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Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

	Abstract

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[PIN⁺] is a prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [PSI⁺]. We previously isolated a truncated form of Rnq1, named Rnq1100, as a [PSI⁺]-eliminating factor in S. cerevisiae. Rnq1100 deletes the N-terminal non-prion domain of Rnq1, and eliminates [PSI⁺] in [PIN⁺] yeast. Here we found that [PIN⁺] is transmissible to Rnq1100 in the absence of full-length Rnq1, forming a novel prion variant [RNQ1100⁺]. [RNQ1100⁺] has similar [PIN⁺] properties as it stimulates the de novo induction of [PSI⁺] and is eliminated by the null hsp104 mutation, but not by Hsp104 overproduction. In contrast, [RNQ1100⁺] inherits the inhibitory activity and hampers the maintenance of [PSI⁺] though less efficiently than [PIN⁺] made of Rnq1–Rnq1100 co-aggregates. Interestingly, [RNQ1100⁺] prion was eliminated by de novo [PSI⁺] induction. Thus, the [RNQ1100⁺] prion demonstrates selfish activity to eliminate a heterologous prion in S. cerevisiae, showing the first instance of a selfish prion variant in living organisms.

	Introduction

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Many proteins can undergo a conformation change to an alternative β-sheet-rich structure in which they often polymerize into amyloid aggregates. A prion is one such class of proteins with the capability to transmit its conformation change to an otherwise non-infectious, non-amyloidogenic protein in an infectious manner (Prusiner 2004). It is well-accepted that prions are transmissible agents caused by the self-propagating conformational change of proteins, behaving as a ‘protein only’ genetic element (Prusiner 1982). Prion amyloids can capture soluble proteins and convert them into an infectious aggregated form (Prusiner 2001). The first characterized prion, mammalian PrP^Sc, is a pathogenic agent causing a series of neurodegenerative disorders, including scrapie (sheep), bovine spongiform encephalopathy (BSE, cow) and chronic wasting (deer and elk) as well as kuru and Creutzfeld-Jacob disease (humans). In fungi, prions have also been characterized as non-Mendelian inheritable elements, notably [PSI⁺], [URE3], and [PIN⁺] in Saccharomyces cerevisiae, and [Het-s] in Podospora anserina (Wickner 1994; Coustou et al. 1997; Sondheimer & Lindquist 2000). The molecular and genetic studies of these fungal prions have greatly facilitated the elucidation of the molecular basis for prion conversion and propagation as well as the general criteria for prionogenicity in a protein's primary structure.

This problem has been approached in many studies of the [PSI⁺] determinant (Cox 1965), which is the prion form (Ter-Avanesyan et al. 1994; Wickner 1994; Zhouravleva et al. 1995; Paushkin et al. 1996) of the polypeptide release factor Sup35 (eRF3) that is essential for terminating protein synthesis at stop codons (Stansfield et al. 1995; Zhouravleva et al. 1995; for a review see Ehrenberg et al. 2007). When Sup35 is in the [PSI⁺] state, ribosomes often fail to release polypeptides at stop codons, causing a non Mendelian trait easily detected by nonsense suppression (Liebman & Sherman 1979; Patino et al. 1996; Paushkin et al. 1996). Auxotrophic markers ade1-14 or ade2-1 (nonsense mutations) have been widely used to select for and study [PSI⁺] since the nonsense suppression by [PSI⁺] is easily selected for on synthetic medium lacking adenine and also prevents the build-up of adenine metabolites that would cause [psi^–] cells (soluble Sup35, no nonsense suppression) to turn red on rich media.

