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Originally published In Press as doi:10.1074/jbc.M500390200 on April 14, 2005 Originally published In Press as doi:10.1074/jbc.M500390200 on April 11, 2005

J. Biol. Chem., Vol. 280, Issue 24, 22809-22818, June 17, 2005
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Modulation of Prion-dependent Polyglutamine Aggregation and Toxicity by Chaperone Proteins in the Yeast Model*

Kavita C. Gokhale{ddagger}, Gary P. Newnam{ddagger}, Michael Y. Sherman§, and Yury O. Chernoff{ddagger}

From the {ddagger}School of Biology and Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332 and the §Department of Biochemistry, Boston University Medical School, Boston, Massachusetts 02118

Received for publication, January 12, 2005 , and in revised form, April 6, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In yeast, aggregation and toxicity of the expanded polyglutamine fragment of human huntingtin strictly depend on the presence of the endogenous self-perpetuating aggregated proteins (prions), which contain glutamine/asparagine-rich domains. Some chaperones of the Hsp100/70/40 complex, modulating propagation of yeast prions, were also reported to influence polyglutamine aggregation in yeast, but it was not clear whether they do it directly or via affecting prions. Our data show that although some chaperone alterations indeed act on polyglutamines via curing endogenous prions, other alterations decrease size and ameliorate toxicity of polyglutamine aggregates without affecting prion propagation. Therefore, the role of yeast chaperones in polyglutamine aggregation and toxicity is not restricted only to their effects on the endogenous prions. Moreover, chaperone interactions with prion and polyglutamine aggregates appear to be of a highly specific nature. One and the same chaperone alteration, substitution A503V in the middle region of the chaperone Hsp104, exhibited opposite effects on one of the endogenous prions ([PSI+], the prion form of Sup35) and on polyglutamines, increasing aggregate size and toxicity in the former case and decreasing them in the latter case. On the other hand, different members of a single chaperone family exhibited opposite effects on one and the same type of aggregates: excess of the Hsp40 chaperone Ydj1 increased polyglutamine aggregate size and toxicity, whereas excess of the other Hsp40 chaperone, Sis1, decreased them. As many stress-defense proteins are conserved between yeast and mammals, these data shed light on possible mechanisms modulating polyglutamine aggregation and toxicity in mammalian cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Expansion of glutamine repeats (poly-Q)1 in certain proteins is responsible for neurodegenerative disorders. The hallmark of poly-Q diseases is the formation of insoluble cytosolic and nuclear inclusions (1). Huntington disease is one of the best known poly-Q disorders (2). It is caused by an expansion of the poly-Q stretch in the essential protein called huntingtin (Htt) to more than 37 amino acids. The length of the poly-Q stretch inversely correlates with the time of onset of the disease and the time of formation of Htt aggregates (3). The role of various types of aggregates in cell toxicity remains a matter of debate (4). Recent models propose that toxicity of poly-Q Htt arises from sequestration of certain essential proteins by Htt aggregates (5–7). The poly-Q stretch is located within the N-terminal (exon 1) region of Htt, which is involved in numerous protein-protein interactions (8). The N-terminal fragment of Htt with poly-Q extensions, when expressed in mice, aggregated and was sufficient to cause Huntington disease-like neurodegeneration (9, 10). This indicates that at least some parameters of poly-Q associated aggregation and toxicity could be reproduced in the experimental assays using only N-terminal poly-Q expanded fragments of Htt.

Several models for studying poly-Q aggregation and toxicity have been developed by using simple organisms such as fruit flies (11), nematode Caenorhabditis elegans (12), and yeast Saccharomyces cerevisiae. Yeast assays usually employ short constructs derived from the poly-Q expanded exon 1 of Htt. Although poly-Q aggregation has been readily observed in yeast (13–17), a yeast-based assay for poly-Q toxicity has not been available until recently. It turned out that efficient cytoplasmic aggregation and toxicity of the chimeric protein, containing the N-terminal region of Htt with expanded poly-Q stretch, fused to green fluorescent protein (GFP), could be detected only in the yeast strains bearing an endogenous yeast QN-rich protein, Rnq1, in its prion form, called [RNQ+], or [PIN+] (18). In the absence of a prion, poly-Q aggregates are rarely found, and toxicity is not seen.

Rnq1, a protein of unknown function, is one of several known yeast proteins containing QN-rich prion domains (19, 20). This group also includes Sup35, Ure2, and New1. Yeast prions are self-perpetuating amyloid-like protein aggregates, which are thought to propagate via a nucleated polymerization process, mediated by interactions between prion domains. By itself, the Rnq1 prion ([PIN+]) is not harmful to yeast cells, but it appears that QN-rich aggregates of Rnq1 "seed" aggregation of the heterologous poly-Q protein, leading to toxicity. The QN-rich domain of New1 in its prion form has also been shown to facilitate aggregation of a poly-Q construct originated from the mammalian mutant ataxin-3 involved in Machado-Joseph disease, although cell toxicity of that protein was not detected in yeast (21). Further analysis has demonstrated that [PIN+]-dependent toxicity of the Htt-derived poly-Q construct in yeast is associated with a defect of endocytosis, possibly via sequestration of some components of the vesicle-assembly machinery by poly-Q aggregates (22). As subcellular localization of Htt in mammalian cells has led to a suggestion that it may play a role in vesicle trafficking (23), it is possible that the yeast model reflects certain features of cell toxicity that are relevant to mammalian Huntington disease.

As many studies suggest a critical relationship between poly-Q aggregation and cell toxicity, chaperone proteins counteracting aggregation are well positioned as likely antagonists of the poly-Q disorders. Indeed, some chaperones of the evolutionary conserved Hsp70 and Hsp40 families counteracted poly-Q aggregation in vitro (14) and antagonize poly-Q aggregation and, in some cases, toxicity in vivo, as seen in Drosophila and cultured mammalian cells (24–27). In yeast, aggregates of the heat-damaged proteins are solubilized and refolded by the chaperone complex, including Hsp104, Hsp70, and Hsp40 (28). Overproduction of some Hsp70 and Hsp40 proteins (14), simultaneous depletion of the Ssa1 and Ssa2 proteins of the Hsp70 family (18), or a point mutation in the Hsp40 protein Ydj1 (18) were shown to counteract poly-Q aggregation in yeast cells. Hsp104 was shown to be essential for poly-Q aggregation in yeast, whereas its overproduction eliminated or reduced poly-Q aggregation at least in some assays (13, 15, 16, 29). However, one concern with most of these experiments is that they did not take into account the prion status of yeast cells. As endogenous yeast prions promote poly-Q aggregation (18), and inactivation or overproduction of yeast chaperones of the Hsp104/Hsp70/Hsp40 complex is known to affect prion propagation (19, 20), chaperones could influence poly-Q aggregates indirectly via alterations in propagation of the endogenous yeast prions. For example, Hsp104 is essential for propagation of [PIN+] (30), as well as other yeast QN-rich prions (31, 32). It is therefore likely that elimination of Hsp104 counteracted poly-Q aggregation simply by eliminating initial [PIN+] "seeds" (18).

