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* Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611 and Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 77030
1 Corresponding author: Department of Molecular Pharmacology and Biological Chemistry, The Feinberg School of Medicine, Northwestern University, Searle 5-474, MC S205, 320 E. Superior St., Chicago, IL 60611.
E-mail: limingli{at}northwestern.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The formation and maintenance of [PSI+] requires interaction of Sup35 with endogenous cellular factors. Molecular chaperones, a group of proteins that exercise protective functions inside the cell by refolding or dis-aggregating denatured proteins, are important components of the cellular machineries required for prion formation and propagation (PIRKKALA et al. 2001). For example, Hsp104, a member of the Hsp100 family, Ssa and Ssb, members of the Hsp70 family, and Ydj1 and Sis1, members of the Hsp40 family, have been implicated in playing important roles in [PSI+] propagation (CHERNOFF et al. 1995; JUNG et al. 2000; KUSHNIROV et al. 2000; JUNG and MASISON 2001; SONDHEIMER et al. 2001; ALLEN et al. 2005). The Hsp90 co-chaperones Sti1 and Cpr7 are also known to influence [PSI+] stability in the context of the mutant ssa1-21 strain harboring an SSA1 point mutation that destabilizes [PSI+] (JONES et al. 2004). Although [PSI+] propagation has been extensively investigated and important insights have been gained regarding the mechanisms of its transmission, the [PSI+] initiation process is less well understood. We have recently reported that two truncation mutants of the heat-shock transcription factor (HSF) strongly influence [PSI+] initiation. An HSF mutant lacking the carboxyl-terminal activation domain, 
CTA-HSF, dramatically increases [PSI+] de novo formation, whereas a mutant lacking the amino-terminal activation domain, NTA-HSF, severely inhibits this process (PARK et al. 2006). Interestingly, CTA-HSF preferentially allows the formation of weak and mosaic [PSI+] variants (PARK et al. 2006). This finding demonstrates that the carboxyl-terminal activation domain of HSF (CTA-HSF) regulates the expression of important factors required for [PSI+] formation and variant determination. To date, five proteins have been conclusively identified as CTA-HSF targets: Hsp90, Sse1, Sti1, Ydj1, and Cpr6 (LIU and THIELE 1999). When Hsp90 was overproduced, no significant effects were observed on preexisting [PSI+] (NEWNAM et al. 1999). Neither overexpression nor deletion of CPR6 had detectable effects on [PSI+] propagation (JONES et al. 2004). Overexpression of YDJ1 cured a weak [PSI+] and a hybrid [PSI+], [PSI+(PS)], but had no notable influence on a strong [PSI+] variant (KUSHNIROV et al. 2000). Overall, STI1 overexpression weakened or destabilized whereas sti1 strengthened [PSI+] (JONES et al. 2004). Recently, Sse1, an Hsp90 co-chaperone and a nucleotide exchange factor (NEF) for the cytosolic Ssa and Ssb proteins, was identified as a novel regulator of [PSI+] propagation (KRYNDUSHKIN and WICKNER 2007). Despite their importance in [PSI+] propagation, whether these CTA-HSF targets play any roles in regulating [PSI+] de novo formation and variant determination has not been investigated. We report here that manipulating the expression levels of individual CTA-HSF target genes can result in dramatic influences on [PSI+] de novo formation. We show that Sse1 not only is important for [PSI+] propagation but also is required for [PSI+] de novo formation and variant determination. In addition, results from mutational analysis also demonstrate that Sse1 correlates the NEF activity of Sse1 with [PSI+] manipulation. Our findings have therefore established Sse1 as an important regulator in [PSI+] biology and raise the possibility that Hsp70 cofactors play critical roles in amyloidoses in higher organisms.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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CTA HSF1, polymerase chain reaction (PCR) was carried out using the 5' primer (5'-AAAGGCCTTAATGAATAGTACACAGGGCAAGGTC-3'), the 3' primer (5'-AGGCACCCCAGGCTTTAC-3'), and pRS305HSF1 as the DNA template. PCR with Taq polymerase (Invitrogen, San Diego) was performed with 1 cycle at 94° for 5 min; 30 cycles at 94° for 30 sec, at 55° for 30 sec, and at 72° for 2 min; and then at 72° for 10 min. The 700-bp PCR product was digested with StuI and XhoI and the resulting 406-bp fragment was ligated to pRS305-HSF1, which had been predigested with StuI and XhoI.