In contrast to the ade1- or ade2-based approach to the [PSI⁺] state, the chromosomal ura3–197 (non-sense) allele provided us with a powerful tool to select for the [psi^–] state (i.e. loss-of-[PSI⁺]), in which yeast cells become viable in the presence of 5-fluoroorotic acid (5-FOA) at concentrations otherwise toxic to [PSI⁺] cells (Kurahashi & Nakamura 2007; Kurahashi et al. 2008). Using this tool, we have isolated a prion inhibitor, Rnq1100, that eliminates [PSI⁺] when overproduced (Kurahashi et al. 2008). Rnq1 is a protein of unknown function and is one of several known yeast proteins containing a QN-rich prion domain, hence named so for rich in asparagine (N) and glutamine (Q) (Sondheimer & Lindquist 2000). Rnq1100 deletes the N-terminal non-prion domain of Rnq1, and inhibits the maintenance of not only [PSI⁺] but also [URE3] and Huntingtin's polyglutamine (polyQ) aggregate in a [PIN⁺] background, but not in a [pin^–] background. Rnq1100, however, does not eliminate [PIN⁺]. These findings are interpreted to indicate that Rnq1–Rnq1100 co-aggregates in the [PIN⁺] state interact with other transmissible and non-transmissible amyloids to destabilize and lose their amyloid form.

To gain further insight into the nature of Rnq1100, we investigated whether or not the [PIN⁺] state of Rnq1 can be transmissible to Rnq1100 in the absence of full-length Rnq1, and whether Rnq1100 alone is sufficient to propagate the transmitted prion state, despite of its inhibitor activity. In this study, we found that [PIN⁺] is transmissible to Rnq1100 when expressed from a strong promoter, but not from a weak promoter, and the resulting prion form of Rnq1100, newly designated [RNQ1100⁺], demonstrates selfishness towards other heterologous prion(s).

	Results

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[PIN⁺] transmission to Rnq1100

Rnq1100 deletes the N-terminal non-QN rich domain of Rnq1, and is thereby highly QN-rich and prone to self-aggregate or co-aggregate with Rnq1 (Kurahashi et al. 2008). Previously, we reported that in [PIN⁺] cells, Rnq1100 is incorporated into the [PIN⁺] aggregates of Rnq1, thereby producing a prion-like co-aggregate (Kurahashi et al. 2008). In this study, we examined the ability of Rnq1100 alone to form a prion in the absence of full-length Rnq1 by constructing several S. cerevisiae strains (the flowchart of strain manipulations is shown in Fig. 1A). Two sets of plasmids (HIS3 marker) expressing Rnq1100 and full-length Rnq1 (control) from a strong (ADH) or a weak (CYC1) promoter were manipulated (note that plasmids are designated with both the promoter and the Rnq1 form, such as pADHp–rnq1100 or pCYC1p–RNQ1) (Mumberg et al. 1995), and transformed into the [PIN⁺] RNQ1 strain (NPK200). As expected, all the transformants were [PIN⁺] (Kurahashi et al. 2008), and their chromosomal RNQ1 gene was subsequently knocked out by transformation with the rnq1 (i.e. rnq1 :: URA3) sequence as described previously (Kurahashi et al. 2008; see Experimental procedures). In parallel, [pin^–] variants (control) from each strain were made by nullifying the chromosomal RNQ1 of NPK200 with rnq1, followed by transformation with Rnq1100 or Rnq1 expression plasmids. Notably, the abundance of Rnq1 synthesized from plasmid pCYC1p–RNQ1 is similar to that from the native chromosome (shown in Fig. 1B, compare NPK494 and NPK521 for [PIN⁺], or NPK525 and NPK520 for [pin^–]), and thereby is useful to interpret the prion-forming ability in wild-type yeast.

View larger version (34K): [in this window] [in a new window]   Figure 1  Transmission of [PIN+] prion state to Rnq1100 in the absence of full-length Rnq1. (A) Schematic diagram of strain manipulations and [PIN+] transmission. The prion state of the indicated strains that were determined in (C) and (D) are shown in square brackets: [+] and [–] indicate the prion and non-prion states, respectively. (B) Rnq1 and Rnq1100 protein levels from cell lysates of the indicated strains were analyzed by Western blotting. The blot was first probed with anti-Rnq1 antibody (top), then stripped and re-probed with anti-Pgk1 antibody as the internal control (bottom). Asterisks indicate degradation or internal initiation products. (C) Prion state monitored by SDD-AGE analysis. The same protein amount from cell lysates of the indicated strains were loaded; Rnq1 and Rnq1100 were detected by immunoblotting using anti-Rnq1 antibody. P and M represent polymer and monomer fractions of Rnq1/Rnq1100, respectively. Molecular weight markers are shown on the left. (D) Prion state monitored by fluorescence microscopy using Rnq1–GFP fusion protein. Rnq1–GFP protein was expressed in the indicated strains from the CUP1 promoter in log-phase culture by adding 50 µM CuSO4 for 6 h, and cells were analyzed by fluorescence microscopy.

