Hsp90-Associated Immunophilin Homolog Cpr7 Is Required for the Mitotic Stability of [URE3] Prion in

Summary

Studies on yeast prions have provided insight into the role of various cellular factors involved in amyloid based disorders. Among the various cellular components the chaperone network has emerged as a crucial regulator of formation and propagation of yeast prions. The chaperone machinery primarily consists of Hsp70 and Hsp90 families of proteins, whose activity is further modulated by multiple co-chaperones. Though it is known that Hsp70s and its co-factors influence prion strength and stability, the role of Hsp90 chaperone machinery in yeast prion propagation is not clear. Here we examine the role of Hsp90 chaperones and show that the C-terminal MEEVD motif on Hsp90, which is required for interaction with tetratricopeptide repeat domain containing co-chaperones, is crucial for propagation of the yeast prion [URE3]. Further study revealed a novel role of the Hsp90 co-chaperone Cpr7 in the propagation of infectious amyloid and thus broadens our understanding about the cellular role of immunophilins.

Introduction

Similar to human prion protein (PrP), many yeast proteins have a tendency to undergo conformational conversion into infectious protein particles known as prions [1,2] [3]. The yeast prions have served as a great model system to understand the mechanistic details of prion induction, propagation, formation of prion variants and roles of various cellular factors in prion maintenance [4,5,6]. Two of the most extensively studied yeast prions [PSI⁺] and [URE3] are formed of native proteins Sup35 and Ure2 respectively. Sup35 is a translation termination factor and Ure2 is involved in nitrogen catabolism to repress nitrogen uptake from poor nitrogen sources, like proline, when a good nitrogen source such as ammonia, glutamine or asparagine, is present. Though the prion-forming domains of yeast proteins do not show a significant amino acid sequence similarity with many amyloid-forming proteins in mammals, various constituents of cellular machinery that influence yeast prions also affect mammalian amyloid based disorders, suggesting a functional conservation of cellular factors with regard to amyloidosis.

Among various cellular factors, heat shock proteins (Hsps), consisting mainly of the Hsp70 family and its co-chaperones, play a major role in the maintenance of yeast prions [7,8,9,10,11,12]. Hsp104, which acts in coordination with the Hsp70 system, is essential for propagation of yeast prions. Also, Hsp104 overexpression leads to [PSI⁺] curing [13,14,15]. When present as the sole source of Ssa Hsp70, Ssa1 antagonizes [URE3], but not [PSI⁺], while cells expressing only Ssa2 show a weak [PSI⁺] phenotype [16]. The overexpression of the Hsp70 co-chaperone Ydj1 or the Hsp70 nucleotide exchange factor (NEF) Sse1 cure [URE3] by a mechanism that requires interaction with Hsp70 [17,18]. Both Sse1 and Fes1 are required for [URE3], and Sse1 also regulates [PSI⁺] induction and propagation in the prion variant specific manner [10,17,19]. Similarly, [SWI⁺] another well studied yeast prion is highly sensitive to alteration in activity of Hsp70 and its co-chaperones [20]. It is believed that chaperones, primarily Hsp70s, play crucial roles in promoting fibril growth and replication required for stable prion propagation [21] [22,23] [16]. Though the role of Hsp70 proteins in yeast prion maintenance has been widely studied, not much is known about Hsp90 function in this regard.

The highly conserved Hsp90 family is essential for cellular growth in all eukaryotes. Hsp90 interacts with about 10% of all cellular proteins and the major clients include kinases, growth hormone receptors [24], transcription factors [25] [26] and signal-transduction factors [27] [28]. Each protomer of dimeric Hsp90 consists of three domains: the N-terminal nucleotide binding domain (NBD), middle domain (MD), and a carboxy-terminal domain (CTD). The Hsp90 dimer exists in a dynamic equilibrium between its open and closed conformation. In the open state Hsp90 is dimerized at its C-terminal domain with N-terminal domains separated. The large-scale conformational changes in Hsp90 upon ATP binding result in dimerization of N-terminal ATPase domains trapping substrate in the substrate binding pocket [29,30]. The charged linker region between the amino terminal and middle domains is crucial for dimerization of Hsp90 at the amino-terminal domain [31].

Hsp90 function is regulated by various other proteins that either modulate its ATPase activity or facilitate interaction with its client proteins. Hsp90 co-chaperones can be broadly divided into those with or without tetratricopeptide repeat (TPR) domains. The TPR domain containing co-chaperones such as Sti1, Cpr6, Cpr7, Cns1, Ppt1 and Tah1 compete for binding at the MEEVD motif present at the C-terminus of Hsp90 [32,33,34] and thus regulate different steps of Hsp90 reaction cycle. The deletion of the highly conserved MEEVD pentapeptide does not cause a growth defect in S.cerevisiae suggesting that even in the absence of the motif, Hsp90 is functional enough to support essential in vivo roles required for cellular viability.

The TPR proteins participate in many cellular processes that include chaperonin activity, phosphatases, transcriptional regulation and cell cycle control [33]. The TPR domain containing Hsp90 co-chaperones influence client protein maturation by regulating Hsp90 ATPase activity, interaction with other cellular partner proteins, and conformational transition among various dynamic states formed during different stages of its reaction cycle. In addition to the TPR domain, Cpr6 and Cpr7 of the cylophilin family also contain a peptidyl-prolyl isomerase (PPIase) domain that catalyzes the cis-trans isomerization of peptide bonds N-terminal to the proline residues in proteins. Deleting the PPIase domain of Cpr7 does not affect yeast growth or Hsp90 activity, and the exact role of its PPIase domain in client maturation remains uncertain [35]. Unlike Cpr6, Cns1, which is essential for yeast cell growth, complements some Cpr7 functions indicating partial functional redundancy among some TPR proteins. Recently, there is emerging interest in the immunophilin family of proteins that includes FK506 binding proteins and cyclophilins for their role in various neurodegenerative diseases [36] [37].