Here, we have systematically examined the effects of various yeast chaperones of the Hsp104/Hsp70/Hsp40 complex on poly-Q aggregation and toxicity in the series of isogenic yeast strains that differ only by prion composition. Our data show that although some chaperone alterations certainly act on poly-Q aggregates indirectly by modulating prion propagation, other alterations appear to influence poly-Q without affecting endogenous yeast prions. Moreover, at least one Hsp104 alteration exhibited differential effects on toxicity of poly-Q aggregates and endogenous prion, and various members of the Hsp70 and Hsp40 families differed from each other in regard to their effects on poly-Q aggregation and toxicity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains—The S. cerevisiae strains used in this study were derivatives of 74-D694, MATa ade1–14 his3 leu2 trp1 ura3 (31). "Strong" [PSI+] derivative OT56, "weak" [PSI+] derivative OT55 (33), and [psi–] derivative OT60 (34) also contain the [PIN+] prion. The [psi– pin–] strain GT17 (34) contains no prions. The [PSI+ pin–] strain GT490 was OT56, cured of [PIN+] by expressing the dominant-negative Hsp104 derivative Hsp104-KT218,620 (35). For this purpose, OT56-derived colonies that retained [PSI+] after pulse induction of Hsp104-KT218,620 expression were then cured of [PSI+] by excess wild-type Hsp104 and checked for retention of [PIN+] (see the description of the [PIN+] assay below). As wild-type Hsp104 usually does not cure [PIN+], isolates becoming [pin–] after excess Hsp104 treatment were identified as originating from the [PSI+ pin–] derivatives. Lack of the aggregated (prion) form of Rnq1 in these derivatives was then confirmed by a differential centrifugation assay (36). The [psi– pin–] strain GT234 (MAT{alpha} ade1–14 his3 leu2 trp1 ura3 lys2) (35) was used as a tester strain for the [PIN+] assay (see below). Yeast cultures were grown at 30 °C unless otherwise noted.

Plasmids—Poly-Q constructs contained the N-terminal portion of the Htt exon 1 with a poly-Q stretch of either 25 residues (Q25 control) or 103 residues (Q103), fused to GFP, and expressed under control of the galactose-inducible PGAL promoter. The high copy 2-µm DNA-based URA3 poly-Q-expressing plasmids, originated from pYES2 (Invitrogen), were described previously (18). The TRP1 plasmids, containing the identical poly-Q constructs and based on pYES (Invitrogen), were constructed by A. Meriin.

The centromeric (CEN) HIS3-based series of PGAL-HSP104 constructs, kindly provided by E. Schirmer and S. Lindquist, included the plasmid pGALSc104 with wild-type HSP104 open reading frame (WT Hsp104) and its mutant derivatives containing single amino acid substitutions A503V or A509D (37). Another series of the CEN PGAL-HSP104 constructs, bearing the URA3 marker, included the plasmid pYSGal-HSP104 with the wild-type HSP104 open reading frame (WT Hsp104; see Ref. 38 and references therein) and the pRS316Gal-based plasmids (35) with dominant-negative mutations in nucleotide binding domains (NBDs): single amino acid substitutions K218T and K620T and double amino acid substitution K218T, K620T (Hsp104-KT). The PGAL-HSP104 clone with the dominant-negative substitution K302N (Library Hsp104) was isolated from the pRS316Gal-based yeast cDNA library, obtained from A. Bretscher (39).

The CEN HIS3 plasmid pRSYDJ1 and CEN TRP1 plasmid pTVSIS1, bearing, respectively, the YDJ1 and SIS1 open reading frames under the strong constitutive PGPD promoter, were kindly provided by S. Lindquist (see Ref. 13 and references therein). The CEN vectors pRS315Ydj1 and pRS315Sis1, bearing, respectively, YDJ1 and SIS1 genes with their endogenous promoters, were a kind gift of D. Cyr. The following plasmids were also used for screening the other Hsps in regard to their effects on poly-Q toxicity: pMC3, carrying PGAL-HSP82 (33); p2UGPD-HSP26, carrying PGPD-HSP26 (gift of S. Lindquist); pRS316K-SSA1, carrying PSSA1-SSA1, and pTEF-SSA1, carrying PTEF-SSA1 (gift of E. Craig; see Ref. 40 and references therein); pGAL-SSA1, carrying PGAL-SSA1 (33); pN2 and pC211, carrying PSSA2-SSA2 and PSSA2-SSA1, respectively (gift of D. Masison; see Ref. 41); pYCL1-GAL-SSA3 and pYCL1-GAL-SSA4, carrying PGAL-SSA3 and PGAL-SSA4, respectively (40); and pRS316GAL-SSB1, carrying PGAL-SSB1 (38).

The CEN LEU2-based plasmid with the PSUP35-SUP35NM-GFP fusion construct (42) and the high copy number URA3 LEU2-d plasmid pEMBL-yex-SUP35-{Delta}3ATG, expressing SUP35C from the endogenous PSUP35 promoter (43), have been described previously. The high copy LEU2 plasmid with the complete SUP35 gene expressed from its own promoter, pSTR7, and the empty control plasmid YEp13 (see Ref. 33 and references therein) were used for [PIN+] assay. CEN plasmids pRS316GAL (URA3), pLA1 (HIS3), pFL39 (TRP1) (Ref. 35 and references therein), pYCL1 (40 and references therein), and high copy URA3 LEU2-d plasmid pEMBL-yex (Ref. 43 and references therein) were used as empty controls wherever needed.

Media—Standard procedures and media were used for yeast transformation and phenotypic assays (see Ref. 44). The non-inducing media contained 2% glucose (Glu). To induce the PGAL promoter, glucose was substituted with 2% galactose (Gal) in the solid media or 2% of each galactose and raffinose (Gal+Raf) in the liquid media. Liquid cultures were grown with at least a 1/5 liquid/flask volumetric ratio on the shaker at 225 rpm.

Plate Assay for Cytotoxicity—To assess cytotoxicity qualitatively, yeast transformants were patched on the Glu medium selective for the plasmid(s), and velveteen replica plated onto the Gal medium of the same composition, to induce the PGAL promoter. This was followed by the second passage on Gal medium via either velveteen replica plating or spotting with a spotter from Replicatech. Differences in growth are better seen after the second passage. At least eight independent transformants were tested in each case for each strain/plasmid combination. All or the vast majority of them did usually show the same results. Typical representatives are shown on the figures.

Quantitative Assay for Cytotoxicity—To measure cytotoxicity quantitatively, yeast cultures were pregrown overnight in the synthetic Glu liquid medium, selective for the plasmid(s), and inoculated at 105 cells/ml into the liquid Gal+Raf medium of the same composition, to induce the PGAL constructs. Aliquots were taken after various periods of incubation and plated onto Glu medium selective for the plasmid(s). Colonies were counted after 3 days of incubation at 30 °C. Two platings of each of two independent cultures for each strain/plasmid combination were performed at each time point. Averages of four counts are shown on the figures. Standard deviations never exceeded 35% of the value for a given time point. Note that the Q103-expressing cultures grew much slower in the –Ura-Trp/Gal (or Gal+Raf) medium than in the –Ura-His/Gal (or Gal+Raf) medium. These differences were reproducible in both plate assay and quantitative assay (for example, see Fig. 5) so that accurate comparisons could be made only between cultures growing in the same conditions.

Assays for [PSI+] and [PIN+]—The presence of the prion form of the Sup35 protein ([PSI+]) was detected by its ability to cause readthrough of the ade1–14 (UGA) mutant allele (31). Sup35 is a translation termination factor, and its aggregation results in termination readthrough. The [psi–] ade1–14 strains are unable to grow on –Ade medium and exhibit a dark red color on organic complete (YPD) medium, whereas [PSI+] ade1–14 strains are able to grow on –Ade medium and exhibit a light pink color on YPD medium.

The presence of [PIN+] in the [psi–] background was tested on the basis of the ability of overproduced Sup35 to induce de novo formation of [PSI+] in the [PIN+] but not [pin–] background (36). The [psi–] strains, tested for [PIN+], were mated to the [psi– pin–] strain GT234, bearing the multicopy plasmid pSTR7 with the SUP35 gene. The [PIN+] diploids, in contrast to the [pin–] diploids, grew on –Ade medium after 10–14 days of incubation due to induction of [PSI+]. In each experiment, 12 colonies originating from four independent cultures were tested for [PIN+] loss.

GFP Detection by Fluorescence Microscopy—GFP fluorescence images were taken using the Olympus BX41 microscope with a x100 objective, with a narrow band GFP filter. Cultures were grown over-night in the synthetic Gal+Raf medium selective for the plasmid(s), as described above for quantitative assays. Only cells showing fluorescence were counted and grouped into different classes based on the patterns observed.

