[PSI+] Prion Transmission Barriers Protect Saccharomyces ... - Genetics

INVESTIGATION

[PSI+] Prion Transmission Barriers Protect Saccharomyces cerevisiae from Infection: Intraspecies ’Species Barriers’ David A. Bateman and Reed B. Wickner1 Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830

ABSTRACT [PSI+] is a prion of Sup35p, an essential translation termination and mRNA turnover factor. The existence of lethal [PSI+] variants, the absence of [PSI+] in wild strains, the mRNA turnover function of the Sup35p prion domain, and the stress reaction to prion infection suggest that [PSI+] is a disease. Nonetheless, others have proposed that [PSI+] and other yeast prions benefit their hosts. We find that wild Saccharomyces cerevisiae strains are polymorphic for the sequence of the prion domain and particularly in the adjacent M domain. Here we establish that these variations within the species produce barriers to prion transmission. The barriers are partially asymmetric in some cases, and evidence for variant specificity in barriers is presented. We propose that, as the PrP 129M/V polymorphism protects people from Creutzfeldt–Jakob disease, the Sup35p polymorphisms were selected to protect yeast cells from prion infection. In one prion incompatibility group, the barrier is due to N109S in the Sup35 prion domain and several changes in the middle (M) domain, with either the single N109S mutation or the group of M changes (without the N109S) producing a barrier. In another, the barrier is due to a large deletion in the repeat domain. All are outside the region previously believed to determine transmission compatibility. [SWI+], a prion of the chromatin remodeling factor Swi1p, was also proposed to benefit its host. We find that none of 70 wild strains carry this prion, suggesting that it is not beneficial.

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RIONS are infectious proteins, with no nucleic acid required for transmission. Most prions are self-propagating amyloids, b-sheet-rich filamentous polymers of a single protein. The mammalian prion causes a uniformly fatal transmissible spongiform encephalopathy (TSE) based on amyloid of PrP, normally a nonessential cell surface GPI-anchored protein that becomes toxic in amyloid form (reviewed in Caughey et al. 2009). Interspecies transmission of TSEs occurs with difficulty, or not at all, as a result of sequence differences between the prion proteins of donor and recipient, a phenomenon called the “species barrier” (Prusiner et al. 1990; Bruce 2003). Human TSEs include the spontaneous form (Creutzfeldt– Jakob disease, CJD) and an infectious form, most famously an epidemic (called Kuru) among the Fore people of New

Copyright © 2012 by the Genetics Society of America doi: 10.1534/genetics.111.136655 Manuscript received October 24, 2011; accepted for publication November 11, 2011 Supporting information is available online at http://www.genetics.org/content/ suppl/2011/11/18/genetics.111.136655.DC1. 1 Corresponding author: National Institutes of Health, Bldg. 8, Room 225, 8 Center Dr. MSC 0830, Bethesda, MD 20892-0830. E-mail: [email protected]

Guinea due to a ritual mortuary cannibal custom (reviewed in Collinge and Alpers 2008). The human gene encoding PrP has a polymorphism with about half of alleles encoding Met at residue 129 and half Val at that site. Remarkably, only rarely do patients heterozygous at this site develop any form of CJD, although both M/M and V/V people are susceptible. It has been suggested that this protective polymorphism was selected to avoid the devastating effects of cannibalism when that phenomenon was more common (Mead et al. 2003). Indeed, the Kuru epidemic selected a new resistant allele at residue 127 (Mead et al. 2009b). The [PSI+] prion (infectious protein) is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor (Wickner 1994; King and Diaz-Avalos 2004; Tanaka et al. 2004), reviewed in Wickner et al. (2010). [URE3] is a prion of Ure2p, a regulator of nitrogen catabolism (Wickner 1994), likewise based on a self-propagating amyloid (Brachmann et al. 2005), and [SWI+] is a prion of the chromatin remodeling factor Swi1p (Du et al. 2008). Each of these prions produces decreased function of the respective protein. Sup35p consists of an N-terminal Q/N-rich domain (N; residues 1–123), a highly charged middle (M; residues

