Prion Formation and Polyglutamine Aggregation Are Controlled by Two Classes of Genes

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI +] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN +]. When fused to GFP and overexpressed in [ps−] [PIN +] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI +] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI +]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI +] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI+] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.


Introduction
Prions are associated with transmissible spongiform encephalopathies, a family of neurological diseases that include Creutzfeldt-Jakob disease in humans, Scrapie in sheep, and the wellpublicized ''Mad Cow'' disease. Transmission of prions occurs when a normally folded protein is converted to an alternate conformation that has the ability to further convert additional molecules of the normal protein into the misfolded infectious form.
Similar to prion strains found in mammals (reviewed in [25]), yeast prions have also been shown to exist in different conformations called ''variants'' [14,[26][27][28][29][30]. The introduction of in vitro generated Sup35 amyloid fibers into yeast not only infects the cells with the prion, proof of the ''protein-only'' hypothesis, but also demonstrates that distinct forms of the in vitro made amyloid cause the appearance of distinct variants that are heritable [31][32].
To further understand how prions are formed and maintained, recent studies have focused on specific host factors that affect the propagation and appearance of yeast prions. Chaperones, which are normally involved in proper protein folding, play a role in prion maintenance and appearance. Hsp104 and Sis1 break prion aggregates into smaller pieces that efficiently segregate into daughter cells, a requirement for prion transmission [33][34][35][36][37][38][39][40][41]. Deletion of the N-terminal activation domain of Hsf1, a heat shock transcription factor, prevents [PSI + ] formation, while deletion of the Hsf1 C-terminal region promotes [PSI + ] appearance [42]. Furthermore, disruption of the non-essential human Hsp110 ortholog, SSE1, or overexpression of HSP82 and HSC82 that encode members of the Hsp90 family of chaperones, dramatically reduces, but does not eliminate, the induction of [PSI + ] caused by the overexpression of Sup35PD [43].
Factors that affect [PSI + ] induction are not limited to chaperones. Deletion of actin cytoskeletal genes, such as SLA1 or SLA2, reduces [PSI + ] induction [44], suggesting that the actin cytoskeleton may play a role in prion appearance. Deletion of the ubiquitin-conjugating enzyme, Ubc4, enhances the de novo appearance of [PSI + ] [45], and exposure to environmental stress can also alter the frequency of [PSI + ] appearance [46].
Here, we identify deletions of several genes (bug1D, bem1D, arf1D, hog1D, las17D, vps5D, and sac6D) that reduce the efficiency with which overexpression of Sup35PD can induce the de novo appearance of [PSI + ]. Deletion of LAS17, VPS5, or SAC6, which are associated with endocytosis and the actin cytoskeleton, not only inhibit [PSI + ] induction, but also suppress the toxicity and aggregation associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease.  [23,46]. Therefore, genes whose deletions enhance or reduce the toxicity associated with overexpression seemed likely to increase or decrease [PSI + ] induction frequency, respectively [46]. We tested 238 deletion strains that enhanced toxicity, 151 that reduced toxicity, and nine other strains studied in Tyedmers et al. [46] (Table S1; [49] were simultaneously cytoduced into the 398 deletion strains as described previously [48]. Following overexpression of Sup35PD-GFP to induce [PSI + ], cells were plated on-Ura where suppression of the plasmid borne ura3-14 nonsense allele allowed [PSI + ], but not [psi 2 ], cells to grow.

Deletions
Six novel deletion strains: bem1D, def1D, scp160D, rpp1aD, spt4D, and pre9D, as well as the expected rnq1D and hsp104D deletions, failed to grow on -Ura (Table 1). Previous work has shown that in the presence of the SUP35 R2E2 allele, which increases the appearance of [PSI + ] without SUP35 overexpression, bem1D and pre9D decreased [PSI + ] appearance [46]. In addition, 29 other deletions showed either low or extremely low induction of [PSI + ] ( Figure 1; Table 1). While strains carrying deletions of SPT4 or YML010C-B failed to express Sup35PD-GFP, all other deletion strains expressed Sup35PD-GFP at similar levels (data not shown).
Deletion strains that decrease the efficiency of [PSI + ] induction (Table 1) were from both enhanced and reduced toxicity groups. This makes sense if we consider that toxic side effects of the deletions could overlay the positive effect on growth rate due to the