Endocytosis Assay—Mid-log phase cells grown in the Gal+Raf medium were concentrated 10 times, incubated for 12 min in the selective Gal+Raf medium with 8 µM FM4-64 (Molecular Probes), washed two times with the same medium without dye, and left at 30 °C with shaking for 45 min (22). FM4-64 staining was detected on the BX41 fluorescence microscope with TRITC filter.

Protein Isolation and Differential Centrifugation—Proteins were isolated from yeast cultures, grown overnight in plasmid-selective synthetic Gal+Raf medium, and analyzed according to the previously published protocol (22) as described below. At least two independent cultures (originated from independent transformants) were checked per each strain/plasmid combination. Cells were collected by centrifugation and disrupted by 10-min vortexing with 600 µM acid-washed glass beads in the lysis buffer (40 mM HEPES (pH 7.5), 50 mM KCl, 1% Triton X-100, 1 mM Na3VO4, 50 mM {beta}-glycerophosphate, 50 mM NaF, 5 mM EGTA, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 1/5 of one proteinase inhibitor tablet, Roche Diagnostics, predissolved in distilled water). Cell debris was removed by centrifugation at 800 x g for 10 min. This step also precipitates some of the Q103 and Q25 protein; the fraction of protein precipitated in these conditions was not influenced by the chaperone composition (not shown). The resulting supernatant was fractionated by centrifugation at 10,000 x g for 10 min (and when specifically mentioned, further subjected to centrifugation at 200,000 x g for 90 min). The pellets were dissolved in the same volume of lysis buffer as respective supernatants. Standard SDS-PAGE loading buffer with 2% SDS was added to both supernatants and dissolved pellets. Samples were denatured by boiling for 5 min, run on SDS-PAGE, transferred to nitrocellulose membrane, and reacted to anti-GFP monoclonal antibody (Sigma) and to the secondary anti-mouse antibody (Sigma). Horseradish peroxidase, tagged to secondary antibody, was detected using Amersham Biosciences ECL detection system. Densitometry was performed on x-ray films by using the Kodak 1D program from a Gel Logic 200 imaging system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of the Yeast Prion [PSI+] and Chaperone Hsp104 on Q103 Toxicity—Our experiments employed a series of the isogenic yeast strains that differed only by prion composition. Prion-containing strains carried [PSI+], the prion isoform of the translation termination factor Sup35, and/or [PIN+], the prion isoform of the Rnq1 protein, the function of which is not known (for review, see Refs. 19, 20, and 36). Both Sup35 and Rnq1 proteins contain QN-rich regions. Strains bearing non-prion isoforms of the same proteins were designated as [psi–] and [pin–], respectively. The poly-Q extended (Q103) N-terminal fragment of human Htt, fused to GFP, is toxic when expressed from the galactose-inducible (PGAL) promoter in the [PIN+] yeast strain but not in the isogenic [pin–] strain lacking a prion (18). We have now demonstrated that [PSI+], the prion form of Sup35, also promotes Q103 toxicity (Fig. 1A). Effects of [PIN+] and [PSI+] on Q103 toxicity appear to be additive (Fig. 1B).



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  		FIG. 1.
Effects of yeast prions and Hsp104 on Q103 toxicity. A, presence of either [PSI+] or [PIN+] prion is required and sufficient for Q103 toxicity. Plates were photographed after 3 days of incubation (second passage) on the medium selective for the poly-Q plasmid (–Ura) and containing galactose to induce the poly-Q constructs. B, additive effects of [PSI+] and [PIN+] on Q103 toxicity. In contrast to the second passage shown in panel A, Q103 causes only a slight inhibition of growth of the [psi– PIN+] and [PSI+ pin–] strains on the 7th day of the first passage on the –Ura/Gal medium but almost completely inhibits growth of the [PSI+ PIN+] strain, indicating that the effects of [PSI+] and [PIN+] on Q103 toxicity are additive. C, excess Hsp104 ameliorates poly-Q toxicity only in [PSI+] cells but not in [psi– PIN+] cells. Strains contained the HIS3-marked Hsp104 overexpressing plasmid (WT Hsp104) or empty control, in addition to a poly-Q construct. Plates were photographed after 7 days of incubation (second passage) on the –Ura-His/Gal medium, which is selective for plasmids and induces poly-Q and Hsp104 constructs. In both A and B, no differences in growth were detected on the Glu medium (data not shown).

 
Overproduction of the yeast chaperone Hsp104 is known to eliminate [PSI+] (31) but not [PIN+] (30). In agreement with these data, we have shown that overproduction of wild-type Hsp104 counteracts Q103 toxicity only in a [PSI+] background and not so when only [PIN+] is present (Fig. 1C).

Mutant Alleles of Hsp104 Counteract Q103 Toxicity—In yeast, toxicity of Q103 appears to be directly associated with its aggregation promoted by an endogenous yeast prion (18). Yeast chaperone protein Hsp104 is required for propagation of the yeast prions [PSI+] (31) and [PIN+] (30). The double Lys-to-Thr substitution at amino acid positions 218 and 620 of Hsp104 (Hsp104-KT; Fig. 2A) inactivates both nucleotide binding domains of Hsp104 (NBD1 and NBD2) and disrupts the function of wild-type Hsp104 in a dominant-negative fashion (31, 45). In contrast to overproduced wild-type Hsp104, mutant Hsp104-KT cures yeast cells not only of [PSI+] (31) but also of [PIN+] (35). As expected, expression of Hsp104-KT counteracts Q103 toxicity not only in the [PSI+] strains but also in the [psi– PIN+] strains, apparently by curing cells of prions (data not shown), thus reproducing an effect of the hsp104 deletion (18). Single dominant-negative NBD mutants KT218 and KT620 also exhibit antitoxicity (data not shown). Surprisingly, we have isolated a presumably wild-type HSP104 clone from a widely used cDNA yeast library, which counteracted Q103 toxicity in both [PSI+] (not shown) and [psi– PIN+] (Fig. 2B) backgrounds. DNA sequencing revealed that this clone of HSP104 actually contained a G-to-C substitution at the nucleotide position 906, resulting in Lys-to-Asn substitution at the amino acid position 302 within NBD1 (Fig. 2A). We have confirmed that the Hsp104-K302N isolate inhibits the thermotolerance function of Hsp104 in a dominant-negative fashion like Hsp104-KT (data not shown) and cures both [PSI+] and [PIN+] prions (Table I), apparently due to inactivation of wild-type Hsp104.


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  		TABLE I
Effects of Hsps on yeast prions in the presence of Q103

Cultures were grown in the conditions inducing both constructs. Aliquots were plated onto non-inducing medium. Resulting colonies were scored for prions as described under "Experimental Procedures."

 



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  		FIG. 2.
Mutant derivatives of Hsp104 antagonize Q103 toxicity. A, domain architecture of the Hsp104 protein and locations of mutations exhibiting an antitoxicity effect. N, M, and C, the N-terminal, middle, and C-terminal regions of Hsp104, respectively. B, the HSP104 clone isolated from the yeast cDNA library (Library Hsp104) reduces Q103 toxicity in the [psi– PIN+] background, in contrast to wild-type (WT) Hsp104. Strains contained the URA3-based Hsp104 plasmid (as shown) or empty control, in addition to the TRP1 poly-Q constructs. Plates were photographed after 3 days of incubation (second passage) on the –Ura-Trp/Gal medium, which is selective for plasmids and induces both Hsp104 and poly-Q constructs. C and D, the antitoxic effect of the HSP104-A503V allele in the [psi– PIN+] background. Strains contained the HIS3-based Hsp104 plasmid (as shown) or empty control, in addition to the URA3 poly-Q constructs. C, plate assay. Plates were photographed after 5 days of incubation (second passage) on the –Ura-His/Gal medium, which is selective for the plasmids and induces both Hsp104 and poly-Q constructs. D, quantitative assay. Cultures were grown in the liquid –Ura-His medium containing galactose and raffinose (Gal+Raf) to induce poly-Q and Hsp104 constructs. The numbers correspond to the concentration of viable cells per ml at the 60-h time point. No differences in growth between any strains were detected on Glu media (not shown).

