrated that the M region of Sup35p makes important and diverse contributions to genetic and biochemical properties of PSI+. Sup35p mutants with a deletion of the M region or with substitutions in place of M can form priors, but these states are strikingly distinct from WT tPSI+] and from each other. A wide variety of prion states and behaviors can be 4:0 4:0 4:0 4:01 7% + + 3:1 56% 2:2 22% 1:3 4% 2:2* + + - + + + + — — + + + — + + + N.T. N.T. conferred on the same C-terminal functional domain and N- terminal prion domain by intervening "auxiliary" sequences. Prion proteins such as PrP and Sup35p aggregate when adopting the prion conformation (2, 37, 45, 46~. However, large-scale aggregation is neither necessary nor sufficient for entry into the prion state. (The former has also been suggested by the analysis of certain URE3 prion variants, ref. 47.) We have shown that M helps maintain Sup35p in the soluble state and, as a result, Sup35AMp is found in the pellet after centrifugation of cell lysates, regardless of its prion state. This finding confirms the special nature of prion state protein. A rpSl~NTc — —~ [PSI~ p~NTC~ IS ~ ~S'] ~ ! - APSIS c E PSI~NTC| USE] I [pS/~NTC~1 04 jlNTC^1 of ~ [p5l~NTC SC-ADE B YPD SC-ADE D SilNTC [psl~NTC T S P T S P _ - Sup35NTC 1 F Tetrad Tetrad 2 Tetrad 3 Fig. 4. Substitution of human topoisomerase I linker forthe Sup35p M region causes meiotic instability. (A) [psi-] and [PSI+] strains containing the linker from human topoisomerase I in place of the M linker of Sup35p were plated onto YPD; [PSI+] and [psi~] were plated for comparison. (B) Growth phenotype of SUP35NTCcells on YPD and SC-ADE. (C) Deletion of HSP104 did not affectthe suppressors/ate of [PSI+]NTC cells. (D) Sup35p solubility assay of [PSI+]NTC and [psi-]NTC (methods as described in Fig. 1). (E) Expression of NT-GFP in [psi~]NT~ and [PSI+]NTC did not induce aggregation. (F) Tetrad dissection of a [PSI+]NTC diploid shows varying numbers of [PSI+]NTC and [psi~]NTC spores. Liu et al. PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16451

A SUP35N9Cx[PSI~ SUP35N9C spore #1 SUP35 spore #1 SUP35 spare #2 SUP35N9C spore #2 D B N9C ~ [ASIA N9C [if spore spore_ T S P T S P T S P T S PT S P T S P ~ Sup35N9C Fig. 5. Substitution of the Hsp90p linker for the Sup35p M region causes distinct genetic behavior. (A) Diploid [PSI+] cells with the SUP35/N9C geno- type grew white on YPD (top row). On sporulation of this diploid, two red colonies and two white colonies were always obtained (following rows). (B) Expression of N9-GFP in the SUP35/N9C diploid [PSI+] strain causes aggrega- tion. (C) Expression of N9-GFP in haploid cells expressing only N9C did not cause aggregation. (D) Sup35p solubility assay of N9C indicates that it pelleted only in the presence of Sup35p in the [PSI+] state. The protein returned to the soluble state after sporulation (methods as described in Fig. 1). Differences between the Sup35AM protein in tPSI+~^M cells and in ~5i-~M cells are not simply a difference between aggregated and nonaggregated states. This point is also dem- onstrated by our experiments with Sup35NTCp. No aggre- gated state was detectable in tPSI+]NTC cells. The prion state of Sup35NTCp may well involve higher-order complexes, but if so, they are clearly different in character from the large complexes of WT Sup35p in the tPSI+] state. The M region is also important for the stabilization of tPSI+] during cell division. Cells with either the SUP35AM or the SUP35NKC replacement genotype could enter a prion state, but this state was not well maintained during mitotic division. Cells with the SUP35NTC genotype could also enter the prion state, and tPSI+iNTC was mitotically stable. However, tPSI+]NTC was not propagated after meiosis with the same fidelity as [PSI+~. Thus, the propagation of prion elements is quite sensitive to changes in the M region. Requirements for the maintenance of the prion during mitotic and meiotic cell division are distinct and M contributes to them both. Altering the M region also had important consequences for prion curing. Because HsplO4p function is sensitive to Gdn HCl, it has been suggested that Gdn HCl treatment cures cells through the inactivation of HsplO4p (48-51), but this hypothesis is controversial (48, 50, 52~. We have identified a prion state, tPSI+]NTC, which can be cured by growth on Gdn HCl but cannot be cured by HSP104 deletion (Table 1~. This finding indicates that curing by Gdn HCl is not solely caused by HsplO4p inacti- vation. The results also suggest that some feature of M strongly 1. Wickner, R. B., Masison, D. C. & Edskes, H. K. (1995) Yeast 11, 1671- 1685. 