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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">04288</article-id><article-id pub-id-type="doi">10.7554/eLife.04288</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group></article-categories><title-group><article-title>Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-17613"><name><surname>Klaips</surname><given-names>Courtney L</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="fn" rid="pa1">¶</xref><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17614" equal-contrib="yes"><name><surname>Hochstrasser</surname><given-names>Megan L</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="pa2">‡</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-20201" equal-contrib="yes"><name><surname>Langlois</surname><given-names>Christine R</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-12746"><name><surname>Serio</surname><given-names>Tricia R</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><label>1</label><institution content-type="dept">Department of Molecular Biology, Cell Biology and Biochemistry</institution>, <institution>Brown University</institution>, <addr-line><named-content content-type="city">Providence</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><label>2</label><institution content-type="dept">Department of Molecular and Cellular Biology</institution>, <institution>University of Arizona</institution>, <addr-line><named-content content-type="city">Tucson</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Kelly</surname><given-names>Jeffery W</given-names></name><role>Reviewing editor</role></contrib><aff><institution>Scripps Research Institute</institution>, <country>United States</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>tserio@email.arizona.edu</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn><fn fn-type="present-address" id="pa1"><label>¶</label><p>Max Planck Institute of Biochemistry, Munich, Germany</p></fn><fn fn-type="present-address" id="pa2"><label>‡</label><p>Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States</p></fn></author-notes><pub-date publication-format="electronic" date-type="pub"><day>09</day><month>12</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e04288</elocation-id><history><date date-type="received"><day>08</day><month>08</month><year>2014</year></date><date date-type="accepted"><day>23</day><month>10</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014, Klaips et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Klaips et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife04288.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.04288.001</object-id><p>The proteostasis network has evolved to support protein folding under normal conditions and to expand this capacity in response to proteotoxic stresses. Nevertheless, many pathogenic states are associated with protein misfolding, revealing in vivo limitations on quality control mechanisms. One contributor to these limitations is the physical characteristics of misfolded proteins, as exemplified by amyloids, which are largely resistant to clearance. However, other limitations imposed by the cellular environment are poorly understood. To identify cell-based restrictions on proteostasis capacity, we determined the mechanism by which thermal stress cures the [<italic>PSI</italic><sup>+</sup>]/Sup35 prion. Remarkably, Sup35 amyloid is disassembled at elevated temperatures by the molecular chaperone Hsp104. This process requires Hsp104 engagement with heat-induced non-prion aggregates in late cell-cycle stage cells, which promotes its asymmetric retention and thereby effective activity. Thus, cell division imposes a potent limitation on proteostasis capacity that can be bypassed by the spatial engagement of a quality control factor.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.001">http://dx.doi.org/10.7554/eLife.04288.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.04288.002</object-id><title>eLife digest</title><p>Proteins must fold into specific shapes to work inside cells, and the misfolding of proteins is associated with a growing number of diseases. For example, prions are misfolded proteins that form insoluble aggregates called amyloids. These aggregates are not easily destroyed and can cause other nearby proteins to misfold and join the amyloid. This process of amyloid assembly leads to progressive diseases such as mad cow disease, Huntington's disease, Alzheimer's disease, and Parkinson's disease, which are collectively known as amyloidoses.