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124–253) domain, and a C-terminal domain (C; residues 254–685). Sup35NM functions normally in the general mRNA degradation system, linking translation termination to mRNA lifetime (Hoshino et al. 1999; Hosoda et al. 2003; Funakoshi et al. 2007), and is necessary and largely sufficient for prion generation and propagation (Teravanesyan et al. 1994; King 2001; King and Diaz-Avalos 2004). However, some variant-specific information requires sequence to residue 137 (Bradley and Liebman 2004). Alterations in Sup35M can also affect prion propagation in subtle ways, but deletion of Sup35M is reported not to interfere with [PSI+] propagation (Liu et al. 2002). Sup35C carries out the essential translation termination function of the protein (Teravanesyan et al. 1994; Stansfield et al. 1995; Zhouravleva et al. 1995). A single prion protein sequence can stably propagate any of several amyloid conformations, resulting in different “prion variants” or “prion strains.” Thus, different isolates of sheep scrapie (a TSE) propagated in mice can show dramatically different incubation periods and different distribution of pathology in the brain, even though the sequence of the infecting prion protein and that of the infected animal are identical in each prion strain. In yeast, prion variants may differ in the intensity of the prion phenotype, stability of prion propagation, response to overproduction or deficiency of chaperones, or differing ability to overcome a species barrier (Derkatch et al. 1996; Chernoff et al. 1999; Kushnirov et al. 2000b; Edskes et al. 2009). Recently, prion variants of [PSI+] and [URE3] that are severely toxic or even lethal have also been found (Mcglinchey et al. 2011). The sequence of the prion protein is the same in different prion variants, but the conformation of the protein in the amyloid is different (Bessen and Marsh 1994; Tanaka et al. 2006), and this conformation is propagated faithfully as the filaments elongate, are severed to make new filaments, and are distributed to daughter cells. The mechanism by which a single protein can faithfully transmit its conformation to another molecule of the same is suggested by the architecture of yeast prions. Infectious amyloid of the prion domains of Ure2p, Sup35p, and Rnq1p (the latter the basis of the [PIN+] prion) each have an inregister parallel b-sheet architecture with multiple folds in the b-sheet along the long axis of the filaments (Shewmaker et al. 2006; Baxa et al. 2007; Wickner et al. 2008a). This type of amyloid, also typical of most pathological human amyloids (Tycko 2011), is characterized by lines of identical amino acid side chains running the length of the filament. The in-register structure is enforced by the favorable interactions between these aligned identical side chains that are possible only if the molecules are in-register. Hydrogen bonds between the side-chain amides of glutamines or asparagines produce a great stabilization of the structure, and serine or threonine residues can likewise form a chain of H-bonds with their side chain 2OH group. Hydrophobic side chains can also have interactions that promote in-register alignment. Of course, a line of charged residues would be unfavorable for in-register structure and charged residues

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are few in these prion domains. Prion variants are proposed to differ in the location of the folds of the sheet (i.e., the turns of the chain). The same favorable interactions of identical side chains that enforce the in-register architecture would force new molecules to assume the same conformation as molecules already in the chain, thus explaining the faithful propagation of prion variants (Wickner et al. 2007, 2008b, 2010). The prion proteins each have a discrete region needed for prion propagation that corresponds roughly to the part of the protein that actually forms amyloid (Teravanesyan et al. 1994; Masison and Wickner 1995; King et al. 1997; Taylor et al. 1999). The prion domain of Sup35p was first defined (Teravanesyan et al. 1994) as the N-terminal residues 1–114 necessary to propagate the original [PSI+] variant (Cox 1965). Later studies showed that residues 1–137 are sufficient to propagate any of several [PSI+] variants (Bradley and Liebman 2004), although several variants were faithfully propagated by 1–64 (Shkundina et al. 2006) or 1–61 (King and Diaz-Avalos 2004). The prion domains of Sup35p of several other Saccharomyces species can form [PSI+] in cerevisiae, but because of sequence differences with S. cerevisiae Sup35p and with each other, there are species barriers to transmission of [PSI+] (Chen et al. 2007; Afanasieva et al. 2011), although which differences are responsible for this barrier have not yet been defined. Mutational studies of the S. cerevisiae prion domain have shown that transmission barriers can be produced by mutations in Sup35p residues 1–33, a very Q/N-rich part of the protein (Depace et al. 1998). Solid-state NMR studies of infectious amyloid of Sup35NM showed in-register parallel b-sheet structure of essentially all of N (1–123) and suggested that this structure extends partially into the M domain (residues 124–253) (Shewmaker et al. 2006, 2009). The N and M domains of Sup35p are known to be far more variable between yeast species than the C domains (Kushnirov et al. 1990, 2000a; Santoso et al. 2000). Previous studies of SUP35 sequences from wild S. cerevisiae strains have revealed several variable sites, including coding changes Q30R, A34T, A42G, Y55H, Q90H, N109S, V147F, G152C, G162D, D169E, T206K, and H255D and a 19amino-acid deletion in the oligopeptide repeats (Jensen et al. 2001; Resende et al. 2003; Supporting Information, Table S4). This deletion was reported to alter the expression of [PSI+] without causing its loss (Resende et al. 2003), but other polymorphisms have not been tested for their effects on prion transmission or prion-forming ability. We have examined the sequence, prion-forming ability, and susceptibility to prion infection of SUP35 in wild isolates of S. cerevisiae. We observe considerable polymorphism, with variations particularly in the N and M domains. While all alleles tested can form prions, we show that there are strong transmission barriers between different Sup35p’s. Surprisingly, residues in the C-terminal part of Sup35N and in M contribute to these transmission barriers. We also find that [SWI+] is not found in our

Table 1 Laboratory strains of S. cerevisiae Strain no. 4828 4830 779-6a 74D-694 74D-694 SWI+

Genotype MATa ade 2-1 SuQ5 trp1 kar1-1 his3 leu2 ura3 sup35::kanMX [PIN+] [psi2] p1215: CEN URA3 SUP35MC MATa ade 2-1 SUQ5 trp1 kar1-1 lys2 leu2 ura3 sup35::kanMX [pin2] [psi2] p1215: CEN URA3 SUP35MC MATa ade 2-1 SUQ5 trp1 kar1-1 his3 leu2 ura3 [PSI+] (Jung and Masison 2001) MATa ade 1-14 trp1-289 his3-200 leu2-3 ura3-52 [swi2] MATa ade 1-14 trp1-289 his3-200 leu2-3 ura3-52 [SWI+]

The entire ORF of SUP35 is deleted in strains 4828 and 4830, and substituted with the kanMX gene conferring resistance to G418.