Many Hsp90 clients first enter into the Hsp70-Hsp40 reaction cycle [38]. The bridge proteins Hop/Sti1 form an intermediate complex with Hsp70 and Hsp90 thus facilitating substrate transfer to Hsp90, which remains in an ATP-free open conformation in the complex [39]. The Hsp90 cycle then begins with ATP binding and displacement of Hop/Sti1 with other TPR containing co-chaperones belonging to peptidyl-prolyl-cis/trans isomerase family such as yeast Cpr6 and Cpr7 [34]. Other non-TPR co-chaperones such as p23/Sba1 and Aha1 influence client maturation by modulating Hps90 conformational changes and its ATPase activity [40,41,42].

What determines substrate specificity and how a large pool of substrates gets partitioned between Hsp70 and Hsp90 is poorly understood. In contrast to the known role of cytosolic Hsp70s, not much is known about the role of Hsp90 and its co-chaperones on yeast prion formation and propagation. Though Hsp90 inhibition has no effect on [PSI⁺], indirect evidence suggests some unknown role of Hsp90 proteins in yeast prions maintenance. Alterations of heat shock transcription factor, whose activity is regulated by Hsp90, influence de novo [PSI⁺] formation and propagation [43]. Similarly, Hsp90 co-chaperones Sti1 and Cpr7 are important for [PSI⁺] curing upon Hsp104 overexpression [44,45]. Furthermore, Cpr7 is involved in maintaining conformation of prion variants [46]. Similarly, other TPR co-chaperone such as overexpressed Sgt2, which is involved in the guided entry of tail-anchored proteins (GET) trafficking pathway, enhances excess Ssa Hsp70 mediated protection of [PSI⁺] from curing by excess Hsp104 [47].

Here, we explore whether Hsp90 plays any role, direct or indirect, on yeast prion propagation. We show that [URE3] propagation is not affected by inhibition of Hsp90 or by deleting either of the genes encoding Hsc82 or Hsp82. Interestingly, though mutations affecting Hsp90 conformational changes have no effect on [URE3], those lacking the C-terminal pentapeptide motif destabilize [URE3], revealing that protein-protein complexes of Hsp90 and its TPR co-chaperones has a crucial role in prion maintenance. Our data suggest that the decrease in [URE3] stability in cells expressing Hsp90ΔMEEVD is due to loss of Hsp90 interaction with Cpr7. Our study uncovers a role of Cpr7 in [URE3] propagation and that it is unique among known Hsp90 co-chaperones in its being required for prion maintenance. Our data suggest that Hsp90 controls the fate of not only its client proteins but also other cellular substrates through the functional modulation of other members of the chaperone machinery.

Results

The conserved C-terminal MEEVD motif of Hsp90 is required for [URE3] stability

It is known that Hsp70 and Hsp90 family proteins influence folding and maturation of many cellular proteins, yet what determines chaperone specificity is not entirely clear. Whether yeast prions that are well known Hsp70 substrates also require Hsp90 remains unclear. In order to explore the role of Hsp90 chaperones on yeast prions, S. cerevisiae strains having deletion of either HSC82 or HSP82 were constructed. As seen in Fig 1A, no significant effect on [URE3] was observed suggesting that the presence of either Hsp90 is enough to support stable prion propagation. Alternatively, the Hsp90 family of proteins may be dispensable for [URE3] propagation.

To address the latter possibility, we cultured yeast [URE3] cells lacking HSC82 (strain SY297) in the presence of the known Hsp90 inhibitor 17-AAG (S1 Fig). The inhibitor competes with ATP for binding to Hsp90 and thus inhibits its activity [48]. Inhibition of Hsp90 leads to Hsp70 overexpression and a defect in the maturation of its client proteins [49]. We first examined the effect of 17-AAG on Hsp70 expression and the steady state level of the Hsp90 client v-Src in yeast strain SY136. The SY136 strain was transformed with plasmid (pRS316P_GAL-FLAG-vSrc) encoding for FLAG-tagged v-Src. The transformants were further cultured in liquid SGal media lacking uracil in the presence and absence of 17-AAG. As expected, incubation with 17-AAG (50μM in DMSO) upregulated Hsp70 expression. Also 17-AAG treated cells showed reduced steady state level of Hsp90 client protein v-Src (S1A Fig) with no significant change in its mRNA levels (S1B Fig). However 17-AAG (100μM in DMSO) mediated inhibition of Hsp90 had no effect on [URE3] stability, suggesting the Hsp90 function crucial for client maturation does not contribute much to [URE3] propagation (S1C Fig).

Hsp90 is essential and thus to examine whether Hsp90 influences [URE3], we monitored prion propagation in yeast cells expressing the previously known Hsp90 mutants, His₆-Hsp82Δ211–264 or His₆-Hsp82ΔMEEVD as the sole source of Hsp90 [34]. Amino acids 211–264 form a part of the charged linker region required for Hsp90 conformational changes and thus cells expressing His₆-Hsp82Δ211–264 as the only Hsp90 grow poorly at 37°C [34]. When combined with cpr7Δ the growth defects are additive even at 30°C. Also, strains expressing a similar construct Hsp90Δ211–263 show reduced activation and lower steady state level of its client v-Src [31]. The highly conserved C-terminal pentapeptide MEEVD is a common interaction site where various Hsp90 co-chaperones possessing TPR domains compete for binding to Hsp90. As seen in Fig 1B, a similar frequency (>99%) of [URE3] colonies was observed in cells expressing wt His₆-Hsp82 or His₆-Hsp82Δ211–264. However, cells expressing His₆-Hsp82ΔMEEVD exhibit an increased [ure-o] phenotype as seen by an increase in the number of red colonies (about 25 ± 5%), which arise from cells that lost [URE3]. The increase in frequency of [ure-o] colonies is not due to differences in steady state abundance of Hsp90, Hsp70, Ydj1, or Sse1, as shown in S2 Fig. The results indicate that the presence of the C-terminal MEEVD motif is required for the stability of [URE3]. Overall the data suggest an important previously unknown role of Hsp90, independent of its client maturation activity, in the propagation of prions.