Author Summary
Certain proteins that exist in functional unaggregated conformers can also form self-perpetuating infectious aggregates called prions. Here we investigate factors involved in the initial switch to the prion form. De novo appearance of the [PSI + ] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by overexpression of the SUP35 gene in the presence of the prion form of the Rnq1 protein, [PIN + ]. When tagged with green fluorescent protein and transiently overexpressed in [psi 2 ] [PIN + ] cells, Sup35 forms fluorescent rings, and cells with these rings give rise to daughter cells that are [PSI + ]. Here, we investigate factors required for this induction of [PSI + ]. Analyses of over 400 gene deletions revealed two classes that reduce [PSI + ] induction: one class forms fluorescent rings normally, and the other does not. Interestingly, the former class enhanced, while the latter class reduced, toxicity associated with the expanded polyglutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. These results suggest that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.  reduced [PSI + ] induction. Furthermore, in some deletions, even the overexpression of YFP alone (without the Sup35 prion domain) causes strong toxicity that must be, therefore, completely unrelated to prion induction frequency [46]. To eliminate the effects of secondary mutations known to have accumulated in library strains [50,51], multiple independent deletions of nineteen of the best candidates that showed no or reduced induction of [PSI + ] ( Table 1) were re-engineered in a wildtype 74-D694 [PIN + ] strain. This 74-D694 genetic background contains a [PSI + ] suppressible ade1-14 allele that provides the ability to directly score for [PSI + ] by examining growth on -Ade. Of the 19 re-engineered deletions, only the six deletions (bre1D, bug1D, bem1D, arf1D, pre9D, and hog1D) that reproducibly reduced the frequency of [PSI + ] induction, relative to the wildtype induction frequency of approximately 7.5 X 10 23 (Figure 2), were pursued further. Since a slow growth phenotype complicates the scoring for [PSI + ], deletions that significantly inhibited growth in the 74D-694 background, like lst7D and swa2D (data not shown), were eliminated from further analysis.

Deletions that show low induction of [PSI
In addition to the above deletions, we made a deletion of LAS17, which was previously shown to inhibit the aggregation of the polyglutamine 103Q repeat [52]. We also made deletions of VPS5 and SAC6, because they, like Las17 and other proteins shown to affect the appearance of [PSI + ], are associated with endocytosis and the actin cytoskeleton. Even though prion propagation is unaffected in these three deletion strains because they were all able to maintain [PSI + ] over many generations after cytoduction (data not shown), vps5D caused a significant decrease in prion induction, and las17D and sac6D strains completely failed to induce [PSI + ] ( Figure 2).
To eliminate deletions that reduced [PSI + ] induction by altering the levels of Sup35, Sup35PD-GFP, or chaperones, we examined these levels in all strains. None of the deletions caused significant changes in Hsp104, Ssa1, Sis1, Sse1, or Ssb1/2 (data not shown). Since Sup35PD-GFP levels were reduced in bre1D strains and Sup35 endogenous levels were decreased in pre9D strains ( Figure  S1), these deletions were dropped from further study.
To eliminate the possibility that the reduced [PSI + ] induction was due to the loss of [PIN + ] during the construction of any of the deletion strains, we showed that the maintenance of [PIN + ] was unaffected by the deletions. Each independently constructed deletion was crossed to a [pin -] strain carrying a plasmid with the CUP1 controlled RNQ1-GFP fusion. When grown on medium containing copper, induction of the resulting diploid containing the RNQ-GFP construct caused the appearance of punctate dots indicative of [PIN + ] (data not shown). Furthermore, we showed that [PIN + ] was maintained in deletion strains by the presence of [PIN + ] characteristic SDS-resistant oligomeric species after 24 hours of Sup35PD-GFP overexpression ( Figure S2). Differences in the migration of Rnq1 SDS-resistant oligomers have been shown to be associated with different [PIN + ] variants [53]. We observed similar migration of Rnq1 oligomeric species in the deletion strains, suggesting that the [PIN + ] prion variants were not altered during construction of the strain or by Sup35PD-GFP overexpression. These results and other properties of the deletions investigated further below are summarized in Table 2. To determine the deletions that inhibit the induction of [PSI + ] and also prevent ring formation, we compared the number of cells that contained rings after 24 hours of expressing Sup35PD-GFP in the seven deletion strains. Ring containing cells in four of the deletions, bug1D, bem1D, arf1D, and hog1D, appeared at levels similar to wildtype (30%, Figure 3A). The rnq1D strains, which are [pin 2 ], always displayed diffuse fluorescence ( Figure 3A), as expected, since [PIN + ] has been previously shown to be required for ring formation [54].
Interestingly, when measured after 24 hrs of Sup35PD-GFP induction, the three deletions associated with endocytosis and organization of the actin cytoskeleton, sac6D, las17D, and vps5D, all showed a significant reduction in cells containing rings. Since by 25 hrs of induction, wildtype, sac6D, and vps5D cultures were in stationary phase, where ring formation peaks [54], the inhibition of ring formation in sac6D and vps5D was not the result of a failure of these cultures to reach stationary phase ( Figure S3). Also, since las17D cells reached saturation phase only after 36 hours, we also measured ring formation in las17D cultures following 50 hrs of induction, well after the culture reached stationary phase. These cultures still showed a 50% reduction in ring formation relative to the wildtype cultures (data not shown).

