Hsp90 is a highly conserved ATP-dependent molecular chaperone that forms a clamp around client proteins. The role of ATP in Hsp90 function is unclear since cell viability requires ATP binding, but not hydrolysis. Here, we present findings that support our hypothesis that after ATP binds, the γ phosphate repositions in a regulated manner to interact with a conserved arginine (R380) and stabilize the closed clamp. We propose that the essential role of ATP in Hsp90 function is structural: ATP is a linker that physically tethers the N and M domains and stabilizes closing. Severing this link by hydrolysis facilitates reopening. Our findings support the idea that R380 is an arginine finger, a motif found in diverse NTPase families, due to its interdomain interaction with ATP. This in turn suggests that for some arginine fingers the nucleotide itself is a structural element important for stabilization of inter-domain or -subunit interactions.

The yeast Sup35 protein misfolds into the infectious [PSI+] prion, which is then propagated by the severing activity of the molecular chaperone, Hsp104. Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression as well as the inactivation of Hsp104. However, it is controversial whether curing by overexpression is due to the dissolution of the prion seeds by the trimming activity of Hsp104 or the asymmetric segregation of the prion seeds between mother and daughter cells which requires cell division. To answer this question, we conducted experiments and found no difference in the extent of curing between mother and daughter cells when half of the cells were cured by Hsp104 overexpression in one generation. Furthermore, curing was not affected by the lack of Sir2 expression, which was reported to be required for asymmetric segregation of the [PSI+] seeds. More importantly, when either hydroxyurea or ethanol were used to inhibit cell division, the extent of curing by Hsp104 overexpression was not significantly reduced. Therefore, the curing of [PSI+] by Hsp104 overexpression is not due to asymmetric segregation of the prion seeds, but rather their dissolution by Hsp104.

Hsp104 propagates the yeast prion [PSI+], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [PSI+] in a process that is not yet understood but may be caused by trimming, which removes monomers from the ends of the amyloid fibers. This curing was shown to depend on both the N-terminal domain of Hsp104 and the expression level of various members of the Hsp70 family, which raises the question as to whether these effects of Hsp70 are due to it binding to the Hsp70 binding site that was identified in the N-terminal domain of Hsp104, a site not involved in prion propagation. Investigating this question, we now find, first, that mutating this site prevents both the curing of [PSI+] by Hsp104 overexpression and the trimming activity of Hsp104. Second, we find that depending on the specific member of the Hsp70 family binding to the N-terminal domain of Hsp104, both trimming and the curing caused by Hsp104 overexpression are either increased or decreased in parallel. Therefore, the binding of Hsp70 to the N-terminal domain of Hsp104 regulates both the rate of [PSI+] trimming by Hsp104 and the rate of [PSI+] curing by Hsp104 overexpression.

Hsp90 is an essential eukaryotic chaperone that regulates the activity of many client proteins. Current models of Hsp90 function, which include many conformational rearrangements, specify a requirement of ATP hydrolysis. Here we confirm earlier findings that the Hsp82-E33A mutant, which binds ATP but does not hydrolyze it, supports viability of S. cerevisiae , although it displays conditional phenotypes. We find binding of ATP to Hsp82-E33A induces the conformational dynamics needed for Hsp90 function. Hsp90 orthologs with the analogous EA mutation from several eukaryotic species, including humans and disease organisms, support viability of both S. cerevisiae and Sz. pombe . We identify second-site suppressors of EA that rescue its conditional defects and allow EA versions of all Hsp90 orthologs tested to support nearly normal growth of both organisms, without restoring ATP hydrolysis. Thus, the requirement of ATP for Hsp90 to maintain viability of evolutionarily distant eukaryotic organisms does not appear to depend on energy from ATP hydrolysis. Our findings support earlier suggestions that exchange of ATP for ADP is critical for Hsp90 function. ATP hydrolysis is not necessary for this exchange but provides an important control point in the cycle responsive to regulation by co-chaperones.