Cpr7 is required for [URE3] stability

As compromising Hsp90 client maturation activity does not affect [URE3], the appearance of [ure-o] cells in the His₆-Hsp82ΔMEEVD strain points toward a crucial role of Hsp90 co-chaperones in prion propagation. In order to examine the role of various Hsp90 co-factors, we created many single-knockout S. cerevisiae strains each lacking an Hsp90 co-chaperone (Sti1, Sba1, Cpr6, Cpr7, Ppt1, Tah1, Hch1 or Aha1) and monitored [URE3]. As shown in Fig 2A the deletion of non-TPR Hsp90 co-chaperones (Sba1, Hch1 or Aha1) had no effect on the prion propagation. Among the TPR domain containing proteins, only deletion of the gene encoding Cpr7 profoundly affects [URE3] stability as seen by the appearance of red colony color as well as poor growth on solid medium lacking adenine (Fig 2A and 2B). To examine whether this effect was [URE3] specific, we further constructed similar single-knockouts in strain 779-6A propagating the other well studied yeast prion [PSI⁺]. In contrast to its requirement for [URE3], lack of Cpr7 had no effect on [PSI⁺] stability which is also in agreement with a previously reported study [50]. Similarly, other knockout strains carrying a single gene deletion encoding one of seven other Hsp90 co-chaperones also show no apparent differences in [PSI⁺] stability (Fig 2C). We also examined the effect of deleting Cpr7 on strong and weak [PSI⁺] variants and found no difference in the prion stability in the presence and absence of Cpr7 (S3 Fig). Thus both weak and strong [PSI⁺] variants remain largely unaffected by Cpr7 deletion which is distinct from the effect of Hsp70 co-chaperone Sse1 where deletion of Sse1 destabilize weak [PSI⁺] but has no effect on strong [PSI⁺] prions [51].

As described in Materials and Methods, the red phenotype in [ure-o] cells is due to repression of ADE2 transcription by Ure2 protein. Any cellular activity that negatively affects ADE2 transcription or Ade2 protein stability, even independently of Ure2, could also produce similar red colony color phenotype as seen for [ure-o]. Thus, it was possible that [URE3] was still present in cpr7Δ cells, but its phenotype was masked by lack of Cpr7. In order to explore these possibilities we transformed [ure-o] colonies from the cpr7Δ strain with a plasmid encoding Cpr7 (pRS316P_CPR7-CPR7). If in cpr7Δ cells the red colony color is independent of [URE3], or the [URE3] phenotype is masked, then the transformants would show white phenotype upon plasmid based complementation of CPR7. Alternatively if the red phenotype of cpr7Δ cells is due to the absence of [URE3], the transformants would continue to show red phenotype as the frequency of spontaneous appearance of [URE3] clones is rare. As shown in S4 Fig, more than 99% of transformants show red colony color phenotype suggesting deletion of gene encoding Cpr7 results in loss of [URE3].

In order to further examine whether [URE3] propagation depends upon the presence of Cpr7, we sporulated a [URE3] diploid strain heterozygous for cpr7Δ (CPR7/cpr7::KanMX) and dissected multiple tetrads. In yeast the chromosomally inherited traits follow Mendelian segregation in 2:2 fashion following meiosis however the cytoplasmically inherited infectious yeast prion particles segregate in a non-Mendelian fashion and are transmitted to all four spores. Upon tetrad dissection of a diploid strain heterozygous for CPR7, [URE3] segregated in a Mendelian 2:2 fashion, as seen by white colony color (Fig 2D). These results suggest the [ure-o] phenotype is linked to one of the segregating genes. All colonies having red colony color, and none of the white colonies, grew normally on a plate containing G418 showing that all [ure-o] colonies lack gene encoding Cpr7. Together our results clearly indicate that stable [URE3] maintenance requires the presence of Cpr7 and that the absence of [URE3] in cpr7Δ cells is not due to synthetic lethality of [URE3] with depletion of Cpr7.

[URE3] loss is not due to altered expression of other chaperones

Modulation of Hsp70 activity can alter yeast prion formation and propagation. Similarly, overexpressing the Hsp70 co-chaperones Ydj1 or nucleotide exchange factor Sse1 destabilize [URE3]. In order to explore whether the loss of [URE3] in cpr7Δ strain is due to alteration in the overall abundance of Hsp70 and/or its co-chaperones, their relative expression level was monitored in [URE3] and [ure-o] cells of wild type and cpr7Δ strains. As seen in Fig 3A, Ydj1, Sse1 and Hsp90 are expressed at similar levels in both wild type and cpr7Δ strains, regardless of [URE3] status. In contrast, the Hsp70 level was increased by about 1.5–2.0 fold in the cpr7Δ strain as compared to that of wild type strains (Fig 3B).

The four Ssa Hsp70 isoforms in S.cerevisiae influence [URE3] propagation, and though they are highly homologous they have distinct effects on yeast prions [16]. In order to examine whether the decrease in [URE3] stability in cpr7Δ strain is due to an increase in Hsp70, wild type strain SY187 was transformed with a plasmid (pRS315P_SSA2-SSA1/2/3or4) encoding one of the four Ssa Hsp70 isoforms. Nevertheless, the Leu⁺ primary transformants expressing an additional Ssa Hsp70 isoform show stable [URE3] phenotype similar to that seen for the wild type strain transformed with pRS315 (Fig 3C). These results suggest that increase in Hsp70 upon CPR7 deletion does not contribute significantly to [URE3] destabilization. Additionally, we previously showed that further subculturing of transformants overexpressing Ssa1 destabilize [URE3] with about 7–8% of cells showing [ure-o] phenotype [52]. However, as the absence of Cpr7 leads to more than 99% of cells showing [ure-o] phenotype, the near complete loss of [URE3] in cpr7Δ cells can be interpreted as being caused by something other than an increase in amount of Ssa1 [52].