</p><p>A series of biological pathways called the proteostasis network control protein integrity in a cell. Under normal conditions or even mildly stressful conditions—such as at slightly increased temperatures—the proteostasis network is able to prevent proteins from misfolding. However, if a cell is placed under lots of stress this network may become overwhelmed and misfolded proteins can accumulate. To date, the proteostasis network has not been linked to the clearance of amyloids.</p><p>A protein called Sup35, which is found in budding yeast, can exist as two different prion forms. Previous studies have shown that briefly heating the yeast cells can ‘cure’ the so-called ‘weak’ form of the prion. The ‘strong’ prion form, however, was thought to be unaffected by elevated temperature. These previous studies had only tested yeast cells that had been dividing for a few generations; it was unknown if cells that had been dividing for longer might respond differently.</p><p>Klaips et al. found that a protein called Hsp104—which helps to fold proteins properly—can break down the amyloid aggregates. This protein is normally only present in small amounts, but heating causes the levels of Hsp104 to rise. Klaips et al. found that the extra Hsp104 protein associated with the aggregates and led to their disassembly. When Hsp104 was prevented from associating with the prions, the aggregates were not cured even if high levels of Hsp104 were present in the cell.</p><p>When budding yeast form new cells, a daughter cell ‘buds’ off from the mother cell. Klaips et al. found that mother cells exposed to heat retain most of the Hsp104 when the cell divides, and this retention allowed Hsp104 to accumulate to a level required for the breakdown of amyloid aggregates. Therefore, under normal conditions, amyloids persist because cell division keeps the amount of Hsp104 below this threshold.</p><p>Previously it had been thought that the physical characteristics of amyloids accounted for their resilience in the face of the cell mechanisms designed to counteract protein misfolding. However, Klaips et al. show that the balance of the different mechanisms involved in proteostasis can be manipulated to create environments where amyloids are either created and maintained or destroyed. Targeting these mechanisms could therefore present new treatment options for amyloidosis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.002">http://dx.doi.org/10.7554/eLife.04288.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>chaperone</kwd><kwd>protein misfolding</kwd><kwd>amyloid</kwd><kwd>prion</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>S. cerevisiae</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000057</institution-id><institution content-type="university">National Institute of General Medical Sciences</institution></institution-wrap></funding-source><award-id>R01 GM069802001</award-id><principal-award-recipient><name><surname>Serio</surname><given-names>Tricia R</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution content-type="university">National Institutes of Health</institution></institution-wrap></funding-source><award-id>F31 AG034754</award-id><principal-award-recipient><name><surname>Klaips</surname><given-names>Courtney L</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000057</institution-id><institution content-type="university">National Institute of General Medical Sciences</institution></institution-wrap></funding-source><award-id>F31 GM099383</award-id><principal-award-recipient><name><surname>Langlois</surname><given-names>Christine R</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Cell division imposes a limit on proteostasis capacity by reducing chaperone accumulation, but chaperone-substrate interactions reverse these events to allow clearance of even chronically misfolded protein amyloids.