Suicidal [PSI+] is a lethal

Ryan P. McGlinchey, Dmitry Kryndushkin, and Reed B. Wickner1

Laboratory of Biochemistry and Genetics, National Institute of Diabetes Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830

Contributed by Reed B. Wickner, February 17, 2011 (sent for review January 28, 2011) [PSI+] is a prion of the essential termination factor codons in ade1-14 or ade2-1 is then sufficiently frequent that the Sup35p. Although mammalian prion infections are uniformly fatal, cells are Ade+ (grow without adenine and are white) instead of − commonly studied [PSI+] variants do not impair growth, leading to Ade (and red). A [PSI+] variant that efficiently inactivated all suggestions that [PSI+] may protect against stress conditions. We Sup35p (“killer [PSI+]”) would be lethal. As a permissive condi- report here that over half of [PSI+] variants are sick or lethal. These tion for killer [PSI+], we express a full-length chromosomal “killer [PSI+]s” are compatible with cell growth only when also SUP35 gene, and, on a plasmid, SUP35C encoding only the es- expressing minimal Sup35C, lacking the N-terminal prion domain. sential part but lacking the NM prion domain. This truncated The severe detriment of killer [PSI+] results in rapid selection of Sup35C cannot be converted to the [PSI+] prion form because it nonkiller [PSI+] variants or loss of the prion. We also report variants lacks the N domain that is essential for prion formation (18) and of [URE3], a prion of the nitrogen regulation Ure2p, that forms the core of the in-register parallel β-sheet structure grow much slower than ure2Δ cells. Our findings give a more re- that constitutes the prion form (19, 20). To detect killer [PSI+], alistic picture of the impact of the prion change than does focus on the plasmid-borne SUP35C must be expressed only enough to “mild” prion variants. keep cells alive but not enough for efficient termination, which − would make them Ade (Fig. S1). With a tetracycline-repressible rep lthough mammalian cause a devastating and uniformly promoter (Ptet ) (21), 10 μg/mL doxycycline sufficiently re- Afatal spongiform encephalopathy (reviewed in ref. 1), yeast pressed the plasmid-encoded Sup35C expression that cells lack- + and fungal prions, as described so far, are compatible with in- ing a chromosomal SUP35 were Ade (white) but could grow definite growth, often at normal or near-normal rates. This ap- slowly (Fig. S2). Doxycycline at these levels does not detectably rep parent contrast has led to proposals that the yeast and fungal slow growth of normal cells. The tet -SUP35C gene was on prions may benefit their hosts, even though prion formation may a URA3 CEN (centromere-containing) vector whose loss could be decrease the activity of the protein as a result of the aggregate/ selected by growth on plates with 5-fluorouracil (FOA; kills URA3 amyloid formation that is the prion change. The [Het-s] prion of cells but not ura3 mutants) (22). Podospora anserina is necessary for proper heterokaryon incom- Using strains 74-D694 or GT159 carrying PGAL1-SUP35NM rep + patibility of het-s with het-S strains (2), so it was proposed that this and URA3 Ptet -SUP35C plasmids, we induced [PSI ] forma- is a prion benefiting the host (3). However, [Het-s] is also part of tion by overexpression of Sup35NM on galactose media and + μ a meiotic drive system, promoting inheritance of het-s by cheating selected Ade clones on plates with 10 g/mL doxycycline. GENETICS on meiosis (4), so the beneficial prion suggestion may be wrong. Artificially overproducing Sup35NM (the prion domain) can [PSI+] is an amyloid prion of the Sup35p produce lethality because all Sup35p is drawn