collection of wild strains, suggesting that it is not adaptive for yeast.

work Protein Sequence Analysis), to determine coding polymorphisms. Wild-strain SUP35 plasmid constructions

Materials and Methods Nomenclature

We refer to the standard laboratory yeast sequence (Kikuchi et al. 1988; Kushnirov et al. 1988; Wilson and Culbertson 1988) as the “reference sequence,” and that of differing wild isolates are called “polymorphs.” A prion originating with the Sup35p sequence of strain G2, for example, but being propagated in a strain expressing only the reference sequence will be designated [PSI+G2]ref, in analogy with similar nomenclature for [URE3] (Edskes et al. 2009). Scoring the [PSI+] prion

Sup35p is a subunit of the translation termination complex, and the incorporation of a large proportion of Sup35p into the prion amyloid filaments makes it inactive, resulting in increased read-through of termination codons. This is measured by read-through of ade2-1, with an ochre termination codon in the middle of the ADE2 gene. In addition to ade2-1, strains carry the SUQ5 weak suppressor mutation, which leaves cells Ade2 unless the [PSI+] prion is also present. Strains and media

The strains used are listed in Table 1 (laboratory strains) and Table S1 (wild strains). All yeast media and plates contained 20 mM copper sulfate unless noted. Rich and minimal media (YPAD and SD) are as described (Sherman 1991). Required nutrients were added to minimal plates. Sequencing the SUP35 of wild strains

Genomic yeast DNA was isolated using the QAIprep spin miniprep kit. Dilutions of 1:10 and 1:100 were made and PCR was performed using Platinum PCR SuperMix (Invitrogen) with primer DB082 with either DB051 or DB089 (Table S2) for S. cerevisiae strains; primers DB093 and DB094 for S. paradoxus; and primers DB095 and DB119 with S. bayanus or S. pastorianus strains. PCR products were cleaned using the QIAquick PCR purification kit (Qiagen) and sequenced (University of Maryland Sequencing facility, UMBI). Sequences were assembled and aligned, and fasta format output files were generated for resulting DNA and translated protein sequences using Codon Code Aligner v. 3.7.1 (CodonCode Corp.). Translated protein sequences were aligned to reference sequences using ClustalW (Net-

Using primers DB082 and DB085, the full SUP35 open reading frame was amplified from the wild strains indicated (in parentheses) and cloned between the Bam1H and XhoI sites of pDB03 to generate pDB73 (strain F9, D19) and pDB86 (strain F8), or into pDB66 replacing Sup35C–GFP to generate pDB88 (strain A9), pDB89 (strain E9), pDB91 (strain F7), and pDB93 (strain G2). A list of all plasmids is included in Table 2. Plasmids were transformed into strain 4828, p1215 loss was selected by growth on 5-fluoroorotic acid (5-FOA) media, and clones that were Ade2 and red on 1/2 YPD were picked. Prions were selected by plating cells at high density on media lacking adenine or by induction with overnight growth with 160 mM copper in liquid YPAD media to overproduce Sup35p and plating on media lacking adenine. Colonies that were Ade+ were streaked on 1/2 YPD and then mated with a lawn of strain 4830 to determine dominance. Isolates that proved to be dominant were streaked on 1/2 YPD containing 5 mM guanidine hydrochloride to isolate cured strains. Generation of 109 mutants

Using PCR with primers DB082, DB129, DB130, and DB133 on plasmid pDB89 (strain E9) and pDB93 (strain G2), we converted the serine 109 residue back to the reference sequence asparagine, generating plasmids pDB102 (strain E9) and pDB103 (strain G2). Using primers DB082, DB131, DB132, and DB133 with plasmid pDB08 (reference Sup35 sequence), the point mutation asparagine to serine at position 109 was made, generating plasmid pDB101. Cytoduction

Cytoplasm may be transformed from one strain to another utilizing the kar1-1 mutation (Conde and Fink 1976), defective for nuclear fusion. Cells fuse, but the nuclei do not fuse, and nuclei separate at the next cell division. However, cytoplasmic mixing has occurred, and so a genetic element (prion or mitochondrial DNA) present in one strain (identified by its nuclear genotype) will be transferred to the other. We use transfer of mitochondrial DNA as a marker of cytoplasmic transfer, and score prion transfer. Wild-strain sequence plasmids were transformed into strain 4830, loss of p1215 was selected by growth on 5-FOA media, and Ade2 transformants were made rhoo by growth on YPAD containing 1 mg/ml ethidium bromide. Donor and recipient

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Table 2 Plasmids used in this study Plasmid

Name

Description

Origin

pDB08 pDB09 pDB03 pDB22 pDB11 pDB61 pDB64 pDB65 pDB66 pDB67 pDB73 pDB81 pDB82 pDB86 pDB88 pDB89 pDB91 pDB93 pDB97 pDB99 pDB100 pDB101 pDB102 pDB103

pH 953 p1215 p1147 pH 610 pH 952 p416TEFSWI1NQ–YFP pKanMXURE2N–GFP (#210) pCUP1SUP35NM–GFP (#211) Cup10 Swi-E D19-1147 A8-210 F9-210 F8-1147 A9-Cup10 E9-Cup10 F7-Cup10 G2-Cup10 E9-610 F9-610 G2-610 Ref-N109S E9-S109N G2-S109N

