Spontaneous Variants of the [RNQ+] Prion in Yeast Demonstrate the Extensive Conformational Diversity Possible with Prion Proteins

Prion strains (or variants) are structurally distinct amyloid conformations arising from a single polypeptide sequence. The existence of prion strains has been well documented in mammalian prion diseases. In many cases, prion strains manifest as variation in disease progression and pathology, and in some cases, these prion strains also show distinct biochemical properties. Yet, the underlying basis of prion propagation and the extent of conformational possibilities available to amyloidogenic proteins remain largely undefined. Prion proteins in yeast that are also capable of maintaining multiple self-propagating structures have provided much insight into prion biology. Here, we explore the vast structural diversity of the yeast prion [RNQ+] in Saccharomyces cerevisiae. We screened for the formation of [RNQ+] in vivo, allowing us to calculate the rate of spontaneous formation as ~2.96x10-6, and successfully isolate several different [RNQ+] variants. Through a comprehensive set of biochemical and biological analyses, we show that these prion variants are indeed novel. No individual property or set of properties, including aggregate stability and size, was sufficient to explain the physical basis and range of prion variants and their resulting cellular phenotypes. Furthermore, all of the [RNQ+] variants that we isolated were able to facilitate the de novo formation of the yeast prion [PSI+], an epigenetic determinant of translation termination. This supports the hypothesis that [RNQ+] acts as a functional amyloid in regulating the formation of [PSI+] to produce phenotypic diversity within a yeast population and promote adaptation. Collectively, this work shows the broad spectrum of available amyloid conformations, and thereby expands the foundation for studying the complex factors that interact to regulate the propagation of distinct aggregate structures.