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CTA-HSF integrated strains, the integrating constructs pRS305-HSF1 or pRS305-CTAHSF1 was digested with ClaI and transformed into a 74D-694 ([psi–][RNQ+]) strain whose chromosomal HSF1 was disrupted with a kanR but contained a pRS416-HSF1 plasmid for viability (PARK et al. 2006). Transformants selected on SC–ura–leu were streaked on SC–leu+5-FOA to eliminate the pRS416-HSF1 and the resulting strains were termed 74D-694-I-wtHSF and 74D-694-I-CTAHSF, respectively.
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strains, the disruption construct of sse1::LEU2 (SHANER et al. 2004) was digested with SacII and PstI. The resulting digestion mixture was transformed into various isogenic strains with different prion backgrounds as shown in Table 2. In all cases, SSE1 disruption was confirmed by immunoblot analysis using a polyclonal Sse1 antibody, a kind gift from J. Brodsky's laboratory. The weak [PSI+] variant, [PSI+]W, was obtained from 74D-694 cells ([psi–][RNQ+]) after transient overexpression of SUP35NMGFP as described (PARK et al. 2006).
Yeast cultures and other genetic manipulations were performed according to the established protocols (SHERMAN 1991).
[PSI+] induction and variant determination:
[PSI+] induction in cells containing pCUP1-NMGFP was performed as described previously (PARK et al. 2006). Briefly, cells were grown in selective media at 30° overnight before diluted into fresh media at a density of 
4 x 106 cell/ml. After an additional 1 hr of growth at 30°, CuSO4 was added to a final concentration of 34 µM. At various induction times, cells were spotted with a fivefold serial dilution onto SC-ade and YPD plates. [PSI+] induction in cells containing pGAL-NMGFP was carried out by growing cells in selective media with 2% raffinose to mid-log phase (A600 = 0.5) before adding galactose to a final concentration of 2% to induce Sup35NMGFP production. As time indicated, cultures were spread onto SC–ade and YPD plates to obtain individual colonies. In all cases, Ade+ colonies were considered as [PSI+] candidates but only GdnHCl-curable Ade+ isolates were scored as [PSI+]. Subsequent [PSI+] confirmation and variant determination were carried out as described (PARK et al. 2006).
For experiments described in Figure 2, isogenic strains of 74D-694-I-wtHSF and 74D-694-I-CTAHSF (see Table 2 for strain descriptions) were cotransformed with either pRS313CUP1-NMGFP and p2UGHSP82 or pRS313CUP1-NMGFP and p426GPDSSE1. To examine the overexpression effect of STI1, the same cells were transformed with pCUP1-NMGFP and pRS423STI1 (a kind gift from D. Masison). [PSI+] de novo formation was analyzed using cell patches as described (DERKATCH et al. 2001). Briefly, individual transformants were patched onto plates selective for the containing plasmids followed by replica plating onto plates containing 70 µM CuSO4. After incubation for 20 hr at 30°, the cell patches were replica plated onto SC–ade to view potential [PSI+] colonies. To quantify [PSI+] de novo formation (Figure 2, right), three individual transformants of each transformation were grown in liquid media selective for the indicated plasmids to early log phase followed by addition of CuSO4 to a final concentration of 34 µM. After 4 hr of induction, cells were counted and spotted onto SC–ade and YPD plates with a fivefold serial dilution. Ade+ colonies that were cured by GdnHCl were scored as [PSI+]. The ratio of [PSI+] to the total number of viable cells (calculated from colonies on the YPD plates) was used to determine the frequency of [PSI+] de novo formation.
