Destabilizing Interactions Among [PSI⁺] and [PIN⁺] Yeast Prion Variants

Corresponding author: Susan W. Liebman, University of Illinois, 900 S. Ashland Ave., Chicago, IL 60607., suel{at}uic.edu (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The yeast Sup35 and Rnq1 proteins can exist in either the noninfectious soluble forms, [psi^-] or [pin^-], respectively, or the multiple infectious amyloid-like forms called [PSI⁺] or [PIN⁺] prion variants (or prion strains). It was previously shown that [PSI⁺] and [PIN⁺] prions enhance one another's de novo appearance. Here we show that specific prion variants of [PSI⁺] and [PIN⁺] disrupt each other's stable inheritance. Acquiring [PSI⁺] often impedes the inheritance of particular [PIN⁺] variants. Conversely, the presence of some [PIN⁺] variants impairs the inheritance of weak [PSI⁺] but not strong [PSI⁺] variants. These same [PIN⁺] variants generate a single-dot fluorescence pattern when a fusion of Rnq1 and green fluorescent protein is expressed. Another [PIN⁺] variant, which forms a distinctly different multiple-dot fluorescence pattern, does not impair [PSI⁺] inheritance. Thus, destabilization of prions by heterologous prions depends upon the variants involved. These findings may have implications for understanding interactions among other amyloid-forming proteins, including those associated with certain human diseases.

THE notion of an infectious protein was first imagined more than 30 years ago to make sense of an odd infectious agent without nucleic acids that appeared to be causing sheep scrapie disease (GRIFFITH 1967 ). Today infectious proteins are known as prions (PRUSINER 1982 ) whether they cause infectious and fatal diseases in animals or heritable variations of traits in yeasts (WICKNER 1994 ). Animal and fungal prions appear to be amyloid protein aggregates that propagate by capturing soluble proteins and converting them into infectious aggregated forms (PRUSINER 2001 ; UPTAIN and LINDQUIST 2002 ). According to the "protein only" hypothesis (PRUSINER 1982 ), the prion protein (PrP) is the sole agent responsible for causing numerous infectious diseases including scrapie, mad cow, kuru, and Creutzfeldt-Jakob, to name a few (PRUSINER 1998 ; MCKINTOSH et al. 2003 ). Many other amyloid-forming proteins are associated with various human neurodegenerative diseases and systemic amyloidoses and do not appear to be infectious.

Surprisingly, different strains of the PrP prion diseases infecting inbred animals have distinct and heritable characteristics such as incubation times and neural degeneration patterns (BRUCE et al. 1989 ; BESSEN and MARSH 1992 ). The notion of these disease strains has been difficult to reconcile with the protein only model, but is easily explained by a virino hypothesis proposing that the diseases and their strains are caused by a very small nucleic-acid-containing agent (DICKINSON and OUTRAM 1988 ). However, demonstrations of what appear to be differently aggregated PrP forms associated with distinct disease strains (BESSEN and MARSH 1994 ; TELLING et al. 1996 ; SAFAR et al. 1998 ) indicate that explaining the disease strains in terms of structural variations of PrP prion molecules may be possible.

The yeast Saccharomyces cerevisiae possesses several amyloid-forming proteins that are infectious, but have no sequence homology with PrP (WICKNER et al. 2001 ). One of these prion proteins, Sup35, is a component of yeast's translation termination factor (STANSFIELD et al. 1995 ; ZHOURAVLEVA et al. 1995 ) and the determinant of the [PSI⁺] prion (TER-AVANESYAN et al. 1994 ; WICKNER 1994 ). Cox first described [PSI⁺] as a non-Mendelian enhancer of stop codon readthrough (nonsense suppression) in yeast already containing a mutant tRNA suppressor (COX 1965 ), and it was later shown that [PSI⁺] could act as a nonsense suppressor on its own (LIEBMAN and SHERMAN 1979 ). It is now known that [PSI⁺] cells have prion aggregates of Sup35 that capture most of the soluble Sup35 molecules and reduce the yeast's translation termination efficiency (PATINO et al. 1996 ; PAUSHKIN et al. 1996 ). In [PSI⁺] cells a fusion between Sup35 and green fluorescent protein (Sup35-GFP) forms a few dots visible by microscopy (PATINO et al. 1996 ), but many more unseen aggregates are likely to be present in each cell (BAILLEUL-WINSLETT et al. 2000 ; CHERNOFF et al. 2002 ).

The normal propagation of the [PSI⁺] prion is disrupted in a variety of ways. Mutations in the Sup35 N-terminal domain (Sup35N) interfere with [PSI⁺] maintenance (DOEL et al. 1994 ; TER-AVANESYAN et al. 1994 ). Deleting or overexpressing HSP104 blocks [PSI⁺] propagation (CHERNOFF et al. 1995 ) and various other chaperones are important for normal [PSI⁺] propagation (CHERNOFF et al. 1999 ; NEWNAM et al. 1999 ; JUNG et al. 2000 ; KUSHNIROV et al. 2000 ; CHACINSKA et al. 2001 ). Growth in the presence of guanidine hydrochloride (GuHCl) also cures cells of [PSI⁺] (TUITE et al. 1981 ).