 
Next, we checked whether Hsp104 mutations in regions other than the NBDs affect poly-Q toxicity. Indeed, two mutant alleles of HSP104 with substitutions in the middle region (Fig. 2A), previously isolated in a genetic screen at the S. Lindquist laboratory (37), HSP104-A503V (Fig. 2, C and D) and HSP104-A509D (not shown), counteracted Q103 toxicity in the [psi– PIN+] strain. Hsp104-A509D also counteracted Q103 toxicity in [PSI+] and cured yeast cells of prions (Table I), whereas Hsp104-A503V by itself was toxic to the [PSI+] strain (see in more detail below) and did not cure [PIN+], as confirmed both in the presence (Table I) and in the absence (data not shown) of Q103. The Hsp104-A503V derivative was also previously shown not to antagonize thermotolerance (37).

As A503V mutation counteracted poly-Q toxicity without curing [PIN+], we have checked its effects on Q103 aggregation in the [psi– PIN+] background. Normally, Q103 forms large "clumps" in about 40% of the [psi– PIN+] cells and either "dots" or both small clumps and dots in the rest of them (Fig. 3, A and B). In contrast, cells co-expressing Hsp104-A503V with Q103 showed essentially no large clumps, and 97% of them contained dots (Fig. 3B). This shows that expression of Hsp104-A503V decreases the size of Q103 aggregates, suggesting a possible mechanism for antitoxicity.

Prion-dependent Q103 aggregation in yeast is associated with a defect of endocytosis, which is one of the likely causes for Q103 toxicity (22). We have monitored the effect of Q103 aggregation on endocytosis by using the lipophilic fluorescent dye FM4-64 (see "Experimental Procedures" for more details). This dye binds to the cell membrane and is internalized through the endocytic pathway. In cells with normal endocytosis, FM4-64 forms a distinct ring around the vacuole at 45 min after dye addition, reflecting the fusion of dye-stained endosomes with the vacuolar membrane. In contrast, cells with large Q103 clumps, and about 50% of cells with both small clumps and dots, contained no such fluorescent rings, indicating a defect in endocytosis. Cells with dots usually exhibited FM4-64 rings, confirming that in the absence of large aggregates, endocytosis was not affected (Fig. 3, C and D). As expression of Hsp104-A503V increased the proportion of cells with dots and almost eliminated large clumps (Fig. 3B), the endocytosis defect was essentially not seen in the presence of Hsp104-A503V (data not shown).

The differential centrifugation assay confirmed that co-expression of Hsp104-A503V with Q103 shifted a portion of the Q103 protein from the fraction pelletable at 10,000 x g to the supernatant fraction (Fig. 3E). At a higher centrifugation speed (200,000 x g), essentially all Q103 was precipitated independently of the presence or absence of Hsp104-A503V, whereas non-expanded Q25 control remained soluble (data not shown). These results are in agreement with the fluorescence microscopy data and show that expression of Hsp104-A503V does not solubilize Q103 aggregates but leads to a decrease in aggregate size so that a higher speed is needed for the efficient precipitation of aggregates. As the total amount of Q103 was not affected by Hsp104-A503V (data not shown), a decrease in aggregate size should be accompanied by an increase in the number of aggregated units. Taken together, our data confirm that Hsp104-A503V ameliorates Q103 toxicity by counteracting formation of the large aggregates, which inhibit endocytosis.



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  		FIG. 3.
Effects of the Hsp104-A503V mutant derivative on poly-Q aggregation and endocytosis in the [psi– PIN+] background. A, types of aggregates observed in Q103 overexpressing cells. Cultures were grown in the liquid Gal+Raf medium to induce the poly-Q construct. Three distinct classes of cells with aggregates were observed: black circle with curve, cells containing large clumps; black circle with two dots, cells containing dots; black circle with two dots and a curve, cells containing both clumps and dots. B, Expression of Hsp104-A503V decreases proportions of cells with clumps and increases the proportion of cells with dots. The average number from two repeats for each of two independent cultures is given in each case. The error bars indicate standard deviations. C and D, clumps but not dots block endocytosis. Ring-like staining of the vacuole with FM4-64 is a marker of normal endocytosis. The majority of cells with dots show rings, whereas the majority of cells with clumps and 50% of cells with both clumps and dots do not, indicating a defect in endocytosis. Note that residual fluorescence of the cells with large clumps in the red (FM4-64) filter does not represent endocytic vesicles but rather results from leakage of GFP fluorescence through the red filter. Data were homogenous for all plasmid combinations. Error bars indicate standard deviations. E, expression of Hsp104-A503V increases the proportion of soluble Q103 protein. Proteins were isolated after 24 h of growth in synthetic Gal+Raf medium selective for plasmids and fractionated as described under "Experimental Procedures." No differences in the proportion of Q103 precipitated at 800 x g were seen (not shown). Proportions of Q103-GFP protein in pellet (P) and supernatant (S) fractions obtained at 10,000 x g were determined by densitometry. Standard deviations are shown.

 
[PSI+]-specific Toxicity of Hsp104-A503V—As mentioned above, expression of Hsp104-A503V by itself inhibited growth of the [PSI+ PIN+] strain OT56 and [PSI+ pin–] strain GT490. Such an effect was not seen in the isogenic [psi– PIN+] strain OT60, confirming that it is specific to [PSI+] rather than to any prion (Fig. 4, A and B). Moreover, the severity of the toxic effect depended on the variant of [PSI+]. The [PSI+ PIN+] strain OT55, which is isogenic to OT56 but bears a weak variant of [PSI+], characterized by the larger proportion of the Sup35 protein remaining in the soluble fraction (40, 46), was not inhibited by expression of Hsp104-A503V to such an extent as was OT56 (data not shown). One possibility is that toxicity of Hsp104-A503V is due to increased sequestration of the soluble Sup35 protein by prion aggregates. As Sup35 is an essential protein working in termination of translation, such sequestration may inhibit growth of the strong [PSI+] variants in which the amount of the remaining soluble Sup35 is already low. To test this hypothesis, a high copy plasmid expressing only the Sup35C domain was introduced into the [PSI+] strain together with the Hsp104-A503V expression construct. Sup35C is completely functional in translation but is not sequestered by the [PSI+] prion aggregates as it lacks the Sup35N prion domain. As expected, Sup35C counteracted toxicity of Hsp104-A503V in the [PSI+] background (Fig. 4C).



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  		FIG. 4.
[PSI+] dependent toxicity of the Hsp104-A503V mutant derivative. A and B, expression of Hsp104-A503V inhibits growth of the [PSI+] cells. A, plate assay. Plates were photographed after 3 days of incubation (second passage) on the –His/Gal medium, used to induce the Hsp104-A503V construct. No differences in growth were detected on Glu medium (not shown). WT, wild type. B, quantitative assay. Cultures were grown in the liquid –His/Gal+Raf medium to maintain and induce the Hsp104-A503V and control constructs. The growth rate of the [PSI+] culture overexpressing Hsp104-A503V is statistically different from the growth rate of all other cultures; differences between other cultures are statistically insignificant. C, Hsp104-A503V toxicity is ameliorated by overproducing Sup35C in [PSI+ PIN+] cells. Cultures contained the PGAL-HSP104-A503V plasmid, high copy SUP35C plasmid, and empty controls in all possible combinations. Plates were photographed after 4 days of incubation (second passage) on the –Ura-His/Gal medium, which is selective for both plasmids and induces the Hsp104-A503V construct. D, the effect of Hsp104-A503V on the Sup35NM-GFP aggregation. Cultures were grown in the Gal+Raf medium, which is selective for both PGAL-HSP104-A503V (or control) and PSUP35-SUP35NM-GFP plasmids, and induces the PGAL -HSP104-A503V construct. All cells expressing Sup35NM-GFP alone show diffused fluorescence, whereas cultures co-expressing Hsp104-A503V with Sup35NM-GFP contain cells with detectable clumps and dots. Percentages of cell types are the averages of two counts, with at least 100 cells counted in each case.