As a way to confirm that the anti-[URE3] effects of depleting Cpr7 were not due to increased expression of Ssa1, we constructed strain NY17 by deleting the gene encoding Cpr7 in strain NY16 that expresses Ssa2 under Ssa2 promoter as the only source of Ssa Hsp70. As seen by red colony color phenotype and poor growth on Ade^- solid growth medium in S5A Fig, depleting Cpr7 caused loss of [URE3]. This loss occurred despite the complete absence of Ssa1, Ssa3 and Ssa4. We examined the abundance of Hsp70 in strains NY16 and NY17 and found it was relatively higher in the cpr7Δ strain (S5B Fig). As elevated Ssa2 abundance supports a stable [URE3] phenotype (S5C Fig), the increase in [ure-o] colonies in strain NY17 must be unrelated to an increase in amount of Ssa2.

The TPR domain of Cpr7 is required for [URE3] propagation

The Hsp90 associated immunophilins Cpr6 and Cpr7 belong to the PPIase family of enzymes and share more than 50% sequence similarity, yet they can act in distinct cellular processes [53] [54]. In order to examine whether Cpr6 can complement the role of Cpr7 with regard to [URE3], the cpr7Δ strain harbouring Cpr7 on a URA3-based plasmid (pRS316P_CPR7-CPR7) was transformed with a plasmid encoding Cpr6 or Cpr7 (pRS413P_TEF-CPR6 or pRS413P_TEF-CPR7, respectively) regulated by the TEF promoter. We then used FOA selection to isolate cells having lost the resident URA3 plasmid. The resulting cpr7Δ strains with plasmids encoding Cpr6 or Cpr7 were grown in liquid medium selecting for plasmid maintenance and then spread onto ½ YPD plates. Colony color was noted after incubation at 30°C for 3–4 days, at which time the ½ YPD plates were replicated onto SD medium lacking histidine (for monitoring plasmid loss) or adenine (for monitoring [URE3]). As shown in Fig 4B, cells expressing Cpr7 colonies were white on ½ YPD and grew well on medium lacking adenine, suggesting that the plasmid expressed Cpr7 functionally complements the chromosomal knockout of CPR7 with regard to [URE3]. In contrast, cells harbouring either an empty plasmid (ev) or a plasmid encoding Cpr6 show similar red colony color on ½ YPD. After continued incubation on adenine deficient growth medium at 30°C, cells expressing Cpr6 grew only poorly, which is similar to cells with the empty plasmid. Thus, Cpr6 was unable to complement Cpr7 function with regard to [URE3] maintenance.

The amino acid identity between the PPIase and TPR domains of Cpr6 and Cpr7 is about 45% and 32% respectively. To examine which domain of Cpr7 is crucial for [URE3] propagation, hybrid proteins, based upon the Cpr6 and Cpr7 as the parent proteins, were constructed in which either the PPIase (6PPI/7TPR) or TPR (7PPI/6TPR) domain of Cpr7 was swapped with that of Cpr6 (Fig 4A) [34] and their ability to support [URE3] was again monitored as described above for Cpr6. As shown in Fig 4B, cells expressing 7PPI/6TPR show red colony color phenotype on ½ YPD medium. The 7PPI/6TPR expressing colonies show either no growth (day 1) or poor growth (day 2) on adenine deficient medium. In contrast, the frequency of white colonies on ½ YPD plate and growth on plates lacking adenine were found to be similar in strains expressing 6PPI/7TPR and wild type Cpr7 suggesting that the TPR domain of Cpr7 is crucial for [URE3] propagation. Furthermore, cells expressing a truncated derivative of Cpr7 lacking the PPIase domain also showed a stable [URE3] phentoype, suggesting peptide-prolyl activity of Cpr7 does not contribute much to the prion stability.

Cns1 restores [URE3] stability in cpr7Δcells

Cns1 is unique among the known TPR containing Hsp90 co-chaperones in being essential for yeast cell growth. The 385 amino acid protein contains three TPR motifs and is a suppressor of the growth defect caused by cpr7Δ, which suggests partial functional redundancy between Cpr7 and Cns1 [55]. In order to explore whether Cns1 complements for the loss of Cpr7 with regard to [URE3], a plasmid encoding Cns1 (pRS426P_CNS1-CNS1) was transformed into cpr7Δ cells pooled from Ade^- medium and transformants were further monitored for [URE3] propagation as described above for Cpr6. As seen before, cells expressing Cpr7 from the constitutive TEF promoter showed stable [URE3] on ½YPD (Fig 5). As expected, cells that lost the Cpr7-encoding plasmid (no growth on histidine deficient medium) show red colony color and no growth on Ade^- medium. As we saw for Cpr7, we found that cells that express Cns1 are white and grow normally on plates lacking adenine, suggesting that Cns1 functionally complements Cpr7 with regard to [URE3] propagation.