Ring-associated toxicity
Ring formation is associated with [PSI + ] appearance [54]. However, it has been shown that cells that contain rings have a higher rate of cell death than those that have diffuse fluorescence [44,47,54]. Therefore, we asked if the deletions might be more toxic to ring bearing cells, which would result in a decrease in [PSI + ] induction. To test this, we micromanipulated individual ring containing cells from selected deletion strains and assessed whether they were viable. Similar to previous findings [47], only 36% of wildtype ring containing cells were viable, whereas 95% of wildtype cells with diffuse cytoplasmic fluorescence were viable ( Figure 3B). In general, the viability of ring containing wildtype, bem1D, and hog1D cells appeared to be similar ( Figure 3B) and therefore cannot explain the strong reduction of de novo induction of [PSI + ]. Ring containing arf1D cells had reduced viability (21%), which could account for the decrease in [PSI + ] induction ( Figure 2). In contrast, bug1D ring cells showed an increase in viability (59%),  Table S3) for a total of three transformants per deletion. The exception was pre9D which was calculated from three transformants from a single independent knock out. Only deletion strains that showed a significant difference in induction frequency compared to wildtype strains (p,0.05 unpaired t-test) were included in the figure. doi:10.1371/journal.pgen.1001386.g002    Table S3) for a total of three transformants per deletion. Bars  but apparently these cells did not efficiently give rise to [PSI + ] cells.
In the absence of rings, there appeared to be no effect on the viability of any of the deletion strains ( Figure 3B, green bars).
Since isolating ring cells in strains with low ring formation was difficult, we focused on one example and found that of the small population of cells in vps5D strains that formed rings, a majority were inviable ( Figure 3B). This suggests that rings in the absence of VPS5 may be harmful to the cell and likely explains the decreased percentage of rings observed in Figure 3A.

