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Molecular and Cellular Biology, May 2008, p. 3313-3323, Vol. 28, No. 10
0270-7306/08/$08.00+0     doi:10.1128/MCB.01900-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A Regulatory Role of the Rnq1 Nonprion Domain for Prion Propagation and Polyglutamine Aggregates ^,

Hiroshi Kurahashi, Masao Ishiwata, Shoichiro Shibata, and Yoshikazu Nakamura^*

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

Received 20 October 2007/ Returned for modification 26 November 2007/ Accepted 27 February 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Prions are infectious, self-propagating protein conformations. Rnq1 is required for the yeast Saccharomyces cerevisiae prion [PIN⁺], which is necessary for the de novo induction of a second prion, [PSI⁺]. Here we isolated a [PSI⁺]-eliminating mutant, Rnq1100, that deletes the nonprion domain of Rnq1. Rnq1100 inhibits not only [PSI⁺] prion propagation but also [URE3] prion and huntingtin's polyglutamine aggregate propagation in a [PIN⁺] background but not in a [pin^–] background. Rnq1100, however, does not eliminate [PIN⁺]. These findings are interpreted as showing a possible involvement of the Rnq1 prion in the maintenance of heterologous prions and polyQ aggregates. Rnq1 and Rnq1100 form a sodium dodecyl sulfate-stable and Sis1 (an Hsp40 chaperone protein)-containing coaggregate in [PIN⁺] cells. Importantly, Rnq1100 is highly QN-rich and prone to self-aggregate or coaggregate with Rnq1 when coexpressed in [pin^–] cells. However, the [pin^–] Rnq1-Rnq1100 coaggregate does not represent a prion-like aggregate. These findings suggest that [PIN⁺] Rnq1-Rnq1100 aggregates interact with other transmissible and nontransmissible amyloids to destabilize them and that the nonprion domain of Rnq1 plays a crucial role in self-regulation of the highly reactive QN-rich prion domain of Rnq1.

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Prions are transmissible agents caused by the self-propagating conformational change of proteins (32). Prions appear to be amyloid protein aggregates that propagate by capturing soluble proteins and converting them into an infectious aggregated form (33). According to the "protein only" hypothesis (32), the prion protein (PrP) is the sole agent responsible for causing numerous infectious diseases, including scrapie (sheep), bovine spongiform encephalopathy (cow), and chronic wasting disease (deer and elk) as well as kuru and Creutzfeld-Jacob disease (humans). In fungi, prions, notably [PSI⁺], [URE3], and [PIN⁺] in Saccharomyces cerevisiae and [Het-s] in Podospora anserina, have also been characterized as non-Mendelian inheritable elements (7, 37, 43). 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.

[PSI⁺] is a prion form of Sup35, which is the eRF3 polypeptide release factor that is essential for terminating protein synthesis at stop codons (39, 45; for a review, see reference 17). When Sup35 is in the [PSI⁺] state, ribosomes often fail to release polypeptides at stop codons, causing a non-Mendelian trait to appear that is easily detected by nonsense suppression (23, 29, 30). To uncover host factors responsible for [PSI⁺] propagation, we have developed a genome-wide genetic selection method for [PSI⁺]-eliminating factors or mutants by use of the chromosomal ura3-197 mutant (21). Based on this selection system, we have selected host factors whose high-level expression on a multicopy plasmid leads to [PSI⁺] elimination. One clone yielded Rnq1100, an N-terminal truncation of Rnq1, and is further examined in this study. Although there are some reports that the maintenance or de novo appearance of one prion is affected by several genetic manipulations such as overexpression of its own prion domain (15, 16), heterologous prion variants (5, 35), or nonprion protein mutants (1), the molecular basis of the action of one prion in inhibiting heterologous prions is not known.

Rnq1 is a protein of unknown function and is one of several known yeast proteins containing a QN-rich prion domain, where the name derives from "rich in asparagine (N) and glutamine (Q)" (37). Rnq1 forms the prion [PIN⁺] (name derived from "[PSI⁺] inducibility") (12, 27, 37), since [PIN⁺] is required for efficient [PSI⁺] production (14) but not for [PSI⁺] propagation (13). Although it is known that several other yeast QN-rich proteins can be attributed to the Pin⁺ phenotype (12), [PIN⁺], also known as [RNQ⁺], always refers to the prion form of Rnq1 in this article. Two models, "seeding" and "titration," have been proposed to explain how heterologous prions, e.g., [PIN⁺], facilitate the de novo appearance of [PSI⁺]. According to the seeding model, a heterologous preexisting protein in the prion conformation is used as a template for the conversion of Sup35 into its prion form, which then proceeds to seed its own rapid and separate aggregation. Importantly, [PIN⁺] also facilitates the de novo appearance of the prion [URE3] and promotes polyglutamine (polyQ) aggregation and toxicity in general (5, 25, 27). Therefore, the seeding model predicts that [PIN⁺] aggregates provide a "friendly" nidus on which the first seeds of a heterologous prion or polyQ amyloid can form (11, 41). The alternative titration model postulates that preexisting heterologous prions or prion-like aggregates capture and inactivate an inhibitor that prevents conversion of Sup35 into a prion (12, 27). As yet, neither model has been proved or disproved.

So far, it has been widely accepted that Rnq1 plays a positive role in facilitating the conversion of other prions in [PIN⁺] cells. In this study, however, we isolated a Rnq1 mutant, Rnq1100, whose overexpression is inhibitory to [PSI⁺] and [URE3] propagation as well as polyQ aggregation. Importantly, this inhibitory Rnq1 truncation lacks the N-terminal non-QN-rich domain, i.e., the nonprion domain, whose functional significance has never been reported. Rather, it has been suggested that the N-terminal portion is dispensable and that the C-terminal QN-rich (prion) domain of Rnq1 (amino acids [aa] 153 to 405) is sufficient to maintain its heritable aggregated state in vivo and its interaction with the prion domain of Sup35 (Sup35NM; see references 37 and 41). This article sheds light on the biological significance of the N-terminal nonprion domain of Rnq1.