Cpr7 interacts physically with Ure2

Cpr7 belongs to the family of immunophilins that are also known to interact with many unfolded substrates to prevent their aggregation and keep them in a folding competent state [54] [56]. We used a pull-down assay using purified His₆-Cpr7 as bait to examine interaction of Cpr7 with Ure2. The cell lysate from wild type [ure-o] cells was fractionated and the supernatant was incubated with Cpr7-bound cobalt based metal affinity resin. The unbound proteins were washed and the bound fraction was probed by immunobloting with antibodies specific against Ure2 or Hsp90. In agreement with previous studies, Hsp90 was identified in fractions bound to His₆-Cpr7 (Fig 6). The deletion of MEEVD leads to a significant decrease in Hsp82 interaction with Cpr7. Interestingly, Ure2 was also detected in the fraction that bound to His₆-Cpr7. The presence of Ure2 in this fraction suggests that Ure2 either interacts with Cpr7 or Hsp82. We thus examined Ure2 interaction with Cpr7 using cell lysate from cells expressing Hsp82ΔMEEVD. If the presence of Ure2 in the fraction bound to Cpr7 is indirectly due to its interaction with Hsp82, the loss of Cpr7-Hsp82 interaction upon deletion of C-terminus MEEVD motif of Hsp82 would lead to a decrease in Ure2 level in the bound fraction. As shown, however, a similar level of Ure2 was detected in the bound fraction obtained using lysates from cells expressing either Hsp82 or Hsp82ΔMEEVD, suggesting that Cpr7 interacts directly with Ure2.

To substantiate this conclusion, we also examined Cpr7 interaction with Ure2 in vivo by using cells expressing His₆-Cpr7. We co-transformed strain SY187 with plasmids expressing His₆-Cpr7 (pRS413P_TEF-His₆-CPR7) and Ure2-GFP (pRS426P_GPD-URE2-GFP) or GFP (pRS426P_GPD-GFP). Transformants were grown further and the lysate was passed over cobalt based metal affinity resin. As shown in S6 Fig, Ure2 was detected in the bound fraction obtained only from the strain co-expressing His₆-Cpr7, again suggesting Cpr7 interacts with Ure2 in vivo. Overall, these results indicate that the role of Cpr7 in [URE3] maintenance could be mediated by its ability to interact directly with Ure2.

Cpr7 enhances Ure2 fibrillation

As Cpr7 interacts with Ure2 and is required for stable [URE3] propagation, we speculated that Cpr7 might have the ability to modulate Ure2 fibrillation required for stable [URE3] maintenance. In order to examine the effect of Cpr7 on amyloid forming tendency of Ure2, we monitored fibrillation of Ure2 in vitro using Thioflavin T (ThT), a dye that forms fluorescent complexes with amyloids, in the presence and absence of Cpr7. As expected ThT fluorescence intensity increases upon incubation with Ure2 at 37°C, suggesting spontaneous fibrillation of Ure2 with a lag of about 2–3 min. Cpr7 and Cpr6 alone did not affect ThT fluorescence intensity. Pre-incubation of Ure2 with Cpr7 did not affect the lag time. However, the rate of amyloid formation and amyloid yield increased significantly, indicating that Cpr7 promotes Ure2 fibrillation. The increase in Ure2 fibrillation was observed only upon incubation with Cpr7 and not its near homolog Cpr6 (Fig 7). The increase in fluorescence intensity was not due to a direct effect of Cpr7 on ThT as fluorescence intensity was similar when similar amounts of pre-aggregated Ure2 protein, with and without Cpr7, was measured (S7A and S7B Fig).

As the isolated TPR domain of Cpr7 supports [URE3], we also tested if this domain influenced ure2 amyloid formation. Interestingly, as seen for Cpr7, the addition of 7TPR to Ure2 similarly enhanced its fibrillation. These results are in agreement with the in vivo results that showed that 7TPR, but not Cpr6, complemented Cpr7 function with regard to [URE3] propagation. Together these results support the conclusion that Cpr7 plays an important role in [URE3] propagation by enhancing Ure2 fibrillation.

Discussion

Studies on yeast prions have formed the basis of understanding mechanistic aspects of the formation and propagation of not only of prions, but also other amyloid based disorders. The insight obtained has broadened our understanding not only of the role of various cellular factors in prion maintenance, but also of basic cellular biology. Though both Hsp70 and Hsp90 families of proteins are central to protein quality control in the cell, most of the cellular factors that influence prion propagation belong to the Hsp70 family and its co-chaperones. Whether prion propagation also requires Hsp90 machinery is not yet clear and the role of Hsp90 and its co-chaperones has been shown only to maintain prion conformation and the curing of [PSI⁺] upon overexpression of Hsp104 [44] [45] [46,50]. In the present study we show that Hsp90, by virtue of its ability to interact with TPR proteins, is critical for yeast prion propagation. Furthermore we show that Cpr7 is a novel cellular factor required for [URE3] maintenance.

Here we investigated the role of Hsp90 and its co-chaperones on [URE3] propagation. The knockout of genes encoding either Hsc82 or Hsp82 has no adverse effect on [URE3]. This is in contrast to the role of Ssa Hsp70, as different Ssa Hsp70 isoforms show distinct effects on [URE3] [16]. Also, the inhibition of Hsp90 ATPase activity with 17-AAG did not alter [URE3] propagation. Together these results suggest that Hsp90 function specific for the maturation of its client proteins is not crucial for [URE3] maintenance. This is also in agreement with previous observations suggesting that Hsp90 is a more specialized chaperone than Hsp70, which acts on a much broader range of substrates in the cell. The Hsp90 mutant lacking charged linker region (amino acids from 211 to about 263) is defective in ATP dependent amino terminal dimerization, shows a lower steady state level of its client v-Src, and subsequent maturation of its client proteins such as glucocorticoid receptor and v-Src [31]. The strain expressing Hsp90Δ211–264 as sole Hsp90 source also supports a stable [URE3] propagation which provides further evidence that [URE3] stability does not require optimal Hsp90 activity.

Hsp90ΔMEEVD, however, which lacks the binding motif for TPR containing co-chaperones, is incapable of propagating [URE3] stably when it is expressed as the only source of Hsp90. It is possible that Hsp90 interaction with its TPR co-chaperones might modulate the latters' functions in a way that is required for maintenance of [URE3]. Alternatively, the loss of Hsp90 interaction might increase affinity of TPR co-chaperones of Hsp90 toward other cellular proteins having a binding motif similar to MEEVD and such new interactions might lead to [URE3] stability. Collectively the data suggest that although the Hsp90 chaperone cycle including client binding and release may not be involved in [URE3] maintenance, the chaperone might influence [URE3] propagation indirectly through the interaction of its C-terminus with partner co-chaperones that can influence [URE3].