into the filaments (5–7), and was suggested to protect against stress (8) or to promote (23); thus, we repressed expression of Sup35NM by over 30 evolution by protecting against stress (9). All these tests of stress generations of growth on glucose to dilute out the Sup35NM resistance were done using the usual [PSI+] variants, which were before looking for colonies that grew poorly or not at all on FOA selected to be compatible with vigorous cell growth (10). medium (loss of Sup35C). Sup35p is an essential subunit, with Sup45p, of the translation We found that of 313 Ade+ guanidine-curable clones from termination factor. Like the mammalian prion “strains” (11, 12), 74-D694, 144 showed slow growth on FOA (“sick [PSI+]”) and [PSI+] has “variants,” several different stably propagated biolo- 24 did not grow at all (killer [PSI+]) (Fig. 1). Similar proportions gically and structurally different forms of the same prion protein of sick and killer [PSI+] were isolated from strain GT159 (Fig. (13, 14). In the usual [PSI+] variants, much of the Sup35p is tied S3). Cells with killer [PSI+] grew very poorly after several days up in amyloid filaments and is unavailable for translation ter- on ½YPD containing 10 μg/mL doxycycline, confirming that mination, but enough is free to keep the cells alive. The shortage failure to grow on FOA is attributable to lack of Sup35C (Fig. of Sup35p increases the misreading of nonsense codons, which 2A) and not, for example, a variant that never loses the URA3 provides the basis for the genetic assay of [PSI+]. plasmid. Most of the colonies that did grow were pink/red and Ure2p is a regulator of nitrogen catabolism that can convert to proved to have lost [PSI+]. On ½YPD lacking doxycycline, killer an amyloid prion called [URE3] (5, 15, 16). Variants of [URE3] [PSI+] cells were red and grew well because of an abundant have also been described (16, 17). supply of Sup35C (Fig. 2B). Here, we show that more than half of [PSI+] variants are lethal The instability of sick and killer [PSI+] is shown in Fig. 3. or highly pathogenic, unlike the variants usually studied. Simi- [PSI+] isolates were streaked on both FOA (Fig. 3A, Left) and larly, we describe abundant [URE3] variants that slow cell ½YPD without doxycycline (Fig. 3A, Center). After several days growth dramatically in a background in which deletion of the on ½YPD, sick and killer cells grew more normally when URE2 gene has no effect on growth. These results show that streaked to FOA (Fig. 3A, Right), showing that toxic [PSI+] acquisition of a yeast prion may be disastrous for yeast. variants are not stable, even in the presence of excess Sup35C. This suggests that toxicity is not limited to undersupply of active Results Sup35p has two domains, the N-terminal (NM) domain, which is necessary and sufficient for prion formation (and has physiolog- Author contributions: R.P.M., D.K., and R.B.W. designed research, performed research, ical function as well, as discussed below) but is dispensable for contributed new reagents/analytic tools, and wrote the paper. growth, and the C-terminal C domain, which is essential for The authors declare no conflict of interest. translation termination, and can perform this function without 1To whom correspondence should be addressed. E-mail: [email protected]. + fi the NM domain. In [PSI ] cells, most of the Sup35p is in la- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ments, and is inactive. Readthrough of premature termination 1073/pnas.1102762108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1102762108 PNAS | March 29, 2011 | vol. 108 | no. 13 | 5337–5341 Downloaded by guest on September 24, 2021 A Single colony