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Strains and manipulations. S. cerevisiae strains used in this study are listed in Table 1. The yeast media used were YPD (1% yeast extract, 2% polypeptone, 2% dextrose), synthetic complete glucose (SC) (0.67% yeast nitrogen base without amino acid [DIFCO] and 2% dextrose), and galactose (SGal) (0.67% yeast nitrogen base without amino acid and 4% galactose) supplemented with adenine, leucine, uracil, histidine, tryptophan, or 5-fluoroorotic acid (5-FOA) when and as required. Yeast cells were grown at 30°C in YPD or in SC and SGal media with appropriate supplements. To monitor colony color based on ade1-14 nonsense suppression or transcriptional (de)repression of the ADE2 expression, yeast cells were grown on YPD plates for 4 days at 30°C. Elimination of the [PSI⁺] or [URE3] element by Rnq1 mutants was examined by transforming cells with plasmids expressing Rnq1 mutants from the CUP1 copper-inducible promoter as follows. Transformants selected by the plasmid-bearing marker were incubated on SC for 3 days and then passaged onto SC medium containing 50 µM CuSO₄ for 3 days and subsequently passaged onto YPD media. Whole-deletion strains of RNQ1 were constructed by transformation with a PCR product of the rnq1::URA3 sequence, which was made by substituting URA3 for KanMX in the rnq1::KanMX strain (American Type Culture Collection catalog no. 4013435; see reference 44). Transformation was performed using Frozen-EZ Yeast Transformation II (ZYMO Research, Orange, CA) according to the manufacturer's instructions.

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TABLE 1. Strains

Plasmids. PolyQ toxicity was monitored using pYES2 (high-copy-number 2µ)-based plasmid expressing green fluorescent protein (GFP) fusion to the polyQ extended (103Q) N-terminal fragment of human huntingtin protein from the galactose-inducible GAL1 promoter (25) (a kind gift from M. Sherman, Boston University School of Medicine, Boston, MA). Other expression plasmids used in this study were constructed from pRS400 series vectors (Stratagene; see reference 36) in which the CUP1 and GPD promoters are placed at the SacI-BamHI site and the CYC terminator is placed at the XhoI-KpnI site. Hence, mutant rnq1 sequences were amplified by PCR using the following primers and cloned into the BamHI-XhoI site. The PCR primers were P1 (5'-GGGGATCCAATGGATACGGATAAGTTAAT-3') and P2 (5'-GGGGCTCGAGTCAGTAGCGGTTCTGGTTGC-3') for wild-type RNQ1; P3 (5'-TGGGGATCCAATGAACACTTTAATGGCAG-3') and P2 for rnq175; P4 (5'-TTTGGATCCAATGGCAGACTCTAAGGG-3') and P2 for rnq179; P5 (5'-AAAGGATCCAATGACACACTCATCAAAT-3') and P2 for rnq1100; P6 (5'-TTTGGATCCAATGTCAATGCTAAGTGG-3') and P2 for rnq1119; P7 (5'-TTTGGATCCAATGCTAAGTGGTTCTGG-3') and P2 for rnq1121; P8 (5'-TTTGGATCCAATGGGTGCTTCCGGCCTG-3') and P2 for rnq1132; P5 and P9 (5'-AAACTCGAGTCATTGACCTTGACCTTGTCCTT-3') for rnq1-101-171; P5 and P10 (5'-AAACTCGAGTCATTGATTTTGACCTTGCTGAT-3') for rnq1-101-197; P5 and P11 (5'-AAACTCGAGTCATTGTCCCTGTTGTTGTTGGT-3') for rnq1-101-262; and P5 and P12 (5'-AAACTCGAGTCATTGTTGCTGCTGCTGACCCT-3') for rnq1-101-319. The resulting fragments were cut with BamHI and XhoI and cloned under the control of the CUP1 promoter in pRS414CUP1p (ARS/CEN, TRP1) (18). Fluorescent tag fusions to Rnq1 mutants were made as follows. GFP, cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) sequences were amplified by PCR from plasmids pEGFP, pECFP, and pEYFP (Clontech) by use of primers P13 (5'-GGGGTCGACATGGTGAGCAAGGGCGAGGAG-3') and P14 (5'-GGGCTCGAGTTACTTGTACAGCTCGTCCA-3'), and their SalI/XhoI digests were cloned into the SalI-XhoI site of pRS413CUP1p (ARS/CEN, HIS3), pRS414CUP1p (ARS/CEN, TRP1), or pRS415CUP1p (ARS/CEN, LEU2). Then, rnq1 mutant sequences were cloned into the BamHI-SalI site by use of BamHI-SalI digests of PCR fragments amplified with primers P1 and P15 (5'-GGGGGTCGACGTAGCGGTTCTGGTTGCCG-3') for RNQ1, P4 and P15 for rnq179, P5 and P15 for rnq1100, and P6 and P15 for rnq1119.

Selection of the [PSI⁺]-eliminating host factor. S. cerevisiae [PSI⁺] cells (NPK265; MATa ade1-14 leu2 ura3-197 his3 trp1) were transformed with a yeast genomic library (Sau3AI partial digests of S. cerevisiae DNA) cloned into the multicopy vector pRS423. His⁺ transformants were selected on SC-His plates by incubation at 30°C for 24 h and replica plated onto SGal-His plus 5-FOA plates as described previously (21). His⁺ 5-FOA-resistant (i.e., Ura^– phenotype) colonies were isolated, and those colored red on YPD passed through the screening as [psi^–] colonies. Plasmids were recovered from these red colonies and were retransformed using NPK265 for confirmation. pS5 is one such plasmid that cures cells of [PSI⁺], i.e., eliminates [PSI⁺].

Fluorescence microscopy. Fluorescence microscopy was performed using a MetaMorph apparatus (Universal Imaging Corporation, Downington, PA) attached to an IX71 microscope (Olympus, Tokyo, Japan). Images were captured with a CoolSNAP HQ cooled charge-coupled-device camera (Photometrics, Munich, Germany). Transformants with plasmids carrying Rnq1 mutants under the control of the inducible CUP1 promoter were grown in SC liquid medium and were induced for Rnq1 expression upon addition of 50 µM CuSO₄.

Induction of [PIN⁺] and [PSI⁺] elements. [PIN⁺] was induced from [pin^–] cells upon transformation with pRS426 (multicopy 2µ URA3)-based plasmid overexpressing Rnq1 from the constitutive strong GPD promoter. Transformants were grown in SC-ura liquid medium for 2 days and then grown in YPD medium. Ura^– colonies (i.e., plasmid segregants) were isolated, and the [PIN⁺] state was confirmed by fluorescence visualization of Rnq1-GFP foci in these cells upon transformation with the monitoring plasmid pRS415CUP1p-RNQ1-GFP and by semidenaturing detergent-agarose gel electrophoresis (SDD-AGE) in the presence of 1% sodium dodecyl sulfate (SDS) (22). [PSI⁺] was induced in [psi^–] cells upon transformation with pRS414 (centromeric ARS/CEN, TRP1)-based plasmid overexpressing Sup35's N-terminal and middle (NM) prion domain from the CUP1 promoter. Transformants were grown in SC-trp liquid medium supplemented with 50 µM CuSO₄ for 2 days and were subsequently grown on SC-ade plates. Ade⁺ colonies were grown on YPD plates, and white or pink colonies were isolated as [PSI⁺] candidates. The [PSI⁺] state was confirmed by curing of [PSI⁺] by guanidine HCl treatments as described previously (40).