Multiple factors also influence the appearance of the [PSI⁺] prion. Overproducing Sup35 (CHERNOFF et al. 1993 ; DERKATCH et al. 1996 ) or just its N domain (DERKATCH et al. 2000 ) induces the de novo appearance of [PSI⁺]. After curing [PSI⁺] by growth in GuHCl, some cells were inducible to the [PSI⁺] state by overproducing Sup35, whereas other cells were completely refractory to [PSI⁺] induction. Genetic analyses of these differences led to the hypothesis that another prion-like factor controls the appearance of [PSI⁺] (DERKATCH et al. 1997 ). That controlling factor, termed [PIN⁺], exists independently of [PSI⁺] (DERKATCH et al. 2000 ) and has been identified (DERKATCH et al. 2001 ) as the prion form of the Rnq1 protein (SONDHEIMER and LINDQUIST 2000 ). In this article, [PIN⁺], also known as [RNQ⁺], always refers to the prion form of Rnq1. Other prions or protein aggregates cause the Pin⁺ phenotype because they facilitate the de novo appearance of [PSI⁺] even in the absence of the [PIN⁺] prion (DERKATCH et al. 2001 ). It has been suggested that preexisting prions act as templates to occasionally facilitate the initial formation of heterologous prions (DERKATCH et al. 2001 ; OSHEROVICH and WEISSMAN 2001 ), which would explain how the presence of one prion could enhance the formation of another. A negative interaction between [PSI⁺] and another yeast prion, [URE3], has also been described (SCHWIMMER and MASISON 2002 ).

In a phenomenon resembling PrP disease strains, excess Sup35 induces at least two distinct types of variants (or strains) of [PSI⁺], weak and strong, in the same genetic background (DERKATCH et al. 1996 ; KING 2001 ; KOCHNEVA-PERVUKHOVA et al. 2001 ; UPTAIN et al. 2001 ). Weak [PSI⁺] differs from strong [PSI⁺] by causing less nonsense suppression and being less stably inherited (DERKATCH et al. 1996 ). Furthermore, cells bearing weak [PSI⁺] contain more nonprion soluble molecules of Sup35 compared to cells bearing strong [PSI⁺] (ZHOU et al. 1999 ; UPTAIN et al. 2001 ). In matings between weak and strong [PSI⁺] haploids, strong [PSI⁺] outcompetes weak [PSI⁺] presumably because weak [PSI⁺] aggregates propagate more slowly than strong [PSI⁺] aggregates do. Furthermore, cells displaying weak [PSI⁺] never reappear in the mitotic or meiotic progeny of these diploids (BRADLEY et al. 2002 ).

[PIN⁺] also exists in distinct variants (or strains; BRADLEY et al. 2002 ). When a [PIN⁺] derivative referred to as the "original" is cured to the [pin^-] state by growth on GuHCl (DERKATCH et al. 1997 ), new [PIN⁺] derivatives appear spontaneously following prolonged storage of the [pin^-] cells (DERKATCH et al. 2000 ). The spontaneous appearance of [PIN⁺] coincides with the appearance of aggregated Rnq1 (DERKATCH et al. 2001 ), and a fusion of Rnq1 and green fluorescent protein (Rnq1-GFP) forms punctate dots in [PIN⁺] cells but is distributed evenly throughout [pin^-] cells (SONDHEIMER and LINDQUIST 2000 ; DERKATCH et al. 2001 ). It was recently shown that three independent spontaneous [PIN⁺] together with the "original" [PIN⁺] compose an array of distinct [PIN⁺] variants in which low, medium, high, or very high levels of [PSI⁺] are induced by overproducing Sup35, in which high [PIN⁺] corresponds to the original [PIN⁺] derivative (BRADLEY et al. 2002 ). In matings between different [PIN⁺] haploids, the [PIN⁺] variants maintaining less soluble Rnq1 outcompete those with more soluble Rnq1. "Very high" contains the most soluble Rnq1 and the remaining order is very high > low > medium > high (BRADLEY et al. 2002 ).

In this article we first describe how expressing Rnq1-GFP in different [PIN⁺] derivatives produces two distinct fluorescence patterns. Cells harboring any of the spontaneously acquired [PIN⁺] variants predominantly display a single-dot (s.d.) Rnq1-GFP pattern, while cells harboring the original [PIN⁺] predominantly display a multiple-dot (m.d.) Rnq1-GFP pattern. These two patterns are properties of the [PIN⁺] variants and are not due to Mendelian or non-Mendelian modifiers. We then show that the presence of s.d. [PIN⁺], unlike m.d. [PIN⁺], causes weak [PSI⁺] to be very unstable. Likewise, the acquisition of [PSI⁺] often eliminates specific s.d. [PIN⁺] variants.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media and cultivation procedures:
Standard yeast media and cultivation were employed (SHERMAN et al. 1986 ) and yeast were grown on yeast extract/peptone/dextrose media (YPD) at 30° unless indicated otherwise. To cure [PSI⁺] and [PIN⁺], yeast were grown on YPD containing 5 mM guanidine hydrochloride (TUITE et al. 1981 ; DERKATCH et al. 1997 ). Transformants were grown on plasmid selective synthetic media with dextrose (SC), glycerol (SGly), or galactose (SGal). [PSI⁺] variants were distinguished by white (strong [PSI⁺]) or pink (weak [PSI⁺]) color on YPD and by the amount of growth on SC medium lacking adenine (SC-Ade). [RHO⁺] derivatives were made [rho^-] by growing on YPD plates containing 40 mg/liter of ethidium bromide (GOLDRING et al. 1970 ).

Cytoductions were performed between [RHO⁺] donors and [rho^-] recipients and either the donor or the recipient contained a nonfunctional KAR1 allele, which reduces the efficiency of nuclear fusion following mating (CONDE and FINK 1976 ). The KAR1 gene was disrupted to produce the kar1-d15 allele as described (VALLEN et al. 1992 ). Cytoductants were selected on SGly medium containing 3 mg/liter of cycloheximide (SGly+Cyh) if the recipient was cycloheximide resistant (cyh^R). Otherwise diploids and cytoductants were selected on SGly medium lacking a nutrient required by the donor, and cytoductants were isolated by a subsequent restreaking on YPD to identify colonies with the recipient's nuclear markers.