CEN SUP35 LEU2 SUP35 promoter CEN SUP35MC URA3 SUP35 promoter CEN LEU2 ADH1 promoter 2m TRP1 GAL1 promoter 2m SUP35NM TRP1 GAL1 promoter CEN SWI1(1–536)–YFP URA3 TEF promoter CEN URE2–GFP KanMX ADH promoter CEN SUP35NM-GFP KanMX CUP1 promoter CEN SUP35C–GFP LEU2 CUP1 promoter CEN SWI1(1–536)–GFP KanMX ADH promoter CEN SUP35 (from strain F9) LEU2 ADH promoter CEN SUP35NM (strain A8) KanMX ADH promoter CEN SUP35NM (strain F9) KanMX ADH promoter CEN SUP35 (strain F8) LEU2 ADH promoter CEN SUP35 (strain A9) LEU2 CUP1 promoter CEN SUP35 (strain E9) LEU2 CUP1 promoter CEN SUP35 (strain F7) LEU2 CUP1 promoter CEN SUP35 (strain G2) LEU2 CUP1 promoter 2m SUP35NM (strain E9) TRP1 GAL1 promoter 2m SUP35NM (strain F9) TRP1 GAL1 promoter 2m SUP35NM (strain G2) TRP1 GAL1 promoter CEN SUP35 N109S LEU2 SUP35 promoter CEN SUP35 (strain E9) S109N LEU2 SUP35 promoter CEN SUP35 (strain G2) S109N LEU2 SUP35 promoter

H. E. Edskes This study This study H. E. Edskes H. E. Edskes Du et al. (2008) Nakayashiki et al. (2005) Nakayashiki et al. (2005) This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

All plasmids carry AmpR.

strains were mixed in water at high density and 0.1 ml was spotted onto a YPAD plate. After 18 hr at room temperature, cells were streaked for single colonies on media selective against the donor strains. Clones are shown to be cytoductants by their growth on glycerol and failure to grow on media selective for diploids. As further tests of a sample confirm, Ade+ cytoductants are judged to have received and propagated [PSI+]. Analysis of [SWI+] in wild strains

SWI1 was amplified from p416TEFSWI1NQ-YFP (Du et al. 2008) by PCR using primers DB087 and DB097 and cloned between the BamHI and XhoI sites of pDB64 (Nakayashiki et al. 2005) resulting in pDB67 (CEN KanMX SWI1–GFP). This plasmid was sequenced and transformed into wild strains using 0.3 mg/ml G418 (Mediatech Inc., Herndon, VA) for plasmid selection and maintenance. Cells were imaged using a Nikon Eclipse TE2000-U spinning disc confocal microscope with 100· NA 1.4 Nikon oil lens with 1.5· magnifier and captured with a Hamamatsu EM-CCD ImagEM digital camera with IPLab version 4.08. The [SWI+] phenotype was assessed by streaking each wild strain on YPAD, YPARaf (1% yeast extract, 2% peptone, 0.04% adenine sulfate, 2% raffinose), YPG (1% yeast extract, 2% peptone, 2% glycerol), and synthetic medium with 2% raffinose (SRaf).

Results Sup35 sequences in wild S. cerevisiae strains

Among the SUP35 genes of 55 wild S. cerevisiae strains (Table S1) (Nakayashiki et al. 2005) sequenced in this study, 13

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infrequent mutations were found in the C domain (residues 254–685), each observed in 5 or fewer strains (Table 3 and Table S3). The M domain (residues 124–253) had a similar number of mutations per residue (8/129 13/431), but at a much higher frequency in the wild strains (Figure 1). All but two strains had the G162D mutation, and other common mutations within the M domain include the D169E mutation found at 22%, P186A mutation at 13%, T206K at 45%, and H225D found at 27% of the S. cerevisiae population sample. Very few mutations within the N domain (1–123) were observed (Table 3 and Table S3); however, the N109S mutation was observed in 31% of the population. Only the N and M domains of the non-cerevisiae SUP35 genes were sequenced. The SUP35 genes of four wild S. bayanus strains had only one common N domain (1–116) mutation P4S, and no M domain mutations compared to the reference sequence (Cliften et al. 2003) (Table 3 and Table S3). Five sequenced SUP35 genes from S. pastorianus wild strains were sequenced and compared to the reference S. pastorianus sequence (Nakao et al. 2009)). Of these sequences the N5I and N8I mutations were found with highest frequency among the five N domain (1–116) mutations and two M domain (117–242) mutations (Table 3 and Table S3). For S. paradoxus, only two mutations were observed compared to the reference sequence (Kellis et al. 2003), Y29S and L84P within the N domain (Table 3 and Table S3). Three major transmission compatibility groups

We found the common N109S, G162D, D169E, P186A, T206K, and H225D mutations most interesting within our wild S. cerevisiae strains (Table 3 and Table S3), since all

Table 3 Polymorphs of Saccharomyces Sup35 proteins Strains of S. cerevisiae

Mutations

B2, C4 A5, A7, A10, A11, B1, B3, B5, B6, B7, B10, C1, C3, C5, E2, E4, E6, E11, F1 B11, F9 E12 E8 F11 F8 A1, A8, B12, E9, F10, F12, G2 E1 E3 A4 F4 F3 C2 E7 E10 A9, B9, F7 A6 A2, F5 B8, F2 A3 A12 B4 F6 E5 G1