Introduction
Protein misfolding disorders refer broadly to a class of human diseases associated with the failure of a protein or peptide to adopt its native, functional conformation [1]. Such misfolding can lead to the formation of fibrillar aggregates called amyloid. Amyloid fibers typically form as a β sheet-rich structure in a self-replicating process [2]. These highly ordered arrangements of β sheets are formed from non-covalent interactions of neighboring polypeptides in which the β strands run perpendicular to the fibril axis [1,3]. This fundamental architecture is shared among a variety of proteins associated with unrelated protein conformational disorders, including Alzheimer's disease and Type II diabetes [4]. Interestingly, significant conformational variation can exist while still maintaining this generic amyloid structure [5]. Such amyloid polymorphism has been most studied in the context of prion strains, but recent data suggest that it is a common feature of many amyloidogenic proteins [6,7,8].
Prion diseases, also called transmissible spongiform encephalopathies (TSEs), represent a subset of protein misfolding disorders that are invariably fatal [9]. These diseases include bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. TSEs develop when the host-encoded prion protein, PrP C , assumes the abnormal β sheet-rich PrP Sc conformation [10]. This infectious structure self-propagates by sequestering native PrP C and templating further conversion to PrP Sc [11,12].
Initial transmission experiments with PrP Sc encountered what is now known as the "species barrier" [13]. This refers to the observation that transmission of PrP Sc between two different species is typically far less efficient than transmission within the same species [14]. This barrier may be partially due to changes in amino acid sequence, but can also be due to changes in the self-propagating structure of the protein itself. Indeed, even within a single species, pathological variation in TSEs and different biochemical signatures of PrP Sc have been observed, leading to the isolation of distinct PrP Sc types [15,16]. These different types of PrP Sc are called prion strains, and represent amyloid conformations of PrP that are structurally unique. In many cases, different prion strains show differences in biochemical properties, such as protease resistance or denaturant sensitivity, which correlate with variation in pathology and the time course of disease [17,18,19,20,21,22]. However, in other cases, prion strains have been isolated that vary in pathology, yet remain biochemically indistinguishable, according to the levels of sensitivity available with current assays [23]. Moreover, while genetic polymorphisms in PrP bias the formation of particular conformations of PrP Sc , a single primary sequence can propagate a multitude of distinct prion strains [14]. Indeed, it has been estimated that the range of heterogeneity seen in samples from patients with sporadic CJD represents over 30 distinct prion strains [24,25]. Clearly, the structural limits of amyloid polymorphism of prion strains are quite large.
Interestingly, functionally distinct prion proteins exist in fungi such as the yeast Saccharomyces cerevisiae [26,27]. Yeast prion proteins share many of the same misfolding and aggregation characteristics as the proteins associated with human protein conformational disorders. As such, yeast has provided a tractable model system to investigate many facets of protein aggregation and prion biology, including that of prion strain diversity. As in mammals, prion strains in yeast (termed prion variants) are conformationally distinct, self-propagating amyloid structures. This formation of amyloid in yeast leads to changes in cellular phenotypes, which typically resemble a loss-of-function phenotype of the prion protein [28,29,30,31]. One of the most well-studied prion proteins in S. cerevisiae is the translation termination factor Sup35. Sup35 is the eRF3 that normally exists in a complex that functions to recognize stop codons in mRNA and facilitate the release of polypeptide chains from ribosomes [32,33]. Conversion of Sup35 into its prion form, [PSI+], establishes a loss-of-function phenotype that is dominant and inherited in a non-Mendelian fashion [31]. In [PSI+] cells, much of the Sup35 is sequestered into prion aggregates, thereby impairing translation termination and causing readthrough of stop codons (also known as nonsense suppression) [34,35].
[PSI+] variants have been broadly classified into categories based on the degree of nonsense suppression [36]. Two wellcharacterized variants are strong [PSI+] and weak [PSI+]. Cells propagating the strong [PSI+] variant exhibit a greater amount of nonsense suppression as compared to cells propagating the weak [PSI+] variant [36]. Studies of strong and weak [PSI+] led to a model that proposed an explanation for how differences in the biochemical properties of these [PSI+] variants correlate with differences in biological phenotypes [5,37,38,39,40,41]. This model posits that decreased fiber stability results in increased fragmentation, thereby giving rise to a greater number of prion seeds, and thus more fibril "free ends" that can recruit and sequester natively-folded Sup35 [39]. Ultimately, the more "free ends" available are hypothesized to correlate to an increased rate of fiber growth that, in the case of the [PSI+] prion, modulates the strength of the nonsense suppression phenotype as the efficiency of translation termination is linked to the size of the soluble, active pool of Sup35 [41]. Interestingly, these trends have been recapitulated with some PrP Sc strains, as lower aggregate stability correlated with a shorter incubation period and earlier onset of disease [42]. However, this correlation between aggregate stability and fiber growth does not explain differences in all PrP Sc strains [21], or even in prion variants of another yeast prion, [RNQ+] [43]. Indeed, even with the [PSI+] prion, there may be multiple ways to acquire phenotypically similar prion variants [39,44,45]. Such differences highlight the remarkable conformational diversity of amyloid and the fact that there may be several ways to generate amyloid variant structures from a single polypeptide sequence.
The  [52]. Recently, variants of α-synuclein, the protein that misfolds and aggregates in Parkinson's disease, have been reported to differentially influence the formation of tau inclusions [53]. However, what properties allow some amyloid structures to promote heterologous cross-seeding more efficiently than others remains unanswered.
Additional variants we obtained and analyzed in this study are distinct from those originally isolated [51]. These findings demonstrate the tremendous amount of conformational diversity that can be generated from a single amyloidogenic protein. By expanding the number of existing [RNQ+] variants, we are poised to better understand what factors dictate the ability of a given prion variant to form, propagate, and cause a particular phenotype. This will provide insight into the structural basis of prion strains and may help elucidate the mechanisms underlying pathological variation in protein misfolding diseases and the species transmission barrier of prion diseases. pEMBL-SUP35 was created through a similar process as a previously described pEMBL-SUP2 plasmid [58]. The SUP35 promoter was first amplified using oligonucleotides 5'-CGCCTCGAGGACGACGCGTCACAGTG and 5'-CCCGGATCCTGTTGCTAGTGGGCAGATATAG, digested with XhoI/BamHI, and ligated into pEMBL-yex4 (2μ, URA3). The SUP35 open reading frame and terminator were then amplified using oligonucleotides 5'-CGCGGATCCACTAGTATGTCGGATTCAAACCAAGG and 5'-GGGGAGCTCGTGATTGAAGGAGTTGAAACCTTGC, digested with BamHI/XbaI, and ligated, thereby disrupting the GAL-CYC1 promoter of pEMBL-yex4.