Scoring for [pin^-], s.d. [PIN⁺], and m.d. [PIN⁺]:
Derivatives of 74-D694 (MATa ade1-14 leu2-1 his3-200 trp1-289 ura3-52; CHERNOFF et al. 1993 ), BY4741 (MATa his3 leu2 met15 ura3; Research Genetics, Huntsville, AL), or c10B-H49 (MAT ade2-1 SUQ5 lys1-1 his3-11,15 leu1 kar1-1 cyh^R; KOCHNEVA-PERVUKHOVA et al. 1998 ) were mated, respectively, to the [psi^-] [pin^-] tester strains SL1010-1A (MAT ade1-14 met8-1 leu2-1 his5-2 trp1-1 ura3-52; ZHOU et al. 1999 ) already transformed with a URA3-based centromeric vector containing a fusion of RNQ1 and GFP (Rnq1-GFP) under the CuSO₄-inducible CUP1 promoter (SONDHEIMER and LINDQUIST 2000 ) or A3099 (MATa ade2-1 SUQ5 lys1-1 his3-11,15 leu1 kar1-1 ura3::KanMX4; a kind gift of S. Lindquist, Whitehead Institute) already transformed with a HIS3-based centromeric vector containing Rnq1-GFP under the CUP1 promoter (I. DERKATCH and S. LIEBMAN, unpublished results). Diploids containing the plasmid were selected on appropriate omission medium and observed on either glass slides using a Zeiss Axioskop 2 equipped with a x40 Plan-Neofluar objective lens or poly-L-lysine-coated glass slides using a Zeiss Axioskop 200M deconvolution workstation equipped with a x100 Plan-Apochromat objective lens. The differences among [pin^-] (all cells show diffuse fluorescence with no dots), s.d. [PIN⁺] (90% of cells display a single fluorescent dot), and m.d. [PIN⁺] (90% of cells have more than one fluorescent dot) are evident even when cells are grown on medium without supplemental CuSO₄ due to residual Cu in the synthetic medium.

Unstable [PSI⁺] cytoductions and matings:
The L1767 unstable [PSI⁺] derivative of 74-D694 was cytoduced into derivatives of L2595, a kar1-d15 cyh^R derivative of L1845 (MAT ade1-14 leu2-1 his3-200 trp1-289 ura3-52; BRADLEY et al. 2002 ). Individual cytoductants were then used as donors in a second round of cytoduction. Each was transformed with pRS415, a LEU2-based centromeric vector (SIKORSKI and HIETER 1989 ), and mated with various [psi^-] [PIN⁺] derivatives of 74-D694 already transformed with pFL44, a URA3-based 2µ vector (BONNEAUD et al. 1991 ) or a URA3-based centromeric vector expressing Rnq1-GFP. The recipients were [RHO⁺] in this set of cytoduction experiments only. Cytoductants were selected on SC-Ura-Leu medium and distinguished by scoring for mating type. Transfer of the pRS415 plasmid, known as plasmiduction (NATSOULIS et al. 1994 ), and [PSI⁺] from the donor distinguished the cytoductant from the original recipient. Cytoductants were then scored for [PSI⁺] stability by spreading on YPD. The presence of red and sectoring colonies and the absence of pink or white colonies indicated that [PSI⁺] was unstable.

[PSI⁺] induction:
To induce the de novo appearance of [PSI⁺], the four derivatives of 74-D694 harboring different [PIN⁺] variants (low, medium, high, and very high) were transformed with the pGAL::SUP35 plasmid (DERKATCH et al. 1996 ). Three transformants of each of the four derivatives were grown in a patch on plasmid-selective SGal-Leu medium for approximately seven generations and then diluted in water and plated on YPD medium at 200 colonies per plate. After 7 days, the numbers of red and non-red (white, pink, or sectored) colonies were counted to give the percentage of [PSI⁺] induction. Approximately 70 non-red colonies from the three transformants of each of the four derivatives were spread on YPD plates. To score these colonies as [psi^-], weak [PSI⁺], or strong [PSI⁺], at least three colonies from each plate were patched on YPD master plates, which were incubated for 3 days and then replica plated on YPD, YPD+GuHCl, SC-Leu, and SC-Ade medium. Patches that became red on YPD+GuHCl were scored as [PSI⁺], and all newly induced [PSI⁺] failed to grow on SC-Leu because they had lost the inducing plasmid. In addition to the color on YPD, the amount of growth on SC-Ade was used to score weak vs. strong [PSI⁺]. Red on YPD and no growth on SC-Ade indicated [psi^-]. Pink on YPD and poor growth on SC-Ade indicated weak [PSI⁺]. White on YPD and good growth on SC-Ade indicated strong [PSI⁺]. To determine if the [PSI⁺] were unstable, additional colony spreads on YPD medium were performed so that no more than 100 colonies were grown on each plate. This was done to ensure that the final colonies were large enough to observe the red sectoring that occurs only in the unstable [PSI⁺] condition.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Two distinct Rnq1-GFP patterns are not caused by Mendelian modifiers:
When Rnq1-GFP was expressed in derivatives bearing the original high [PIN⁺], multiple fluorescent dots (m.d.'s) formed in 90% of the cells (Fig 1). In contrast, Rnq1-GFP formed a single fluorescent dot (s.d.) in 90% of the cells bearing any one of the eight spontaneously acquired [PIN⁺], including low, medium, and very high [PIN⁺]. Therefore, we use s.d. [PIN⁺] to collectively refer to these eight spontaneously acquired [PIN⁺] variants.