Deletion strains affect aggregation and toxicity of polyglutamine
We next asked if our deletion strains also affect the [PIN + ] dependent aggregation of the polyglutamine (polyQ) expanded repeat found in the mutant huntingtin protein associated with Huntington's disease. Since las17D strains, carrying a galactose inducible expanded polyQ repeat (103Q) fused to GFP, were previously shown to delay 103Q-GFP aggregate formation compared to wildtype strains [52], we tested our other deletions for similar aggregation patterns. When wildtype [PIN + ] cells expressed 103Q-GFP for approximately one to two hours, 82% of cells displayed strong fluorescent puncta, while [pin -] cells showed mostly cells with diffuse fluorescence (73%) and a minor population that contained a few faint fluorescent foci on a diffuse background (27%; Figure 4A and 4B). Similar analyses of [PIN + ] strains with bug1D, bem1D, arf1D, and hog1D deletions resulted in a reduced number of cells that contained strong fluorescent puncta ( Figure 4B, green bars) and an increased number of cells that had faint fluorescent foci with a diffuse background ( Figure 4B, purple bars). As in the previous study [52], [PIN + ] las17D strains had barely any strong fluorescent foci. Examination of [PIN + ] vps5D and [PIN + ] sac6D strains also showed a strong reduction in cells having bright foci and a similar distribution of cells with faint foci vs. no foci as seen in wildtype [pin -] strains.
We next examined the effect of these deletions on toxicity associated with expression of the expanded polyglutamine protein in the presence of the [PIN + ] prion [57]. Our controls verified earlier findings [57] that wildtype [PIN + ] cells carrying a galactose inducible 103Q-GFP plasmid show toxicity compared to cells carrying the non-toxic 25Q-GFP plasmid. As expected, polyglutamine expressing cells that are either [pin -] or rnq1D did not exhibit toxicity, whereas wildtype [PIN + ] cells were sick ( Figure 4C). Since previous studies have correlated the presence of polyQ aggregates with cell toxicity [57], the lack of strong polyQ fluorescent foci in the class of deletions that also reduced ring formation after Sup35PD-GFP overexpression (las17D, vps5D, and sac6D; Figure 4A and B) nicely explained the observed decrease in polyQ toxicity. Interestingly, the class of deletions that reduced [PSI + ] induction, but not ring formation (bug1D, bem1D, arf1D, and hog1D), increased the frequency of cells containing faint polyQ-GFP fluorescent foci on diffuse backgrounds and clearly enhanced 103Q toxicity.

Discussion
We scored 398 gene deletions, previously identified by their ability to increase or decrease toxicity caused by overexpression of Sup35PD-GFP [46], for effects on the induction of [PSI + ] and established effects for four of the deletions (see Table 2 summarizing all results). Since there is not always a strong correlation between the toxicity associated with SUP35 overexpression and the induction of [PSI + ], we tested all of the enhanced and reduced toxicity candidates found in the previous study [46] for [PSI + ] induction. We observed that induction of [PSI + ] was inhibited by deletions that either reduced (arf1D and bem1D) or enhanced (bug1D and hog1D) toxicity. Possibly the rings or other less visible aggregates have an altered property in the presence of bug1D or hog1D that inhibits detectable [PSI + ] appearance and causes toxicity. It has been shown that rings cause toxicity by titrating Sup35 and/or Sup45 (the essential release factor that binds to Sup35) into aggregates and away from the ribosome [46]. The toxicity associated with bug1D and hog1D during [PSI + ] induction could be an enhancement of this effect or could be by the formation of a different type of toxic [PSI + ] intermediate.
We also examined three deletions (las17D, vps5D, and sac6D), which were chosen for their association with endocytosis and the actin cytoskeleton, and found that they reduce [PSI + ] induction. While las17D and vps5D were not included in the library originally scored for SUP35PD-GFP toxicity, the sac6D BY4741 library strain was not associated with changes in toxicity [46]. We found that sac6D inhibits [PSI + ] induction in both the deletion library (BY4741; data not shown) and the 74-D694 (Figure 2)