rnq1

Next, we examined the [PIN⁺] state by monitoring Rnq1 or Rnq1100 aggregates using semi-denaturing detergent-agarose gel electrophoresis in the presence of 1% SDS (SDD-AGE; Kryndushkin et al. 2003; Liebman et al. 2006). Western blot analysis indicated that SDS-stable polymers are formed in NPK486, NPK488, and NPK494, but not in NPK496 or the other strains tested (Fig. 1C). These findings indicated that [PIN⁺] is transmissible to Rnq1100 expressed from the strong ADH promoter, but not to Rnq1100 expressed from the weak CYC1 promoter. Again, Rnq1100 expressed from the weak CYC1 promoter was undetectable in NPK496 and NPK526 (Fig. 1C).

Further, we examined the [PIN⁺] state by monitoring Rnq1 or Rnq1100 aggregates using a fusion of Rnq1 and green fluorescent protein (Rnq1–GFP) expressed from the CUP1 promoter in the presence of CuSO₄. Rnq1–GFP formed punctate foci in NPK486, NPK488, and NPK494, whereas it showed mostly cytoplasmic, dispersed fluorescence (i.e., [pin^–]) in NPK496 and the other strains tested (note that the appearance of a few aggregates in [pin^–] cells is probably due to the overproduction of Rnq1 protein and the tendency of Rnq1100 to self-aggregate), with the exception of NPK521 ([PIN⁺] control) (Fig. 1D). These results confirm that the [PIN⁺] state is transmissible to Rnq1100 expressed from the ADH promoter, not from the CYC1 promoter, in the absence of full-length Rnq1. The putative prion form of Rnq1100 transmitted from the [PIN⁺] state of Rnq1 is tentatively referred to as [RNQ1100⁺] since it shows shared as well as distinct properties compared to [PIN⁺] (see below).

Genetic evidence for [RNQ1100⁺] prion

The non-Mendelian inheritance of [RNQ1100⁺] was examined by tetrad analysis of [RNQ1100⁺]/[rnq1100^–] heterozygous diploid made by crossing the [RNQ1100⁺] strain (NPK488 carrying the HIS3-marked Rnq1100 plasmid) with the [rnq1100^–] strain (NPK576 carrying the TRP1-marked Rnq1100 plasmid). The control [rnq1100^–]/[rnq1100^–] homozygous diploid was also made by crossing NPK576 with the [rnq1100^–] strain (NPK524 carrying the HIS3-marked Rnq1100 plasmid). The flowchart of strain manipulations is shown in Fig. 2A. After sporulation of these diploids, two asci each were examined by SDD-AGE analysis. As shown in Fig. 2B, the [RNQ1100⁺]/[rnq1100^–] heterozygous diploid contained SDS-stable Rnq1100 polymers, representing the [RNQ1100⁺] prion. Of eight spores derived from [RNQ1100⁺]/[rnq1100^–] diploid, one spore (B-4) lost both HIS3- and TRP1-marked Rnq1100 plasmids, thus showing the [pin^–] state, while the other spores contained either plasmid or both, thus showing the [RNQ1100⁺] state. The [RNQ1100⁺] state was also confirmed by the fluorescence study (as above) using Rnq1–GFP (data not shown) as well as by the Pin assay (Fig. 2C and see below). Spores derived from the control [rnq1100^–]/[rnq1100^–] homozygous diploid were essentially [rnq1100^–], while the one spore (D-2) contained SDS-stable Rnq1100 polymers, though less abundant. Further study using Rnq1–GFP, however, detects a diffused fluorescence with some foci in the D-2 spore as found in [rnq1100^–] (data not shown), suggesting that the observed polymers in D-2 does not represent the [RNQ1100⁺] prion but a non-prion aggregate formed by overexpression.