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on [PSI+] de novo formation (shown in Figure 3), an SSE1 disruption strain of 74D-694 ([psi–][RNQ+]) and the isogenic wild-type strain ([psi–][RNQ+]) were transformed with pCUP1-NMGFP and the resulting transformants were assayed for [PSI+] de novo formation using CuSO4 as an inducer as described above. [PSI+] determination and calculation of [PSI+] appearance percentage were also carried out as described above. At least three transformants were used for each experiment and results from three independent transformations were summarized.
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SDS–PAGE and immunoblot analyses:
Yeast cells grown overnight in either YPD (Sse1 immunoblot analysis) or selective media to mid-log phase ([PSI+] induction experiments) were harvested by centrifugation at 3000 rpm for 3 min followed by washing once with sterile water. The washed cells were used for preparing total protein extracts by the ethanol lysis method (PARK et al. 2006). The resulting protein extracts were analyzed by SDS–PAGE and immunoblot analysis as described previously (PARK et al. 2006). Antibodies used in this study were kindly provided by S. Lindquist (Hsp104, Hsp90, and Rnq1), E. Craig (Ssa, Ssb, and Sis1), J. Brodsky (Sse1), and S. Liebman (Sup35).
Semidenaturing agarose gel electrophoresis:
Crude protein extracts prepared from isogenic strains of 74D-694 [PSI+]S[RNQ+], [PSI+]W[RNQ+], and their corresponding sse1 derivatives were subjected to SDS–PAGE analysis according to BAGRIANTSEV et al. (2006) with minor modifications. Briefly, yeast cultures were grown in liquid YPD media to A600 of 1.5–2.0 and harvested by centrifugation at 3000 rpm for 3 min. After washing once with sterile water, the cell pellet was suspended in the extraction buffer containing 50 mM Tris–HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 5% glycerol, 8 µg/ml aprotinin, 8 µg/ml leupeptin, 10 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (Roche protease inhibitor complete mini, 1/2 tablet/10 ml). After homogenization for 4 x 1 min in a bead beater with 0.5-mm glass beads, the crude lysates were centrifuged at 600 x g for 1 min at 4°. The supernatant was incubated for 7 min in the sample buffer containing 50 mM Tris–HCl (pH 6.8), 5% glycerol, 2% SDS, and 0.05% bromophenol blue at room temperature before being loaded onto a 1.5% horizontal agarose gel as described (BAGRIANTSEV et al. 2006).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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CTA-HSF dramatically influences the de novo formation of [PSI+] and Sse1 is a CTA-HSF target, we decided to examine whether manipulating the expression levels of SSE1 would have any effects on [PSI+] de novo formation. [PSI+] can arise spontaneously from [psi–] cells with a very low frequency (TUITE et al. 1981). However, [PSI+] formation can be greatly enhanced by Sup35 overproduction in the presence of another prion, [RNQ+] (CHERNOFF et al. 1993; DERKATCH et al. 1997). To examine if overexpression of SSE1 would affect [PSI+] de novo formation, we transformed 74D-694 ([psi–][RNQ+]) cells with pGAL-NMGFP, a galactose-inducible plasmid expressing a GFP fusion of the N-terminal and middle regions of Sup35 (1–265 aa), and p426GPDSSE1, a strong constitutive SSE1 expression plasmid. The resulting transformants were analyzed for [PSI+] de novo formation. Since the GAL promoter is tightly regulated, it was possible to analyze how Sse1 overproduction could kinetically affect [PSI+] de novo formation. As shown in Figure 1A, Sse1 overproduction dramatically stimulated [PSI+] formation throughout the time course of study. After 72-hr induction with galactose, the average [PSI+] formation in cells containing the SSE1 overexpression plasmid p426GPDSSE1 was 8.7% whereas cells containing the empty vector p426GPD had an average [PSI+] formation rate of 0.06%, an 145-fold difference. The stimulatory influence of Sse1 on [PSI+] formation was also evident when Sup35NMGFP was overproduced under the regulation of a CUP1 promoter (pRS313CUP1-NMGFP). Upon CuSO4 addition, the number of Ade+ colonies derived from cells containing pRS313CUP1-NMGFP and p426GPDSSE1 was significantly greater than that derived in cells containing pRS313CUP1-NMGFP and p426GPD (Figure 1C, top). However, the stimulatory effect of Sse1 on [PSI+] formation was less dramatic when the CUP1 promoter was used. After a 4-hr induction, the difference in [PSI+] formation in the presence or absence of Sse1 overproduction was 10-fold. This smaller difference is likely due to the fact that the CUP1 promoter is not tightly regulated (TAMAI et al. 1994; HAHN et al. 2004). We also examined the pattern of Sup35NMGFP fluorescence in the presence of overproduced Sse1. As shown in Figure 1B, the observed Sup35NMGFP fluorescent foci in cells containing p426GPDSSE1 (20%) is significantly more than that of cells containing the empty vector p426GPD (0.2%). We conclude that overproduction of Sse1 promotes both Sup35NMGFP aggregation and [PSI+] formation.