Protein analysis. Centrifugation analysis and SDS-polyacrylamide gel electophoresis (SDS-PAGE) were carried out as described elsewhere (8). For separation of low-molecular-weight proteins, tricine-based SDS-PAGE (anode buffer [pH 8.9], 0.2 M Tris-HCl; cathode buffer [pH 8.25], 0.1 M Tris-HCl, 0.1 M tricine, 0.1% SDS) was performed (34). SDD-AGE and protein blotting by semidry transfer were performed as described previously (22). Immunoprecipitations were performed as described elsewhere (38). Immunoblot experiments were performed using rabbit polyclonal antibodies against full-length Rnq1 (prepared in our laboratory) (21), Rnq1's N-terminal first 100-aa polypeptide (Rnq1NTD [prepared in our laboratory]), full-length Sis1 (prepared in our laboratory), and GFP (catalog no. 8367; Clontech) as well as mouse monoclonal antibodies against GFP (catalog no. A11120; Molecular Probes) and Pgk1 (catalog no. A-6457; Molecular Probes) for a loading control.

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Selection for a [PSI⁺]-eliminating factor: the N-terminal truncation of Rnq1. The chromosomal marker ura3-197 (incorporating a nonsense UGA allele) is a useful tool of selection for [PSI⁺]-eliminating factors or mutants as 5-FOA-resistant colonies (21). Here we selected for genes whose high-level expression is inhibitory to [PSI⁺] by transforming ura3-197 [PSI⁺] cells (NPK265) with a high-copy-number yeast genomic library (HIS3-bearing vector pRS423). His⁺ 5-FOA-resistant transformants were selected for on SGal-His plates containing 5-FOA. The initial 5-FOA-resistant isolates were first screened for a true [PSI⁺]-eliminating phenotype according to the production of red colonies on YPD based on the use of the ade1-14 nonsense allele. The auxotrophic marker ade1-14 is widely used to study [PSI⁺], since nonsense suppression by [PSI⁺] allows cells to grow on synthetic media lacking adenine and prevents the buildup of adenine metabolites that would cause [psi^–] cells (soluble Sup35, no nonsense suppression) to turn red on rich media. Plasmids were isolated from these candidates, and upon retransformation, plasmid pS5 passed the screening as a bona fide [PSI⁺]-eliminating factor (Fig. 1A). Importantly, the resulting [psi^–] phenotype remained unchanged upon segregation of pS5 from the transformant (see Fig. S1A in the supplemental material), indicating that pS5 persistently cured cells of [PSI⁺].

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FIG. 1. Rnq1100, the truncated Rnq1 mutant that eliminates [PSI+]. (A) [PSI+] elimination by a Rnq1100-expressing plasmid. [PSI+]-based nonsense readthrough was determined by nonsense suppression of an ade1-14 allele on YPD. The [PSI+] strain (NPK265 [PIN+]) was transformed with an empty vector (vec.; pRS423 [multicopy plasmid with HIS3 marker]) or with pS5. Transformants were selected on SC-His after 3 days and regrown on YPD for 4 days. [PSI+] and [psi–] control cell results are also shown. (B) Schematic presentation of genes cloned in pS5. Open arrows indicate protein-coding sequences. The bold bars indicate segments subcloned on pS5-1 and pS5-2. The ability (+) or inability (–) of these two clones to eliminate [PSI+] is indicated on the right. (C) Rnq1100 product visualized by immunoblotting. The same amounts of protein from whole-cell lysates of NPK265 carrying plasmid pRS425 (empty vector [vec.]) and carrying pS5-1 were separated by SDS-PAGE, and transfer membranes were probed with anti-Rnq1 antibody. The asterisk denotes the position of Rnq1100. (D) A set of N-terminal and C-terminal deletions of Rnq1. Numbers represent the amino acid positions with respect to the first Met codon. Open triangles denote the internal Met codons not examined in this study, and closed triangles indicate the Met residue from the translational products examined in this study. The elimination (+) or nonelimination (–) of [PSI+] by these truncations, as indicated in Fig. 2, is summarized on the right hand side. (E) Schematic diagram of non-QN-rich and QN-rich regions in the Rnq1 protein. The frequency of QN residues was calculated from every 10-aa interval. QN-rich subregions are indicated in red roman numerals. Note that the amino acid positions are shown in the same scale in panels D and E for comparison.

The pS5 insert contains the RNQ1 sequence lacking the N-terminal 195 nucleotides and the complete sequences for FUS1 and YCL026C-B (Fig. 1B). Subclone analysis revealed that the 1.2-kb fragment containing the N-terminal-truncated rnq1 (i.e., pS5-1), but not the 2.6-kb fragment containing FUS1 and YCL026C-B (i.e., pS5-2), cured NPK265 cells of [PSI⁺] upon transformation (Fig. 1B). Cell lysates from NPK265 cells transformed with pS5 and empty pRS423 vector (control) were analyzed by Western blotting using anti-Rnq1 antibody after SDS-PAGE. The data showed that the pS5-bearing transformant produces an excess of truncated Rnq1 with a molecular mass of about 30 kDa. The 43-kDa full-length Rnq1 protein is also shown (Fig. 1C). These results indicate that overexpression of the N-terminally truncated Rnq1 eliminates [PSI⁺].