Prion Color Assay
The yeast strain 74-D694 harbors the ade1-14 allele having a premature nonsense mutation that can be used to easily monitor the [PSI+] status of cells [62]. Soluble Sup35 in [psi-] cells functions to faithfully terminate translation at the premature stop codon. As such, these cells are unable to complete the adenine biosynthetic pathway, cannot grow on medium lacking adenine, and appear red when grown on a rich medium, such as YEPD, due to the accumulation of a metabolic intermediate in the pathway. Conversely, the aggregation of Sup35 in [PSI+] cells results in readthrough of the nonsense mutation in ade1-14, thereby allowing cells to grow on medium lacking adenine. The extent of nonsense suppression can vary depending on the prion variant that propagates and the associated degree of Sup35 sequestration. When Sup35 is efficiently sequestered in strong [PSI+] cells, colonies are white and grow robustly on SD-ade. In contrast, weak [PSI+] cells do not sequester Sup35 as efficiently and are pink in color on YEPD and grow less well on SD-ade. As described previously, RRP can functionally replace Sup35. Analogous to Sup35, RRP co-aggregates with Rnq1 in [RNQ+] cells, but remains soluble and is functional in translation termination in [rnq-] cells [57]. The RRP construct in 74-D694 allows [RNQ+] variants to be distinguished phenotypically in the same manner as [PSI+] variants. Before evaluating color phenotypes, YEPD plates were moved from 30°C to 4°C for at least one day to allow the colony color to develop.

Spontaneous [RNQ+] Formation
The spontaneous formation of [RNQ+] was quantified using a [rnq-] strain expressing RRP and following previously described methods with some modifications [57,63]. This strain was transformed with pRS415-ura3-197, a kind gift from Dr. Yoshikazu Nakamura, that carries an allele of URA3 with a nonsense mutation (UGA) at W197, which is suppressed in [PSI+] and [RNQ+] RRP cells [64]. Eight single transformants were independently grown in SD-leu to an OD 600 of ~1.6. 150μl of each culture was plated onto SD-ade-ura medium to select for colonies that could suppress both the ade1-14 and ura3-197 alleles. These plates were incubated at 30°C overnight, moved to 4°C for two weeks (as cold has been shown to enhance de novo prion formation [46]), and then incubated at 30°C for another two weeks. All colonies that had acquired the white or pink phenotype indicative of ade1-14 and ura3-197 suppression were counted and spotted onto YEPD, YEPD containing 3mM GdnHCl, and SD-ade. Colonies were scored as true [RNQ+] if growth on YEPD+3mM GdnHCl resulted in a permanent color change from white or pink to red, thereby demonstrating curability of the [RRP+] phenotype.
The total number of cells plated on the SD-ade-ura medium for each independent culture was determined by plating 200μl of a 1:10,000 dilution of the overnight culture onto SD-leu, which selected for the presence of pRS415-ura3-197. The total number of cells plated on SD-ade-ura for each culture was calculated by multiplying the number of colonies counted on SD-leu by a dilution factor of 52,500, calculated as follows: (# of colonies on SD-leu/200μl) * 10,000 * 150μl * 7 SD-ade-ura plates.

Mitotic Stability of [RNQ+]
The mitotic stability of [RNQ+] was determined as previously described [43]. Independent colonies of each [RNQ+] variant expressing RRP were grown overnight in YEPD to an OD 600 of 2.5. 250μl of a 1:10,000 dilution of each culture was plated onto 13cm diameter YEPD plates. Mitotic loss of the [RNQ+] prion was scored as any red colony or any colony exhibiting red sectoring. Over 1,500 colonies were counted for each [RNQ+] variant. The percentage of mitotic loss was calculated by dividing the number of red and red sectoring cells by the total number of cells.
[RNQ+] variants were characterized as mitotically stable if the percentage of prion loss was less than 0.5%. Additionally, cells were grown in YEPD medium overnight and normalized by OD 600 , followed by spotting 5-fold serial dilutions of cells onto ¼YEPD, YEPD+3mM GdnHCl, and SD-ade plates. Plates were incubated at 30°C for 3 days for the rich media and 7 days for SD-ade.