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To further investigate whether this [PSI+]-promoting effect was caused indirectly by modulating other cellular factors in response to Sse1 overproduction, we examined the expression levels of several molecular chaperones known to influence [PSI+]: Hsp104, Ssa1/2, Ssb1/2, and Sis1. Since Sse1 has been identified as an Hsp90 co-chaperone (LIU and THIELE 1999), we also examined the expression levels of Hsp90 and its co-chaperone Sti1. As shown in Figure 1D, all examined molecular chaperones displayed similar expression levels in cells with or without overproduction of Sse1, except Hsp104, which increased about twofold in cells overproducing Sse1.
Next, we examined if other CTA-HSF targets—Hsp90, Sti1, Cpr6, and Ydj1—would have effects similar to Sse1 on [PSI+] de novo formation. Interestingly, as shown in Figure 2, striking differences were observed for different CTA-HSF targets. In contrast to Sse1, overexpression of HSP82 severely inhibited [PSI+] formation (Figure 2). Similar results were also obtained when HSC82 was overexpressed (data not shown). As shown in Figure 2, overproduction of Sti1 also strongly inhibits [PSI+] formation. However, elevated expression levels of Cpr6 and Ydj1 had no significant effects on [PSI+] de novo formation (data not shown). The same experiments were also conducted for CTA-HSF cells. As shown in Figure 2, in all cases, CTA-HSF cells gave rise to significantly more Ade+ colonies than wt-HSF cells did, confirming our previous report that CTA-HSF stimulates [PSI+] de novo formation (PARK et al. 2006). However, the inhibitory effects upon Hsp90 and Sti1 overproduction and the stimulatory effect upon Sse1 overproduction were also evident in CTA-HSF cells (Figure 2). Since overproduction of each individual CTA-HSF target resulted in similar effects in both wt-HSF and CTA-HSF cells, it is unlikely that the observed effects by Hsp90, Sti1, or Sse1 overproduction were caused by the deletion of the CTA of HSF. Our results thus demonstrate that different components of the Hsp90 complex play distinct roles in [PSI+] de novo formation.
The effects of SSE1 disruption on [PSI+] formation and variant determination:
To further analyze the role of Sse1 in [PSI+] regulation, we next examined the effect of SSE1 disruption on [PSI+] de novo formation. A chromosomal SSE1 disruption strain (sse1) was created in 74D-694 ([psi–][RNQ+]). After transformation with pCUP1-NMGFP, isogenic sse1 and wild-type cells were assayed for [PSI+] induction. If Sse1 is a [PSI+]-promoting factor, SSE1 disruption would be expected to abolish or reduce [PSI+] formation. As shown in Figure 3A, the number of [PSI+] colonies was indeed greatly reduced in sse1 cells when compared to that of wild-type cells. To examine if sse1 also affects [PSI+] variant determination, Ade+ colonies formed in sse1 and isogenic wild-type cells were randomly picked and streaked onto YPD plates to view their colony appearances. Interestingly, all Ade+ cells formed in sse1 cells had the appearance of very weak [PSI+] variants, which were unstable and appeared undifferentiated (Figure 3B). This appearance is similar to that of an unstable, undifferentiated [PSI+] variant ([PSI+]U) formed in CTA-HSF cells (PARK et al. 2006). A careful, side-by-side comparison of [PSI+]U cells formed in CTA-HSF and sse1 cells under identical induction conditions, however, revealed distinct colony morphologies. As shown in Figure 3C, the CTA[PSI+]U colonies are more distinctly sectored but the sse1[PSI+]U colonies do not have clear sectoring boundaries. Multiple independent [PSI+] induction experiments were conducted and no uniformly colored white or pink colonies were observed in sse1 cells. Despite repeated efforts, we were not able to transfer [PSI+]U formed in sse1 cells to wild-type [psi–] cells through mating/sporulation. sse1[PSI+]U is easily lost during the mating process to yield [psi–] diploids. Thus, [PSI+]U formed in sse1 cells is extremely unstable and poorly transmitted. In comparison, CTA[PSI+]U can be easily transmitted to wild-type cells as [PSI+]W (PARK et al. 2006).