View larger version (27K): [in this window] [in a new window]   Figure 2  Tetrad analysis of non-Mendelian determinant [RNQ1100+]. (A) A diagram of strain manipulation. NPK488 ([RNQ1100+] MATa) and NPK524 ([rnq1100-] MATa) were mated with NPK576 ([rnq1100–] MAT). The resulting diploids were sporulated and two asci were dissected respectively. The parentheses indicate the HIS3 and TRP1 markers representing pRS413ADHp–rnq1100 and pRS414ADHp–rnq1100 plasmids, respectively: for example, (+/–) means the strain has the HIS3-marked plasmid but not the TRP1-marked plasmid. (B) The Rnq1100 prion state of heterozygous diploids and their progeny monitored by SDD-AGE analysis. The same protein amount of the indicated strain lysates was loaded, and Rnq1100 was detected by immunoblotting using anti-Rnq1 antibody. (C) The Rnq1100 prion state of the strains indicated in (A) was monitored by Pin+ activity. The Sup35-NM domain was overproduced, and cells were spotted on YPD (for control) and SC-ade plate as the arrangement in (A).

RNQ1100

MAT

RNQ1100

rnq1

RNQ1100

RNQ1100

Pin⁺ phenotype transmitted to [RNQ1100⁺] prion

The most remarkable phenotype of the [PIN⁺] prion is an increase in the de novo induction of [PSI⁺], hence referred to as Pin⁺ (Derkatch et al. 1997). To examine whether the Pin⁺ phenotype is associated with [RNQ1100⁺] or not, all strains constructed above were transformed with plasmid pCUP1p-NM, a pRS414 (TRP1)-based plasmid over-expressing Sup35's NM prion domain from the CUP1 promoter (Kurahashi et al. 2008). Transformants were grown in SC–trp–his liquid medium supplemented with 50 µM CuSO₄ for 2 days (about 48 h), subsequently spotted on YPD and SC-ade plates in fivefold serial dilutions, and then grown for 3 days on YPD or 7 days on SC-ade or SC-ade-his (to secure the presence of HIS3-marked plasmid pADHp-rnq1100). As shown in Fig. 3, NPK486, NPK488, and NPK494, but not NPK496, showed increased de novo induction of [PSI⁺] prion, indicating that the Pin⁺ phenotype is successfully transmitted from Rnq1 to the [RNQ1100⁺] prion. It is noteworthy that the induction frequency is similar between [RNQ1100⁺] and [PIN⁺] made of full-length Rnq1 (see Fig. 3B). Interestingly, Ade⁺ colonies borne from NPK488 lost the original [RNQ1100⁺] prion (described shortly).

View larger version (24K): [in this window] [in a new window]   Figure 3  Pin+ activity monitored by de novo appearance of [PSI+] colonies. (A) The Sup35-NM domain was overproduced in the indicated strains, and cells were spotted on YPD (for control), SC-ade and SC-ade-his plates after fivefold serial dilutions. Pin+ activity is summarized on the right-hand side. (B) The [PSI+]-induction frequency. The values are expressed as the mean of three independent experiments with standard deviations.