View larger version (99K): In this window In a new window Download PPT slide   Figure 1. Phenotypes of m.d. [PIN+] and s.d. [PIN+] Rnq1-GFP patterns. Derivatives of 74-D694 harboring m.d. [PIN+] (high) or s.d. [PIN+] (the low variant is shown here) were mated with a [psi-] [pin-] derivative of SL1010-1A bearing a plasmid with the Rnq1-GFP fusion under a CuSO4-inducible promoter. The diploids were grown in medium containing 50 µM CuSO4 prior to imaging. One typical cell is shown filtered for GFP-emitted light (GFP), in differential interference contrast mode (DIC), and as an overlay of the GFP and DIC images (MERGE). Each cell is shown at seven different focal points separated by 1-µm distances in the z-axis (dist.). GFP images were deconvolved with the Zeiss nearest-neighbor algorithm from an original Z-stack of 75 images collected every 200 nm. Note that 10% of cells containing s.d. [PIN+] displayed two or more dots, while cells containing m.d. [PIN+] displayed a uniform range of dots from 1 up to 10.

To determine whether the distinction between the m.d. and s.d. Rnq1-GFP patterns was controlled by nuclear or cytoplasmic factors, we utilized cytoduction, an incomplete form of yeast mating that results in the transfer of cytoplasm, but not of the nucleus, from a donor cell into a recipient cell (CONDE and FINK 1976 ). Because the m.d. and s.d. patterns were faithfully transferred by cytoduction into [pin^-] recipients, the difference between them is not controlled by a nuclear gene: from the eight spontaneous s.d. [PIN⁺] donors, 24 of 24 cytoductants were s.d. [PIN⁺], and from the original high m.d. [PIN⁺] donor 3 of 3 cytoductants were m.d. [PIN⁺] (Fig 2). The distinct Rnq1-GFP patterns could reflect differences among the m.d. and s.d. [PIN⁺] variants themselves (i.e., different prion forms of Rnq1). Alternatively, the m.d. [PIN⁺] pattern might result from the presence of a non-Mendelian modifier, e.g., "prion X," which was cured on the GuHCl medium along with the original high [PIN⁺] prior to the appearance of s.d. [PIN⁺].

View larger version (103K): In this window In a new window Download PPT slide   Figure 2. Genetic analyses of m.d. [PIN+] and s.d. [PIN+] Rnq1-GFP patterns. At each step cells were scored as m.d. [PIN+] or s.d. [PIN+] by mating with a [pin-] strain expressing the Rnq1-GFP fusion (see MATERIALS AND METHODS). Derivatives of 74-D694 including the "original" high m.d. [PIN+] (L1749 in the laboratory collection) and eight spontaneous s.d. [PIN+] including low, medium, and very high (L1941, L1943, L1945, L1947, L1949, L1951, L1953, and L1955), were cytoduced into a [psi-] [pin-] derivative of c10B-H49. Next (left), m.d. [PIN+] derivatives of c10B-H49 were cytoduced into a [psi-] [pin-] derivative of BY4741. The resulting m.d. [PIN+] derivatives of BY4741 were grown for >20 generations and then cytoduced into the eight s.d. [PIN+] derivatives of c10B-H49, including low, medium, and very high (L2337, L2338, L2339, L2340, L2341, L2343, L2347, and L2348). Also (right) the m.d. [PIN+] derivatives of c10B-H49 were cytoduced into a [psi-] rnq1 derivative of BY4741. The resulting [pin-] (rnq1) derivatives of BY4741 were grown for >20 generations and then cytoduced into the eight s.d. [PIN+] derivatives of c10B-H49.

The two distinct Rnq1-GFP patterns are not caused by other non-Mendelian modifiers:
When s.d. [PIN⁺] cells received cytoplasm from m.d. [PIN⁺] donors, 54 of 54 cytoductants from 22 independent trials were scored as m.d. [PIN⁺] (Fig 2). Also, in a "reverse" experiment (not shown) in which an m.d. [PIN⁺] [rho^-] derivative of 74-D694 (L2345) received cytoplasm from five different s.d. [PIN⁺] donors (L2397, L2398, L2399, L2401, and L2402), 15 of 15 cytoductants remained m.d. [PIN⁺]. To control for the positive transfer of s.d. [PIN⁺] in the reverse experiment, a [pin^-] [rho^-] derivative of 74-D694 (L2346) received cytoplasm from five different s.d. [PIN⁺] donors and 14 of 15 cytoductants became s.d. [PIN⁺]. Therefore, m.d. [PIN⁺] always outcompeted s.d. [PIN⁺] in cytoplasmic mixing experiments.

This result is consistent with either the prion X hypothesis or the [PIN⁺] variants hypothesis. On the one hand, the hypothetical prion X in the m.d. [PIN⁺] cells would be expected to be dominant over the absence of prion X in s.d. [PIN⁺] cells. But on the other hand, the m.d. [PIN⁺] Rnq1-GFP pattern would be expected to overshadow the s.d. [PIN⁺] pattern if the two [PIN⁺] variants coexisted in the same cell. The m.d. [PIN⁺] variant probably even propagates faster and thus outcompetes s.d. [PIN⁺] variants since the high de novo [PSI⁺] induction phenotype associated with the m.d. [PIN⁺] variant outcompeted the low, medium, or very high phenotypes associated with three different s.d. [PIN⁺] variants (BRADLEY et al. 2002 ).