The Sup35 prion protein of budding yeast has been reported to undergo phase separation to form liquid droplets both at low pH in vitro and when energy depletion decreases the intracellular pH in vivo. It also has been shown using purified proteins that this phase separation is driven by the prion domain of Sup35 and does not re-quire its C-terminal domain. In contrast, we now find that a Sup35 fragment consisting of only the N-terminal prion domain and the M-domain does not phase separate in vivo; this phase separation of Sup35 requires the C-terminal domain, which binds Sup45 to form the translation termination complex. The phase-separated Sup35 not only colocalizes with Sup45 but also with Pub1, a stress granule marker protein. In addition, like stress granules, phase separation of Sup35 appears to require mRNA since cycloheximide treatment, which inhibits mRNA release from ribosomes, prevents phase separation of Sup35. Finally, unlike Sup35 in vitro, Sup35 condensates do not disassemble in vivo when the intracellular pH is increased. These results suggest that, in energy-depleted cells, Sup35 forms supramolecular assemblies that differ from the Sup35 liquid droplets that form in vitro.

Human J-domain protein (JDP) DnaJB6 has a broad and potent activity that prevents formation of amyloid by polypeptides such as polyglutamine, A-beta, and alpha-synuclein, related to Huntington's, Alzheimer's, and Parkinson's diseases, respectively. In yeast, amyloid-based [PSI+] prions, which rely on the related JDP Sis1 for replication, have a latent toxicity that is exposed by reducing Sis1 function. Anti-amyloid activity of DnaJB6 is very effective against weak [PSI+] prions and the Sup35 amyloid that composes them, but ineffective against strong [PSI+] prions composed of structurally different amyloid of the same Sup35. This difference reveals limitations of DnaJB6 that have implications regarding its therapeutic use for amyloid disease. Here, we find that when Sis1 function is reduced, DnaJB6 represses toxicity of strong [PSI+] prions and inhibits their propagation. Both Sis1 and DnaJB6, which are regulators of protein chaperone Hsp70, counteract the toxicity by reducing excessive incorporation of the essential Sup35 into prion aggregates. However, while Sis1 apparently requires interaction with Hsp70 to detoxify [PSI+], DnaJB6 counteracts prion toxicity by a different, Hsp70-independent mechanism.

The accumulation of misfolded proteins as amyloids is associated with pathology in dozens of debilitating human disorders, including diabetes, Alzheimer’s, Parkinson’s, and Huntington’s diseases. Expressing human amyloid-forming proteins in yeast is toxic, and yeast prions that propagate as infectious amyloid forms of cellular proteins are also harmful. The yeast system, which has been useful for studying amyloids and their toxic effects, has provided much insight into how amyloids affect cells and how cells respond to them. Given that an amyloid is a protein folding problem, it is unsurprising that the factors found to counteract the propagation or toxicity of amyloids in yeast involve protein quality control. Here, we discuss such factors with an emphasis on J-domain proteins (JDPs), which are the most highly abundant and diverse regulators of Hsp70 chaperones. The anti-amyloid effects of JDPs can be direct or require interaction with Hsp70.

[PSI+] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI+] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI+] prions typically affect viability only modestly, so [PSI+] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI+] propagation, yet [PSI+] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI+] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI+] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI+] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI+] prions.

The yeast prion [URE3] propagates as a misfolded amyloid form of the Ure2 protein. Propagation of amyloid-based yeast prions requires protein quality control (PQC) factors, and altering PQC abundance or activity can cure cells of prions. Yeast antiprion systems composed of PQC factors act at normal abundance to restrict establishment of the majority of prion variants that arise de novo While these systems are well described, how they or other PQC factors interact with prion proteins remains unclear. To gain insight into such interactions, we identified mutations outside the Ure2 prion-determining region that destabilize [URE3]. Despite residing in the functional domain, 16 of 17 mutants retained Ure2 activity. Four characterized mutations caused rapid loss of [URE3] yet allowed [URE3] to propagate under prion-selecting conditions. Two sensitized [URE3] to Btn2, Cur1, and Hsp42, but in different ways. Two others reduced amyloid formation in vitro Of these, one impaired prion replication and the other apparently impaired transmission. Thus, widely dispersed sites outside a prion's amyloid-forming region can contribute to prion character, and altering such sites can disrupt prion propagation by altering interactions with PQC factors.