None G162D D59-77, G162D Q14H, G162D Q10H, G162D N109S, G162D N109S, G162D, D169E, P186A, T206K, E216D, H255D, Y467H N109S, G162D, D169E, P186A, T206K, H225D N109S, G162D, D169E, T206K, H225D N109S, G162D, D169E, Q201H, T206K, H225D N109S, G162D, D169E, Q201H, T206K, H225D, N426Y N109S, G162D, D169E, T206K, H225D N109S, G162D, D169E, T206K, H225D, S428R, K439M N109S, G162D, D169E, T206K, H225D, A676S, K679E N109S, G162D, P186A, T206K, H225D, K324P N109S, G162D, T206K, H225D N109S, G162D, T206K, H225D, G678D G162D, T206K G162D, T206K, V402A, S428R, G449A G162D, T206K, V402A G162D, T206K, V402A, N480T G162D, T206K, E560G G162D, T206K, S210L G162D, A676S, G678D G162D, N426Y G162D, K324T, K679E G162D, K679E

Strains of S. bayanus D4 D8 D9 D10

Mutations P4S P4S P4S NA

Strains of S. pastorianus D6 D7 D12 D5 D11

Mutations N5I, N8I N5I, N8I, Q41H, T161I, D225N P4S, N5I, N8I, A54S N5I, N8I N8I

Strains of S. paradoxus C7, C6, C8, C9, C10, 11C, D1, D3 D2 C12

Mutations None L84P Y29S, L84P Y29S

these mutations lie well outside the region (residues 1–33) commonly proposed to determine the transmission barrier between Sup35 molecules with different sequences (Depace et al. 1998). We transformed laboratory strain 4828 [sup35D p(URA3 SUP35C)] with plasmids generated from these wild S. cerevisiae SUP35 genes, selected cells that had lost the URA3 plasmid on FOA, and named these laboratory strains with the corresponding wild-strain code. This generated strains A9 and F7 (both G162D, T206K), E9 and A8 (both N109S, G162D, D169E, P186A, T206K, H225D), G2 (N109S, G162D, D169E, T206K, H225D), and F8 (N109S, G162D, D169E, P186A, T206K, E216D, H225D, Y467H), as listed in Table 3 and Table S3. The D19 allele, found in

strains B11 and F9, lacked residues 59–77 and had the G162D mutation (Table 3 and Table S3). These strains were tested to determine if [PSI+] could be transmitted into these mutants from a strain with the reference sequence. Cytoduction, a transfer of cytoplasm between strains without transfer of DNA plasmids or nuclei, was used in this test as the closest parallel to mammalian transmission events. Because the donor and recipient Sup35 molecules are only transiently coexpressed, this type of experiment gives the best opportunity to determine transmission barriers, with the least likelihood of confusion resulting from prion variant changes being selected. Data in Table 4 show that there is no transmission barrier between the reference sequence and recipients A9 or F7,


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Figure 1 Distribution of mutations in Sup35p of wild strains of S. cerevisiae. The width of the vertical lines marking the location of mutations is in rough proportion to their frequency (Table S3). The box on the left is a region in which mutation can block [PSI+] transmission (Depace et al. 1998). Boldface, underlined text indicates those mutations observed most frequently. The location of the octapeptide repeats is shown as ......

indicating that the nearly universal mutation G162D does not produce a transmission barrier. However, there is a substantial barrier to transmission to strains that have either the D19 mutation or strains with the N109S and multiple M-domain mutations (Table 4, recipients A8, E9, F8, G2, D19). The cytoductants were isolated and used as donors to [psi2] cells with the reference Sup35 sequence, to confirm that Ade2 was due to loss of [PSI+] and not, for example, simply a decrease in the amount of amyloid such that there was no prion phenotype. The D19 Ade2 donors did not transmit [PSI+] back to a strain with the reference sequence confirming that [PSI+ref] was indeed lost in the D19 host (Table 5). The same result was obtained with the F8 and G2 Ade2 donors, the latter differing from the reference sequence only by N109S and M-domain changes. The E9 donors, differing in sequence slightly from both G2 and F8, showed some retention of infectivity for the reference sequence, indicating that it was losing, but had not completely lost [PSI+]. De novo [PSI+] derivatives of strains expressing the polymorphic sequences were generated for both the D19 and the G2 strains by transiently overexpressing the same polymorphic Sup35p and selecting Ade+ clones. These Ade+ clones were shown to be [PSI+] by their being dominant, by being efficiently cured by growth on rich medium containing 5 mM guanidine, and by the appearance of fluorescent foci when transformed with plasmids expressing the corresponding Sup35NM fused to GFP (Figure 2). This conclusion was confirmed by the efficient transmission of [PSI+] by cytoduction to cells carrying the same SUP35 allele (Table 6). Cytoductions between these new [PSI+] isolates and cells expressing other Sup35p polymorphs, along with data in Table 4, indicate that there are at least three main [PSI+] incompatibility groups (Table 6). Cytoduction from [PSI+] arising in any polymorph into a [psi2] cell expressing the same polymorph was efficient, but cytoduction into a [psi2] recipient expressing a different polymorph was quite inefficient— sometimes completely blocked. [PSI+D19] propagated poorly in cells expressing another polymorph, most likely due to a structural rearrangement of the NM domain due to such a large loss of amino acids within this domain. Similarly, [PSI+G2] propagated best with other N109S containing mutant strains, although some transmission (30%) was observed to the A9, F7, reference, and D19 sequences. We transformed the [PSI+ref]ref, [PSI+G2]G2, and [PSI+D19]D19 strains with the various polymorph NM–GFP