We also examined the steady-state levels of several molecular chaperones in sse1 cells. As shown in Figure 3D, there were no significant changes in expression of the examined molecular chaperones, including Ssa1/2, Ssb, Hsp104, Sis1, Sti1, and Hsp90. Our data suggest that the lack of Sse1 is responsible for the observed reduction in [PSI+] formation and deficiency in variant determination.
Mutational analysis of SSE1 on [PSI+] variant determination:
As a nucleotide exchange factor, Sse1 stimulates ADP release from ADP-bound Hsp70 to allow it to rebind to ATP and thus promotes the Hsp70 cycle. This ADP/ATP exchange is essential for refolding thermally denatured luciferase (DRAGOVIC et al. 2006; RAVIOL et al. 2006). The nucleotide exchange function of Sse1 requires the ATP-binding domain and the substrate-binding domain (DRAGOVIC et al. 2006; RAVIOL et al. 2006; SHANER et al. 2006). Mutations predicted to abolish ATP hydrolysis in Sse1 do not affect its function in vivo or in vitro, while those that block nucleotide binding also prevent the binding to Hsp70 (DRAGOVIC et al. 2006; RAVIOL et al. 2006; SHANER et al. 2006). For example, Sse1-K69Q, a mutation that inhibits ATP hydrolysis in other Hsp70s, retains Hsp70-binding capability; Sse1-G233D, however, cannot bind to ATP or Hsp70 and Sse1-PBD, which contains only the carboxyl terminal peptide (substrate)-binding domain, is likewise inactive in vivo (SHANER et al. 2004; SHANER et al. 2006). To test if the observed Sse1 effects on [PSI+] variant determination are due to the chaperone's function as an Hsp70 NEF, we analyzed [PSI+] variants formed in the cells whose endogenous SSE1 was disrupted but which contain a CEN plasmid expressing one of the three described sse1 mutants. Ade+ colonies formed in these mutant cells were randomly picked and streaked onto YPD to view their colony appearances. As shown in Figure 3E, cells expressing wild-type SSE1 and SSE1-K69Q gave rise to [PSI+] variants from very weak to very strong. Cells expressing SSE1-G233D or SSE1-PBD gave rise only to [PSI+]U, an unstable [PSI+] variant with the same colony appearance as that of [PSI+]U formed de novo in sse1 cells. To exclude the possibility that the differences that we observed among the different sse1 mutants were due to differences in their expression levels or stabilities, the steady-state levels of individual Sse1 mutant proteins were estimated by immunoblot analysis using a polyclonal antibody recognizing Sse1. As shown in Figure 3F, there was no reactive Sse1 band in protein extracts prepared from the sse1 cells transformed with an empty vector (lane 2), confirming that the chromosomal SSE1 was disrupted. However, immunoblot of protein extracts prepared from sse1 cells containing p414TEFSSE1, p414TEFSSE1K69Q, or p414TEFSSE1G233D indicated that all SSE1 alleles were expressed at similar levels. Since ectopic expression of these SSE1 alleles in wild-type cells gave rise to [PSI+] variants with a full spectrum from very weak to very strong, the effects of these sse1 mutant alleles on [PSI+] variant establishment are not dominant (data not shown). Taken together, our data suggest that the binding of Sse1 to Hsp70, with concomitant NEF activity, is essential for establishing a full spectrum of distinct [PSI+] variants.