Hsp90 is a highly conserved molecular chaperone important for the activity of many client proteins. Hsp90 has an N-terminal ATPase domain (N), a middle domain (M) that interacts with clients and a C-terminal dimerization domain (C). "Closing" of dimers around clients is regulated by ATP binding, co-chaperones, and post-translational modifications. ATP hydrolysis coincides with release of mature client and resetting the reaction cycle. Humans have two Hsp90s: hHsp90α and hHsp90β. Although 85% identical, hHsp90β supports Hsp90 function in yeast much better than hHsp90α. Determining the basis of this difference would provide important insight into functional specificity of seemingly redundant Hsp90s, and the evolution of eukaryotic Hsp90 systems and clientele. Here, we found host co-chaperones Sba1, Cpr6 and Cpr7 inhibited hHsp90α function in yeast, and we identified mutations clustering in the N domain that considerably improved hHsp90α function in yeast. The strongest of these rescuer mutations accelerated nucleotide-dependent lid closing, N–M domain docking, and ATPase. It also disrupted binding to Sba1, which prolongs the closed state, and promoted N–M undocking and lid opening. Our data suggest the rescuer mutations improve function of hHsp90α in yeast by accelerating return to the open state. Our findings imply hHsp90α occupies the closed state too long to function effectively in yeast, and define an evolutionarily conserved region of the N domain involved in resetting the Hsp90 reaction cycle.

Fes1 is a conserved armadillo repeat-containing Hsp70 nucleotide exchange factor important for growth at high temperature, proteasomal protein degradation and prion propagation. Depleting or mutating Fes1 induces a stress response and causes defects in these processes that are ascribed solely to disruption of Fes1 regulation of Hsp70. Here, we find Fes1 was essential for degradation of gluconeogenic enzymes by the vacuole import and degradation (Vid) pathway and for cell wall integrity (CWI), which is crucial for growth at high temperature. Unexpectedly, Fes1 mutants defective in physical or functional interaction with Hsp70 retained activities that support Vid and CWI. Fes1 and the Fes1 mutants bound to the Vid substrate Fbp1 in vitro and captured Slt2, a signaling kinase that regulates CWI, from cell lysates. Our data show that the armadillo domain of Fes1 binds proteins other than Hsp70, that Fes1 has important Hsp70-independent roles in the cell, and that major growth defects caused by depleting Fes1 are due to loss of these functions rather than to loss of Hsp70 regulation. We uncovered diverse functions of Fes1 beyond its defined role in regulating Hsp70, which points to possible multi-functionality among its conserved counterparts in other organisms or organelles.

Heat shock protein 90 (Hsp90) is a highly conserved ATP-dependent molecular chaperone that is essential in eukaryotes. It is required for the activation and stabilization of more than 200 client proteins, including many kinases and steroid hormone receptors involved in cell-signaling pathways. Hsp90 chaperone activity requires collaboration with a subset of the many Hsp90 cochaperones, including the Hsp70 chaperone. In higher eukaryotes, the collaboration between Hsp90 and Hsp70 is indirect and involves Hop, a cochaperone that interacts with both Hsp90 and Hsp70. Here we show that yeast Hsp90 (Hsp82) and yeast Hsp70 (Ssa1), directly interact in vitro in the absence of the yeast Hop homolog (Sti1), and identify a region in the middle domain of yeast Hsp90 that is required for the interaction. In vivo results using Hsp90 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Hsp70 in cell lysates. In vitro, the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface.