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vectors. The refNM–GFP produced dots in each of the hosts (Figure 2), indicating that there is some interaction of the prion domains of the reference Sup35 with each polymorph amyloid, even though those interactions did not generally lead to prion propagation. At the other extreme, the G2NM–GFP showed dots only in the homologous host, showing that these interactions are asymmetric. The D19NM–GFP showed dots with the homologous amyloid, as well as with the reference amyloid (Figure 2). These results indicate some sequence specificity in association with the NM–GFP fusions with amyloids, with a preference for the same sequence. Asymmetry and prion variant specificity of transmission barriers

Although [PSI+G2]G2 was transmitted to 5 of 25 cytoductants expressing the reference sequence, [PSI+ref]ref was not transmitted to any of 86 cytoductants expressing the G2 polymorph (Tables 4 and 6). Although [PSI+G2]G2 was successfully transmitted in 8 of 22 cytoduction events into a strain expressing D19, [PSI+D19]D19 was not transmitted in any of 10 cytoduction events to a G2 strain (Table 6). Thus transmission barriers can be asymmetric for [PSI+] as was previously found for [URE3] (Edskes et al. 2009).

Table 4 Cytoductions of [PSI+] from the reference sequence to polymorphic Sup35s Donor

779-6a Ref Seq PSI+

Ref Seq [psi2]

Recipient

Ade+ cytoductants

Total cytoductants

% Ade+

Reference A9 F7 A8 E9 G2 F8 D19

80 64 37 0 16 0 0 13

80 66 37 30 86 86 86 101

100 97 100 0 19 0 0 13

Reference A9 F7 A8 E9 G2 F8 D19

0 0 0 0 0 0 0 0

30 30 28 30 30 21 25 42

0 0 0 0 0 0 0 0

The [PSI+] donor (top) was strain 779-6a and the [psi2] donor (bottom) was 4828. The [psi2] recipient was 4830 in all cases.

Table 5 Cytoduction from Ade+ and Ade2 cytoductants (in strain 4830) from Table 4 back to strain 4828 expressing the reference sequence and lacking the prion Donor Cytoductant

Figure 2 Interaction of Sup35p polymorphs with each other. In [PSI+], strains of one polymorph is expressed Sup35NM–GFP of another (or the same) polymorph, and the appearance of the GFP fluorescence as dots indicates association of the indicated NM–GFP with the Sup35 prion aggregates. Strain 4828 with each prion (rows) was transformed with plasmids expressing the polymorph NM–GFP (columns) fusion proteins from the ADH1 promoter and cells were examined as described in methods. GFP expressed alone (and not fused to Sup35) is evenly distributed in the cytoplasm. A8NM– GFP does differ by one residue (P186A) from the G2 sequence.

Although [PSI+D19]D19 was transmitted to only 1 of 10 cytoductants expressing the reference sequence (Table 6), [PSI+ref]D19 was transmitted to 97 of 110 cytoductants expressing the reference Sup35p (Table 5). This result suggests a variant-dependent transmission barrier. The variant that originated in cells expressing the reference sequence transmitted poorly to a D19 recipient (Table 4), but “remembers” its origins and returns more easily to the reference sequence Sup35p than does a prion originating in a D19 strain (Table 5). N109S and M-domain mutations each affect [PSI+] transmission barrier

To determine the effect of the N109S mutation, point mutations were created in the reference sequence to generate this specific point mutation, and corrective mutations were introduced into the E9 and G2 sequences (S109N mutation). The resulting strains were used to determine the transmission of [PSI+]. Table 7 indicates that the [PSI+ref]ref meets some resistance to infection of the N109S point mutant. In addition, correcting the N109S in E9 and G2 strains back to the reference sequence allows more ready infection by [PSI+ref]ref. In addition, this corrective mutation in E9 and G2 hindered the transmission of [PSI+G2]G2, indicating that the single-residue difference results in a transmission barrier in the context of G2 or E9, as it does in the context of the reference sequence. The same data also indicate that the M-domain mutations found in many polymorphs can produce a transmission barrier without differences in the N or C domains (Table 7). Although

Ref Ade2 Ref Ade+ A Ref Ade+ B Ref Ade+ C Ref Ade+ D D19 Ade2 A D19 Ade2 B D19 Ade+ A D19 Ade+ B D19 Ade+ C D19 Ade+ D A9 Ade+ A A9 Ade+ B E9 Ade2 A E9 Ade2 B E9 Ade+ A E9 Ade+ B F8 Ade2 A F8 Ade2 B F8 Ade2 C G2 Ade2 A G2 Ade2 B

Recipient Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref

Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq

Ade+ cytoductants

Total cytoductants

% Ade+

0 40 54 32 45 0 0 20 38 14 25 10 55 12 12 22 12 0 0 0 0 0

25 40 54 32 45 30 45 23 42 20 25 19 65 36 47 33 20 50 23 22 16 35

0 100 100 100 100 0 0 87 90 70 100 53 85 33 26 67 60 0 0 0 0 0

The Ade2 D19, G2 and F8 donors produced only Ade2 cytoductants suggesting that the donors did not carry an unexpressed [PSI+]. The Ade2 E9 donors produced a proportion of Ade+ cytoductants indicating that [PSI+] was being lost, but had not been completely lost in the E9 strain.