 
Next, we checked whether Hsp104-A503V influences the size of the Sup35 prion aggregates. These experiments employed the construct containing the Sup35N (prion) and Sup35M (middle) domains fused to GFP (Sup35NM-GFP). Inclusion of this tagged protein into the prion aggregates makes them detectable by fluorescence microscopy (36, 47). In the [PSI+] cells, Sup35NM-GFP, expressed from the endogenous PSUP35 promoter, usually shows a diffused fluorescence pattern (35, 42), due to the fact that the Sup35PSI+ aggregates are usually so small in these conditions that they can hardly be recognized. However, in the presence of Hsp104-A503V, the same PSUP35-SUP35NM-GFP construct produced large detectable clumps or dots (Fig. 4D). Thus, expression of Hsp104-A503V increased the size of Sup35 aggregates. Possibly, increased Sup35 aggregation contributes to cell toxicity. Such an effect of Hsp104-A503V would be similar to consequences of Sup35 (or Sup35N/NM) overproduction in the [PSI+] background, which results in both the appearance of large detectable Sup35 clumps (47) and cell toxicity (43, 46, 48). Therefore, it appears that Hsp104-A503V exhibits opposite effects on the Q103 and Sup35 aggregates, decreasing aggregate size and toxicity in the former case and increasing them in the latter case.

Differential Effects of the Hsp40 and Hsp70 Chaperones on Q103 Toxicity—Hsp40 and Hsp70 chaperones cooperate with Hsp104 in solubilization and refolding of heat-damaged proteins (28). One of the major yeast Hsp40 chaperones, Sis1, is also involved in [PIN+] propagation (49), whereas overproduction of the other Hsp40 chaperone, Ydj1, has been shown to antagonize poly-Q aggregation in some yeast models (14). Yeast Hsp70 proteins of the Ssa subfamily aid in propagation of the yeast prion [PSI+] (33, 40, 50) and exhibit differential effects on the other yeast prion, [URE3] (41, 51). Proteins of the other cytosolic Hsp70 subfamily, Ssb, consistently antagonize [PSI+] in various assays (38, 40, 52). In contrast, Hsp26 and Hsp90 chaperones did not show any detectable effects on [PSI+] (33).

We have checked the effects of individually overproduced Hsps on the prion-dependent Q103 toxicity in yeast. Among all yeast Hsp70 proteins tested (Ssa1, -2, -3, and -4, and Ssb1), only overproduction of Ssa4 counteracted Q103 toxicity in the [PSI+ PIN+] strain (Fig. 5A), although it exhibited no detectable antitoxicity in the [psi– PIN+] or [PSI+ pin–] backgrounds and did not cure [PSI+] at a significant level (Ref. 40 and data not shown). Overproduction of Hsp26 or Hsp90 did not exhibit any antitoxic effect on Q103, independently of which prions were present (data not shown). The Hsp40 chaperones Ydj1 and Sis1 did not affect Q103 toxicity at any detectable level in the [PSI+] strains but exhibited opposite effects on Q103 toxicity when overproduced in the [psi– PIN+] strain. An extra copy of YDJ1, expressed from either the strong constitutive PGPD promoter (Fig. 5, B and D) or the endogenous PYDJ1 promoter (not shown), increased Q103 toxicity. In contrast, an extra copy of SIS1, expressed from either PGPD (Fig. 5, C and E) or endogenous PSIS1 promoter (not shown), counteracted Q103 toxicity.



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  		FIG. 5.
Effects of Hsp40 and Hsp70 chaperones on Q103 aggregation and toxicity. A–C, plate assays. Plates were photographed after 7 days of incubation (second passage) on the –Ura-Leu/Gal medium (Ssa4), 7 days of incubation (second passage) on the –Ura-His/Gal medium (Ydj1), or 3 days of incubation (second passage) on the –Ura-Trp/Gal medium (Sis1). These media select for the plasmids and induce the Q103 (and in panel A, also Ssa4) constructs; Ydj1 and Sis1 constructs are constitutively expressed. No differences in growth were detected on Glu media (not shown). No effects of Ydj1 or Sis1 on toxicity were detected in the presence of [PSI+] (not shown). Ssa1, -2, and -3 overexpressors did not exhibit any detectable effect on toxicity in the same conditions, and none of the Ssa constructs influenced toxicity in the isogenic [psi– PIN+] and [PSI+ pin–] strains at detectable levels (data not shown). D and E, quantitative assay. Cultures were grown in the liquid –Ura-His/Gal+Raf (Ydj1) or –Ura-Trp/Gal+Raf (Sis1) media, which are selective for the plasmids and induce the poly-Q constructs. Differences between Q103 cultures and cultures co-expressing Ydj1 or Sis1 with Q103 were statistically significant after 3 and 6 days of incubation, respectively. The numbers show the concentration of viable cells at these time points. F and G, excess Ydj1 increases the proportion of cells with Q103 clumps, whereas excess Sis1 decreases it. Cultures were grown in the liquid –Ura-His/Gal+Raf (Ydj1) or –Ura-Trp/Gal+Raf (Sis1) medium overnight. Designations are the same as in Fig. 3. Error bars indicate standard deviations. H and I, excess Ydj1 decreases the proportion of soluble Q103 (H), whereas excess Sis1 decreases it (I). Proteins were isolated from the [psi– PIN+] strain. Procedure was same as described in the legend for Fig. 3 (for panel E). Differences between the proportion of soluble protein in Q103 controls in H and I are apparently due to different media used.

 
Fluorescence microscopy analysis confirmed that co-expression of Ydj1 with Q103 in the [psi– PIN+] background increased the proportion of the endocytosis-defective cells with large Q103 clumps (Fig. 5F). Likewise, the proportion of Q103 protein that is insoluble and pelletable at 10,000 x g was increased in the presence of excess Ydj1 (Fig. 5H). In contrast, the [psi– PIN+] cultures co-expressing Sis1 with Q103 exhibited a decrease in the proportion of the endocytosis-defective cells with large clumps, in comparison with those expressing Q103 alone. This was accompanied by an increase in the proportion of the cells with dots or with both dots and small clumps (Fig. 5G) and by a decrease in the proportion of the Q103 protein pelletable at 10,000 x g (Fig. 5I). Neither excess Ydj1 nor excess Sis1 affected total levels of Q103 (data not shown). Excess Sis1 did not lead to any detectable loss of [PIN+] (Table I), confirming that the effect of Sis1 is not due to loss of a prion. Taken together, these data suggest that excess Ydj1 exacerbates the toxic effect of Q103 by increasing the size of Q103 aggregates, whereas excess Sis1 counteracts toxicity by decreasing aggregate size.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Role of Endogenous Prions in Poly-Q Aggregation and Toxicity—Our data confirm previous observations showing that the presence of endogenous yeast QN-rich proteins in a prion form promotes poly-Q aggregation (18, 21) and toxicity (18) and demonstrate that [PSI+], a prion form of Sup35, is promoting toxicity of the Htt-derived poly-Q construct in the same way as does [PIN+], a prion form of Rnq1 (Fig. 1A). These results, implicating endogenous QN-rich prions as susceptibility factors in the poly-Q disorders, could be relevant to mammalian cells, containing a lot of proteins with potentially prionogenic QN-rich domains (see Ref. 53).

Prion domains of Sup35, and possibly of Rnq1, are composed of "aggregation modules," or QN-rich stretches, and "propagation modules," in the case of Sup35, oligopeptide repeats (20, 54). QN-rich stretches form stable intermolecular {beta}-sheets, leading to the generation of aggregates with fibrillar morphology (1), characteristic for both prions and poly-Q proteins such as Htt. It is possible that QN-rich prion aggregates serve as aggregation seeds for the poly-Q derivatives of Htt (18). Propagation modules are required for propagation of aggregates in the cell divisions via repetitive cycles of the chaperone-mediated aggregate "shearing," generating new seeds (20, 54). Poly-Q fragments of Htt apparently lack propagation capabilities of their own (54) so that in the absence of endogenous prions, poly-Q aggregates are not only rarely formed but also incapable of persisting in yeast cells. Possibly, association with prions confers a self-perpetuating capability to the whole prion/poly-Q complex. Direct biochemical assays confirm that aggregated Q103 is associated with Rnq1 in the yeast cells (22).