The results from multiple single knockout strains of Hsp90 co-chaperones reveal that Cpr7 is unique among various Hsp90 co-chaperones with regard to its role in the maintenance of mitotically stable [URE3] variant in our yeast strain. Similar to many of the other yeast prions, variants of [URE3] also occur in S. cerevisiae, and whether or not the essential role of Cpr7 in [URE3] stability is extendable to the other [URE3] variants remains to be explored.

Among the various Hsp90 co-chaperones that facilitate different steps of the Hsp90 reaction cycle, only Cpr7 deletion affects [URE3], suggesting a specific and not a general function of Hsp90 could be essential for [URE3] stability. This is also supported by the observation that cells expressing Hsp82ΔMEEVD and not Hsp82Δ211–264 show [URE3] loss, which points toward a specific role of the C-terminus of Hsp90 in [URE3] propagation. Cpr7 deletion also alters nucleotide dependent Cpr6 interaction with Hsp90, however, as Hsp82Δ211–264, which also leads to a similar effect, does not destabilize [URE3], the alteration of Hsp90-Cpr6 interaction could not be the cause of [URE3] loss from cpr7Δ cells [34].

Collectively the results suggest that Cpr7 influences [URE3] propagation directly by participating in processes required for efficient [URE3] propagation, rather than indirectly by influencing Hsp90 function. The possibility that Cpr7 impacts [URE3] directly is in agreement with previous reports showing that cyclophilins could interact with substrate directly to assist their folding and prevent aggregation. Similar to the role of Cpr7 in [URE3] propagation, another related immunophilin FKBP52, present in brain, induces aggregation of the pathological mutant of Tau, Tau-P301L [36,57], suggesting that the role of Cpr7 in [URE3] is not an isolated example of the significance of immunophilins in amyloidosis, but is widespread in higher eukaryotes including mammals.

Yeast cells express several TPR domain containing proteins, but how these interact with their specific partner proteins and influence a specific function of the interacting partner is not clearly understood. Though Cpr7 is primarily an Hsp90 co-chaperone, previous studies suggests that Cpr7 also interacts with Hsp70 [50,58]. Similarly Cpr6 is also known to interact with Hsp70 proteins. Similar to other Hsp90 TPR co-chaperones, such interaction could be mediated by the TPR domain which is known to interact with EEVD-like motifs. However in contrast to the known influence of several other TPR co-chaperones on Hsp70 activity or function, the significance of Cpr6 or Cpr7 interaction on wild type Ssa Hsp70 activity has still not been clearly shown [59]. Indeed, while Cns1 stimulates Ssa1 ATPase activity by about 8-fold, no stimulation was seen by Cpr7 [60]. Similarly, another Hsp90 co-chaperone Sti1 that interacts with Hsp70 strongly stimulates Ssa Hsp70 ATPase activity and helps in the substrate transfer from Hsp70 to Hsp90 [61]. Since our study shows that Cns1 complements Cpr7 function with regard to [URE3], and Cns1, but not Cpr7, stimulates Hsp70 activity, it is more likely that such a profound effect of cpr7Δ on [URE3] stability is not mediated entirely through Hsp70. This idea is further supported by the fact the Cpr7 influences Ure2 fibrillation in the absence of any additional cellular factor.

Among the various knockout strains, although cpr7Δ decreased [URE3] stability significantly, the lack of effect by its nearly homologous cyclophilin Cpr6 suggests a clear distinction between in vivo roles of these closely related Hsp90 associated cyclophilins. This is also in agreement with previous reports showing non-redundancy in Cpr6 and Cpr7 functions such as their ability to support yeast growth [53]. By using Cpr6 and Cpr7-based hybrid proteins, we find that the TPR domain acts as the regulatory domain to govern functional distinctions between Cpr7 and Cpr6 with regard to [URE3]. As the PPIase domain of Cpr7 is dispensable for [URE3], and a hybrid protein containing the TPR domain from Cpr7 supports stable [URE3] propagation, the distinct action of the two Hsp90 co-chaperones could be due to their different chaperone activity independent of the PPIase function. Stable prion propagation requires continuous growth and breakage of fibrils to generate more seeds for further fibril growth and transmittance to daughter cells. Previous studies show that Cpr7 has more potent chaperone activity than Cpr6, thus it is possible that Cpr7 might be more efficient to help break larger fibrils into smaller ones and hence able to generate more seeds required for [URE3] propagation.

Another TPR co-chaperone, Cns1 restores some Cpr7-dependent activities such as the effect on cell growth, negative regulation of heat shock factors (HSF) and maturation of glucocorticoid receptor [55,62]. Our study now reveals that overexpressed Cns1 also complements Cpr7 function for [URE3] propagation. The HSF and glucocorticoid receptor require Hsp90 and thus Cpr7 deletion could affect these substrates through modulation of the Hsp90 reaction cycle. It is known that Cns1 restores altered Cpr6-Hsp90 interaction upon loss of Cpr7 [34], however, as discussed above, the complementation of Cpr7 function by Cns1 for [URE3] is not due to restoration of Cpr6 interaction with Hsp90, but to redundancy in other Cpr7 functions required for [URE3] stability. As indicated above, Hsp90 client binding function is not crucial for [URE3] stability and the effect of Cpr7 deletion on [URE3] was not caused by altering Hsp90 interaction with Cpr6. Also, we did not find a direct interaction of Hsp90 with Ure2, but did see a direct influence of Cpr7 on Ure2 fibrillation in the absence of Hsp90. Collectively, our results suggest that it is most likely that the effect of Cpr7 deletion on [URE3] is independent of Hsp90 and due to a loss of specific function of Cpr7 in cpr7Δ strains. Together our data further suggest that Cns1 could also perform functions independent of Hsp90, and that functional redundancy between Cns1 and Cpr7 is not only limited to their roles as Hsp90 co-chaperones.