The Hsp90 chaperone is regulated by many cochaperones that tune its activities, but how they act to coordinate various steps in the reaction cycle is unclear. The primary role of Saccharomyces cerevisiae Hsp70/Hsp90 cochaperone Sti1 (Hop in mammals) is to bridge Hsp70 and Hsp90 to facilitate client transfer. Sti1 is not essential, so Hsp90 can interact with Hsp70 in vivo without Sti1. Nevertheless, many Hsp90 mutations make Sti1 necessary. We noted that Sti1-dependent mutations cluster in regions proximal to N-terminal domains (SdN) or C-terminal domains (SdC), which are known to be important for interaction with Hsp70 or clients, respectively. To uncover mechanistic details of Sti1-Hsp90 cooperation, we identified intramolecular suppressors of the Hsp90 mutants and assessed their physical, functional, and genetic interactions with Hsp70, Sti1, and other cochaperones. Our findings suggest Hsp90 SdN and SdC mutants depend on the same interaction with Sti1, but for different reasons. Sti1 promoted an essential Hsp70 interaction in the SdN region and supported SdC-region function by establishing an Hsp90 conformation crucial for capturing clients and progressing through the reaction cycle. We find the Hsp70 interaction and relationship with Sti1/Hop is conserved in the human Hsp90 system. Our work consolidates and clarifies much structural, biochemical, and computational data to define in vivo roles of Sti1/Hop in coordinating Hsp70 binding and client transfer with progression of the Hsp90 reaction cycle.

Genetic screens using Saccharomyces cerevisiae have identified an array of Hsp40 (Ydj1p) J-domain mutants that are impaired in the ability to cure the yeast [URE3] prion through disrupting functional interactions with Hsp70. However, biochemical analysis of some of these Hsp40 J-domain mutants has so far failed to provide major insight into the specific functional changes in Hsp40-Hsp70 interactions. To explore the detailed structural and dynamic properties of the Hsp40 J-domain, 20 ns molecular dynamic simulations of 4 mutants (D9A, D36A, A30T, and F45S) and wild-type J-domain were performed, followed by Hsp70 docking simulations. Results demonstrated that although the Hsp70 interaction mechanism of the mutants may vary, the major structural change was targeted to the critical HPD motif of the J-domain. Our computational analysis fits well with previous yeast genetics studies regarding highlighting the importance of J-domain function in prion propagation. During the molecular dynamics simulations several important residues were identified and predicted to play an essential role in J-domain structure. Among these residues, Y26 and F45 were confirmed, using both in silico and in vivo methods, as being critical for Ydj1p function.

Polyglutamine (polyQ) aggregates are associated with pathology in protein-folding diseases and with toxicity in the yeast Saccharomyces cerevisiae Protection from polyQ toxicity in yeast by human DnaJB6 coincides with sequestration of aggregates. Gathering of misfolded proteins into deposition sites by protein quality control (PQC) factors has led to the view that PQC processes protect cells by spatially segregating toxic aggregates. Whether DnaJB6 depends on this machinery to sequester polyQ aggregates, if this sequestration is needed for DnaJB6 to protect cells, and the identity of the deposition site are unknown. Here, we found DnaJB6-driven deposits share characteristics with perivacuolar insoluble protein deposition sites (IPODs). Binding of DnaJB6 to aggregates was necessary, but not enough, for detoxification. Focal formation required a DnaJB6-Hsp70 interaction and actin, polyQ could be detoxified without sequestration, and segregation of aggregates alone was not protective. Our findings suggest DnaJB6 binds to smaller polyQ aggregates to block their toxicity. Assembly and segregation of detoxified aggregates are driven by an Hsp70- and actin-dependent process. Our findings show sequestration of aggregates is not the primary mechanism by which DnaJB6 suppresses toxicity and raise questions regarding how and when misfolded proteins are detoxified during spatial segregation.

The [URE3] yeast prion is the self-propagating amyloid form of the Ure2 protein. [URE3] is cured by overexpression of several yeast proteins, including Ydj1, Btn2, Cur1, Hsp42, and human DnaJB6. To better understand [URE3] curing, we used real-time imaging with a yeast strain expressing a GFP-labeled full-length Ure2 construct to monitor the curing of [URE3] over time. [URE3] yeast cells exhibited numerous fluorescent foci, and expression of the GFP-labeled Ure2 affected neither mitotic stability of [URE3] nor the rate of [URE3] curing by the curing proteins. Using guanidine to cure [URE3] via Hsp104 inactivation, we found that the fluorescent foci are progressively lost as the cells divide until they are cured; the fraction of cells that retained the foci was equivalent to the [URE3] cell fraction measured by a plating assay, indicating that the foci were the prion seeds. During the curing of [URE3] by Btn2, Cur1, Hsp42, or Ydj1 overexpression, the foci formed aggregates, many of which were 0.5 μm or greater in size, and [URE3] was cured by asymmetric segregation of the aggregated seeds. In contrast, DnaJB6 overexpression first caused a loss of detectable foci in cells that were still [URE3] before there was complete dissolution of the seeds, and the cells were cured. We conclude that GFP labeling of full-length Ure2 enables differentiation among the different [URE3]-curing mechanisms, including inhibition of severing followed by seed dilution, seed clumping followed by asymmetric segregation between mother and daughter cells, and seed dissolution.