Thermal Stability Assay
To analyze the stability of Rnq1 aggregates, cells were washed and lysed with 425-600μm acid-washed glass beads (Sigma-Aldrich) by vortexing in buffer containing 100mM Tris-HCl pH 7.5, 200mM NaCl, 1mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol, 0.5mM dithiothreitol (DTT), 50mM Nethylmaleimide (NEM), 3mM phenylmethylsulfonyl fluoride (PMSF), and Roche complete mini protease inhibitor cocktail for 3 min at high speed at 4°C twice with a 5 min incubation on ice in between. After adding an equal volume of RIPA buffer (50mM Tris-HCl pH 7.0, 200mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) following lysis, cell debris was removed by centrifugation at 3,300g for 15 seconds. Precleared lysates were incubated in sample buffer (50mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 100mM DTT) and treated for 5 minutes across a temperature gradient ranging between 45-95°C, and also at 25°C and 100°C. Samples were analyzed by SDS-PAGE. When subjected to higher temperatures, Rnq1 aggregates are solubilized and able to enter the SDS-PAGE gel. Protein separated by SDS-PAGE was then transferred to PVDF membrane for western blotting with an anti-Rnq1 antibody. ImageJ was used to quantify the resulting bands. All readings were normalized to the 100°C band, and the results were plotted using Origin 8.1 software.

Semi-Denaturing Detergent Agarose Gel Electrophoresis
SDD-AGE was performed as previously described with slight modifications [57]. Yeast cells were lysed by vortexing with glass beads as described above in buffer containing 25mM Tris-HCl pH 7.5, 100mM NaCl, 1mM EDTA, Roche complete mini protease inhibitor, 0.5mM DTT, 3mM PMSF, 5μg/mL pepstatin, and 40mM NEM. Cell debris was cleared by centrifugation at 3,300g for 30 sec. Lysates were incubated with sample buffer (60mM Tris-HCl pH 6.8, 5% glycerol, 2% SDS) for 7 minutes at room temperature and 40μg of protein was separated on a 1.5% Tris-glycine agarose gel. Protein was transferred to PVDF membrane overnight and analyzed by western blot using an anti-Rnq1 antibody.

[PSI+] Induction
The de novo formation of [PSI+] was monitored as previously described [43,57]. Yeast propagating different [RNQ+] prion variants were transformed with pEMBL-SUP35. At least three independent overnight cultures were started for each prion variant in SD-ura and grown to an OD 600 of 0.6-1.5. Each culture was diluted roughly 1:8,000 in water before plating 250μl onto 13cm diameter ¼YEPD plates and incubated at 30°C for 5 days, followed by overnight incubation at 4°C for color development.
[PSI+] colonies were scored as any white/ pink colonies or colonies having white/pink sectoring. More than 1,200 colonies were scored for each [RNQ+] variant. Previous studies have reported that only ~12% of the white/ pink sectoring colonies are the result of nonheritable amyloids that cause Sup35 over-expression-dependent nonsense suppression, while the rest contain bona fide [PSI+] [57,65]. As an alternative means of assessing [PSI+] induction, overnight cultures were normalized by OD 600 and spotted in 5-fold serial dilutions onto ¼YEPD, YEPD+3mM GdnHCl, and SD-ade. Plates were incubated at 30°C for 3 days for the rich media and 9 days for SD-ade.
To characterize the [PSI+] variants that formed, ~300 individual [PSI+] colonies of each [RNQ+] variant were spotted onto ¼YEPD, YEPD+3mM GdnHCl, and SD-ade. Plates were incubated at 30°C for 3 days for the rich media and 6 days for SD-ade. Based primarily on growth on SD-ade, [PSI+] variants that were confirmed curable after transient growth on YEPD +3mM GdnHCl were classified as one of the following: very weak (≤3 colonies in the spot), weak (growth covering up to 50% of the spot), medium (growth covering 50-90% of the spot), or strong (dense growth covering >90% of the spot in addition to white colonies present on ¼YEPD).

Microscopy
When expressed in [RNQ+] cells, Rnq1-GFP decorates Rnq1 aggregates to form fluorescent foci in prion variantspecific patterns, while remaining diffuse in [rnq-] cells [48,55]. Cells were transformed with the copper inducible pRS316CUP1-RNQ1(153-405)-GFP, a kind gift from Dr. Susan Lindquist, as used in previous studies [48,55]. Overnight cultures were grown to an OD 600 of ~1.0 in SD-ura before backdiluting to an OD 600 of 0.2 in SD-ura containing 50μM CuSO 4 for ~2.5 hours prior to imaging. Samples were prepared on agar pads (3% wt/vol in liquid SD-ura) and plated directly onto VWR 3in x 1in, 1mm thick microscope slides. Fisherbrand No.#1.5 microscope coverslips were secured with nail polish. Images were collected using a Zeiss Axiovert 200 Inverted Microscope equipped with a Zeiss 100x/1.4 NA oil objective. Slidebook 5.0 (Intelligent Imaging Innovations) was used to analyze captured images and deconvolve (no neighbors algorithm) GFP Z-stacks.