hsp104

RNQ1100

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RNQ1100

hsp104

View larger version (30K): [in this window] [in a new window]   Figure 4  Effect of Hsp104 on the [RNQ1100+] prion. (A) Effect of the hsp104 mutation on [PIN+] (NPK486 and NPK521) and [RNQ1100+] (NPK488) prions. The indicated strains were examined by SDD-AGE analysis using anti-Rnq1 antibody. P and M represent polymer and monomer fractions of Rnq1/Rnq1100, respectively. (B) Effect of Hsp104 overproduction on [PIN+] (NPK486 and NPK521) and [RNQ1100+] (NPK488) prions. [PIN+] and [RNQ1100+] strains were transformed with Hsp104-overproducing plasmid pGPDp–HSP104, and grown on SC-leu-his plate for 3 days, in which plasmids carrying RNQ1, rnq1100 and HSP104 were retained. After SC-leu-his liquid culture for 24 h, cells were grown in SC-his containing leucine, and plasmid pGPDp-HSP104 segregants were selected and examined by SDD-AGE analysis as indicated above. (C) Western blot analysis of hsp104 strains. Cell lysates used in (A) were subjected to SDS-PAGE and probed with anti-Rnq1 (top panel), anti-Hsp104 (middle panel) and anti-Pgk1 (bottom panel) antibodies. (D, E) Western blot analysis of [PIN+] and [RNQ1100+] strains exposed to Hsp104 overproduction as shown in (B). Plasmid pGPDp-HSP104 segregants were selected and examined in (D), while pGPDp-HSP104-bearing transformants were examined in (E).

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Inhibitory action of [RNQ1100⁺] on [PSI⁺]

Finally we examined whether [RNQ1100⁺] exerts the same inhibitory action on [PSI⁺] as Rnq1100 in the [PIN⁺] state. For this purpose, the NPK488 strain ([RNQ1100⁺], [psi^–]) and its non-prion (control) strain NPK524 ([rnq1100^–] or [pin^–], [psi^–]) were mated to different [PSI⁺] strains containing [pin^–], rnq1 allele (NPK527); [pin^–], RNQ1 allele (NPK519) or [PIN⁺], RNQ1 allele (NPK550). The resulting diploids were scored for their [PSI⁺] phenotype by the ade1–14 (nonsense)-based colony color on YPD plates. Most importantly, the [pin^-] rnq1/[RNQ1100⁺] rnq1100 heterozygous diploid (NPK527 x NPK488), containing only Rnq1100, produced both red and white colonies, while the isogenic [pin^–] rnq1/[rnq1100^–] rnq1100 heterozygous diploid (NPK527 x NPK524) never produced red colonies (Fig. 5). Notably, the frequency of red colonies, that is, [PSI⁺] elimination, from the [pin^–] rnq1/[RNQ1100⁺] rnq1100 diploid (NPK527 x NPK488) is significantly lower than that from [PIN⁺] RNQ1/[RNQ1100⁺] rnq1100 heterozygous diploid (NPK550 x NPK488), in which Rnq1100 is rather incorporated into [PIN⁺] Rnq1-aggregates giving rise to strong [PSI⁺] elimination, as shown previously (Kurahashi et al. 2008) (Fig. 5). The low frequency of [PSI⁺] elimination was not reversed in the [pin^–] RNQ1/[RNQ1100⁺] rnq1100 heterozygous diploid (NPK519 x NPK488) (Fig. 5). These findings are interpreted to indicate that the [RNQ1100⁺] prion inhibits the maintenance of [PSI⁺], but less efficiently compared to [PIN⁺] made of Rnq1–Rnq1100 co-aggregates. Importantly, the white colonies retaining [PSI⁺] lost [RNQ1100⁺] (5/5) when monitored by SDD-AGE and Rnq1–GFP foci analyses (data not shown), suggesting that [PSI⁺] and [RNQ1100⁺] are incompatible.

View larger version (41K): [in this window] [in a new window]   Figure 5  [PSI+]-elimination ability of [RNQ1100+] prion. The [RNQ1100+] [psi–] strain (NPK488) was mated with the indicated [PSI+] strains, and the resulting diploids were plated on YPD to monitor the appearance of [psi–] red colonies. [PSI+] and [psi–] control colonies are also shown.