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>The proper folding of proteins is essential to cellular homeostasis, and an extensive collection of protein quality control (PQC) pathways, known as the proteostasis network, has evolved to protect nascent and metastable proteins from misfolding and to reactivate or remove proteins that have already misfolded (<xref ref-type="bibr" rid="bib65">Powers et al., 2009</xref>; <xref ref-type="bibr" rid="bib96">Wolff et al., 2014</xref>). The PQC network is tailored to buffer protein folding in a distinct homeostatic niche but can adapt when these buffering thresholds are exceeded by elevating the expression of PQC factors, including proteases and molecular chaperones, to clear accumulating misfolded proteins (<xref ref-type="bibr" rid="bib55">Morimoto, 2008</xref>; <xref ref-type="bibr" rid="bib65">Powers et al., 2009</xref>). In cases such as thermal stress, these corrections are sufficient to restore balance, but in others such as aging, misfolded proteins assemble into ordered amyloid aggregates, which persist and dramatically alter cellular physiology by inducing disease (<xref ref-type="bibr" rid="bib88">Tuite and Serio, 2010</xref>; <xref ref-type="bibr" rid="bib90">Voisine et al., 2010</xref>; <xref ref-type="bibr" rid="bib82">Taylor and Dillin, 2011</xref>; <xref ref-type="bibr" rid="bib39">Kim et al., 2013</xref>). This proteostasis collapse has been linked to the unique ability of amyloids to incorporate and conformationally convert like protein to the misfolded state and to their high thermodynamic stability (<xref ref-type="bibr" rid="bib8">Chiti and Dobson, 2006</xref>; <xref ref-type="bibr" rid="bib32">Jahn and Radford, 2008</xref>). Together, these properties are thought to enhance the production and restrict the resolution of the misfolded protein to the point that the buffering capacity and adaptability of the proteostasis network is chronically exceeded.</p><p>Despite this natural upper boundary on proteostasis capacity, the heterologous overexpression of molecular chaperones in <italic>Caenorhabditis elegans</italic>, mice, <italic>Drosophila</italic>, yeast, and tissue culture-cell models of amyloidoses reduces proteotoxicity (<xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref>; <xref ref-type="bibr" rid="bib55">Morimoto, 2008</xref>; <xref ref-type="bibr" rid="bib4">Broadley and Hartl, 2009</xref>; <xref ref-type="bibr" rid="bib29">Holmes et al., 2014</xref>). While these observations are often interpreted as evidence of amyloid resolution, existing protein has not been demonstrated to transition from an amyloid to a non-amyloid form in any of the studies. Instead, two correlations have been observed where the reduced proteotoxicity has been linked to a change in amyloid state. Either amyloid accumulation is enhanced by chaperone overexpression (<xref ref-type="bibr" rid="bib16">Douglas et al., 2009</xref>; <xref ref-type="bibr" rid="bib12">Cushman-Nick et al., 2013</xref>), or amyloid accumulation is reduced. In the few cases where the mechanism has been determined, the reduction in amyloid accumulation results from an inhibition of amyloid assembly by the overexpressed chaperone (<xref ref-type="bibr" rid="bib43">Kobayashi et al., 2000</xref>; <xref ref-type="bibr" rid="bib71">Schaffar et al., 2004</xref>; <xref ref-type="bibr" rid="bib79">Tam et al., 2006</xref>; <xref ref-type="bibr" rid="bib75">Shorter and Lindquist, 2008</xref>; <xref ref-type="bibr" rid="bib94">Winkler et al., 2012</xref>). Thus, even the specific overexpression of individual chaperones is unable to extend the proteostasis upper boundary in vivo to resolve protein amyloids.</p><p>Although these targeted interventions have yet to succeed, studies conducted under conditions that reduce amyloid amplification indicate that amyloid clearance may not represent an insurmountable obstacle. For example, repressing expression of an amyloidogenic protein can reverse established toxicity and, at least in some cases, clear existing amyloid (<xref ref-type="bibr" rid="bib97">Yamamoto et al., 2000</xref>; <xref ref-type="bibr" rid="bib52">Mallucci et al., 2003</xref>; <xref ref-type="bibr" rid="bib47">Lim et al., 2011</xref>). In addition, expression of a dominant-negative mutant also promotes disassembly of wild-type amyloid in vivo (<xref ref-type="bibr" rid="bib15">DiSalvo et al., 2011</xref>). Together, these observations suggest that amyloid clearance mechanisms exist in vivo, and indeed amyloid resolution is biochemically feasible in vitro using purified chaperones such as yeast Hsp104, alone or in combination with its co-chaperones Hsp40, Hsp70, and small heat shock proteins (<xref ref-type="bibr" rid="bib30">Inoue et al., 2004</xref>; <xref ref-type="bibr" rid="bib74">Shorter and Lindquist, 2004</xref>, <xref ref-type="bibr" rid="bib50">Lo Bianco et al., 2008</xref>; <xref ref-type="bibr" rid="bib75">Shorter and Lindquist, 2008</xref>). What limitations, then, restrict the ability of cells to expand proteostasis capacity to effectively resolve continuously expressed wild-type protein amyloids in vivo?