Role of the Chaperone Hsp104 in Poly-Q Aggregation and Toxicity—Numerous previous studies have shown that the presence of Hsp104 is required for poly-Q aggregation in yeast (13, 15, 16, 18). Hsp104 overproduction reduced poly-Q aggregation at least in some yeast strains (13) and in the heterological model of C. elegans (29). However, Hsp104 is required for propagation of all known endogenous yeast prions, including [PSI+] (31) and [PIN+] (30). Apparently, it serves as a major factor promoting aggregate shearing (20). Therefore, previously reported effects of Hsp104 on poly-Q aggregates could be mediated by its effects on the endogenous prions.

Indeed, NBD mutations (both the previously described Hsp104-KT218,620 and the newly found Hsp104-K302N) and at least one mutation in the middle region of Hsp104 (Hsp104-A509D) inhibited both poly-Q toxicity and Hsp104 function in prion maintenance (Fig. 2B and Table I). Loss of poly-Q toxicity was accompanied by a loss of the endogenous prion(s), suggesting prion elimination as a major cause of the toxicity relief. In contrast, overproduction of wild-type Hsp104, which is known (30, 31) and confirmed (Table I) to solubilize and eliminate [PSI+] but not [PIN+], reduced poly-Q toxicity only in the [PSI+] strains but not in the [psi– PIN+] strains (Fig. 1C), suggesting that excess Hsp104 influenced poly-Q via prion elimination.

However, in the case of Hsp104-A503V mutant, toxicity relief (Fig. 2, C and D) was not accompanied by elimination of a prion (Table I) or loss of the thermotolerance function of Hsp104 (37). The antitoxicity effect of Hsp104-A503V was apparently due to a decreased size of poly-Q aggregates (Fig. 3, B, C, and E); expression of Hsp104-A503V reduced the proportion of the cells with large poly-Q clumps (Fig. 3B), which exhibit a defect in endocytosis (Fig. 3, C and D), a landmark of prion-dependent poly-Q toxicity in yeast (22). Thus, our data provide the first evidence that at least some mutant derivatives of Hsp104 are capable of modulating poly-Q aggregation without eliminating yeast prions.

Differential Effects of Hsp104-A503V on Poly-Q and [PSI+] Aggregates—In contrast to the observations Schirmer et al. (37) made in a different genotypic background, expression of the Hsp104-A503V protein in our strains did not lead to temperature sensitivity (data not shown). However, Hsp104-A503V was incompatible with the presence of the strong variant of [PSI+] prion (Fig. 4, A and B). This effect was counteracted by simultaneous expression of the Sup35C region, incapable of aggregation due to the lack of prion domain (Fig. 4C). Thus, incompatibility between Hsp104-A503V and [PSI+] was most likely due to increased sequestration of functional Sup35 by prion aggregates. Indeed, the size of the Sup35 aggregates is increased in the [PSI+] strain expressing Hsp104-A503V (Fig. 4D). It is known that decreased Hsp104 function increases the size of the Sup35 prion clumps without detectable inhibition of growth (35). Probably, this is due to the fact that shearing-defective Sup35 aggregates are quickly lost from the population. As such loss apparently does not occur in the case of Hsp104-A503V, it seems likely that this mutant protein derivative promotes aggregate growth by facilitating prion conversion rather than by inhibiting aggregate shearing. The gain-of-function effect of Hsp104-A503V also agrees with the observations that it remains functional in thermotolerance (37) and does not impair [PIN+] maintenance (Table I). Recent data suggest that in addition to aggregate shearing, Hsp104 promotes Sup35 aggregation in certain in vitro assays (55).

Possible Molecular Mechanisms of the Hsp104-A503V Effects—According to previous data from the S. Lindquist laboratory (56), the A503V mutant exhibits increased background ATPase activity but cannot further increase ATP hydrolysis at NBD1 in response to substrate binding at the C terminus. We propose that these alterations of the Hsp104 function have different consequences, depending on the type of aggregates Hsp104 encounters. In the case of non-perpetuating poly-Q aggregates, increased background ATPase activity may lead to increased aggregate shearing, and therefore, to decreased aggregate size. Hsp104 interaction with the self-perpetuating (prion) aggregates of Sup35 is more complex. One possibility is that the inability of mutant Hsp104 to undergo fine tuning of the NBD action results in shifting the balance between its shearing and "conversion-promoting" activities, which facilitates aggregate growth and sequestration of the newly synthesized Sup35 protein by aggregates.

Differential Effects of Hsp40 Chaperones on Poly-Q Aggregation and Toxicity—Yeast Sis1 and Ydj1, functionally distinct heat shock proteins of the Hsp40 family, are homologs of the human Hsp40 proteins Hdj1 and Hdj2, respectively. In the Drosophila model of a poly-Q disease, both proteins suppressed neurodegeneration, but Hdj1 had a stronger effect than Hdj2 (25). Interestingly, overexpression of Hdj2 increased inclusion formation by Htt in the mammalian COS7 cells (57). However, yeast Ydj1, a homolog of Hdj2, has previously been shown to suppress rather than increase formation of insoluble aggregates of poly-Q expanded exon 1 of Htt in yeast (14). The effect of Sis1 on poly-Q aggregation in yeast has not been studied previously.

In our system, excess Sis1 decreased Q103 aggregate size (Fig. 5, G and I) and counteracted Q103 toxicity (Fig. 5, C and E), similar to the effect of its mammalian homolog Hdj1 in other models. Intact Sis1 is required for the maintenance of the [PIN+] prion (49); however, excess Sis1 did not cure cells of [PIN+] (Table I), ruling out a possibility that antitoxicity was due to loss of a prion. In contrast to Sis1, excess Ydj1 increased Q103 aggregate size (Fig. 5, F and H), similar to the effect of its mammalian homolog Hdj2 at least in some assays (57), and increased Q103 toxicity (Fig. 5, B and D). This agrees with the previous observation that Q103 aggregation is decreased in the strain carrying the ydj1–159 mutation (18). Interestingly, our preliminary data indicate that Sis1 and Ydj1 also differ from each other in regard to their effects on the yeast prion [PSI+].2 Differences between our results and previous reports on the effects of yeast Ydj1 on poly-Q (14) are probably due to differences in the poly-Q constructs employed, yeast strains, and/or prion compositions. For instance, our plate assay has not detected any effects of Hsp40s on poly-Q toxicity in the presence of both [PSI+] and [PIN+] prions simultaneously, possibly because toxicity was too strong to see an effect clearly (data not shown). As differential effects of yeast Hsp40s on poly-Q aggregation and toxicity, observed in our [psi– PIN+] strain, parallel differences between their human homologs detected in at least some higher eukaryotic models, one could argue that the yeast assay used in our work better reproduces patterns of interactions between poly-Q aggregates and chaperones in the homologous mammalian systems, in comparison with the yeast assays used by other groups. Also, our data suggest that at least some differences between members of the Hsp40 family in patterns of their interactions with poly-Q aggregates could be conserved throughout evolution from yeast to humans.

Do Hsp70 Chaperones Influence Poly-Q Toxicity?—Both yeast Hsp40 proteins studied in this report, Sis1 and Ydj1, are thought to be co-factors of the Hsp70 chaperones of the Ssa subfamily (58). Yeast Ssa1 was shown to suppress the formation of insoluble poly-Q aggregates in yeast cells (14). However, simultaneous depletion of two members of the Ssa subfamily, Ssa1 and Ssa2, decreased prion-dependent Q103 aggregation in our system (18), and excess of any Ssa protein promoted rather than decreased aggregation of the yeast prion protein Sup35 in our hands (33, 40).

