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

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.

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 rnq1Δ75; P4 (5′-TTTGGATCCAATGGCAGACTCTAAGGG-3′) and P2 for rnq1Δ79; P5 (5′-AAAGGATCCAATGACACACTCATCAAAT-3′) and P2 for rnq1Δ100; P6 (5′-TTTGGATCCAATGTCAATGCTAAGTGG-3′) and P2 for rnq1Δ119; P7 (5′-TTTGGATCCAATGCTAAGTGGTTCTGG-3′) and P2 for rnq1Δ121; P8 (5′-TTTGGATCCAATGGGTGCTTCCGGCCTG-3′) and P2 for rnq1Δ132; 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 rnq1Δ79, P5 and P15 for rnq1Δ100, and P6 and P15 for rnq1Δ119.

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.

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⁺].

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., Δ75, Δ79, Δ100, Δ119, Δ121, and Δ132) (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 Rnq1Δ132 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 Rnq1Δ100, 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 Rnq1Δ100 product (data not shown). When the presumed AUG codon for Met101 was changed to UUG, the resulting construct Rnq1Δ100ΔM failed to synthesize Rnq1Δ100 (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 Rnq1Δ100 (see Fig. S2 in the supplemental material).

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 Rnq1Δ100 (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 rnq1Δ100 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 rnq1Δ100 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 Rnq1Δ100 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.

The most likely candidate affecting Rnq1Δ100-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 Rnq1Δ100, 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 Rnq1Δ100, 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 Rnq1Δ100 and the [PIN⁺] state, the [PSI⁺] [PIN⁺] strain NPK294 (ade1-14), which is cured of [PSI⁺] by Rnq1Δ100, 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 Rnq1Δ100 (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 Rnq1Δ100 (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 Rnq1Δ100'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 Rnq1Δ100.

[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 Rnq1Δ100'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 rnq1Δ100 plasmid; without exception, all the transformants became [psi⁻] (data not shown). These findings indicate that Rnq1Δ100 cures [PSI⁺] independently of the presence of different [PIN⁺] variants.

Rnq1Δ100 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 Rnq1Δ100 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 rnq1Δ100-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 Rnq1Δ100 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 Rnq1Δ100 (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 Rnq1Δ100 (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).

Effect of Rnq1Δ100 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 Rnq1Δ100 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/Rnq1Δ100-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 Rnq1Δ100 (see Fig. S4D in the supplemental material). These observations suggest that Rnq1Δ100 does not affect [PIN⁺] itself.

[PIN⁺] is not affected by Rnq1Δ100.

Following the observation reported above, we extensively examined the [PIN⁺] state in Rnq1Δ100-expressing cells. GFP fusions to Rnq1, Rnq1Δ100, and two truncated Rnq1 mutants, Rnq1Δ79 and Rnq1Δ119 (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, Rnq1Δ100-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 Rnq1Δ100-GFP in the [pin⁻] strain (Fig. 5A). In these experiments, [PSI⁺] or [psi⁻] did not affect the fluorescence pattern. Interestingly, Rnq1Δ79-GFP and Rnq1Δ119-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 Rnq1Δ100 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 Rnq1Δ79 but not in Rnq1Δ119.

Rnq1Δ100-GFP aggregates formed in [pin⁻] and [PIN⁺] cells were investigated by SDD-AGE. As shown in Fig. 5B, Rnq1Δ100-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 Rnq1Δ100 protein was associated with 100,000 × g pellets even in [pin⁻] cells. These findings indicate that Rnq1Δ100 exists in an SDS-sensitive self-aggregated form in [pin⁻] (Fig. 5C).

Direct interaction between Rnq1 and Rnq1Δ100 in the absence of [PIN⁺].

When Rnq1-YFP and Rnq1Δ100-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 Rnq1Δ100 are able to coaggregate independently of [PIN⁺] or [pin⁻]. It is likely that Rnq1 joins preexisting Rnq1Δ100 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 Rnq1Δ100 (lacking CFP) (data not shown). Consistently, centrifugation analysis of homogenates from [pin⁻] cells expressing Rnq1 alone or with Rnq1Δ100 revealed that pelletable Rnq1 aggregation is enhanced by coexpression of Rnq1Δ100 (Fig. 6B).

Finally, a direct association between Rnq1 and Rnq1Δ100 was confirmed by immunoprecipitation analysis. First, Rnq1Δ100-GFP was expressed in [pin⁻] (NPK50) and [PIN⁺] (NPK294) cells, and total Rnq1Δ100 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 Rnq1Δ100-GFP in both [pin⁻] and [PIN⁺] strains. Similarly, when Rnq1-GFP and Rnq1Δ100 were coexpressed and total Rnq1 protein was immunoprecipitated by anti-GFP antibody, Rnq1Δ100 associated with Rnq1-GFP in both [pin⁻] and [PIN⁺] strains (Fig. 6D).

The interaction of Rnq1 and Rnq1Δ100 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 Rnq1Δ100-associated proteins from the experiments reported above were analyzed with an anti-Sis1 antibody. In both cases, Sis1 associated with Rnq1/Rnq1Δ100 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 Rnq1Δ100, both Rnq1 and Rnq1Δ100 associated with Sis1 in the presence of [PIN⁺] (Fig. 6E). These findings confirm that the Rnq1/Rnq1Δ100 coaggregate in [PIN⁺] represents a prion form of Rnq1 and that Rnq1/Rnq1Δ100 coaggregates in [pin⁻] are structurally distinct from the [PIN⁺] aggregate and are not recognized by Sis1.

Cellular colocalization of Rnq1Δ100 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 Rnq1Δ100 aggregates colocalize with either newly induced or already established [PSI⁺] Sup35NM aggregates upon coinduction of Rnq1Δ100-CFP and NM-YFP synthesis in [psi⁻] and [PSI⁺] strains. The data show that all NM-YFP foci colocalized perfectly with Rnq1Δ100-CFP foci in [psi⁻] cells (Fig. 7B). In [PSI⁺] cells, although the NM-YFP foci disappeared 24 h after induction of Rnq1Δ100-CFP (data not shown), Rnq1Δ100-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/Rnq1Δ100 coaggregates.

[Supplemental material]

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