In vitro, Cns1 is unable to prevent aggregation of citrate synthase, and also fails to promote refolding of unfolded RNase T1, suggesting that unlike Cpr7, Cns1 not only lacks PPIase activity but also chaperone function for some Cpr7 substrates [60] [54]. Though Cns1 fails to chaperone RNase T1 or Citrate Synthase, it might still be able to influence Ure2 aggregation. As Cns1 lacks PPIase activity, the restoration of [URE3] propagation upon Cns1 overexpression in cpr7Δ strain could be through its TPR domain. Though the TPR domain of Cpr7 is only about 10% identical with that of Cns1, the functional complementation of Cpr7 by Cns1 shown in previous and present studies suggests that despite low identity, the structural region critical for Cpr7 function remains partially conserved between the two TPR proteins.

The pull down assay using Cpr7 as a bait protein reveals a novel interaction of Cpr7 with Ure2. Our in vitro ThT assay shows that in the absence of other additional cellular factors, Cpr7 is able to promote Ure2 fibrillation. Though the mechanism by which Cpr7 promotes Ure2 fibrillation is not clear, it is possible that similar to Hsp70s, Cpr7 may act as a chaperone to promote solubilisation of preformed amyloid and during the process breaks a larger fibril into smaller fibrils and thus generates more seeds for further fibrillation. Cpr7 thus might be essential for the prion replication required for stable transmittance of [URE3] prion seeds into daughter cells. Alternatively, in the absence of any additional factor Ure2 assembly could be partitioned between on-pathway and off-pathway intermediates, and the presence of Cpr7 might shift the process more towards the formation of on-pathway intermediates for amyloid formation. Also, as cells expressing the Hsp90ΔMEEVD mutant show [URE3] loss, it is possible that Hsp90 interaction with Cpr7 might modulate its chaperone activity either through inducing conformational changes in Cpr7 or yet other unknown mechanism required for [URE3] maintenance. Hsp90 is previously known to induce conformational changes of its co-chaperone Ppp5 that further activates its phosphatase activity [63]. Alternatively, the retargeting of Hsp90 TPR co-chaperones in the Hsp90ΔMEEVD strain to other cellular factors possessing motifs similar to MEEVD might influence [URE3] stability. Thus, intact Hsp90 could be supporting [URE3] stability by sequestering Cpr7 from binding to its alternative partner protein that in turn might destabilize the prion.

Our data reveal a novel role of cyclophilin Cpr7 in yeast prion [URE3] propagation. Thus, in addition to Hsp70 and its co-factors, Hsp90 and some of its co-chaperones also modulate the maintenance of this yeast prion. The role of immunophilins in amyloid based human diseases such as Alzheimer’s diseases, is emerging, and thus our finding that yeast immunophilins also modulate a yeast prion in a manner required for its efficient propagation provides a genetically tractable system for further understanding the role of immunophilins in amyloidosis. Why [PSI⁺] does not require TPR co-chaperone Cpr7 remains to be investigated. Interestingly, cellular factors required for stable [URE3] maintenance, such as Ssa2, are not essential for [PSI⁺]. Similarly, Hsp104 overexpression efficiently cures [PSI⁺] but not [URE3]. Though more is needed to be explored, it is tempting to speculate that the distinct chaperone requirement for [URE3] and [PSI⁺] could be due a specific requirement of Hsp90 chaperone machinery in [URE3] stability.

Materials and Methods

Strains and plasmids

Strains and plasmids used are described in Tables 1 and 2, respectively. The wild type strain SY187 (MATa, kar1-1, P_DAL5::ADE2, his3Δ202, leu2Δ1, trp1Δ63, ura3-52) encodes ADE2 gene regulated by the DAL5 promoter for monitoring [URE3]. The cpr6Δ (NY14) and cpr7Δ strains (NY01) were constructed by genomic integration of cpr6::KanMX or cpr7::KanMX cassette, respectively, into SY187 using homologous recombination. The hsp82Δ (SY295) and hsc82Δ (SY297) strains were constructed using strain MR195 or MR194 (Kind gift from Dr. Masison’s lab), respectively, using standard yeast genetic manipulations. Strain NY02 containing double deletion of HSC82 and HSP82 and harbouring plasmid pRS316P_HSP82-HSP82 was constructed from SY295 using standard yeast genetic methods. The strains NY03, NY04, NY05, NY06 and NY07 were made by shuffling pRS316P_HSP82-HSP82 in strain NY02 with pRS413P_TEF-His₆-HSP82, pRS413P_TEF-His₆-HSP82(ΔMEEVD), pRS413P_TEF-His₆-HSP82(Δ211–264), pRS413P_TEF-HSP82 and pRS413P_TEF-HSP82(ΔMEEVD), respectively. The knockout strain for other Hsp90 co-chaperones (Sti1, Sba1, Cpr6, Cpr7, Ppt1, Tah1, Hch1 or Aha1) was constructed as described above for the cpr7Δ strain using standard genetic techniques.