One could suggest that Hsp40 chaperones act on poly-Q aggregates via modulating activity of the Hsp70 proteins, which are known to interact with Hsp40s. However, our data show that only one of the four members of the S. cerevisiae Hsp70-Ssa subfamily, Ssa4, counteracts Q103 toxicity when overproduced. Moreover, the antitoxic effect of Ssa4 could be seen clearly only in the yeast strains containing both prions, [PIN+] and [PSI+] simultaneously (Fig. 5A). Differential effects of various members of the Hsp40 and Hsp70 families on Q103 confirm that chaperone interactions with poly-Q aggregates are of a highly specific nature and suggest caution in interpreting the previous data in which only some members of the family were studied. For example, the effect of the ssa1/2 deletion (18) could be not due to elimination of Ssa1 and Ssa2 per se but due to the increased fraction of Ssa4 in the total Ssa pool. Interestingly, although all Ssa proteins act in the same direction on [PSI+] (40), Ssa1 and Ssa2 differ from each other in their effects on the other yeast prion, [URE3] (41). Further investigations are needed to establish the molecular basis of the differential effects of Ssa chaperones on protein aggregates.

Conclusions—Not only [PIN+], a prion form of Rnq1 protein, but also [PSI+], a prion form of Sup35 protein, promote toxicity of poly-Q protein in the yeast model. This suggests that preexisting endogenous QN-rich aggregates manifest themselves as susceptibility factors in poly-Q diseases, probably by providing nuclei for poly-Q aggregation.

Effects of the yeast chaperone Hsp104 on poly-Q aggregation and toxicity are not restricted to its role in propagation of endogenous yeast prions. Expression of the Hsp104-A503V mutant decreases the size of poly-Q aggregates and ameliorates poly-Q toxicity without eliminating an endogenous yeast prion. In contrast, Hsp104-A503V increases the size of the Sup35 prion aggregates, leading to synthetic lethality between the A503V mutation and [PSI+], a prion form of Sup35. Our data provide the first evidence for the synthetic lethality between the chaperone mutation and a prion.

Chaperone interactions with poly-Q aggregates are of a highly specific nature as different members of one and the same chaperone family may exhibit different effects. Ydj1, a chaperone of the yeast Hsp40 family, increased prion-dependent poly-Q aggregation and toxicity, whereas Sis1, another yeast Hsp40 protein, decreased the size of poly-Q aggregates and antagonized poly-Q toxicity. Among yeast Hsp70 proteins, only Ssa4 was capable of counteracting poly-Q toxicity.

Taken together, our data establish yeast prion-based experimental model as a cheap and effective experimental assay for studying the chaperone modulation of poly-Q aggregation and toxicity. As many stress-defense proteins are conserved between yeast and mammals, our data shed light on possible mechanisms modulating poly-Q aggregation and toxicity in mammalian cells.


   FOOTNOTES
 
* This work was supported by grants from The Huntington's Disease Society of America and from the National Institutes of Health (Grant R01GM58763) (to Y. O. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: School of Biology, Georgia Institute of Technology, 310 Ferst Dr., Atlanta, GA 30332-0230. Tel.: 404-894-1157; Fax: 404-894-0519; E-mail: yury.chernoff{at}biology.gatech.edu.

1 The abbreviations used are: poly-Q, polyglutamine; Htt, huntingtin protein; GFP, green fluorescent protein; Hsp, heat shock protein; Q25, normal polyglutamine stretch of 25 glutamine residues in exon 1 of huntingtin; Q103, expanded polyglutamine stretch of 103 glutamine residues in exon 1 of huntingtin; PGAL, PGPD, PTEF, PSSA1, PSSA2, and PSUP35, promoters of the yeast GAL1, GPD1, TEF1, SSA1, SSA2, and SUP35 genes, respectively; NBD, nucleotide binding domain; Glu, Gal, and Gal+Raf, media containing glucose, galactose, or galactose and raffinose, respectively, as a carbon source; FM4-64, lipophilic fluorescent dye; Sup35N, M and C, N-proximal, middle, and C-proximal regions of the Sup35 protein, respectively; CEN, centromeric; TRITC, tetramethylrhodamine isothiocyanate. Back