The influence of Sse1 on [PSI+] propagation:
We also investigated the role of Sse1 in [PSI+] propagation. In agreement with a recent report (KRYNDUSHKIN and WICKNER 2007), we observed that overproduction of Sse1 has no effect on preexisting [PSI+], either [PSI+]S or [PSI+]W (data not shown). Disruption of SSE1, however, dramatically influences [PSI+] propagation. Figure 4A (left) shows representative streaks of isogenic 74D-694 cells of [PSI+]S, [PSI+]W, and their corresponding SSE1 disruption derivatives. SSE1 disruption was confirmed by immunoblot analysis (Figure 4A, right). The sse1 derivatives of [PSI+]S had a redder colony appearance than that of the [PSI+]S control (Figure 4A, left), suggesting that either a new [PSI+] variant was reestablished upon sse1 or the [PSI+]W-like phenotype was a different readout of [PSI+]S in sse1 genetic background. Upon SSE1 disruption, [PSI+]W appeared as red as [psi–] cells (Figure 4A, left). This indicates that either sse1 cured [PSI+]W or the [PSI+]W phenotype was masked in the sse1 background. For further clarification, we crossed the sse1 derivatives of both [PSI+]S and [PSI+]W with isogenic [psi–] cells containing the wild-type SSE1 gene. As shown in Figure 4B, the diploids derived from sse1[PSI+]S x [psi–] had the [PSI+]S colony appearance, indicating that disruption of SSE1 in [PSI+]S did not change [PSI+]S into a weaker variant. The pink appearance is thus a modified readout of [PSI+]S in sse1 background. This conclusion was further supported by results from sporulation of the sse1/SSE1 diploids of [PSI+]S, which gave rise to four spores with a ratio of 2:2 of [PSI+]S to [PSI+]W-like spores (Figure 4B). The [PSI+]W-like spores were verified to be sse1 cells as the colonies were smaller and they were able to grow on SC–leu media (SSE1 was disrupted by a LEU2 marker). In this regard, it has been reported that sse1 null mutants grow more slowly than isogenic wild-type cells (LIU et al. 1999). In contrast to masking the [PSI+]S phenotype, results from similar crosses of [PSI+]W to [psi–] indicate that sse1 had caused [PSI+]W loss. As shown in Figure 4B, diploids derived from sse1[PSI+]W and [psi–] had the [psi–] colony appearance. Upon sporulation, they gave rise to four spores that were all [psi–], indicating that [PSI+] was eliminated in the sse1 cells. Subsequent analysis confirmed that none of the spores were able to grow in SC–ade media (data not shown).
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on the polymer sizes of Sup35:[PSI+]U cells have the average size of Sup35 polymers similar to that of [PSI+]W cells but with a much lower yield. This is likely due to the fact that some [PSI+] elements were lost during the culturing process as a result of [PSI+]U instability or of the lower amount of Sup35 polymers contained in the sse1[PSI+]U cells.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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A fascinating phenomenon of prion biology is the existence of "strains" or variants. There are multiple PrPSc forms described, which are associated with distinct prion diseases varying in symptoms, incubation times, and brain pathologies (PRUSINER 1998). All identified yeast prions are able to exist in multiple variants (DERKATCH et al. 1996; SONDHEIMER and LINDQUIST 2000; SCHLUMPBERGER et al. 2001). Despite decades of effort, it remains unclear how a prion polypeptide adopts multiple heritable conformations. Identification of cellular factors responsible for establishing multiple prion conformations will likely shed light on the underlying mechanism required for prion variant determination. Although sse1 cells are capable of propagating the strong variant of [PSI+] (Figure 4A), we have not been able to obtain uniformly colored pink or white colonies from sse1 cells upon Sup35 overproduction. Only [PSI+]U was permitted to form de novo in sse1 cells. Thus Sse1 is an important [PSI+] variant determinant. Our finding that the physical association of Hsp70 with Sse1 is required for establishing a full spectrum of [PSI+] variants suggests that the concerted action of Sse1–Ssa and/or Sse1–Ssb might be a key to [PSI+] variant determination. In this regard, it has been shown that overproduced Sup35-HA is physically associated with Ssa and Ssb both in vivo and in vitro (ALLEN et al. 2005).