The genes encoding Cpr6 or Cpr7 were cloned under their respective native promoters in pRS316 to construct pRS316P_CPR6-CPR6 or pRS316P_CPR7-CPR7, respectively. Similarly, pRS413P_TEF-CPR6 and pRS413P_TEF-CPR7 were constructed to express Cpr6 or Cpr7 respectively from the TEF promoter. Overlap PCR was carried out to construct genes encoding hybrid proteins 6PPI/7TPR or 7PPI/6TPR. The 6PPI/7TPR encodes amino acids (a.a.) 1–176 of Cpr6 and 198–393 of Cpr7. Similarly 7PPI/6TPR encodes a.a. 1–197 of Cpr7 and 177–371 of Cpr6. The full length PCR amplified product was further digested with BamHI and XhoI, and subcloned into plasmid pRS413P_TEF. Similarly plasmid pRS413P_TEF-7TPR, encoding the TPR domain of Cpr7 (a.a. 193–393)(henceforth 7TPR), was constructed by restriction digestion (BamHI and XhoI) followed by ligation of a PCR amplified gene product encoding the TPR domain of Cpr7 into plasmid pRS413P_TEF digested with similar restriction enzymes. For immunoblot and protein purification of Cpr7 and Cpr6, a short tract of Hexa-His-tag was added at the 5’ end of gene encoding Cpr7 or Cpr6 using standard PCR-based recombinant technology. For expression in E.coli, pET29bHTV-CPR6, pET29bHTV-CPR7 or pET29bHTV-7TPR was constructed to encode from 5’ to 3’ direction, a Hexa-His tag, and a TEV protease recognition site followed by the gene encoding CPR6, CPR7 or 7TPR, respectively.

Media and growth conditions

Media and growth conditions are as described previously [64]. ½ YPD medium contains 0.5% yeast extract, 2% peptone, 2% dextrose and 2% agar. Liquid YPAD is similar to ½ YPD except it contains 1% yeast extract and 200mg/L adenine. SD and SGal are synthetic dextrose and synthetic galactose minimal media, respectively. Cells were grown at 30°C unless otherwise stated.

Diploid formation and tetrad analysis

The heterologous diploid (CPR7/cpr7::KanMX) was constructed by mating SY187 [URE3] MATα with NY01 MATa strain (cpr7Δ[ure-o]) and selecting on adenine deficient medium containing G418 (200 μg/ml). Diploids were further streaked to pure colonies on ½ YPD plates and further confirmed by replica plating onto adenine deficient SD plate containing G418. Diploids were sporulated on a 2% potassium acetate plate and tetrad dissection was carried out on ½ YPD.

Monitoring [URE3] and [PSI⁺]

Gln3 is an activator of DAL5 promoter. When cells are grown in the presence of a good nitrogen source such as standard ammonium containing media, Ure2 binds to transcription factor Gln3 and represses the DAL5 promoter. To monitor [URE3], our strains encode ADE2 controlled by the DAL5 promoter. In [ure-o] (lacking [URE3]) cells, functional Ure2 represses transcription of ADE2 so cells do not grow in the absence of adenine and remain red on limiting adenine media. [URE3] cells contain predominantly inactive Ure2, which relieves repression of DAL5 and allows expression of Ade2. Thus [URE3] cells grow on media lacking adenine and remain white on limiting adenine media.

Sup35 is a translation termination factor and thus its conversion to the functionally inactive [PSI⁺] form leads to suppression of ade2-1 nonsense allele. This suppression also requires the weak tRNA suppressor Sup16 (encoded by SUQ5), which alone does not suppress ade2-1. Thus, in cells propagating [PSI⁺], enhanced nonsense suppression of ade2-1 leads to the synthesis of Ade2. In contrast, [psi^-] cells lack functional Ade2 and thus are unable to grow in the absence of externally added adenine and remain red when grown on limiting adenine.

The prion variants of [URE3] and strong [PSI⁺] in our strains are of unknown origin. Our strains 1566 and 1567 are kind gift from Dr. Daniel Masision, and contain the [PSI⁺]^Sc4 and [PSI⁺]^Sc37 variants respectively that were introduced by cytoduction from J. Weissman strains JW127 and JW129 [51].

Protein purification

For Cpr7 purification, the plasmid pET29bHTV-CPR7 was transformed into Escherichia coli strain Rosetta 2(DE3) (Invitrogen). The pre-grown culture of optical density at 600nm (O.D._600nm) ~0.6 was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at 18°C. Cells were lysed and the protein was purified from supernatant using cobalt based Talon metal affinity resin. Cpr7 was eluted with 300mM imidazole and further incubated overnight with His₆-TEV (molar ratio, Cpr7/His-TEV:20/1) protease at 4°C. The sample was extensively dialyzed and further incubated with cobalt metal affinity column and the His₆-tag cleaved Cpr7 protein was collected as unbound fraction. The protein was further purified using anion exchange (Mini Q) chromatography. Protein purity was verified using SDS-PAGE and identity was confirmed using Mass spectrometry. Cpr6 and 7TPR were purified using the procedure described above for Cpr7.

The plasmid pKT41-Ure2 (kind gift from Dr. Reed Wickner, National Institute of Health, Bethesda) encodes Ure2 with an N-terminal Hexa-His-tag. The plasmid was transformed into E.coli strain Rosetta 2(DE3) and Ure2p was purified using cobalt based Talon metal affinity resin as described before [52].

Ure2 fibrillation assay

Thioflavin T (ThT) (500μM) was added to 48 μM of purified Ure2 with and without Cpr6, Cpr7 or 7TPR (30 μM each) in a 96 well plate. The plate was incubated at 37°C with a shaking speed of 900 rpm in linear mode in a multimode plate reader (TECAN infinite M200 PRO). Fluorescence kinetics was measured after every 15 minutes with emission wavelength of 485 nm upon excitation at 450 nm. Each experiment was repeated at least three times.

Western analysis

Cells were lysed using lysis buffer (phosphate-buffered saline with 0.2% TritonX-100 and protease inhibitor cocktail) by vortexing with glass beads and lysate was fractionated into supernatant and pellet. About 10 μg of total protein was separated on sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. Anti His₆-tag antibodies (Pierce Biotechnology) were used to capture His₆-Cpr7. For Pull-down assay, purified His₆-Cpr7 was bound over cobalt based metal affinity resin and yeast lysate was passed through the resin. The resin was washed and the bound fractions were probed with desired antibodies.

Supporting Information

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