Serial N-terminal shortening of Rnq1. The observed 30-kDa polypeptide of truncated Rnq1 might be synthesized from an internal AUG (methionine) codon present in the cloned RNQ1 sequence. There are six potential AUG codons (at amino acid positions 76, 80, 101, 120, 122, and 133) whose translation initiation is capable of producing Rnq1 polypeptides ranging from 29 to 35 kDa (Fig. 1D). To determine the translation initiation site that eliminated [PSI⁺], we designed six serial N-terminal truncations such that each of them initiated at the presumed AUG codon (i.e., 75, 79, 100, 119, 121, and 132) (Fig. 1D). These truncated Rnq1s were expressed from the CUP1 copper-inducible promoter on a centromeric pRS414-based vector. These constructs were transformed into NPK265 cells. and the steady-state levels of Rnq1 products were determined by Western blot analysis (Fig. 2A, left). Each deletion construct synthesized truncated Rnq1 of the correct size. (The Rnq1132 product gave a reproducibly lower immunosignal than the others did, presumably due either to its weak immunoreactivity to the antibody used or to inefficient translation from codon 133.) Most importantly, only Rnq1100, but not the others, eliminated [PSI⁺] upon transformation of NPK265 cells in the presence of 50 µM CuSO₄ (as shown by the production of red coloring on rich media; Fig. 2B). In the absence of CuSO₄, most cells remained [PSI⁺], though a few red colonies or sectors occasionally appeared due to leaky expression from the CUP1 promoter (see Fig. S2A in the supplemental material). The size of the Rnq1 product from pS5-1 also coincides with the Rnq1100 product (data not shown). When the presumed AUG codon for Met101 was changed to UUG, the resulting construct Rnq1100M failed to synthesize Rnq1100 (Fig. 2A, left) or to eliminate [PSI⁺] (data not shown). These data indicate that Met101 functions as the sole internal translation start site in pS5 and that Met76 and Met80 do not function as translation start sites for some unknown reason. Moreover, the curing frequency of [PSI⁺] was dependent on the cellular abundance of Rnq1100 (see Fig. S2 in the supplemental material).

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FIG. 2. Expression of Rnq1 deletion products and their ability to eliminate [PSI+]. (A) Western blot analysis to detect expression of Rnq1 deletion products driven by the CUP1 promoter. Total cell extracts made from NPK265 transformed with the deletions listed in Fig. 1D or with an empty vector were separated by SDS-PAGE, blotted, and probed with anti-Rnq1 antibody. Rnq1132 reproducibly displayed a low immunosignal, while Rnq1-101-171 and Rnq1-101-197 displayed no immunosignal. The designation Rnq1100M is used to indicate that the AUG start codon (Met101) of Rnq1100 was mutated to an UUG codon, resulting in impaired synthesis of Rnq1100. Proteins were separated by SDS-PAGE (left) or by tricine buffer-based SDS-PAGE (right) for better separation of short polypeptides (see reference 34). Pgk1 was used as an internal control. (B and C) [PSI+] elimination upon expression of Rnq1 N-terminal truncations (B) and C-terminal truncations (C) in NPK265 cells ([PSI+] [PIN+]) from the CUP1 promoter. NPK265 transformants with pRS414CUP1p (TRP1 marker) plasmids bearing the indicated Rnq1 mutants were selected on SC-trp plates supplemented with 50 µM CuSO4 after 3 days, passaged onto YPD media, and grown for 4 days.

Serial C-terminal shortening of Rnq1. The Rnq1 sequence consists of a N-terminal non-QN-rich domain (aa 1 to 131) and a C-terminal QN-rich prion domain (aa 132 to 405) (28). The latter has been shown to be sufficient to maintain a heritable aggregated state of [PIN⁺] in vivo and its interaction with Sup35NM in vitro (41, 42). The C-terminal QN-rich region can be further divided into five QN-rich subregions, namely, subregions I through V, when tallied for the percentage of QN within every 10-aa window (Fig. 1E). To examine the minimum requirement of QN-rich subregions for [PSI⁺] elimination, we designed four C-terminal truncations such that each of them removes an additional subregion (rnq1-101-319, rnq1-101-262, rnq1-101-197, and rnq1-101-171; Fig. 1D). When these truncations were expressed from the CUP1 promoter, two small products, Rnq1-101-171 and Rnq1-101-197, were not detectable by Western blot analysis, while other deletion products were detected; note that there was reproducibly a decreased amount of Rnq1-101-262 and Rnq1-101-319 (Fig. 2A, right). Upon transformation, Rnq1-101-262 and Rnq1-101-319 as well as Rnq1100 (i.e., Rnq1-101-405) successfully cured NPK265 cells of [PSI⁺] in the presence of 50 µM CuSO₄ (as indicated by the production of red coloring on rich media; Fig. 2C). Therefore, the presence of at least three QN-rich subregions (subregions I to III) is sufficient to block [PSI⁺] activity, although it is notable that the two short polypeptides Rnq1-101-171 (subregion I) and Rnq1-101-197 (subregions I and II) might be too unstable to allow determinations of their functional significance.

Requirement of [PIN⁺] to eliminate [PSI⁺]. Prions exhibit different conformations called "variants" or "strains" within identical genetic backgrounds. [PSI⁺] variants are characterized by variable nonsense suppression results indicated by the colony color on YPD: the presence of pink coloring represents weak [PSI⁺] suppression, while white coloring indicates strong [PSI⁺] suppression. Likewise, [PIN⁺] variants are characterized as "very high," "high," "medium," and "low" [PIN⁺] variants (for their corresponding levels of efficiency in induction of [PSI⁺]) (5). The NPK265 strain used as described above is a strong [PSI⁺] variant. When the rnq1100 expression plasmid was introduced into another strong [PSI⁺] strain (NPK50), the transformant remained, unexpectedly, [PSI⁺] (as indicated by white coloring; Fig. 3A). Likewise, when two weak (i.e., indicated by pink coloring) [PSI⁺] strains, NPK197 and NPK293, were transformed with the rnq1100 plasmid, the [PSI⁺] state of the latter, but not that of the former, was cured (Fig. 3A). These observations indicate that the inhibitory effect of Rnq1100 is not affected by the strength of the [PSI⁺] strains but might be affected by other genetic allele(s) of strains used in these experiments.

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FIG. 3. Requirement of [PIN+] for Rnq1100 to eliminate [PSI+]. (A) [PSI+] elimination by Rnq1100 independent of [PSI+] strains. Two strong [PSI+] variants (NPK265 and NPK50) and two weak [PSI+] variants (NPK197 and NPK293) were transformed with pRS414CUP1p (vec.) or pRS414CUP1p-Rnq1100 (rnq1100). The transformants were selected from SC-trp containing 50 µM CuSO4 after 3 days and subsequently passaged on YPD media for 4 days. (B) The [PIN+] element is associated with [PSI+] elimination by Rnq1100. [PIN+]/[pin–] states of the indicated strains were visualized with Rnq1-GFP fusion protein. Rnq1-GFP under the control of the CUP1 promoter was induced by 50 µM CuSO4 for 6 h in liquid SC culture. The top panels show DIC (differential interference contrast) images, and the bottom panels show fluorescent images of Rnq1-GFP. (C) Turning on and off of Rnq1100's action by switching the [PIN+] state. Two sets of isogenic [PIN+]/[pin–] strains harboring [PSI+], with one set consisting of NPK294 and NPK299 and the other set consisting of NPK50 and NPK300, were transformed with the empty vector and the rnq1100 plasmid, and the [PSI+] state was examined according to colony color as described above. Strains: NPK294, [PSI+] [PIN+] RNQ1+; NPK299, [PSI+] [pin–] rnq1::URA3 derived from NPK294; NPK50, [PSI+] [pin–] RNQ1+; NPK300, [PSI+] [PIN+] RNQ1+ derived from NPK50.