Overproduction or deficiency of many chaperones and other cellular components cure the yeast prions [PSI+] (formed by Sup35p) or [URE3] (based on Ure2p). However, at normal expression levels, Btn2p and Cur1p eliminate most newly arising [URE3] variants but do not cure [PSI+], even after overexpression. Deficiency or overproduction of Hsp104 cures the [PSI+] prion. Hsp104 deficiency curing is a result of failure to cleave the Sup35p amyloid filaments to make new seeds, whereas Hsp104 overproduction curing occurs by a different mechanism. Hsp104(T160M) can propagate [PSI+], but cannot cure it by overproduction, thus separating filament cleavage from curing activities. Here we show that most [PSI+] variants arising spontaneously in an hsp104(T160M) strain are cured by restoration of just normal levels of the WT Hsp104. Both strong and weak [PSI+] variants are among those cured by this process. This normal-level Hsp104 curing is promoted by Sti1p, Hsp90, and Sis1p, proteins previously implicated in the Hsp104 overproduction curing of [PSI+]. The [PSI+] prion arises in hsp104(T160M) cells at more than 10-fold the frequency in WT cells. The curing activity of Hsp104 thus constitutes an antiprion system, culling many variants of the [PSI+] prion at normal Hsp104 levels.

Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone. To properly chaperone its clientele, Hsp90 proceeds through an ATP-dependent conformational cycle influenced by posttranslational modifications (PTMs) and assisted by a number of co-chaperone proteins. Although Hsp90 conformational changes in solution have been well-studied, regulation of these complex dynamics in cells remains unclear. Phosphorylation of human Hsp90α at the highly conserved tyrosine 627 has previously been reported to reduce client interaction and Aha1 binding. Here we report that these effects are due to a long-range conformational impact inhibiting Hsp90α N-domain dimerization and involving a region of the middle domain/carboxy-terminal domain interface previously suggested to be a substrate binding site. Although Y627 is not phosphorylated in yeast, we demonstrate that the non-conserved yeast co-chaperone, Hch1, similarly affects yeast Hsp90 (Hsp82) conformation and function, raising the possibility that appearance of this PTM in higher eukaryotes represents an evolutionary substitution for HCH1.