To distinguish between these possibilities, m.d. [PIN⁺] cytoplasm was donated to an rnq1 derivative that is capable of maintaining the putative prion X but not [PIN⁺]. We then tested whether these rnq1 cytoductants indeed contained a prion X that could convert s.d. [PIN⁺] into m.d. [PIN⁺] (Fig 2). However, when the rnq1 cytoductants were used as donors to cytoduce s.d. [PIN⁺] recipients, 99 of 99 cytoductants from 33 independent trials remained s.d. [PIN⁺]. Therefore, because m.d. [PIN⁺] always dominates over s.d. [PIN⁺] in cytoplasmic mixing experiments and requires continuous expression of RNQ1 to do so, the m.d. pattern is a property of the "original" high [PIN⁺] variant and is not due to a non-Mendelian modifier of s.d. [PIN⁺].

Certain [PSI⁺] isolates that carry s.d. [PIN⁺] are very unstable:
We used the premature stop codon allele ade1-14 (CHERNOFF et al. 1995 ) to monitor nonsense suppression caused by [PSI⁺]. In [psi^-] ade1-14 cells, translation is efficiently terminated at the premature stop codon and therefore adenine is not produced. Consequently, [psi^-] cells do not grow on medium lacking adenine, and they accumulate a red-colored by-product of adenine biosynthesis on rich medium (FISHER 1969 ). [PSI⁺] cells occasionally read through (suppress) the premature stop codon in ade1-14, and thus they are able to grow on medium lacking adenine and become white or pink on YPD, depending on the amount of nonsense suppression.

Overproducing Sup35 induces [PSI⁺] (CHERNOFF et al. 1993 ). If newly induced [PSI⁺] colonies are observed immediately after induction, many show red sectoring indicating some loss of [PSI⁺]. Upon restreaking, however, these colonies achieve full stability (I. DERKATCH and S. LIEBMAN, unpublished data). Indeed, while experimenting on the de novo appearance of [PSI⁺] in m.d. [PIN⁺] and [pin^-] derivatives of the yeast strain 74-D694 (DERKATCH et al. 1997 , DERKATCH et al. 1998 , DERKATCH et al. 1999 , DERKATCH et al. 2000 , DERKATCH et al. 2001 ), we induced only three [PSI⁺] derivatives that never achieved full stability among thousands of stable [PSI⁺] isolates. We refer to these three [PSI⁺] derivatives as unstable [PSI⁺] since approximately half of their progeny failed to inherit [PSI⁺] (Table 1), an 100-fold increase in [PSI⁺] loss compared to the 0.5% loss observed for normal weak [PSI⁺] (DERKATCH et al. 2000 ). These unstable [PSI⁺] derivatives never achieved the stable [PSI⁺] state (Table 1). This was true even after streaking one of the derivatives 10 consecutive times (Fig 3A).

View larger version (66K): In this window In a new window Download PPT slide   Figure 3. Unstable [PSI+]. (A) The final plate in a series of 10 consecutive streakings of unstable [PSI+] L1767. Each colony is either pink-red sectored or wholly red. (B) Outline of the cytoductions demonstrating that unstable [PSI+] is caused by s.d. [PIN+]. The L1767 unstable [PSI+] was cytoduced into [pin-], m.d. [PIN+], or rnq1 derivatives of L2595. The result of each en masse cytoduction into the L2595 derivatives was analyzed by streaking cells from SGly + Cyh to YPD to check the stability of many [PSI+] cytoductants; one representative colony is shown. Cytoduction into a [pin-] derivative of L2595 (L2598) always resulted in retention of the unstable [PSI+] condition because s.d. [PIN+] was transmitted and maintained in the cytoductants. Cytoduction of unstable [PSI+] into either m.d. [PIN+] (L2595) or its rnq1 derivative (L2667) always resulted in conversion to a stable [PSI+] state because s.d. [PIN+] is outcompeted by m.d. [PIN+] and s.d. [PIN+] cannot be maintained in rnq1 derivatives. Cytoducing the L1768 and L1769 unstable [PSI+] and four more unstable [PSI+] induced in the presence of low [PIN+] (see below) into the three L2595 derivatives shown here gave identical results.

Among the three unstable [PSI⁺] isolates, two appeared upon overproducing Sup35 in the m.d. [PIN⁺] derivative and the other one appeared after overproducing Sup35 following prolonged incubation of a [pin^-] derivative. Surprisingly though, each of the three unstable [PSI⁺] derivatives had acquired an s.d. [PIN⁺] factor. This is the first of many correlations that we report between the unstable [PSI⁺] condition and the presence of an s.d. [PIN⁺]. Although [PSI⁺] was unstable in these derivatives, the s.d. [PIN⁺] was stably maintained. The s.d. [PIN⁺] was found in all mitotic progeny examined (30 unstable [PSI⁺] and 30 [psi^-] colonies) after 10 consecutive streakings of an unstable [PSI⁺] derivative.

Weak [PSI⁺] prions are destabilized by s.d. [PIN⁺] variants:
The unstable [PSI⁺] condition was maintained upon cytoduction of the three unstable [PSI⁺] derivatives into [psi^-] [pin^-] recipients (Fig 3B). This eliminates the possibility that the unstable [PSI⁺] condition results from a Mendelian mutation. Upon cytoduction of the unstable [PSI⁺] derivatives into a [psi^-] m.d. [PIN⁺] recipient, a stable weak [PSI⁺] state emerged (Fig 3B). Since the recipient's m.d. [PIN⁺] outcompetes the donor's s.d. [PIN⁺], one way to explain these results is that the unstable [PSI⁺] condition results from a destabilizing force associated with s.d. [PIN⁺]. Alternatively, m.d. [PIN⁺] might actively stabilize the [PSI⁺]. To distinguish between these possibilities, we cytoduced the unstable [PSI⁺] derivatives into a [psi^-] rnq1 recipient, where neither m.d. nor s.d. [PIN⁺] can be maintained. Since the unstable [PSI⁺] condition was converted to the stable weak [PSI⁺] state in the rnq1 recipients (Fig 3B), m.d. [PIN⁺] is not required to stabilize [PSI⁺]. Rather, s.d. [PIN⁺] appears to be the destabilizing force.