The most likely candidate affecting Rnq1100-mediated inhibition is [PIN⁺], since it is a prion aggregate form of Rnq1 and potentially interacts with Sup35 during the de novo induction of [PSI⁺]. A fusion of Rnq1 and green fluorescent protein (Rnq1-GFP) forms punctate foci in [PIN⁺] cells but is evenly distributed throughout [pin^–] cells (37). The [PIN⁺] state of the four [PSI⁺] strains (NPK265, NPK293, NPK50, and NPK197) was monitored using Rnq1-GFP expressed from the CUP1 promoter in the presence of CuSO₄. The former two strains, whose [PSI⁺] state was eliminated by Rnq1100, formed single-dot (s.d.) or multidot (m.d.) Rnq1-GFP foci (i.e., [PIN⁺]), while the latter two strains, whose [PSI⁺] state was insensitive to Rnq1100, formed cytoplasmic dispersed Rnq1-GFP (i.e., [pin^–]) (Fig. 3B). Further, we examined the [PIN⁺] state by monitoring Rnq1 aggregates by use of SDD-AGE (22). Western blot analysis showed that Rnq1 formed SDS-stable polymers in the former two variants but not in the latter two variants (see Fig. S3A in the supplemental material), which corresponded to the fluorescent data.

To firmly establish the direct relationship between the [PSI⁺]-eliminating activity of Rnq1100 and the [PIN⁺] state, the [PSI⁺] [PIN⁺] strain NPK294 (ade1-14), which is cured of [PSI⁺] by Rnq1100, was changed to [pin^–] by nullifying the chromosomal RNQ1 gene by use of rnq1::URA3 (see Materials and Methods). The resulting [pin^–] NPK299 strain became tolerant to Rnq1100 (Fig. 3C). In contrast, upon conversion of the [pin^–] NPK50 strain to [PIN⁺] by high-level expression of Rnq1 from the strong constitutive GPD promoter, the resulting [PIN⁺] NPK300 strain was cured of [PSI⁺] by Rnq1100 (Fig. 3C). Again, Rnq1-GFP fluorescence microscopy and SDD-AGE analyses confirmed the [PIN⁺] or [pin^–] state in these strains (see Fig. S3 in the supplemental material). These results clearly show that the [PIN⁺] state is a prerequisite for Rnq1100's ability to eliminate [PSI⁺], suggesting a possible involvement of the Rnq1 prion in the maintenance of [PSI⁺] (discussed below).

Effect of [PIN⁺] strain differences on [PSI⁺] elimination by Rnq1100. [PIN⁺] strains are also distinguished by their fluorescent pattern of Rnq1-GFP foci (mostly single large fluorescent dots versus multiple small fluorescent dots per cell). To examine whether the [PIN⁺] strain difference affected Rnq1100's ability to cure [PSI⁺], [PSI⁺] strains were generated from five [psi^–] [PIN⁺] variants (L1749, high m.d.; L1943, low s.d.; L1945, medium s.d.; L1767psi^–, high s.d.; L1953, very high s.d.; kind gifts from S. Liebman) by overproducing Sup35's prion (NM) domain. Since most of these [PIN⁺] variants are known to destabilize weak [PSI⁺] (6), we isolated three independent [PSI⁺] variants, one weak [PSI⁺] and two strong [PSI⁺], from each [PIN⁺] strain and examined them. These variants were transformed with the rnq1100 plasmid; without exception, all the transformants became [psi^–] (data not shown). These findings indicate that Rnq1100 cures [PSI⁺] independently of the presence of different [PIN⁺] variants.

Rnq1100 is poisonous to [URE3] prions in the [PIN⁺] state. [URE3] is a prion form of Ure2 (43), which is a regulator of nitrogen metabolism (24). We examined whether or not Rnq1100 is inhibitory to [URE3] by using the test strains developed by Reed Wickner and colleagues (4). Ure2 binds to the transcription factor Gln3 and negatively regulates a range of downstream factors, including the allantoate permease Dal5. In the test strains, the ADE2 gene is placed under the control of the DAL5 promoter (Fig. 4A). An active Ure2 makes such a strain Ade^– and red on YPD. Therefore, [URE3] clones are white and [ure-o] (indicating the absence of [URE3]) clones are red (Fig. 4B). In this assay, upon transformation with the rnq1100-expressing plasmid, [URE3] [pin^–] cells (NPK302) remained white whereas [URE3] [PIN⁺] cells (NPK304) turned red (Fig. 4B). The resulting red phenotype remained unchanged upon segregation of the plasmid from the transformant (see Fig. S1B in the supplemental material), suggesting that Rnq1100 is inhibitory to [URE3] in the [PIN⁺] background. This was confirmed with a [pin^–] derivative of NPK304 in which the chromosomal RNQ1 gene was knocked out by rnq1::URA3 (NPK346; see Materials and Methods) and thus was rendered insensitive to Rnq1100 (as indicated by white coloring; Fig. 4B). Moreover, it was also confirmed that when a [pin^–] state of NPK302 was converted to [PIN⁺] by Rnq1 overexpression by use of the strong constitutive GPD promoter, the resulting [PIN⁺] cells (NPK435) became curable of [URE3] by Rnq1100 (Fig. 4B). The [PIN⁺] or [pin^–] state of the test strains was confirmed by Rnq1-GFP fluorescence microscopy and SDD-AGE analyses (see Fig. S3 in the supplemental material).

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FIG. 4. [PIN+]-dependent elimination of [URE3] by Rnq1100. (A) Schematic representation of the ADE2 color assay designed to detect the presence of the [URE3] prion (4). Ure2, the protein determinant of [URE3], is a negative regulator of the Gln3 transcription factor that activates the DAL5 promoter. The indicated reporter contained the ADE2 gene under the control of the DAL5 promoter. A [ure-o] strain had active Ure2, which prevented ADE2 transcription and gave rise to red colonies on YPD (top). A [URE3] strain had inactive Ure2, which allowed for ADE2 transcription and gave rise to white colonies on YPD (bottom). (B) [URE3] elimination by Rnq1100 in the presence of [PIN+]. Two sets of isogenic [PIN+]/[pin–] strains harboring [URE3] were transformed with empty vector (vec.) and the rnq1100 plasmid, and the [URE3] state was examined according to colony color. Strains: NPK304, [URE3] [PIN+] RNQ1+; NPK346, [URE3] [pin–] rnq1::URA3 derived from NPK304; NPK302, [URE3] [pin–] RNQ1+; NPK435, [URE3] [PIN+] RNQ1+ derived from NPK302. [URE3] and [ure-o] control cells results are also shown.