Human chaperone DnaJB6, an Hsp70 cochaperone whose defects cause myopathies, protects cells from polyglutamine toxicity and prevents purified polyglutamine and Aβ peptides from forming amyloid. Yeast prions [URE3] and [PSI] propagate as amyloid forms of Ure2 and Sup35 proteins, respectively. Here we find DnaJB6 protected yeast cells from polyglutamine toxicity and cured yeast of both [URE3] prions and weak variants of [PSI] prions, but not strong [PSI] prions. Weak and strong variants of [PSI] differ only in the structural conformation of their amyloid cores. In line with its anti-prion effects, DnaJB6 prevented purified Sup35 from forming amyloid at 37 oC, which produces predominantly weak [PSI] variants when used to infect yeast, but not at 4 oC, which produces mostly strong [PSI] variants. Thus, structurally distinct amyloids composed of the same protein were differentially sensitive to the anti-amyloid activity of DnaJB6, both in vitro and in vivo. These findings have important implications for strategies using DnaJB6 as a target for therapy in amyloid disorders. ________________________________________ In many mammalian diseases, including Alzheimer’s, Parkinson’s and Huntington’s diseases, type 2 diabetes and prion diseases, tissue pathology is associated with accumulation of amyloid, an insoluble, highly ordered fibrous protein aggregate. Elevating expression of protein chaperones, which help other proteins adopt and maintain their native conformations, reduces toxicity associated with amyloidogenic proteins in different model systems (1). In particular, Hsp70 and its Hsp40 (J-protein) co-chaperone partners provide broad protective effects. A major role of Hsp70 and Hsp40 is to prevent aggregation of misfolded proteins, but they also protect cells from toxicity caused by amyloid-forming proteins without preventing formation of aggregates (2,3). These observations led to a view that toxicity is due to something other than amyloid itself, such as non-amyloid oligomeric aggregates, and that the chaperones might neutralize toxic effects by promoting incorporation of such aggregates into more benign amyloid fibers. J-proteins are defined by a signature J-domain that mediates physical interaction with the ATPase regulatory domain of Hsp70. J-proteins have amplified through evolution more than other chaperone families. For example, Saccharomyces cerevisiae and humans each encode about a dozen Hsp70s, but yeast encodes 22 J-proteins while humans have roughly 50. This difference suggests that any additional roles for Hsp70 demanded by increases in cell complexity and differentiation were met, in part, by an expansion in the number http://www.jbc.org/cgi/doi/10.1074/jbc.M115.700393 The latest version is at JBC Papers in Press. Published on December 23, 2015 as Manuscript M115.700393 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jauary 4, 2016 hp://w w w .jb.org/ D ow nladed from DnaJB6b is selective in blocking amyloid formation 2 and diversity of J-proteins that regulate Hsp70 activity (4). Human J-proteins are categorized into DnaJA, DnaJB and DnaJC families on the basis of structural similarities (4). The two major cytosolic yeast Hsp40 J-proteins Ydj1 and Sis1 are most closely related to the DnaJA and DnaJB families, respectively. Elevating expression of DnaJB6b and DnaJB8, but not other DnaJ proteins, blocks aggregation and toxicity of Huntington's related polyglutamine (polyQ) in human cells (5). DnaJB6b is a cytosolic splice variant of the C-terminally extended DnaJB6a that is localized in the nucleus and does not block toxicity. Its protective effect does not depend on J-domain function, which reflects Hsp70-independent activity and is consistent with the inability of Hsp70 to confer similar protection (5,6). Purified DnaJB6 blocks incorporation of polyQ peptides into preformed aggregates (5-7), and sub-stoichiometric amounts of DnaJB6 prevent both the nucleation of Aβ peptides into amyloid and the incorporation of Aβ into pre-existing amyloid fibers (8). Thus, it has been proposed that DnaJB6 protects cells from toxic effects of amyloid-forming proteins by binding to peptides and oligomers that have high propensity to form amyloid and preventing their maturation into amyloid. This mechanism is distinct from those that relieve toxicity without preventing aggregation in that it neutralizes putatively toxic oligomers and blocks formation of amyloid that might continue to promote production of toxic species. Yeast prions provide a genetic model of pathogenic amyloids useful for investigating effects of chaperones on amyloid in vivo (9). Stable propagation of prions in dividing yeast cells requires continued fragmentation of amyloid fibers into new propagons, or seeds, that are passed with cytoplasm to daughter cells (10). This fragmentation depends on the normal cellular functions of the protein disaggregation machinery that is driven by Hsp104 in cooperation with Hsp70 and its