2. Patino, M. M., Liu, J. J., Glover, J. R. & Lindquist, S. (1996) Science 273, 622-626. 3. Paushkin, S. V., Kushnirov, V. V., Smirnov, V. N. & Ter-Avanesyan, M. D. (1996) EMBO ]. 15, 3127-3134. 4. Lindquist, S. (1997) Cell 89, 495-498. 5. Eaglestone, S. S., Cox, B. S. & Tuite, M. F. (1999) EMBO J. 18, 1974-1981. 6. True, H. L. & Lindquist, S. L. (2000) Nature (London) 407, 477-483. 7. Zhouravleva, G., Frolova, L., Le Goff, X., Le Guellec, R., Inge-Vechtomov, S., Kisselev, L. & Philippe, M. (1995) EMBO J. 14, 4065-4072. 16452 1 www.pnas.org/cgi/doi/10.1073/pnas.252652099 influences interactions with HsplO4p. In this respect, it is intriguing that 24 of 24 of the positively charged amino acids in M are lysines (Table 1~. Polylysine binds HsplO4p with high affinity and triggers a series of changes in ATP hydrolysis and HsplO4p conformation (36~. The deletion/insertion mutations we used also exhibit some conformational flexibility and lysine richness. However, the effect of HsplO4p on the prior-state conversions of these proteins differed. There must be something more than the mere presence of lysines and conformational flexibility that influences the interactions of the M region with HsplO4p. For example, length, specific sequence elements, residue spacings, and conformational predisposition could all influence these interactions. The Sup35N9C protein showed an intriguing genetic property that was entirely unexpected. Unlike other M alterations, Sup35N9Cp was incapable of entering a tPSI+~-like state until cells expressing it were mated to cells already containing WT Sup35p in that state. Further, Sup35N9Cp could not support the prion state on its own. Once the two proteins were separated by sporulation, SUP35N9C cells reverted to a Lpsi-] phenotype and the Sup35N9C protein returned to the soluble state. Thus, Sup35N9Cp can participate in a heritable phenotypic change caused by a protein-only mechanism that exhibits Mendelian segregation, a striking departure from ordinary prion behavior. The M region is not required for entry into the prion state. Yet in the context of proteins with the same N-terminal prion- determining region and C-terminal functional region, substitu- tion of M with artificial linker regions confers a rich variety of genetic and biochemical characteristics to the prion state. Pro- teins with very different physical properties can undergo self- perpetuating conformational changes in state, but produce similar phenotypes (Sup351\Mp, Sup35NTCp); proteins with similar physical characteristics can display very different genetic properties (WT Sup35p, Sup35N9Cp). Indeed, were it not for the fact that (i) the prior-determining nature of the N region of Sup35p has been extensively characterized by genetic, biochem- ical and cell biological methods, and (<ii) the self-perpetuating changes in function obtained with each of our different mutants were acquired from WT Sup35p that was in its well- characterized prion state, it might be hard to argue that the changes in function we observed were caused by self- perpetuating prion conformations. We have created prion variants with unusual properties by deliberate, artificial manipulations. We have no direct evidence that proteins with such properties exist in nature. However, given the divergence of prion sequences, which is particularly great in the M region, it seems reasonable to suppose that proteins that have such properties might well have appeared. Thus, we suggest that there may be many prior-like, self-perpetuating states not recognizable as such by the prior-defining criteria used to date. 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