Effect of Rnq1100 on the polyQ aggregate and toxicity. Expansion of polyQ tracts in certain proteins is responsible for neurodegenerative disorders (31). Huntington's disease, one of the best-known polyQ disorders, is caused by an expansion of a polyQ stretch in huntingtin to more than 37 glutamines, leading to amyloid-like fibers similar to yeast prions (31). Sherman and coworkers have found that polyQ (103Q) aggregation is toxic to yeast in the [PIN⁺] strain and constructed a yeast-based assay for determining polyQ toxicity (see reference 25 and Fig. S4A in the supplemental material). Using this system, we found that Rnq1100 appeared to reduce the toxicity (see Fig. S4A in the supplemental material) as well as the formation of 103Q-GFP foci (see Fig. S4B in the supplemental material).

It is known that 103Q-related toxicity and 103Q-GFP aggregates disappear upon nullification of the chromosomal RNQ1 gene (25). This phenotype of polyQ aggregates is in sharp contrast to that seen with yeast prions, i.e., [PSI⁺] and [URE3], that have not been cured by rnq1 deletion. Keeping this in mind, we examined the [PIN⁺] state by monitoring Rnq1 aggregates using SDD-AGE. Western blot analysis showed that Rnq1 forms SDS-stable polymers in 103Q/Rnq1100-expressing cells, as it does in 103Q-expressing cells (see Fig. S4C in the supplemental material), although the level of Rnq1 expression was slightly reduced in the presence of Rnq1100 (see Fig. S4D in the supplemental material). These observations suggest that Rnq1100 does not affect [PIN⁺] itself.

[PIN⁺] is not affected by Rnq1100. Following the observation reported above, we extensively examined the [PIN⁺] state in Rnq1100-expressing cells. GFP fusions to Rnq1, Rnq1100, and two truncated Rnq1 mutants, Rnq179 and Rnq1119 (as controls), were expressed in cells in the presence or absence of [PSI⁺] and/or [PIN⁺] prions. Rnq1-GFP formed foci with no diffuse fluorescence in [PIN⁺] strains, whereas it was diffusely distributed in [pin^–] strains (Fig. 5A). Surprisingly, Rnq1100-GFP formed s.d. foci not only in [PIN⁺] but also in [pin^–] cells, although a bright diffuse background was also visible in the latter, representing partially soluble Rnq1100-GFP in the [pin^–] strain (Fig. 5A). In these experiments, [PSI⁺] or [psi^–] did not affect the fluorescence pattern. Interestingly, Rnq179-GFP and Rnq1119-GFP exhibited distinct patterns; although both of them were diffusely distributed in [pin^–] cells, the latter, but not the former, displayed strong s.d. foci in [PIN⁺] cells (Fig. 5A). These findings suggest that Rnq1100 is able to self-aggregate in [pin^–] cells and to join preexisting Rnq1 aggregates in [PIN⁺] and that the ability to join preexisting Rnq1 aggregates is somehow eliminated in Rnq179 but not in Rnq1119.

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FIG. 5. Efficiency of Rnq1100 to self-aggregate or join preexisting Rnq1 aggregates. (A) Fluorescence microscopy reveals the presence or absence of foci for GFP fusions to Rnq1 and its truncation mutants in [pin–] and [PIN+] cells. Plasmids carrying the indicated GFP fusions under the control of the CUP1 promoter were transformed into [pin–] or [PIN+] strains, and each transformant was grown in SC broth containing 50 µM CuSO4. Strains: NPK51, [psi–] [pin–]; NPK50, [PSI+] [pin–]; NPK200, [psi–] [PIN+]; and NPK294, [PSI+] [PIN+]. (B) SDS-stable Rnq1100 polymers formed in [PIN+] cells. [PIN+] or [pin–] cells expressing Rnq1100-GFP (corresponding to labels a to d in panel A) were harvested and lysed, and the same amounts of protein from lysates were analyzed by SDD-AGE (1% SDS). Rnq1-GFP was detected by immunoblotting using polyclonal rabbit anti-GFP antibody. (C) Distribution of Rnq1100 between supernatant (S) and pellet (P) after centrifugation at 100,000 x g from whole lysates (W) from the indicated strains (corresponding to labels a to d in panel A). Rnq1100 and Pgk1 (control) were probed by anti-Rnq1 antibody (top panel) and anti-Pgk1 antibody (bottom panel), respectively. The presence of the majority of Pgk1 in the soluble fraction indicates that soluble and aggregated proteins were successfully separated.

Rnq1100-GFP aggregates formed in [pin^–] and [PIN⁺] cells were investigated by SDD-AGE. As shown in Fig. 5B, Rnq1100-GFP aggregates from [PIN⁺] displayed SDS-resistant polymers, while those from [pin^–] were easily solubilized with 1% SDS independently of the [PSI⁺] state. Using differential centrifugation of cell homogenates, we found that more than half of the Rnq1100 protein was associated with 100,000 x g pellets even in [pin^–] cells. These findings indicate that Rnq1100 exists in an SDS-sensitive self-aggregated form in [pin^–] (Fig. 5C).

Direct interaction between Rnq1 and Rnq1100 in the absence of [PIN⁺]. When Rnq1-YFP and Rnq1100-CFP were coexpressed in [PIN⁺] (NPK294) cells from the CUP1 promoter, they formed strong s.d. foci against a dark (i.e., no diffuse fluorescence) background and these aggregates completely colocalized (Fig. 6A). In [pin^–] (NPK50) cells, they formed small multiple aggregates against a light, diffusely distributed fluorescent background, and again these aggregates completely colocalized (Fig. 6A). These findings suggest that Rnq1 and Rnq1100 are able to coaggregate independently of [PIN⁺] or [pin^–]. It is likely that Rnq1 joins preexisting Rnq1100 aggregates in [pin^–], since Rnq1-YFP alone did not form significant foci in [pin^–] cells (or only a small percentage of cells, if any, exhibited small dots) but formed punctate dots in about half of the [pin^–] cells upon coexpression of Rnq1100 (lacking CFP) (data not shown). Consistently, centrifugation analysis of homogenates from [pin^–] cells expressing Rnq1 alone or with Rnq1100 revealed that pelletable Rnq1 aggregation is enhanced by coexpression of Rnq1100 (Fig. 6B).