2 S. Müller, J. Patterson, and Y. Chernoff, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank E. Craig, D. Cyr, S. Lindquist, D. Masison, and E. Schirmer for the Hsp constructs, A. Meriin for the poly-Q constructs, R. Dulebohn for the help in some experiments, and S. Müller and J. Patterson for unpublished data.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Perutz, M. F. (1999) Trends Biochem. Sci. 24, 58–63[CrossRef][Medline] [Order article via Infotrieve]
  2. The Huntington's Disease Collaborative Research Group (1993) Cell 72, 971–973[CrossRef][Medline] [Order article via Infotrieve]
  3. Martindale, D., Hackam, A., Wieczorek, A., Ellerby, L., Wellington, C., McCutcheon, K., Singaraja, R., Kazemi-Esfarjani, P., Devon, R., Kim, S. U., Bredesen, D. E., Tufaro, F., and Hayden, M. R. (1998) Nat. Genet. 18, 150–154[CrossRef][Medline] [Order article via Infotrieve]
  4. Sherman, M. Y., and Goldberg, A. L. (2001) Neuron 29, 15–32[CrossRef][Medline] [Order article via Infotrieve]
  5. Perez, M. K., Paulson, H. L., Pendse, S. J., Saionz, S. J., Bonini, N. M., and Pittman, R. N. (1998) J. Cell Biol. 143, 1457–1470[Abstract/Free Full Text]
  6. Kazantsev, A., Preisinger, E., Dranovsky, A., Goldgaber, D., and Housman, D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11404–11409[Abstract/Free Full Text]
  7. Nucifora, F. C., Jr., Sasaki, M., Peters, M. F., Huang, H., Cooper, J. K., Yamada, M., Takahashi, H., Tsuji, S., Troncoso, J., Dawson, V. L., Dawson, T. M., and Ross, C. A. (2001) Science 291, 2423–2428[Abstract/Free Full Text]
  8. Li, S., and Li, X. (2004) Trends Genet. 20, 146–154[CrossRef][Medline] [Order article via Infotrieve]
  9. Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S. W., and Bates, G. P. (1996) Cell 87, 493–506[CrossRef][Medline] [Order article via Infotrieve]
  10. Davies, S. W., Turmaine, M., Cozens, B. A., DiFiglia, M., Sharp, A. H., Ross, C. A., Scherzinger, E., Wanker, E. E., Mangiarini, L., and Bates, G. P. (1997) Cell 90, 537–548[CrossRef][Medline] [Order article via Infotrieve]
  11. Jackson, G. R., Salecker, I., Dong, X., Yao, X., Arnheim, N., Faber, P. W., MacDonald, M. E., and Zipursky, S. L. (1998) Neuron 3, 633–642
  12. Faber, P. W., Alter, J. R., MacDonald, M. E., and Hart, A. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 179–184[Abstract/Free Full Text]
  13. Krobitsch, S., and Lindquist, S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1589–1594[Abstract/Free Full Text]
  14. Muchowski, P. J., Schaffar, G., Sittler, A., Wanker, E. E., Hayer-Hartl, M. K., and Hartl, F. U. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7841–7846[Abstract/Free Full Text]
  15. Cao, F., Levine, J. J., Li, S. H., and Li, X. J. (2001) Biochim. Biophys. Acta 1537, 158–166[Medline] [Order article via Infotrieve]
  16. Kimura, Y., Koitabashi, S., Kakizuka, A., and Fujita, T. (2001) Genes Cells 6, 887–897[Abstract]
  17. Muchowski, P. J., Ning, K., D'Souza-Schorey, C., and Fields, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 727–732[Abstract/Free Full Text]
  18. Meriin, A. B., Zhang, X., He, X., Newnam, G. P., Chernoff, Y. O., and Sherman, M. Y. (2002) J. Cell Biol. 157, 997–1004[Abstract/Free Full Text]
  19. Chernoff, Y. O. (2001) Mutat. Res. 488, 39–64[CrossRef][Medline] [Order article via Infotrieve]
  20. Chernoff, Y. O. (2004) Trends Biotechnol. 22, 549–552[CrossRef][Medline] [Order article via Infotrieve]
  21. Osherovich, L. Z., and Weissman, J. S. (2001) Cell 106, 183–194[CrossRef][Medline] [Order article via Infotrieve]
  22. Meriin, A. B., Zhang, X., Miliaras, N. B., Kazantsev, A., Chernoff, Y. O., McCaffery, J. M., Wendland, B., and Sherman, M. Y. (2003) Mol. Cell. Biol. 23, 7554–7565[Abstract/Free Full Text]
  23. Velier, J., Kim, M., Schwarz, C., Kim, T. W., Sapp, E., Chase, K., Aronin, N., and DiFiglia, M. (1998) Exp. Neurol. 152, 34–40[CrossRef][Medline] [Order article via Infotrieve]
  24. Warrick, J. M., Chan, H. Y., Gray-Board, G. L., Chai, Y., Paulson, H. L., and Bonini, N. M. (1999) Nat. Genet. 23, 425–428[CrossRef][Medline] [Order article via Infotrieve]
  25. Chan, H. Y., Warrick, J. M., Gray-Board, G. L., Paulson, H. L., and Bonini, N. M. (2000) Hum. Mol. Genet. 9, 2811–2820[Abstract/Free Full Text]
  26. Kazemi-Esfarjani, P., and Benzer, S. (2000) Science 287, 1837–1840[Abstract/Free Full Text]
  27. Cummings, C. J., Sun, Y., Opal, P., Antalffy, B., Mestril, R., Orr, H. T., Dillmann, W. H., and Zoghbi, H. Y. (2001) Hum. Mol. Genet. 10, 1511–1518[Abstract/Free Full Text]
  28. Glover, J. R., and Lindquist, S. (1998) Cell 94, 1–20[CrossRef][Medline] [Order article via Infotrieve]
  29. Satyal, S. H., Schmidt, E., Kitagawa, K., Sondheimer, N., Lindquist, S., Kramer, J. M., and Morimoto, R. I. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5750–5755[Abstract/Free Full Text]
  30. Derkatch, I. L., Bradley, M. E., Zhou, P., Chernoff, Y. O., and Liebman, S. W. (1997) Genetics 147, 507–519[Abstract]
  31. Chernoff, Y. O., Lindquist, S. L., Ono, B., Inge-Vechtomov, S. G., and Liebman, S. W. (1995) Science 268, 880–884[Abstract/Free Full Text]
  32. Moriyama, H., Edskes, H. K., and Wickner, R. B. (2000) Mol. Cell Biol. 20, 8916–8922[Abstract/Free Full Text]
  33. Newnam, G. P., Wegrzyn, R. D., Lindquist, S. L., and Chernoff, Y. O. (1999) Mol. Cell Biol. 19, 1325–1333[Abstract/Free Full Text]
  34. Bailleul, P. A., Newnam, G. P., Steenbergen, J. N., and Chernoff, Y. O. (1999) Genetics 153, 81–94[Abstract/Free Full Text]
  35. Wegrzyn, R. D., Bapat, K., Newnam, G. P., Zink, A. D., and Chernoff, Y. O. (2001) Mol. Cell. Biol. 21, 4656–4669[Abstract/Free Full Text]
  36. Chernoff, Y. O., Uptain, S. M., and Lindquist, S. L. (2002) Methods Enzymol. 351, 499–538[Medline] [Order article via Infotrieve]
  37. Schirmer, E. C., Homann, O. R., Kowal, A. S., and Lindquist, S. (2004) Mol. Biol. Cell 15, 2061–2072[Abstract/Free Full Text]
  38. Chernoff, Y. O., Lindquist, S. L., Newnam, G. P., Kumar, J., Allen, K., and Zink, A. D. (1999) Mol. Cell. Biol. 19, 8103–8112[Abstract/Free Full Text]
  39. Liu, H., Krizek, J., and Bretscher, A. (1992) Genetics 132, 665–673[Abstract]
  40. Allen, K. D., Wegrzyn, R. D., Chernova, T. A., Müller, S., Newnam, G. P., Winslett, P. A., Wittich, K. B., Wilkinson, K. D., and Chernoff, Y. O. (2005) Genetics 169, 1227–1242[Abstract/Free Full Text]
  41. Schwimmer, C., and Masison, D. C. (2002) Mol. Cell. Biol. 22, 3590–3598[Abstract/Free Full Text]
  42. Bailleul-Winslett, P. A., Newnam, G. P., Wegrzyn, R. D., and Chernoff, Y. O. (2000) Gene Expr. 9, 145–156[Medline] [Order article via Infotrieve]
  43. Ter-Avanesyan, M. D., Kushnirov, V. V., Dagkesamanskaya, A. R., Didichenko, S. A., Chernoff, Y. O., Inge-Vechtomov, S. G., and Smirnov, V. N. (1993) Mol. Microbiol. 7, 683–692[Medline] [Order article via Infotrieve]
  44. Sherman, F. (2002) Methods Enzymol. 350, 3–41[Medline] [Order article via Infotrieve]
  45. Schirmer, E. C., Queitsch, C., Kowal, A. S., Parsell, D. A., and Lindquist, S. (1998) J. Biol. Chem. 273, 15546–15552[Abstract/Free Full Text]
  46. Derkatch, I. L., Chernoff, Y. O., Kushnirov, V. V., Inge-Vechtomov, S. G., and Liebman, S. W. (1996) Genetics 144, 1375–1386[Abstract]
  47. Patino, M. M., Liu, J. J., Glover, J. R., and Lindquist, S. (1996) Science 273, 622–626[Abstract]
  48. Chernoff, Y. O., Inge-Vechtomov, S. G., Derkatch, I. L., Ptyushkina, M. V., Tarunina, O. V., Dagkesamanskaya, A. R., and Ter-Avanesyan, M. D. (1992) Yeast 7, 489–499
  49. Sondheimer, N., Lopez, N., Craig, E. A., and Lindquist, S. (2001) EMBO J. 20, 2435–2442[CrossRef][Medline] [Order article via Infotrieve]
  50. Jung, G., Jones, G., Wegrzyn, R. D., and Masison, D. C. (2000) Genetics 156, 559–570[Abstract/Free Full Text]
  51. Roberts, B. T., Moriyama, H., and Wickner, R. B. (2004) Yeast 21, 107–117[CrossRef][Medline] [Order article via Infotrieve]
  52. Chacinska, A., Szczesniak, B., Kochneva-Pervukhova, N. V., Kushnirov, V. V., Ter-Avanesyan, M. D., and Boguta, M. (2001) Curr. Genet. 39, 62–67[CrossRef][Medline] [Order article via Infotrieve]
  53. Michelitsch, M. D., and Weissman, J. S. (2000) Proc. Natl. Acad. Sci. U. S. A. 22, 11910–11915
  54. Osherovich, L. Z., Cox, B. S., Tuite, M. F., and Weissman, J. S. (2004) PLoS Biol. 10.1371/journal.pbio.0020086[CrossRef][Medline] [Order article via Infotrieve]
  55. Shorter, J., and Lindquist, S. (2004) Science 304, 1793–1797[Abstract/Free Full Text]
  56. Cashikar, A. G., Schirmer, E. C., Hattendorf, D. A., Glover, J. R., Ramakrishnan, M. S., Ware, D. M., and Lindquist, S. L. (2002) Mol. Cell 9, 751–760[CrossRef][Medline] [Order article via Infotrieve]
  57. Wyttenbach, A., Sauvageot, O., Carmichael, J., Diaz-Latoud, C., Arrigo, A. P., and Rubinsztein, D. C. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2898–2903[Abstract/Free Full Text]
  58. Bukau, B., and Horwich, A. L. (1998) Cell 92, 351–366[CrossRef][Medline] [Order article via Infotrieve]

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