We have previously shown that CTA-HSF cells preferentially give rise to a weak [PSI+] variant that is unstable, CTA[PSI+]U (PARK et al. 2006). Although Sse1 is a CTA-HSF target (LIU and THIELE 1999) and is important for [PSI+] variant establishment (Figure 3), the effect of CTA-HSF on [PSI+] variant formation is unlikely for several reasons due to the lack of Sse1. First, the [PSI+]U cells formed in sse1 cells have a unique colony appearance that is distinct from that of [PSI+]U formed in CTA-HSF cells. As shown in Figure 3C, although both CTA[PSI+]U and sse1[PSI+]U have a colony appearance similar to very weak [PSI+] variants, their detailed colony morphologies are different. Second, sse1[PSI+]U is much less stable than CTA[PSI+]U. Third, ecotopic expression of SSE1 driven by promoters of variable strength cannot change the preference of [PSI+]U formation in CTA-HSF cells (data not shown). Thus, the preference of [PSI+]U formation in CTA-HSF cells is likely caused by an unknown factor(s) other than Sse1. It is also possible that the imbalance of molecular chaperones in the CTA-HSF cells is the responsible factor.
We have shown that overproduction of Sti1 and either isoform of Hsp90 severely inhibited [PSI+] de novo formation whereas overproduction of Sse1 dramatically stimulated it (Figure 2). Overproduction of Cpr6 or Ydj1, however, exhibited no significant effects. Our finding demonstrates that different Hsp90 co-chaperones have distinct effects on [PSI+] de novo formation and variant determination. This finding is intriguing as the Hsp90 complex is responsible for the maturation of a large number of client proteins and is an important target for anticancer therapy. The effects of individual components of the Hsp90 complex on [PSI+] de novo formation may be due to their dynamic association with the Hsp90 complex. It is also possible that the Hsp90 co-chaperones exercise distinct functions when they are or are not in association with Hsp90. In this regard, it has been shown that Sti1 and Cpr7 can also form stable complexes with Hsp104 both in vivo and in vitro (ABBAS-TERKI et al. 2001). How such dynamic associations of Sti1 and Cpr7 with Hsp90 and Hsp104 affect [PSI+] formation and propagation, however, has not been investigated. Modulating the levels of one Hsp90 co-chaperone might also affect the overall structure and integrity of the Hsp90 complex and thus affect their associated cofactors and client proteins. For example, overproduction of Hsp90 might cause more Sse1–Ssa1 complex to be associated with Hsp90, thus reducing the Sup35 associated Sse1–Ssa1 complex. As a consequence, [PSI+] de novo formation is reduced. However, increasing the expression levels of Sse1 would promote the nucleotide exchange of ADP to ATP of Ssa and thus enhance [PSI+] formation. Although it is less likely, our results cannot rule out the possibility that the Hsp90 complex directly associates with Sup35 to affect [PSI+] de novo formation.
A recent study by KRYNDUSHKIN and WICKNER (2007) showed that overproduction of Sse1 can efficiently cure [URE3]. However, results from both KRYNDUSHKIN and WICKNER (2007) and our independent study (data not shown) indicate that overproduction of Sse1 has no detectable effect on preexisting [PSI+], including both [PSI+]S and [PSI+]W variants. The effect of sse1 on [PSI+] is more complicated. Disruption of SSE1 weakens [PSI+]S and completely eliminates [PSI+]W (KRYNDUSHKIN and WICKNER 2007; Figure 4). How altered SSE1 expression levels affect [RNQ+] has not been systematically investigated. However, neither overexpression nor disruption of SSE1 has detectable effects on the particular [RNQ+] variant in the 74D-694 strains used in this study (data not shown). Taken together, the Hsp110 chaperone Sse1 is not only a prion-specific but also a variant-specific factor that plays important and distinct roles in maintaining various prion elements in yeast.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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, NMGFP
cells of 74D-694 ([psi–][rnq–]) and the resulting diploids and spores are shown.