Furthermore, one such rnq1-passaged stable weak [PSI⁺] remained stable upon cytoduction into [psi^-] recipients that were either [pin^-] or m.d. [PIN⁺], but emerged as unstable [PSI⁺] with varying degrees of instability upon cytoduction into [psi^-] recipients carrying s.d. [PIN⁺] variants (Table 2). Similar results were obtained using an rnq1-passaged weak [PSI⁺] that had never before been unstable. Interestingly, introducing either of these weak [PSI⁺] often caused the loss of medium and very high s.d. [PIN⁺] (Table 2).

An unstable [PSI⁺] derivative harboring an s.d. [PIN⁺] was also mated with [psi^-] derivatives that were [pin^-], s.d. [PIN⁺], or m.d. [PIN⁺] (data not shown). Mating with the m.d. [PIN⁺] resulted in conversion to a stable weak [PSI⁺] state as expected since the m.d. [PIN⁺] outcompetes the s.d. [PIN⁺] from the unstable [PSI⁺] parent. Mating with the [pin^-] or with any of the s.d. [PIN⁺] derivatives resulted in retention of s.d. [PIN⁺] and the unstable [PSI⁺] condition. Thus, these results together with the cytoduction experiments shown in Fig 3B and Table 2 indicate that weak [PSI⁺] prions are destabilized by the presence of an s.d. [PIN⁺].

Unstable [PSI⁺] can be induced in the presence of s.d. [PIN⁺]:
We hypothesized that since the unstable [PSI⁺] condition results from a destabilizing force associated with s.d. [PIN⁺], unstable [PSI⁺] should arise in derivatives containing s.d. [PIN⁺], but not in derivatives containing m.d. [PIN⁺]. To test this, [PSI⁺] was induced by overproducing Sup35 in four [psi^-] derivatives that each contained a different [PIN⁺] variant. As expected, the frequency of induced [PSI⁺] correlated with the previously determined strengths of the different [PIN⁺] variants (BRADLEY et al. 2002 ). In addition, only stable weak or strong [PSI⁺] states appeared in the presence of m.d. [PIN⁺], while unstable [PSI⁺] and stable strong [PSI⁺] appeared in the presence of low and medium s.d. [PIN⁺] (Table 3). Levels of instability varied among unstable [PSI⁺] induced in the presence of low and medium s.d. [PIN⁺] (Fig 4). Strong [PSI⁺] isolates that were induced and propagated in the presence of s.d. [PIN⁺] were no more unstable than those of m.d. [PIN⁺] origin (data not shown). Thus only weak [PSI⁺] are subject to destabilization by s.d. [PIN⁺]. We refer to cells that are weak [PSI⁺] and unstable simply as unstable [PSI⁺] and we refer to cells that are weak [PSI⁺] and stable simply as weak [PSI⁺].

View larger version (47K): In this window In a new window Download PPT slide   Figure 4. Colonies harboring newly induced [PSI+]. (A and B) Unstable [PSI+] induced in the presence of low s.d. [PIN+] display various degrees of instability as indicated by the amount of red sectors in each colony. (C) Weak [PSI+] induced in the presence of the m.d. [PIN+] variant are always stable and never display red sectors.

Paradoxically, weak [PSI⁺] were frequently induced in cells carrying medium s.d. [PIN⁺]. We found, however, that these weak [PSI⁺] had always become [pin^-], while all newly induced unstable [PSI⁺] retained the s.d. [PIN⁺] (Table 3). The appearance of [PSI⁺] also frequently caused the loss of very high [PIN⁺]. The loss of low or high [PIN⁺] never occurred. In addition, loss of [PIN⁺] was strictly associated with the appearance of [PSI⁺] since [psi^-] colonies from the same experiment, which had been treated with excess Sup35 but did not become [PSI⁺], never lost [PIN⁺] (36 of 36 from each of the four [PIN⁺] derivatives). Upon cytoduction into [psi^-] rnq1 recipients, each of four tested unstable [PSI⁺] isolates induced in the presence of an s.d. [PIN⁺] emerged as stable weak [PSI⁺], as did the three original unstable [PSI⁺] (see Fig 3 legend).

An unusual type of weak [PSI⁺] was frequently induced in the very high [PIN⁺] derivative. These extraordinary weak [PSI⁺] frequently converted to strong [PSI⁺]. A similar phenomenon in which weak [PSI⁺] apparently give rise to strong [PSI⁺] has been observed previously (KOCHNEVA-PERVUKHOVA et al. 2001 ). Streaking colonies containing the unusual weak [PSI⁺] gave two types of colonies: white strong [PSI⁺] colonies, which, upon restreaking, gave rise only to the same white colonies; and pink-white sectored colonies, which, upon restreaking, gave rise to nearly equal amounts of both pink-white sectored colonies and white colonies. The unusual pink-white sectoring [PSI⁺] was maintained upon cytoduction into [psi^-] recipients that were [pin^-], m.d. [PIN⁺], or rnq1. Thus, the very high [PIN⁺] was not required to maintain this [PSI⁺] state. Possibly, this unusual [PSI⁺] is a [PSI⁺] variant that requires the specific seeding of very high [PIN⁺] to appear, but that once established can be propagated independently of very high [PIN⁺].