Sis1 and nucleotide exchange factor co-chaperones (11-18). Different prions depend on this machinery to different degrees, so impairing disaggregation machinery function by altering activity of these components can affect their propagation differently (9,19). For example, by competing with Sis1, elevating expression of Ydj1 efficiently cures cells of [URE3] prions, which are composed of Ure2 protein, but not [PSI] prions composed of Sup35 (20,21), and elevating Hsp104 has the opposite effects on these prions (11,21). The general ability of DnaJB6 to interfere with formation of amyloid composed of different proteins suggests it could act as an anti-prion factor in yeast, but in a manner mechanistically distinct from the yeast chaperones. Indeed, we find here that expressing DnaJB6b in yeast cures cells of [URE3] prions in an apparently direct manner that is clearly distinct from that of curing by Ydj1. Importantly, DnaJB6b also cured "weak" variants of [PSI], but it was ineffective against phenotypically stronger [PSI] variants. Thus, antiamyloid activity of DnaJB6b in vivo was limited by structural characteristics of different amyloids composed of the same protein. We further show that this limitation is related to its ability to block formation of Sup35NM amyloid in vitro only under conditions that produce material that is thermodynamically similar to Ure2 amyloid and that generates weak [PSI] prions when used to infect yeast. These findings provide further insight into the remarkable properties of DnaJB6b, and raise important concerns regarding use of DnaJB6 as a target for therapies in amyloid diseases. EXPERIMENTAL PROCEDURES Yeast strains and growth conditions– Yeast strains used are isogenic to 1075 (MATa, kar1-1, PDAL5::ADE2, his3∆200, leu2∆1, trp1∆63, ura352, SUQ5, [URE3]) (22). 779-6A is the same but has ade2-1 in place of PDAL5::ADE2, is [ure-o] and has endogenous strong [PSI] and [PIN], both of uncertain origin. [PSI] prion variants were cytoduced into a [rho] version of 779-6A that had been cured of prions by passage on medium containing 3 mM guanidine-HCl to generate strains 1566, 1567, 1586 and 1587. Strains 1566 and 1567 were made using strains JW129 [PSI] (strong) and JW127 [PSI] (weak) as prion donors (23). Similarly, strains 1586 and 1587 were made using L2892 [PSI] (weak, here designated SL[PSI]) and L2885 [PSI] (strong, here designated SL[PSI]) as prion donors (24). Strains 930 and MR502, for testing complementation of SIS1 and YDJ1 in vivo functions, respectively, have been described (25). YPAD contains 1% yeast extract, 2% peptone, 400 mg/L (excess) adenine and 2% dextrose. To monitor prion phenotypes cells were grown on by gest on Jauary 4, 2016 hp://w w w .jb.org/ D ow nladed from DnaJB6b is selective in blocking amyloid formation 3 1/2YPD (0.5% yeast extract, 2% peptone, 2% dextrose), which contains a limiting but undefined amount of adenine. To maintain plasmids, cells were grown on minimal synthetic dextrose (SD) media, which contain 2% dextrose and 0.7% yeast nitrogen base (YNB) supplemented with appropriate nutrients. SRaf is 2% raffinose, 0.7% YNB, and SGal is 2% galactose, 2% raffinose, 0.7% YNB. Limiting adenine in selective media was 10 mg/L. Solid media are the same, but contain 2% agar (Difco). Unless otherwise noted, cells were grown at 30 oC. Plasmids– Plasmids used are listed in Table 1. pRU4 is a centromeric LEU2-marked plasmid containing the GAL1 promoter and the CYC1 terminator. It was made by replacing the GPD promoter in plasmid p415-GPD (26) with a PCRgenerated GAL1 promoter fragment amplified from plasmid pGCH17 (14) using SacI/BamHI. The human J-protein cDNA clones used as template in PCR reactions were gifts from H. Kampinga. J-protein open reading frames were cloned as BamHI/SalI fragments into either p414GPD, p415-GPD, or p415-TEF (26), and as BamHI/XhoI into pRU4. Plasmids pRU69, pRU70 and pRU71, used for expressing C-terminal Histagged DnaJB6b, DnaJB6b-D33N and DnaJB6b∆ST in E. coli, respectively, were made by cloning the coding regions into pET28b as NcoI/XhoI fragments. Restriction sites and/or C-terminal cMyc tags were added by using PCR. Point mutations were introduced by site-directed mutagenesis. Plasmid p1339, for expression of His-tagged Sup35NM (the prion-determining and adjacent charged domains of Sup35) in E. coli, was described previously (27). Plasmid pKT41, for expression of His-tagged Ure2 in E. coli, was described previously (28). Plasmid pSL1066, for expression of Sup35NM-GFP under the control of the CUP1 promoter in yeast, was described previously (29). Plasmid pYES2-HttQ103GFP, for the expression of Huntingtin polyQ103 fused to GFP under the control of the GAL promoter in yeast, was described previously (30,31). Plasmid pMR18, used for expression of GSTHsp104 in E. coli, is the HSP104 open reading frame cloned into pET41c (Novagen) as a PshAI/XhoI fragment. Plasmid pET-SSA1, for expression of Ssa1p in E. coli is the