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FIG. 6. Fluorescence microscopy and immunoprecipitation analysis of Rnq1/Rnq1100 complexes. (A) Colocalization of Rnq1 and Rnq1100 as visualized by fluorescent foci. The [PSI+] [pin–] strain (NPK50) and [PSI+] [PIN+] strain (NPK294) were transformed with pRS415CUP1p-Rnq1100-CFP and pRS413CUP1p-Rnq1-YFP. These transformants were grown to early log phase and supplemented with 50 µM CuSO4 followed by growth for 6 h. Shown are DIC (differential interference contrast) images and fluorescent images representing Rnq1100-CFP (green) and Rnq1-YFP (red) and merged images (yellow shows colocalization). (B) Distribution of Rnq1 between supernatant (S) and pellet (P) after centrifugation at 100,000 x g of whole lysates (W) from [pin–] cells (NPK50) in the presence or absence of Rnq1100. NPK50 cells transformed with empty vector or Rnq1100-expressing vector were analyzed by centrifugation, as shown in Fig. 5C. (C) Immunoprecipitation of Rnq1100 complexes. [pin–] (NPK50) and [PIN+] (NPK294) cells carrying pRS415CUP1p-Rnq1100-GFP were harvested as described for panel A, and cell lysates were subjected to immunoprecipitation using a mouse monoclonal anti-GFP antibody or no antibody (N). Associated proteins were resolved by SDS-PAGE and detected by Western blotting using an anti-Rnq1 antibody and an anti-Sis1 antibody. (D) Immunoprecipitation of Rnq1 complexes. [pin–] (NPK50) and [PIN+] (NPK294) cells carrying pRS415CUP1p-Rnq1100 and pRS413CUP1p-Rnq1-GFP were harvested and subjected to immunoprecipitation analysis as described for panel C. (E) Immunoprecipitation of Sis1 complexes. [pin–] (NPK50) and [PIN+] (NPK294) cells carrying pRS415CUP1p-Rnq1100 were harvested and subjected to immunoprecipitation analysis using anti-Sis1 antibody and preimmune (PI) antibody as described for panel C.

Finally, a direct association between Rnq1 and Rnq1100 was confirmed by immunoprecipitation analysis. First, Rnq1100-GFP was expressed in [pin^–] (NPK50) and [PIN⁺] (NPK294) cells, and total Rnq1100 protein was immunoprecipitated with anti-GFP antibody. The associated proteins were analyzed by SDS-PAGE followed by blotting with anti-Rnq1 antibody (Fig. 6C). Rnq1 associated with Rnq1100-GFP in both [pin^–] and [PIN⁺] strains. Similarly, when Rnq1-GFP and Rnq1100 were coexpressed and total Rnq1 protein was immunoprecipitated by anti-GFP antibody, Rnq1100 associated with Rnq1-GFP in both [pin^–] and [PIN⁺] strains (Fig. 6D).

The interaction of Rnq1 and Rnq1100 with Sis1 depends on the presence of [PIN⁺]. Previous studies showed that Sis1 coimmunoprecipitates with Rnq1 from cell lysates and that the association between Sis1 and Rnq1 depends on the presence of [PIN⁺] (38). Sis1 is a member of the Hsp40 chaperone family and is required for [PIN⁺] maintenance (38), probably through catalyzing generation of [PIN⁺] seeds (2). Blots of Rnq1- and Rnq1100-associated proteins from the experiments reported above were analyzed with an anti-Sis1 antibody. In both cases, Sis1 associated with Rnq1/Rnq1100 in [PIN⁺] cells but not in [pin^–] cells (Fig. 6C and D). When total Sis1 protein was immunoprecipitated by anti-Sis1 antibody from lysates of [pin^–] (NPK50) or [PIN⁺] (NPK294) cells expressing Rnq1100, both Rnq1 and Rnq1100 associated with Sis1 in the presence of [PIN⁺] (Fig. 6E). These findings confirm that the Rnq1/Rnq1100 coaggregate in [PIN⁺] represents a prion form of Rnq1 and that Rnq1/Rnq1100 coaggregates in [pin^–] are structurally distinct from the [PIN⁺] aggregate and are not recognized by Sis1.

Cellular colocalization of Rnq1100 and Sup35NM aggregates. It has been reported that Sup35NM aggregates that appear during [PSI⁺] induction in [psi^–] strains always colocalize with [PIN⁺] aggregates consisting of full-length Rnq1; however, Sup35NM aggregates that are established in [PSI⁺] do not always colocalize with [PIN⁺] aggregates (11). We independently confirmed the observation by fluorescence microscopic analysis using Rnq1-CFP and NM-YFP induced in [psi^–] and [PSI⁺] strains (Fig. 7A). Then, we further investigated whether the Rnq1100 aggregates colocalize with either newly induced or already established [PSI⁺] Sup35NM aggregates upon coinduction of Rnq1100-CFP and NM-YFP synthesis in [psi^–] and [PSI⁺] strains. The data show that all NM-YFP foci colocalized perfectly with Rnq1100-CFP foci in [psi^–] cells (Fig. 7B). In [PSI⁺] cells, although the NM-YFP foci disappeared 24 h after induction of Rnq1100-CFP (data not shown), Rnq1100-CFP and NM-YFP foci occasionally, but not always, colocalized 6 h after the induction (Fig. 7B). These findings are interpreted as indicating a transient association of Sup35NM with Rnq1/Rnq1100 coaggregates.

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FIG. 7. Conditional colocalization of Sup35NM with Rnq1 or Rnq1100 aggregates. (A) The [psi–] [PIN+] strain (NPK200 [upper row]) and [PSI+] [PIN+] strain (NPK294 [lower two rows]) were transformed with pRS415CUP1p-Rnq1-CFP and pRS413CUP1p-NM-YFP. These transformants were grown to early log phase and supplemented with 50 µM CuSO4 followed by growth for 6 h. Shown are DIC (differential interference contrast) images and fluorescent images of CFP (green) and YFP (red) and merged images. (B) The same strains as described above were transformed with pRS415CUP1p-Rnq1100-CFP and pRS413CUP1p-NM-YFP and examined for the presence of all fluorescent foci.