Loss of [RNQ1100⁺] prion state upon [PSI⁺] induction

The above finding raises the question of why [PSI⁺] cells induced de novo by [RNQ1100⁺] (in Fig. 3) could be propagated, and not inhibited by [RNQ1100⁺]. Since these [PSI⁺] colonies were generated on SC-ade plates depleted of histidine, which is a selection marker of the Rnq1100-maintainer plasmid pADHp–rnq1100 (see Fig. 3), the apparent lack of inhibition is not due to a segregation of pADHp–rnq1100. The presence of [PSI⁺] in Ade⁺ colonies appeared from NPK488 was first confirmed in the absence of plasmid pCUP1p-NM (Fig. 6B), as well as by the prion-curing experiment using guanidine hydrochloride (Fig. 6C). Then, we checked the [RNQ1100⁺] prion state in de novo induced [PSI⁺] cells. Three independent [PSI⁺] colonies induced in NPK488 were isolated and their cell lysates were examined by SDD-AGE. Immunoblotting by anti-Rnq1 antibody revealed that Rnq1100 no longer forms a polymer but rather remains as a monomer (Fig. 6D; the weakness in the intensity of NPK521 compared with the other strains is due to the weak expression of Rnq1 from the native promoter). The same results were confirmed by the fluorescent study using Rnq1–GFP (data not shown).

View larger version (86K): [in this window] [in a new window]   Figure 6  Loss of [RNQ1100+] prion upon induction of [PSI+]. (A, B) The Ade+ color assay of three independent [PSI+] colonies [one strong [PSI+] (white) and two weak [PSI+] (pink)] de novo induced by Sup35 NM overproduction in NPK488 ([RNQ1100+]) strain. These colonies lost pCUP1p-NM plasmid and were grown on YPD (B) as indicated in (A). (C) Curing of [PSI+]. The same [PSI+] colonies were treated with 3 mM guanidine hydrochloride and grown on YPD. (D) Loss of [RNQ1100+] prion. The same [PSI+] colonies were examined for the [RNQ1100+] state by the SDD-AGE analysis. The Rnq1 and Rnq1100 were detected by immunoblotting using anti-Rnq1 antibody. P and M represent polymer and monomer fractions of Rnq1/Rnq1100, respectively.

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RNQ1100

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
[PIN⁺] is the prion form of the Rnq1 protein, which has unknown function. Rnq1's N-terminal is non-QN rich and is considered to be a non-prion domain. In our previous study, however, we found that the N-terminal non-prion domain truncation of Rnq1, Rnq1100, can be incorporated into the preexisting [PIN⁺] aggregate of Rnq1 and inhibits the maintenance of other prions, [PSI⁺] and [URE3], as well as toxic polyQ aggregates (Kurahashi et al. 2008). These findings were interpreted to indicate that Rnq1–Rnq1100 co-aggregates in [PIN⁺] state interact with other transmissible and non-transmissible amyloids to destabilize and lose their amyloid form. These results raised a question of whether Rnq1100 alone forms a prion or not. In this study, we found that the [PIN⁺] prion can be transmissible to Rnq1100 in the absence of the full-length Rnq1 protein, but only when overproduced by the strong ADH promoter, and not by the weak CYC1 promoter. Therefore, abundant expression of Rnq1100 is a prerequisite for transmission from [PIN⁺]. The resulting prion form of Rnq1100, designated [RNQ1100⁺], shares the same properties as the [PIN⁺] prion of full-length Rnq1 as it stimulates the de novo induction of [PSI⁺], and is eliminated by the null hsp104 mutation but not by Hsp104 overproduction. However, unlike [PIN⁺], [RNQ1100⁺] hampers the maintenance of [PSI⁺] as does Rnq1–Rnq1100 co-aggregates in the [PIN⁺] state. Therefore, Rnq1100 forms a selfish prion that excludes other prions and amyloid aggregates. To our knowledge, this is the first instance of a selfish prion variant in living organisms.

Rnq1's C-terminal is highly QN-rich. We suggest that upon removal of the non-QN rich (non-prion) domain of Rnq1, Rnq1100 gains a strong tendency to interact with other QN-rich proteins. Thus, the [RNQ1100⁺] prion aggregate might bind to the growing tip of a heterologous prion aggregate including [PSI⁺] and [URE3], and block its rapid growth, leading to its destabilization and loss. It was apparently controversial that [RNQ1100⁺] stimulates the de novo induction of [PSI⁺] but hampers the maintenance of existing [PSI⁺]. This inconsistency was solved by the finding that [PSI⁺] and [RNQ1100⁺] prions are incompatible each other and upon induction of [PSI⁺] by [RNQ1100⁺]-mediated Pin action, [RNQ1100⁺] is eliminated. The molecular basis of this ‘sacrificial’ cross-seeding of prion propagation remains to be investigated. Moreover, it is of interest to investigate whether or not the selfish prion nature of [RNQ1100⁺] can be cross transmitted to newly-induced [PSI⁺] prions.