5-FOA ½ YPD 5-FOA from ½ YPD

Non-toxic PSI+

Sick PSI+

Sick PSI+

Killer PSI+

Fig. 1. Screening of Ade+ colonies for killer [PSI+]. Isolated Ade+ colonies Killer PSI+ were restreaked three times on −Ade −Ura + 10 μg/mL doxycycline before being stamped on FOA. Circled colonies have killer [PSI+]. The fractions of PSI+ ½ YPD from 5-FOA isolates with mild, sick, and killer [ ] are shown below. B C D Non-toxic Sick PSI+ Sick PSI+ PSI+ Sup35p and/or that the toxic [PSI+] variants are inherently un- stable. Streaking sick [PSI+] isolates on ½YPD after selection on − FOA showed a mixture of small white (Ade+ Ura ) and large − − pink/red (Ade Ura ) colonies (Fig. 3B, Upper). These small, white, poorly growing colonies, on restreaking on ½YPD, formed a mixture of small and larger white Ade+ colonies and pink/red − Ade colonies, with the latter having lost [PSI+] (Fig. 3B, Lower). Thus, selection for prion loss or alteration attributable to prion toxicity, and likely inherent prion instability, makes isola- + tion of stable killer [PSI ] apparently impossible. ½YPD ½YPD ½YPD Growth of sick [PSI+] isolates with 3 mM guanidine (which PSI+ A PSI+ cures [PSI+]) (24) cured the prion, as shown by cells becoming Fig. 3. Toxicity and instability of [ ] isolates. ( ) Sick and killer [ ] − − − μ candidates were streaked on ½YPD and on FOA plates. After 2 d of growth Ade on the Ade Ura + 10 g/mL doxycycline plates. Once B–D PSI+ + on ½YPD, strains were restreaked on FOA plates. ( ) Individual sick [ ] cured of killer [PSI ], these cells could grow well on FOA plates and nontoxic [PSI+] colonies were streaked on ½YPD from FOA. (Lower) or on ½YPD with doxycycline, similar to those shown in Fig. 3A. Single colonies restreaked on ½YPD are indicated. This shows that the toxicity is indeed attributable to a variant of [PSI+]. Transformation of presumed [PSI+] cells with a plasmid expressing Sup35NM-GFP confirmed the presence of prion color of yeast colonies (16). Strain BY241 was transformed with aggregates (Fig. 4). An apparent correlation between [PSI+] the LEU2 centromeric plasmid pVTG12 expressing Ure2N-GFP toxicity and the number of Sup35NM-GFP aggregates was noted (a fusion of the Ure2p prion domain with GFP) under control (Fig. 4), but the instability of the killer [PSI+] makes this con- of the native URE2 promoter. Low-level expression of Ure2N- clusion tentative. GFP does not induce or cure [URE3] (25) but does allow dis- Cytoduction (cytoplasmic transfer) of killer [PSI+] into tinguishing true prion isolates from chromosomal mutants by a strain not expressing Sup35C is expected to be lethal. In fact, the colocalization of Ure2N-GFP with Ure2p prion aggregates, “nontoxic [PSI+]” and sick [PSI+] produced [PSI+] cytoductants, forming fluorescent foci in [URE3] cells. Strain BY241/pVTG12 whereas their guanidine-cured derivatives produced mostly [psi-] spread on −Ade −Leu plates was grown for 5 d at 30 °C. Colo- + cytoductants (Table S1). The killer [PSI ] donors were clearly nies were analyzed for the presence of [URE3] prion by fluo- unstable, with the prion being lost or changing to a mild form rescent microscopy and by curing on medium with 5 mM GuHCl. (Table S1). Confirmed [URE3] isolates were then streaked on ½YPD for + In contrast to [PSI ], toxicity associated with the [URE3] growth comparison, together with the same strain before prion prion (of Ure2p) can be observed directly on isolation of spon- induction (Fig. 5). Surprisingly, most spontaneous [URE3] var- + taneous prion clones. Strain BY241 [ure-o] [PIN ] carries ADE2 iants were strikingly smaller compared with the parent strain, under control of the DAL5 promoter to monitor activity of with colonies that lost [URE3], or with the parent strain with its Ure2p, thus allowing detection of the prion state based on the URE2 gene deleted (Fig. 5). This shows that the markedly slow growth of these [URE3] variants is not a result of functional inactivation of Ure2p but, rather, was attributable to some toxic A B action of the prion form. These slow-growing prion variants were + Doxycycline - Doxycycline all unstable, producing subclones that had lost the prion as well as less toxic variants (Fig. 5 G and H), perhaps as a result of selection for loss of toxicity. Non-toxic Killer Non-toxic Killer [psi-] [PSI+] [PSI+] [psi-] [PSI+] [PSI+] Discussion We constructed a screen to detect lethal variants of [PSI+] specifically avoiding the overexpression of Sup35p. [PSI+]is Fig. 2. Killer [PSI+] strains are sick because of Sup35p deficiency. Cells car- known to be lethal in cells overexpressing full-length Sup35p or − + rying killer [PSI+], a nontoxic [PSI+], and a [psi ] control were streaked on Sup35NM (the prion domain) (13, 26). In [PSI ] cells over- ½YPD with (A) or without (B) doxycycline (10 μg/mL). expressing full-length Sup35p, most of the other translation