Prion and polyglutamine formation and maturation
Time lapse examination of individually micromanipulated [psi 2 ] [PIN + ] cells, containing an endogenously tagged Sup35-GFP fusion and transiently overexpressing Sup35PD-GFP from a plasmid, previously showed that a fluorescent ring initially forms at the cell periphery and then internalizes around the vacuole. Later, such cells with rings give rise to [PSI + ] daughter cells with fluorescent foci ( Figure 5A) [44,55].
In this paper, we identified two classes of gene deletions that reduce the de novo induction of stable [PSI + ] but differ in effects on ring formation. Class I deletions (bug1D, bem1D, arf1D, and hog1D) form Sup35PD-GFP rings at approximately wildtype levels ( Figure 3). Class II deletions (las17D, vps5D, and sac6D) have a significantly reduced number of cells with Sup35PD-GFP rings (Figure 3).
The existence of these two deletion classes suggests that the prion formation pathway can be inhibited at different steps. In the case of class I deletions, problems in peripheral and internal ring formation were not detected (data not shown), suggesting that these genes are important for ring containing cells to transmit heritable [PSI + ] aggregates to daughter cells ( Figure 5A). In the case of class II deletions, peripheral rings form infrequently, suggesting that these genes are important in the initial formation of the ring even in the presence of [PIN + ] ( Figure 5A). While rings contained in vps5D (class II) cells have reduced viability, it is unlikely that ring formation is so toxic that cells die before the ring appears because there is no corresponding increase in toxicity in non-ring containing cells ( Figure 3B).
The formation of polyglutamine aggregates, upon overexpression of Q103:GFP in a [PIN + ] strain, is not associated with ring formation. Instead, large bright aggregates are observed within one to two hours of induction [52]. All of our deletions affected polyglutamine aggregation, but the type of effect differed based on  I deletions (bem1D, bug1D, arf1D, and  hog1D) caused a decreased level of bright fluorescent aggregates and an increase in diffuse cells with faint foci (Figure 4A and 4B).
Interestingly, these deletions enhanced the toxicity associated with 103Q-GFP ( Figure 4C). In the presence of the class II deletions (las17D, vps5, and sac6D) 103Q-GFP usually remained diffuse, and fluorescence (gray bars) were calculated from over 300 cells from at least one transformant from each independent knock out line for a total of three transformants. C. Wildtype [PIN + ], wildtype [pin 2 ], rnq1D, and the indicated [PIN + ] deletion strains were transformed with either the 103Q-GFP or 25Q-GFP plasmid and grown to late log phase in glucose (uninducing) media. Cells were serially diluted 20-fold and plated on media that either induced (right column; galactose containing media) or did not induce (left column; glucose containing media) the overexpression of the 25Q-GFP or 103Q-GFP plasmid. White arrowhead indicates the lowest dilution displaying growth. Deletion strains in (C) are in the 74D-694 background except sac6D, which is in the BY4741 [PIN + ] background as indicated. Pictures shown are representative of the multiple independent deletion lines tested for 103Q-GFP toxicity. doi:10.1371/journal.pgen.1001386.g004 very few cells had bright aggregates ( Figure 4B). Additionally, all class II deletions suppressed polyglutamine toxicity ( Figure 4C).
The formation of large huntingtin aggregates in mammals was initially thought to be the cause of huntingtin-mediated cell death (reviewed in [59]), but emerging evidence suggests that these large aggregates are neuroprotective ( [60], reviewed in [61]) and toxicity is due to a soluble pool of oligomeric conformers [62][63]. Also in yeast, small multiple foci of 103Q appear to be more toxic than a large aggregate [64,65]. Thus, we propose that class I genes are important for the formation of large protective aggregates but not small toxic oligomers ( Figure 4B), and that when a class I gene product is absent, the reduction of large protective aggregates permits the increased propagation of deadly soluble oligomers.
In contrast, in cells with class II deletions, 103Q-GFP toxic oligomers or aggregates may form only rarely. While the [PSI + ] and polyglutamine formation pathways are not directly comparable, we propose that class II genes affect the initial steps of polyglutamine formation, and the formation of large protective aggregates (promoted by class I genes) is a downstream event ( Figure 5B).