	Experimental procedures

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Strains and manipulations

The original strains used in this study are: NPK200 ([psi^–] [PIN⁺] MATa ade1–14 leu2 ura3 his3 trp1), NPK51 ([psi^–] [pin^–] MATa ade1–14 leu2 ura3 his3 trp1), NPK519 ([PSI⁺] [pin^–] MAT ade1–14 leu2 ura3 his3), NPK527 (rnq1 :: URA3 derivative of NPK519), NPK550 ([PIN⁺] derivative of NPK519, see Kurahashi et al. 2008), NPK569 ([psi^–] [pin^–] MAT ade1–14 leu2 ura3 his3), and NPK576 ([psi^–] [rnq1100^–] MAT ade1–14 leu2 ura3 his3 trp1 rnq1 :: URA3 [pADHp-rnq1100 (TRP1 marker)]). The other strains derived from these are described in Fig. 1A. Whole deletions of HSP104 (hsp104 :: LEU2) or RNQ1 (rnq1 :: URA3) were performed as described previously (Kurahashi & Nakamura 2007; Kurahashi et al. 2008). Media and other manipulations including transformation, tetrad analysis and fluorescence microscopy are as described previously (Kurahashi & Nakamura 2007; Kurahashi et al. 2008).

Plasmids

Plasmids used are pRS400 series vectors (Stratagene) and their expression derivatives (designated by the promoter, such as pADHp, pCYC1p, pGPDp, or pCUP1p) containing the ADH, CYC1, GPD, or CUP1 promoter and the CYC1 terminator for expression of exogenous sequences (Mumberg et al. 1995; Kurahashi et al. 2008). pADHp–RNQ1, pADHp–rnq1100, pCYC1p–RNQ1, or pCYC1p–rnq1100 were constructed by cloning BamHI–XhoI fragments of RNQ1 or rnq1100 (Kurahashi et al. 2008) into the BamHI–XhoI site of pADHp (ARS/CEN, HIS3 or TRP1 marker) or pCYC1p (ARS/CEN, HIS3 marker). The Hsp104-expression plasmid pGPDp-HSP104 is descried previously (Ishiwata et al. 2009). The Rnq1–GFP and Sup35 NM domain expression plasmid, pCUP1p–Rnq1–GFP (ARS/CEN, TRP1, or LEU2 marker) and pCUP1p–NM (ARS/CEN, TRP1, or LEU2 marker) are described previously (Kurahashi et al. 2008).

Induction of [PSI⁺] element

[PSI⁺] was induced in [psi^–] cells upon transformation with pCUP1p–NM (TRP1 or LEU2 marker). Transformants were grown in SC medium lacking selective nutrients and supplemented with 50 µM CuSO₄ for 2 or 3 days and were subsequently spotted on SC-ade, SC-ade-his and YPD plates (for control), and then grown for 7 days on SC-ade and SC-ade-his, or for 3 days on YPD.

Protein analysis

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and SDD-AGE were carried out as described previously (Kurahashi & Nakamura 2007). The immunoblot experiments were performed using anti-Rnq1 antibody (Kurahashi & Nakamura 2007), anti-Hsp104 antibody (Affinity Bioreagents) and anti-Pgk1 antibody (Molecular Probes).

	Acknowledgements

 
We thank Colin G. Crist for critical reading of the manuscript and valuable comments. This work was supported in part by grants from The Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and the BSE Control Project of the Ministry of Agriculture, Forestry and Fisheries of Japan.

	Footnotes

 
Communicated by: Hiroji Aiba

^* Correspondence: nak{at}ims.u-tokyo.ac.jp

	References

Top Abstract Introduction Results Discussion Experimental procedures References

 
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Received: 25 September 2008
Accepted: 25 February 2009

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