5338 | www.pnas.org/cgi/doi/10.1073/pnas.1102762108 McGlinchey et al. Downloaded by guest on September 24, 2021 specifically designed to avoid these artificial situations to de- termine if there were lethal variants of [PSI+] at normal Sup35p expression levels. Full-length Sup35p was expressed from the with the normal SUP35 promoter and context. Sup35NM was overproduced from a GAL1 promoter to induce the appearance of [PSI+] at high frequency, but cells grew on dextrose, repressing the GAL1 promoter, for over 30 generations before they were tested for requiring the Sup35C expressed from the tet promoter. Thus, little or no Sup35NM was expressed, and full-length Sup35p was just normally expressed at the point that lethality was observed. Our screen for killer [PSI+] was specifically designed to detect one kind of toxicity, namely, that attributable to sequestration of nearly all the Sup35p in the amyloid filaments so that translation termination is unduly impaired. The fact that expression of Sup35C allows the killer [PSI+] cells to grow indicates that limi- tation of translation termination is at least part of the toxicity. It is possible, however, that there exist other [PSI+] variants with toxicity on another basis, and therefore not detected in our screen. Part of the killer [PSI+] toxicity may be attributable to adsorption of another protein by the Sup35p amyloid. Mild [PSI+] amyloid is known to adsorb Hsp70s and other chaperones (27). Our finding that more than half of [PSI+] variants are sick or lethal highlights the danger to the cell of acquisition of a prion. Although [PSI+] has been reported to be advantageous under certain growth conditions (8, 9), reproducing these results has been problematic (28). Moreover, the absence of [PSI+] in wild strains (29–31) shows that it is not advantageous overall. Because the Sup35 prion domain in yeast and the corresponding N-terminal part of human Sup35p are important for normal mRNA degra- dation (32, 33) and another function (34), it is likely conserved for these purposes and prion formation in yeast may be viewed as a rare molecular degenerative disease. Moreover, yeast cells ap- parently view [PSI+] and [URE3] as stress conditions, because

they induce heat-shock when infected (35, 36). GENETICS Why is [PSI+] usually nontoxic in laboratory strains? First, the most toxic variants would not be recovered without a special selection scheme. Second, the toxicity of these killer variants (and their possible inherent instability) results in frequent se- lection of more benign variants and cells that have lost the prion. We find that [URE3] can dramatically slow cell growth in a strain whose growth is not slowed by deletion of the URE2 gene. [URE3] has also been suggested to be an advantage to yeast (37).

+ [URE3] is also not found in wild strains (31), however, and the Fig. 4. Multiple Sup35NM-GFP foci for killer [PSI ] and single foci for benign + Ure2p prion domain is important for Ure2p function in stabilizing [PSI ]. (A) Newly induced 74-D694 [PSI+] colonies that could not grow on FOA + plates (killer [PSI ]) were transformed with the pSupNM-GFP plasmid directly the protein against degradation in vivo (38). Taken together with from −Ade −Ura + doxycycline plates. Transformants were selected only for our work, these results show that [URE3] is also a rare molecular the presence of pSupNM-GFP and were highly heterogeneous by color. degenerative disease. White and sectoring transformants appeared as a result of the loss of the There may be prions that are beneficial to yeast or other Sup35C plasmid, and their [PSI+] has become benign. Red transformants still organisms, as we first suggested for [Het-s] (3). There may in- carry the Sup35C-expressing plasmid and contained [PSI+] (probably killer deed be variants of [PSI+] or [URE3] that aid yeast in some way. [PSI+]), as was shown by fluorescent microscopy analysis. (B and C) Red + + Such variants should be found in the wild under the condition in transformants from A of two independent killer [PSI ] variants contain [PSI ] fi fi D which they are a bene t (39). Our ndings also raise the ques- and show cells with multiple Sup35NM-GFP foci. ( ) Typical white trans- tion of whether there are benign variants of the mammalian formants from A and benign 74-D694 [PSI+] isolates show cells with mostly single Sup35NM-GFP foci. Six benign [PSI+] and four killer [PSI+] isolates were prion disease. transformed as described in A. Transformants of both [PSI+] types that tend to retain the Sup35C plasmid were compared, and about 2,000 individual Methods + cells containing [PSI ] were analyzed for the presence of multiple Sup35NM- Strains and Media. Strains 74-D694 (MATa ade1-14 ura3-52 leu2-2 his3-200 − + GFP foci per cell by fluorescent microscopy analysis. About 50% of putative trp1-289 [psi ][PIN ]) and GT159 (MATa ade1-14 his3-Δ200 leu2-3,112 lys2 − + killer [PSI+] cells had multiple foci, compared with only about 10% of benign trp1−Δ ura2-52 [psi ][PIN ]), carrying pH952 (CEN TRP1 PGAL1-SUP35NM) rep + [PSI+] cells under these conditions. and pRPM02 (CEN URA3 Ptet -SUP35C), were used for selecting killer [PSI ] and sick [PSI+] variants. Strain 4755 (MATα lys2 leu2 ura3 kar1 ade2-1 SUQ5 ρo [psi−]) was used as a cytoduction recipient. BY241 (MATa leu2 trp1 ura3 termination factor subunit, Sup45p, is drawn into the aggregates PDAL5ADE2 PDAL5CAN1 kar1) was used for studies of [URE3]. because it interacts with the nonamyloid part of Sup35p, the C- Synthetic dextrose (SD) medium contains 6.7 g/L Yeast Nitrogen Base without amino acids (Difco), 20 g/L dextrose, and 20 g/L agar. Adenine- terminal domain. If Sup35NM is overexpressed, the normal limiting medium (½YPD) has 0.5 g/L yeast extract, 20 g/L peptone, 20 g/L (lower) amount of full-length Sup35p is entirely drawn into the dextrose and 20 g/L agar. Plates for selection were SD plates to which were amyloid filaments and the cells suffer or die (23). Our screen was added the strain’s other required components (e.g., amino acids). FOA plates