Rate of spontaneous [RNQ+] formation
In analyzing the conformational diversity of the [RNQ+] prion, we first wanted to quantify the rate of spontaneous [RNQ+] formation and compare it to what was previously described for  induction and the pattern of Rnq1-GFP fluorescence [51,55]. To compare these two sets of [RNQ+] variants phenotypically, we spotted serial dilutions of normalized cells onto ¼YEPD and SD-ade plates ( Figure 1C) phenotypes. For instance, many E1 colonies were dark pink with light pink sectors, while many B1 colonies were white with light pink sectors. These phenotypes can be contrasted with true mitotic loss, which is represented by sectoring to red. Interestingly, upon subsequent restreaking, the sectoring phenotypes of A5 and C3 stabilized into homogenous populations of colonies (data not shown).  the transformed plasmid, cells were plated onto ¼YEPD plates. Any pink/white colonies or colonies with pink/white sectoring were scored as [PSI+], and the rate of [PSI+] formation was calculated as the number of those colonies divided by the total number of cells plated (Figure 2A). For comparison, we also confirmed the rates of [ in place of RRP as described above. These yeast strains propagated other [RNQ+] variants that had spontaneously formed in our initial screen. To monitor [PSI+] induction capacity, we over-expressed Sup35 as above and spotted normalized numbers of cells onto ¼YEPD and SD-ade plates ( Figure 2D) Figure  2E). Interestingly, an overwhelming majority of our [RNQ+] variants (22 out of 28) were categorized as inducing [PSI+] at low or medium levels. We identified E5 as an outlier in the data set in displaying robust growth on SD-ade, even greater than that of s.d. very high [RNQ+].

Rnq1-GFP aggregation patterns of [RNQ+] variants
In addition to [PSI+] induction levels, fluorescence microscopy was also utilized to identify the original [RNQ+] variants as potentially distinct structures [55]. With expression of an inducible Rnq1(153-405)-GFP fusion construct [48,55] [55,68]. We recapitulated these findings for s.d. low [RNQ+], but also found that m.d. high [RNQ+] displayed a third Rnq1-GFP pattern as the dominant population ( Figure 3A). This pattern was characterized by many petite foci (p.f.) that were much smaller and fainter in intensity than those observed with the s.d. and m.d. patterns.
When analyzing the Rnq1-GFP aggregation patterns of our stable and unstable [RNQ+] variants, we found that these variants often did not fall into a strict bimodal classification of s.d. or m.d. (Figure 3B-C). Instead, we observed a gradient of patterns, which varied in the relative proportions of s.d., m.d., and p.f. cells (

Spontaneous [RNQ+] variants propagate thermally stable amyloid structures
In vitro studies of Sup35 suggested that strong [PSI+] arises from amyloid fibers that have a relatively shorter protected core as compared to Sup35 aggregates of weak [PSI+] [40]. This difference in amyloid core length was found to positively correlate with fiber stability: the longer core of weak [PSI+] resulted in greater stability than strong [PSI+] [39]. Similarly, we found previously that more stable amyloid fibers of Rnq1-PFD produced weaker prion variants of [RNQ+], while less stable  [43]. The resultant differences in phenotype with different [PSI+] and [RNQ+] variants were initially attributed to the ability of fibers to produce more free ends that are required for sequestering and converting monomeric protein [40]. Thus, less stable fibers produce more active "seeds" that convert soluble protein to the prion conformer faster, resulting in stronger biological phenotypes.
We were interested in determining whether the stability of our stable and unstable [RNQ+] variants would show a similar relationship with either their [RRP+] phenotype or efficiency in [PSI+] induction. Using thermal denaturation as an indicator of stability, we subjected cell lysates of our [RNQ+] variants to a temperature gradient and analyzed the samples by SDS-PAGE and western blot to determine the temperature at which 50% of the Rnq1 protein was liberated from the aggregates. Aggregates of all 12 [RNQ+] variants were shown to be very thermal stable with melting temperatures (T m ) ranging from 80-95°C ( Figure 4A-B