E9 S109N and G2 S109N are better recipients for [PSI+ref]ref than the uncorrected polymorphs, each still shows a significant barrier. The combination of N109S and the M-domain mutations produces a much greater barrier than would be expected if they acted independently; the barriers are synergistic. We note also that while the identical E9 and G2 appeared significantly different as recipients of [PSI+ref] in the data in Table 4, the difference is much smaller in the experiment in Table 7, done with the same donor and recipients, suggesting that this is simply experimental variation. [SWI+] is absent from wild strains: Like [PSI+], it has been suggested that [SWI+] benefits yeast (Du et al. 2008). If [SWI+] is indeed a benefit, it should be found in wild strains (Nakayashiki et al. 2005). All 76 wild strains were streaked in patches on media reported to prevent growth in the presence of the [SWI+] prion (Du et al. 2008). Strains C3, C8, and D7 were unable to grow on YPG media (Figure S1); however, these YPG-negative strains could not be cured with guanidine. These strains also lacked the presence of GFP dots with plasmid pDB67, expressing Swi1(1–536)– GFP, as did all the other wild strains tested (Figure S2). The control [SWI+] strain seemed to be unstable, losing its inhibited growth property on raffinose and YPG and needed to be counterselected for control tests. It was also difficult to observe GFP dots with the control strain over prolonged growth on YPD. We concluded that [SWI+] prion is not found in our wild strains and that in our hands the [SWI+] prion is somewhat unstable.


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Table 6 Cytoductions from polymorphic [PSI+] donors Donor

Recipient

G2 Red [psi2]

G2 Ade+ [PSI+G2]G2

D19 Red [psi2]

D19 Ade+ [PSI+D19]D19

G2 E9 F8 Reference A9 F7 D19 G2 E9 F8 Reference A9 F7 D19 D19 G2 E9 F8 A9 F7 Reference D19 G2 E9 F8 A9 F7 Reference

Ade+ cytoductants 0 0 0 0 0 0 0 34 25 32 5 10 8 8 0 0 0 0 0 0 0 7 0 0 0 4 0 1

Total cytoductants 35 35 23 36 35 46 18 36 28 35 20 32 35 22 35 45 30 32 32 35 36 8 10 10 10 8 10 10

% Ade+ 0 0 0 0 0 0 0 94 89 91 25 31 23 36 0 0 0 0 0 0 0 88 0 0 0 50 0 10

The donors were strain 4828 expressing polymorph Sup35 and [PSI+] originating in that polymorph. [psi2] recipients were strain 4830 expressing the indicated Sup35p polymorphs.

Discussion Following the description of an apparently beneficial prion of Podospora anserina (Coustou et al. 1997), it was reported that the [PSI+] prion protected cells from elevated temperature or high concentrations of ethanol (Eaglestone et al. 1999). Subsequent work showed that there was no consistent protection against heat, ethanol, or any of a large number of conditions tested and that for most strains under most conditions [psi2] was preferable to [PSI+] (True and Lindquist 2000). However, some strains were reported to be modestly benefited by [PSI+], and it was suggested that these effects could help yeast evolve by allowing them to survive stress (True and Lindquist 2000). However, although using the same strains, others were unable to reproduce the reported [PSI+] advantages (Namy et al. 2008), casting doubt on the [PSI+]-as-benefit proposal. Efforts to show induction of [PSI+] formation by stress conditions were unsuccessful with the wild-type Sup35p sequence (Tyedmers et al. 2008). Using an artificial Sup35p with high spontaneous [PSI+] formation, a small increase in [PSI+] formation was observed under several stress conditions, but in four of six such conditions, [PSI+] made cell growth worse (Tyedmers et al. 2008), suggesting that this is not an adaptive response.

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Table 7 N109S is responsible for part of the species barrier in wild strains Donor

Recipient

Ade+ cytoductants

Total cytoductants

% Ade+

779-6a [PSI+ref]ref

Reference Ref N109S E9 E9 S109N G2 G2 S109N Reference Ref N109S E9 E9 S109N G2 G2 S109N

49 93 8 112 1 22 5 18 35 14 36 9

50 123 102 130 67 33 50 85 45 95 40 85

98 76 8 86 1 67 10 21 78 15 90 11

[PSI+G2]G2

The [psi2] recipients were strain 4828 expressing the indicated Sup35p.