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[PIN⁺] is the prion form of the Rnq1 protein, which has an unknown function. Rnq1's N terminal is non-QN-rich and is thought to be a nonprion domain. On the other hand, Rnq1's C terminal is highly QN-rich and is believed to be sufficient to maintain a heritable [PIN⁺] state and facilitate the de novo appearance of other prions, including [PSI⁺] (41, 42). In this study, however, we found that the N-terminal nonprion domain is not completely dispensable but is important for the proper functioning of [PIN⁺]. Rnq1100, an N-terminal nonprion domain truncation of Rnq1, inhibits the maintenance of other yeast prions, namely, [PSI⁺] and [URE3], as well as huntingtin's polyQ aggregate, in [PIN⁺] cells but not in [pin^–] cells. These findings are interpreted as indicating that the Rnq1 prion is not only involved in the de novo appearance of [PSI⁺] and other prions but is also engaged in the maintenance of these prions and polyQ aggregates.

The C terminal of Rnq1 contains five QN-rich subregions, and at least the first three of these subregions initiated from Met101 are sufficient for [PSI⁺] elimination. Importantly, Met101, the translation start site, is critical for the inhibitory activity of truncated Rnq1. Rnq1 products synthesized from other nearby Met codons, such as Met80 and Met120, are not inhibitory even when these are shorter or longer by only 21 or 19 aa. Therefore, the very specific protein configuration adopted by Rnq1100 is likely required for the activity to eliminate [PSI⁺].

How does Rnq1100 inhibit or affect [PSI⁺], [URE3], and polyQ aggregate? A possible key to the answer to this question might be its strong activity or tendency to self-aggregate or coaggregate with Rnq1 independently of the [PIN⁺] or [pin^–] state. In [PIN⁺], Rnq1100 is completely integrated into preexisting Rnq1 aggregates; coexpressed Rnq1-YFP and Rnq1100-CFP aggregates colocalize, and these coaggregates represent SDS-stable polymers. Despite the impeded propagation of [PSI⁺], [URE3], and polyQ aggregates, the Rnq1 prion form [PIN⁺] itself is not significantly affected by Rnq1100. This means that the Rnq1/Rnq1100 coaggregate in [PIN⁺] cells is a heritable non-Mendelian element. Indeed, Sis1, a member of the Hsp40 chaperone known to associate with the Rnq1 aggregate only when cells are [PIN⁺], was also found in the Rnq1/Rnq1100 coaggregate. One might speculate that the Rnq1/Rnq1100 coaggregate in [PIN⁺] sequesters a component required for [PSI⁺] and [URE3]. One such candidate is Hsp104, which breaks up amyloid filaments to generate prion seeds for efficient prion transmission (19, 26, 30). However, the titration of Hsp104 cannot explain the observed differential influences of Rnq1100 on [PIN⁺] and the other prions.

Recently, Liebman and coworkers constructed a set of Rnq1 truncations (42). These are mostly different from the constructs presented here, except for Rnq1 truncation between positions 133 and 405 (Rnq1132 in our nomenclature and Rnq1-N2 in their nomenclature). Considering both studies, it is likely that Rnq1-133-405 is not inhibitory to other prions and is sufficient to propagate [PIN⁺]. They also noticed, in experiments employing cytoduction, a cytoplasmic mixing technique routinely used to transmit yeast prions, that high levels of [PIN⁺] were transferred with only minimal efficiency to Rnq1-N2 (aa 133-405), which contains the entire presumptive prion domain. In contrast, transfer of [PIN⁺] to Rnq1-N1 (aa 172-405), which lacked the QN-rich subregion I, was quite efficient (42). This apparent contradiction can be explained by assuming that Rnq1-N2 (aa 133 to 405) may, at least in part, harbor the partially disabled activity of [PIN⁺] transmission.

The alternative, more likely explanation would be that the Rnq1/Rnq1100 coaggregate binds to the growing tip of a prion aggregate and blocks its rapid growth, leading to its destabilization and loss (Fig. 8). According to this scenario, in [pin^–] cells, Rnq1100 associates with Rnq1, forming non-SDS-stable nonprion aggregates (Fig. 8A). However, in [PIN⁺] cells, Rnq1100 associates with the Rnq1 prion amyloid and probably undergoes a conformational change, forming SDS-stable prionogenic aggregates, including Sis1 (Fig. 8B). Because Sis1 binds to Rnq1 polymers with Ssa1, a Hsp70-family chaperone protein (38), Rnq1/Rnq1100/Sis1 coaggregates might also contain Ssa1. In accordance with this model, colocalization, though transient, of Sup35NM with Rnq1/Rnq1100 aggregates was observed upon induction of Rnq1100-CFP and NM-YFP fluorescent proteins in [PSI⁺] [PIN⁺] cells (see Fig. 7B). A similar model was proposed to explain the Pnm (from "[PSI⁺] no more") phenotype of Sup35 mutants (9) and the curing of [URE3] upon overexpression of the Ure2 prion domain fusion to GFP (15, 16); this is referred to as the "capping" model (10).

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FIG. 8. The capping model. The effects of Rnq1100 in [pin–] cells (A) and [PIN+] cells (B) are schematically presented.

In sum, our findings strongly favor the capping model to explain the curing of preexisting [PSI⁺] and [URE3] prions by the Rnq1/Rnq1100 prion amyloid and to explain how the N-terminal non-QN-rich (nonprion) region of Rnq1 plays an essential role for modulating the C-terminal prion domain. Regarding the latter prediction, it is worth mentioning that Sup35 prion domain aggregates more quickly than the complete Sup35 protein both in vivo and in vitro, suggesting that the C domain of Sup35 regulates the highly reactive Sup35 prion domain (20), as now proposed for Rnq1 and [PIN⁺]. Rnq1100 serves as a strong inhibitor of yeast prions (with the exception of [PIN⁺]) and will prove useful for elucidating a network of intermolecular interactions required for the [PIN⁺] "inducer" phenotype as well as heterologous prion maintenance.

 
We thank R. B. Wickner, Y. O. Chernoff, S. Liebman, K. Ito, and M. Y. Sherman for the gift of strains and plasmids, C. G. Crist for critical reading of the manuscript and valuable comments, and K. Oishi for editing the manuscript.

This work was supported in part by grants from The Ministry of Education, Sports, Culture, Science and Technology of Japan (MEXT) and from the BSE Control Project of the Ministry of Agriculture, Forestry and Fisheries of Japan.

 
* Corresponding author. Mailing address: Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5307. Fax: 81-3-5449-5415. E-mail: nak{at}ims.u-tokyo.ac.jp

Published ahead of print on 10 March 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, May 2008, p. 3313-3323, Vol. 28, No. 10
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