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BD FH

Fig. 5. Spontaneously formed [URE3] clones with dramatically slowed growth and mitotic instability. BY241 (A), BY241 Δure2::TRP1 (B), BY241 individual spontaneously formed [URE3] colonies (C–F), and second streaks of small BY241 [URE3] colonies (G and H) from E and F. All strains were grown on ½YPD for 3 d before the pictures were taken.

were prepared as described (40). Prion curing was carried out by streaking to restreaked on the same media and left to grow for 4 d. Colonies were single colonies on rich plates containing 3 mM guanidine HCl. streaked or replica-plated to FOA and incubated for 3–4d.

Construction of Plasmids. Sup35C was amplified using oligos forward (5′ GG- Aggregation of Sup35NM and Ure2N Fusion Proteins. Several clones containing GGGATCCAACAATGTTTGGTGGTAAAGATCACG 3′) and reverse (5′ GGGGT- killer [PSI+]or“benign [PSI+]” were transformed with centromeric pSup35NM- TTAAACTTTACTCGGCAATTTTAACAATTTTAC 3′), cut with BamHI and PmeI, GFP directly from selection plates (−Ade −Ura + doxycycline). The trans- and inserted into pCM189 (21) cut with the same enzymes. This CEN URA3 formants on the selection plate (−Leu with limiting adenine) were very het- rep plasmid (pRPM02) has the structure: tet promoter-BamHI-ATG-SUP35C- erogeneous by color as a result of partial loss of the Sup35C plasmid. White ter-PmeI. The CEN TRP2 plasmid pH952 contains SUP35NM under the control and red transformants were compared by fluorescent microscopy analysis. of a GAL promoter (gift from Herman Edskes, National Institutes of Health, Bethesda, MD). For detection of prion aggregates in vivo, centromeric Cytoduction. Transfer of [PSI+] by cytoplasmic mixing was carried out as plasmids pVTG12 expressing Ure2N-GFP fusion under control of the native previously described (41). Donor and recipient cells were mixed and grown URE2 promoter (25) and pH126 Sup35NM-GFP (31) were used. on yeast extract-peptone-adenine-dextrose agar at 30 °C for 7 h. The in- cubated mixture was then streaked on media selecting against the donor. Induction of [PSI+] Formation. Strain 74-D694 or strain GT159 was transformed − − with pRPM02 and pH952 and selected on Ura Trp media. Induction was ACKNOWLEDGMENTS. We thank Herman Edskes for pH952, our colleagues fi performed in liquid media containing 2% (wt/vol) galactose and 1% raf nose for critical reading of the manuscript, and Frank Shewmaker for fruitful and grown for 2–3 d. Cells (∼106) were plated on synthetic complete −Ade discussions. This work was supported by the Intramural Program of the −Ura + 10 μg/mL doxycycline. Ade+ colonies appearing after 5 d were National Institute of Diabetes Digestive and Kidney Diseases.

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