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Previous work has shown that the presence of one prion, such as [PIN⁺], can positively affect the appearance of other prions like [PSI⁺] (DERKATCH et al. 2001 ; BRADLEY et al. 2002 ). Four different [PIN⁺] variants that each allow for different amounts of [PSI⁺] induction upon Sup35 overproduction maintain distinct ratios of soluble to aggregated Rnq1 protein (BRADLEY et al. 2002 ). We have now shown that these [PIN⁺] direct the formation of either s.d. or m.d. Rnq1-GFP patterns. The possibility that these differences are not due to inherent differences between the prion variants, but rather reflect the influence of other heterologous prions, has been eliminated, addressing for the first time the possibility that multiple heterologous prions cause the apparent difference between prion strains. The presence of s.d. [PIN⁺] drastically destabilizes weak [PSI⁺], and likewise the acquisition of [PSI⁺] often eliminates s.d. [PIN⁺] variants. These findings indicate that the [PSI⁺] and [PIN⁺] prions interact more extensively than previously thought. Curiously, these interactions occur only for specific combinations of prion variants (Table 4). Previous descriptions of antagonistic interactions affecting the phenotypes of two prions, [URE3] and [PSI⁺], including an increase in the frequency of [URE3] loss from undetectable loss in the absence of [PSI⁺] to an 1% loss in the presence of [PSI⁺] (SCHWIMMER and MASISON 2002 ), may represent a phenomenon similar to that reported here.

The numbers of Sup35-GFP or Rnq1-GFP dots detected upon overproduction of the fusions do not appear to correlate with the actual numbers of heritable prion seeds. For example, while only one or two Sup35-GFP dots are often visible in [PSI⁺] cells (BAILLEUL-WINSLETT et al. 2000 ; CHERNOFF et al. 2002 ), 60 heritable seeds have been predicted by genetic tests (EAGLESTONE et al. 2000 ). Possibly, the heritable seeds of [PSI⁺] and [PIN⁺] are numerous smaller aggregates that either coalesce into one or two large dots in the presence of overproduced GFP fusion proteins (CHERNOFF et al. 2002 ) or are mostly not associated with the GFP fusions since the fusion proteins might concentrate around only a few seeds in each cell. Nevertheless, the Rnq1-GFP patterns do suggest that more heritable seeds are associated with m.d. [PIN⁺] compared to s.d. [PIN⁺]. The members of the s.d. [PIN⁺] collection were each outcompeted by m.d. [PIN⁺] and were indistinguishable by the Rnq1-GFP test. Although each s.d. [PIN⁺] tested did destabilize weak [PSI⁺] in cytoplasmic mixing experiments, some differences in their interactions with [PSI⁺] were observed (Table 4). The induction of [PSI⁺] led to the appearance of unstable [PSI⁺] in low and medium s.d. [PIN⁺], but induction in very high s.d. [PIN⁺] did not. Furthermore, the de novo induction of [PSI⁺] and the introduction of [PSI⁺] through cytoduction frequently caused the elimination of medium and very high s.d. [PIN⁺]. In contrast, low s.d. [PIN⁺], like high m.d. [PIN⁺], remained stable under these conditions.

That [PIN⁺] aggregates allow for the de novo induction of [PSI⁺] (DERKATCH et al. 2001 ; BRADLEY et al. 2002 ) and yet cause the elimination of weak [PSI⁺] suggests that the interactions between Sup35 and Rnq1 may persist over a longer period than previously thought (DERKATCH et al. 2000 ). Two models have been proposed to explain how [PIN⁺] aggregates could allow for the de novo appearance of [PSI⁺] (DERKATCH et al. 2001 ; OSHEROVICH and WEISSMAN 2001 ). The seeding model invokes direct interactions between different prion proteins by proposing that preexisting prions template the conversion of a heterologous protein into its prion form. In the titration model, instead of invoking direct interactions among different prion aggregates, preexisting prions are portrayed as sequestering a protein that inhibits the conversion of the other protein into its prion form. Current evidence favors the seeding model. Even a large excess of Sup35 is not sufficient to induce the appearance of [PSI⁺] in the absence of other prions (DERKATCH et al. 1997 ). Also, genome-wide mutagenesis failed to inactivate the hypothesized inhibitor protein (DERKATCH et al. 2001 ). Moreover, there is no general correlation between how frequently different [PIN⁺] variants enhance [PSI⁺] and [URE3] induction, a correlation that would be expected if each [PIN⁺] variant simply sequestered an inhibitor protein (BRADLEY et al. 2002 ). In addition, colocalization of [PIN⁺] prion aggregates and newly induced [PSI⁺] prion aggregates has been detected using cyan fluorescent protein and yellow fluorescent protein tagged alleles of Rnq1 and Sup35 (I. DERKATCH and S. LIEBMAN, unpublished data).