Since assessment of marginal and variable phenotype differences cannot answer the question of prion benefit or detriment to yeast, several other approaches have been used. We noted that since severely pathogenic viruses and prions (e.g., chronic wasting disease of elk and deer) are readily found in nature, a beneficial infectious element will be found in nearly all wild strains (Nakayashiki et al. 2005). The absence of [PSI+] and [URE3] from the 70 wild strains examined indicates that they are quite detrimental (Nakayashiki et al. 2005). Here, we show that the same strains lack the [SWI+] prion, indicating that it too is a rare disease, not an adaptive change. The barrier to transmission between mammalian species (the “species barrier”) is a consequence of sequence differences between the PrP proteins. Sheep and elk/deer develop prion diseases in the wild and some sheep have PrP alleles that make them resistant to infection (Hunter et al. 1996). The M/V129 PrP polymorphism in the human population has the effect of protecting against prion disease, with heterozygotes being resistant, and it is argued that it is just this protection that has produced the “balancing selection” for the polymorphism (Mead et al. 2003). The Kuru epidemic apparently selected a nearby mutation that confers resistance to transmissible spongiform encephalopathies (Mead et al. 2009a). Here we show that the polymorphism of the Sup35p sequence in wild yeast likewise produces barriers to transmission within S. cerevisiae. This clearly protects the cells from the spread of the prion, and we suggest that it is this protection that resulted in the selection of polymorphism for these sequence variants. The mutations producing these barriers are in the N and M domains of Sup35, outside the region involved in translation termination (Teravanesyan et al. 1993). Whether these mutations also affect the mRNA turnover function of the NM region of Sup35 (Hoshino et al. 1999; Hosoda et al. 2003; Funakoshi et al. 2007) is as yet unclear because the sequence requirements for that function have not yet been explored. A change that benefited the yeast by improving one of these nonprion functions of Sup35p might be expected to take over the population, not be present

as a polymorphism. However, if any of these polymorphs took over the population, there would be no protection from prion infection. The fact of this polymorphism argues that its presence is selected to protect against prion infection, as in the case of the PrP 129M/V polymorphism (Mead et al. 2003). The part of the Sup35p prion domain that determines barriers to transmission between different sequences has been assigned to residues 2–33 on the basis of mutagenesis studies of the laboratory S. cerevisiae allele (Depace et al. 1998). We find that in wild S. cerevisiae strains, the barrier is provided by either a deletion in the repeat region (residues 59–77) or by a combination of N109S and a group of mutations in the M domain between residues 162 and 225, each well outside the region determined by mutation. Previous studies showed that making a [PSI +ref]ref strain express both Sup35ref and Sup35D19 eliminated the phenotype of [PSI+], but did not eliminate the prion (Resende et al. 2003). When [PSI+ref]ref cells were made to express only Sup35D19, the [PSI+] phenotype (Ade+) was lost, but it was not determined whether this was eliminating the prion or simply interfering with the phenotype (Resende et al. 2003). We find that [PSI+ref] introduced from a cell expressing only the reference Sup35p is lost if the recipient expresses only D19. This constitutes a transmission barrier and is consistent with the results of Resende et al. (2003). Deletions or substitutions in the M domain have been shown to affect [PSI+] in subtle ways, but no prion variants failed to be transmitted to SUP35DM strains (Liu et al. 2002). A T341A mutation in the C domain appears to block [PSI+] propagation (Kabani et al. 2011), but this was not found in wild strains. We find that the specific changes found in the E9 polymorph (and often in other wild strains)—G162, D169E, T206K H225—are sufficient to significantly block transmission of [PSI+] from the reference sequence. More dramatically, the single N109S mutation produces a substantial barrier to transmission of [PSI +ref]ref. Since many point mutations in the N and M parts of Sup35 do not produce barriers to [PSI+] transmission (Depace et al. 1998; Liu et al. 2002), it is likely that these variants were selected to produce the transmission barrier that we observe, suggesting that acquisition of [PSI+] is undesirable. The finding that mutations in the M region from G162 to H225 can affect prion transmission efficiency is consistent with our previous evidence that there is some in-register b-sheet structure in M, although we were unable to further localize this structure (Shewmaker et al. 2006, 2009). The interpretation of [PSI+] as a disease is consistent with an array of other accumulating evidence. The presence of [PSI+] or [URE3] induces the synthesis of the “stress proteins,” Hsp104 and Hsp70, suggesting that cells view these prions as a stress (Jung et al. 2000; Schwimmer and Masison 2002). Ability of Ure2p to form a prion is not conserved in S. castellii, Candida glabrata, or Kluyveromyces

lactis (Edskes et al. 2009, 2011; Safadi et al. 2011), although their putative prion domains are quite similar in sequence. If prion formation were advantageous, it would be widely conserved. The fact that the prion domains of Sup35p and Ure2p have other well-established functions, unrelated to prion formation, suggests again that these domains are not present for the purpose of the very rare prion formation (Hoshino et al. 1999; Hosoda et al. 2003; Funakoshi et al. 2007; Shewmaker et al. 2007). The finding that [PSI+] can kill cells by inactivating Sup35p and that [URE3] can be extremely toxic (by an as-yet unknown mechanism) again suggests that these prions are not adaptive (Mcglinchey et al. 2011). Certain mutants in the Hsp40-encoding SIS1 gene make even the usual “mild [PSI+]” become lethal, further indicating the potential toxicity of this prion (Kirkland et al. 2011). Further work will be needed to explore the mechanisms of prion pathogenesis in yeast.

Acknowledgments We thank Donna MacCallum for kindly sending strains J041047 and J940610 and Herman Edskes for several plasmids. We are grateful to Zhiqiang Du and Liming Li for kindly providing p416TEFSWI1NQ-YFP and strains 7D694 and 7D-694SWI+. This work was supported by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases.

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GENETICS Supporting Information http://www.genetics.org/content/suppl/2011/11/18/genetics.111.136655.DC1

[PSI+] Prion Transmission Barriers Protect Saccharomyces cerevisiae from Infection: Intraspecies ‘Species Barriers’ David A. Bateman and Reed B. Wickner

Copyright © 2012 by the Genetics Society of America DOI: 10.1534/genetics.111.136655

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