Actin and prion formation
Although endocytosis is relatively unaffected in bem1D, bug1D, arf1D, hog1D, las17D, vps5, and sac6D strains, six out of seven of our deletion mutants display fragmented vacuoles ( Figure S4) [66,67]. Possibly, vacuole fragmentation may affect the perivacuolar deposition site for aggregated proteins, called IPOD [68], which has been suggested to play a role in prion formation [55,56]. Some of the deletion library strains we screened induced [PSI + ] normally but had fragmented vacuoles (Table SI, asterisk; [66]), suggesting that intact vacuoles are not necessarily a requirement for efficient prion appearance. Vacuole fragmentation has been shown to be associated with microtubular defects [69] as well as fluctuations in the soluble actin pool [67]. We showed that treatment with the microtubule disrupting drugs Nocodazole ( Figure S4B and S4C) and Thiabendazole (data not shown) at concentrations that did not inhibit Sup35PD-GFP induction do not affect [PSI + ] appearance. Conversely, the actin disrupting drug Latrunculin A [70] and act1-R117A alleles [44] do inhibit [PSI + ] induction. Furthermore, many of the deletions identified in this study are involved with actin ( Table 2), suggesting that actin organization plays a critical role in the aggregation of prion and polyglutamine proteins.
Possibly, proper actin organization on the cell periphery is required for the initial formation of the [PSI + ] ring or polyglutamine oligomers, where as actin organization elsewhere in the cell could be required for downstream events such as the formation of a propagating [PSI + ] conformer or the formation of large protective polyglutamine aggregates. Actin could possibly be involved in facilitating the addition of monomer to newly formed aggregates. In assaying for [PSI + ], ring cells containing functional monomer not yet integrated into an aggregate would appear to be [psi 2 ].
Our data suggests that prion and polyglutamine formation involves a multi-step process that is dependent upon actin organization. Interestingly, six of the seven proteins identified here have mammalian homologues ( Table 2), suggesting that similar mechanisms may be involved in aggregation and oligomerization of QN-rich proteins in higher eukaryotes. Further elucidation of how actin nucleation contributes to prion induction will not only shed light on how toxic oligomeric species are formed, but also could provide clues to the molecular mechanisms underlying many human aggregating neurodegenerative diseases.

Cultivation procedures
Yeast strains were cultivated using standard media and growth protocols [71] and grown at 30 o C except when indicated. Complex media contained 2% dextrose (YPD), and synthetic complete media contained the required amino acids and 2% dextrose (SD) or 2% galactose (SGal).

Curing of BY4741 deletion strains
Pre-existing prions were cured by growing strains on media containing low levels of guanidine hydrochloride (GuHCl), which cures through the inactivation of Hsp104 [72,73]. Strains were spotted onto YPD plates containing 5mM GuHCl and repeated two to three additional times to ensure that prions were cured.

Preparation of the kar1 plasmid donor and introduction of plasmids and prions into deletion strains
Prions and plasmids were introduced into the kar1 plasmid donor strain. To make a [PSI + ] kar1 plasmid donor strain to test for [PSI + ] maintenance in the deletion strains, the kar1 plasmid donor strain was crossed to either a weak [PSI + ] (L1759) or strong [PSI + ] (L1763) strain containing Sup35PD-GFP. kar1 plasmiductants containing [PSI + ] were chosen as described in Manogaran et al. [48], and confirmed to contain [PSI + ] by the formation of Sup35PD-GFP aggregates after 16 hours. The [PSI + ] kar1 plasmid donor was mated to the BY4741 deletion strains, deletion strain cytoductants were confirmed by testing for auxotrophic markers, and the presence of [PSI + ] Sup35PD-GFP aggregate formation was examined as described above [54].
To test for the induction of [PSI + ], prions and plasmids were introduced into the kar1 plasmid donor strains by crossing to either a [PIN + ] (L1749 high [PIN + ]) or [pin -] strain (L2910) containing the Sup35PD-GFP and ura3-14 plasmids. [PIN + ] kar1 plasmid donor strains were confirmed as above and tested for the presence of [PIN + ] by the formation of Sup35PD-GFP fluorescent rings after 24 hours of induction on copper [54]. Cytoduction of plasmids and prions into the BY4741 library deletion strains has been described previously [48]. To ensure reproducibility, cytoductions were performed in duplicate.