The m.d. [PIN⁺] was previously reported to not affect any phenotypes of weak or strong [PSI⁺], including stability of inheritance (DERKATCH et al. 2000 ). The unstable [PSI⁺] condition described here demonstrates that s.d. [PIN⁺], in contrast to m.d. [PIN⁺], does impair weak [PSI⁺] inheritance. What might be the difference among [PIN⁺] variants that determines whether or not they cause weak [PSI⁺] to become unstable? Using the framework of the seeding model, the unstable [PSI⁺] condition could be explained by a prolonged direct interaction between the prion proteins in addition to the initial seeding event. Possibly, s.d. [PIN⁺] aggregates sequester [PSI⁺] seeds into larger co-aggregates, thus inhibiting their segregation into daughter cells and resulting in instability. Indeed, an allele of Sup35 lacking residues 22–69 was recently shown to lose [PSI⁺] in the absence of selection because it formed much larger aggregates than normal [PSI⁺] (BORCHSENIUS et al. 2001 ). Since aggregation-prone proteins sharing common features such as polyglutamine stretches have been shown to co-aggregate (KAZANTSEV et al. 1999 ), the modest similarity between the Sup35 and Rnq1 prion proteins (SONDHEIMER and LINDQUIST 2000 ) may enable them to directly interact. However, s.d. [PIN⁺] variants are stably inherited when propagated in the presence of [PSI⁺] even though they cause the frequent loss of [PSI⁺]. This could be explained within the direct interaction model if the [PSI⁺] aggregates concentrate around only a few of the [PIN⁺] aggregates, thus leaving the rest of the [PIN⁺] seeds to continue propagating. Furthermore, if [PSI⁺] aggregates also maintain long-term interactions with m.d. [PIN⁺], the [PSI⁺] instability may not be observed simply because m.d. [PIN⁺] seeds are much more widely distributed throughout the cytoplasm compared to s.d. [PIN⁺] seeds.

Even though there is good evidence that prions directly interact to facilitate each other's appearance, the same phenomenon does not necessarily explain the unstable [PSI⁺] condition. An equally valid model depicts s.d. [PIN⁺] sequestering a factor, "protein Y," which is necessary for the stable propagation of weak [PSI⁺]. Possibly, protein Y is a chaperone protein that both weak [PSI⁺] and s.d. [PIN⁺] compete for, and must interact with, to propagate normally. For example, protein Y might be required for partitioning large prion aggregates into heritable seeds, a function proposed to explain why Hsp104 (CHERNOFF et al. 1995 ) is essential for [PSI⁺] maintenance (PAUSHKIN et al. 1996 ; WEGRZYN et al. 2001 ). Alternatively, protein Y could aid the conversion from the [psi^-] to [PSI⁺] form in the presence of [PSI⁺] seeds or protect [PSI⁺] from being completely disaggregated by Hsp104. Both have been proposed to explain why the Ssa proteins are required for proper [PSI⁺] maintenance and why they protect [PSI⁺] from elimination by Hsp104 (NEWNAM et al. 1999 ; JUNG et al. 2000 ). In fact, three chaperone proteins, Ssa1, Sis1, and Ydj1, are known to co-immunoprecipitate with the prion form of Rnq1, and Sis1 mutations render cells incapable of maintaining [PIN⁺] or cause a switch from an s.d.-like pattern to an extreme form of an m.d.-like pattern with numerous fast-moving dots (SONDHEIMER et al. 2001 ; LOPEZ et al. 2003 ).

The strong [PSI⁺] form of Sup35, which is inherently more stable than weak [PSI⁺] (DERKATCH et al. 1996 , DERKATCH et al. 2000 ), is not destabilized by s.d. [PIN⁺]. Possibly, strong [PSI⁺] still interacts with s.d. [PIN⁺] but not in a complete enough manner to lead to its destabilization, or strong [PSI⁺] is not prone to destabilization caused by reduced protein Y activity. Both of these possibilities are consistent with the idea that strong [PSI⁺] variants, because they are more stable than weak [PSI⁺] variants (DERKATCH et al. 1996 , DERKATCH et al. 2000 ), are likely to have more heritable seeds than weak [PSI⁺] (EAGLESTONE et al. 2000 ). Weak [PSI⁺] was recovered from unstable [PSI⁺] whenever s.d. [PIN⁺] was eliminated. In addition, the unstable [PSI⁺] condition was generated whenever s.d. [PIN⁺] was combined with weak [PSI⁺] even if the weak [PSI⁺] had never before been unstable. Therefore, cells in the unstable [PSI⁺] condition contain the weak [PSI⁺] variant, not a new unstable [PSI⁺] variant. The s.d. [PIN⁺] variants caused weak [PSI⁺] to become unstable whether [PSI⁺] was induced de novo in s.d. [PIN⁺] derivatives or introduced by cytoduction into s.d. [PIN⁺] derivatives. Interestingly, although stable weak [PSI⁺] isolates were frequently obtained among cells originally bearing either medium or very high s.d. [PIN⁺], in each such isolate the initial medium or very high s.d. [PIN⁺] had been lost.

In parallel to the above results, de novo induction of strong [PSI⁺] or introduction of weak [PSI⁺] through cytoduction was often associated with the loss of medium and very high s.d. [PIN⁺]. Low s.d. [PIN⁺], like high m.d. [PIN⁺], was not eliminated upon acquiring [PSI⁺]. Since all [PIN⁺] variants stably propagate in [psi^-], stable, or unstable [PSI⁺] backgrounds, it appears that the loss of these specific medium and very high s.d. [PIN⁺] variants is promoted during the early stages of [PSI⁺] appearance.

As noted elsewhere (BRADLEY et al. 2002 ), methods to eliminate prions may be evolutionary adaptations that allow organisms to avoid acquiring too many prions. The fact that the interactions described here are highly specific for different prion variants suggests that prion variants provide additional levels to the control of prion proliferation. These results suggest that complex interactions may exist among various other amyloid-forming proteins such as those associated with Alzheimer's, Huntington's, and Parkinson's diseases.

	FOOTNOTES

¹ Present address: Department of Chemistry, University of Florida, Gainesville, FL 32611.

	ACKNOWLEDGMENTS

We thank I. Derkatch for allowing us to cite unpublished work, S. Lindquist for providing yeast strains and plasmids, and T. Cahill and J. Gavin-Smyth for comments about the manuscript. This work was partially supported by a grant from the National Institutes of Health (GM-56350) to S.W.L.

Manuscript received July 18, 2003; Accepted for publication August 11, 2003.

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