SDD-AGE reveals variant-specific differences in aggregate size distribution
Previous studies with the [PSI+] and [RNQ+] prions have shown that the SDS-resistant, higher molecular weight protein
[ aggregates can be resolved from the monomeric species and visualized using semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) and western blot [37,54]. Furthermore, distinct prion variants can show different sizes of aggregates by SDD-AGE. For instance, many weak [PSI+] cell lysates harbor noticeably larger Sup35 aggregates than strong [PSI+] [37,39]. Slight differences also exist between some of the previously characterized [RNQ+] variants [54]. Therefore, we asked whether SDD-AGE analysis of our 12 stable and unstable [RNQ+] variants would reveal any additional differences in structural properties of the Rnq1 aggregates. , and G4 contained a species that migrated slightly faster. A5 exhibited minor differences in aggregate distribution across multiple trials. Interestingly, B1 P/W and B1 W displayed modest, but reproducible differences as Rnq1 aggregates of B1 W were slightly larger than those found in B1 P/W lysates, despite having formed initially in the same colony. In striking contrast, E1 was the most unique and showed the bulk of the aggregated Rnq1 propagating in larger structures as compared to the other variants. Moreover, the aggregates of medium [RNQ+] consistently appeared much fainter in intensity, a property also observed with m.d. high [RNQ+] (data not shown).

Discussion
In this study, we describe the isolation and characterization of several novel [RNQ+] variants that formed spontaneously in vivo. In the process of isolating these variants, we were able to estimate the rate of spontaneous [RNQ+] formation to be   [71]. Similarly, others have described an "unspecified" [PSI+] phenotype that was characterized by white colonies that sectored to pink, which gave rise to progeny carrying weak [PSI+], strong [PSI+], or unspecified [PSI+] [67]. In contrast to the "cloud" hypothesis, these authors consider an alternative possibility in which a single [PSI+] structure responsible for the unspecified [PSI+] phenotype is able to undergo a conformational maturation process into more than one distinct [PSI+] variant. Similarly, fibers of α-synuclein that were formed in vitro were recently shown to undergo a maturation process and form distinct aggregate conformations over time [53]. Both the "cloud" and "maturation" models may be applicable to our unstable [RNQ+] variants. Presumably, a host of factors, which also include competition between prion variants [51] and strain mutation [72], may play a role in dictating how prions propagate and which variants ultimately emerge phenotypically.
Many groups have speculated on the role of [PSI+] in nature, either as a harmful pathogenic state [73] or as a beneficial mechanism of generating heritable phenotypic diversity in response to stressful or shifting environmental conditions [74,75,76,77]. In the latter case, it is hypothesized that [PSI+]mediated nonsense suppression allows translation of coding sequences downstream of stop codons, thereby creating novel gene products that may facilitate the evolution of new traits [76,78]. While reduced fidelity in translation termination could be deleterious, cells forming particular [PSI+] variants may in fact produce heritable, advantageous phenotypes that allow survival of the population without requiring immediate genetic change. Indeed, when cells were subjected to a range of environmental stresses, a correlation was found between the severity of stress and the frequency of [PSI+] formation, suggesting that cells may induce [PSI+] when a rapid adaptive response is needed [75]. With the discovery of [PIN+] as an essential factor for de novo [PSI+] formation, a two-prion system of epigenetic translation regulation was proposed [50]. The findings that we present here highlight the extent of conformational diversity that exists for the [RNQ+] prion. Such structural variation of [RNQ+] may contribute to differentially modulating the switch into a [PSI+] state, and influence the expansive phenotypic diversity observed with [PSI+]-dependent traits [76].
By isolating a number of novel [RNQ+] variants and adding to those previously described, we show the widespread variability in structures that the Rnq1 protein can assume. A high number of conformational possibilities have also been shown for [PSI+] [71,73], making the classification into strong and weak [PSI+] variants overly simplistic. We speculate that such diversity is not limited to these prion proteins, but may exist with many amyloidogenic proteins. Indeed, it was recently suggested that PrP may form over 30 distinct prion strains in humans [24]. Therefore, studying the numerous structural variants of [RNQ+] may help elucidate what biochemical properties or cellular factors contribute to the prion variant that propagates and how it manifests phenotypically.