Induction of [PSI + ] in cytoduced deletion strains
Cytoduced BY4741 deletion strains, containing [PIN + ] and both Sup35PD-GFP (HIS3) and ura3-14 (LEU2) plasmids, were spotted onto plasmid selective SD-His-Leu plates plus 50 mM copper sulfate and grown for approximately two days. Strains were resuspended in 300 mL of sterile water and either spotted onto SD-Leu (grown two days) to assess growth, or SD-Leu-Ura (grown at room temperature for five to seven days) to score for [PSI + ] induction. Induction experiments on all 398 yeast deletion library strains were repeated six times to ensure reproducibility. Spots that exhibited no or reduced growth on SD-Leu-Ura, compared to controls, were chosen as candidate genes that affect [PSI + ] appearance (Table 1).

Re-engineering of candidate strains
Genetic recombination was used to replace candidate genes ( Table 1) with HIS3 in [PIN + ] 74-D694. Primers (Table S2), adjacent to sequences flanking the 59 or 39 ends of the candidate gene, were used to PCR amplify the HIS3 gene. PCR products were transformed and His + transformants were confirmed for insertion of the HIS3 gene in the correct locus. Two to three independent knockout lines (Table S3) were obtained for each deletion, except for pre9D. To confirm the presence of [PIN + ], deletion strains were mated to a [pin -] tester strain containing a RNQ1-GFP plasmid. Diploids were checked for the formation of fluorescent Rnq1-GFP aggregates after induction overnight. To test whether las17D, vps5D, or sac6D maintain [PSI + ], the deletions were cytoduced with [PSI + ] and checked for growth on -Ade.

Scoring the induction frequency of [PSI + ]
Re-engineered deletion strains were transformed with the Sup35PD-GFP (LEU2) plasmid and grown in SD-Leu plus copper sulfate liquid media for 24 hours. After induction, approximately 10,000 cells were plated on SD-Ade, and a 50-fold dilution of cells was plated on SD+12. Colony counts were obtained from at least one transformant from each independent knock out (see Table S3) on SD+12 vs. SD-Ade. Colony counts from at least three transformants were used to determine the induction frequency.

Sup35PD-GFP ring formation in re-engineered deletion strains
After 24 hours of induction, cells were examined for the formation of GFP fluorescent rings [54] using a Zeiss Axioskop2 deconvolution workstation equipped with either a X40 Plan-Neofluar or X100 Plan-Apochromat objective lens (Zeiss). Approximately 300 cells were counted from at least one transformant from each independent knock out, for a total of three transformants.

Determining cell viability of ring-containing cells
Ring containing cells were simultaneously visualized and micromanipulated onto 2% sterile Noble Agar slabs. Slabs were transferred onto YPD media and grown for one to two days.
Aggregation and toxicity of polyglutamine in re-engineered deletion strains Strains containing the galactose inducible 25Q or 103Q GFP fusion plasmids were grown overnight in SD-Ura and then washed in water approximately four times to remove residual glucose. To score for aggregation, washed cells were grown in liquid Gal-Ura for one to two hours with shaking and then examined for GFP aggregates. To score for toxicity, log phase uninduced washed cells were serially diluted 20-fold and spotted onto SD-Ura or SGal-Ura. Figure S1 Certain deletion strains have decreased levels of Sup35 and Sup35PD-GFP. [PIN + ] wildtype or deletion strains containing Sup35PD-GFP were grown for 22 hours to an approximate OD 600 3.0 at 30uC in the presence of 50 mM copper. All strains are in the 74D-694 background except sac6D, which is in the BY4741 background. Equivalent amounts of cell lysates were loaded per lane and subjected to SDS-PAGE (see Text S1). Lanes in the left panel were run on the same gel. Blotted proteins were incubated either with anti-GFP antibody, anti-Sup35 C-terminal antibody, or anti-PGK antibody (as indicated). Found at: doi:10.1371/journal.pgen.1001386.s001 (0.24 MB TIF) Figure S2 The [PIN + ] variant in the deletion strains appears to be unaffected. [PIN + ] wildtype and deletion strains, containing Sup35PD-GFP, were grown for 24 hours in copper media, and equivalent amounts of protein were loaded on a 1.5% agarose gel and subjected to SDD-AGE [53]. Blotted proteins were incubated with an antibody against the Rnq1 protein.