</p><p>To identify cell-based limitations on proteostasis capacity, we focused on the mechanisms controlling persistence of the yeast prion [<italic>PSI</italic><sup><italic>+</italic></sup>], the alternative, self-templating, amyloid form of the Sup35 protein (<xref ref-type="bibr" rid="bib10">Cox, 1965</xref>; <xref ref-type="bibr" rid="bib62">Patino et al., 1996</xref>; <xref ref-type="bibr" rid="bib63">Paushkin et al., 1996</xref>; <xref ref-type="bibr" rid="bib25">Glover et al., 1997</xref>; <xref ref-type="bibr" rid="bib40">King et al., 1997</xref>). In this study, we report that a transient thermal stress surprisingly leads to the complete disassembly of existing Sup35 amyloid. This process requires the accumulation of heat-induced non-prion protein aggregates in cells primarily at the later stages of the cell cycle. The engagement of Hsp104 with these substrates, and its inability to resolve them before cell division, leads to asymmetric retention of the chaperone in cells that experienced the thermal stress. As a result, Hsp104 accumulates to a level that is sufficient to resolve amyloid aggregates. Thus, the kinetics of substrate engagement by a PQC factor and its partitioning during cell division impose cell-based limitations on proteostasis capacity.</p></sec><sec sec-type="results" id="s2"><title>Results</title><sec id="s2-1"><title>Sup35 amyloid is resolubilized by Hsp104 following thermal stress</title><p>Under normal growth conditions, [<italic>PSI</italic><sup><italic>+</italic></sup>] propagates faithfully (<xref ref-type="bibr" rid="bib10">Cox, 1965</xref>; <xref ref-type="bibr" rid="bib14">Derkatch et al., 1996</xref>). However, at elevated temperatures where the PQC capacity is increased, [<italic>PSI</italic><sup><italic>+</italic></sup>] becomes destabilized in a Sup35 conformation-specific manner. For example, the more thermodynamically stable but less efficiently propagated [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> variant is quantitatively ‘cured’ (i.e. converted to the non-prion [<italic>psi</italic><sup>−</sup>] state) at elevated temperature in comparison with [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup>, whose propagation is unaltered under the same conditions (<xref ref-type="bibr" rid="bib11">Cox et al., 1988</xref>; <xref ref-type="bibr" rid="bib14">Derkatch et al., 1996</xref>; <xref ref-type="bibr" rid="bib35">Jung et al., 2000</xref>; <xref ref-type="bibr" rid="bib80">Tanaka et al., 2006</xref>; <xref ref-type="bibr" rid="bib58">Newnam et al., 2011</xref>). This curing of [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> was linked to the inhibition of the molecular chaperone Hsp104 (<xref ref-type="bibr" rid="bib58">Newnam et al., 2011</xref>), an observation that is seemingly counter to the idea that proteostasis capacity increases in response to stress (<xref ref-type="bibr" rid="bib56">Morimoto, 2011</xref>). However, in this study, stationary phase cultures were only briefly diluted into fresh medium to re-establish exponential growth before exposure to elevated temperature (<xref ref-type="bibr" rid="bib58">Newnam et al., 2011</xref>). Because stationary phase alters chaperone expression and blocks [<italic>PSI</italic><sup><italic>+</italic></sup>] curing at elevated temperature (<xref ref-type="bibr" rid="bib23">Gasch et al., 2000</xref>; <xref ref-type="bibr" rid="bib58">Newnam et al., 2011</xref>), residual effects from the growth phase switch could alter the interaction between Sup35 aggregates and PQC factors. Therefore, we revisited the effects of elevated temperature on [<italic>PSI</italic><sup><italic>+</italic></sup>] propagation, beginning with exponentially growing cultures.</p><p>To monitor transitions from the prion [<italic>PSI</italic><sup><italic>+</italic></sup>] to the non-prion [<italic>psi</italic><sup>−</sup>] state, we used yeast strains encoding a premature termination codon (PTC) in the <italic>ADE1</italic> gene. In [<italic>PSI</italic><sup><italic>+</italic></sup>] strains, Sup35 is functionally compromised, leading to stop-codon read-through and the formation of white or pink colonies on rich medium, but in [<italic>psi</italic><sup>−</sup>] strains, termination is faithful at the PTC, leading to the formation of red colonies on rich medium (<xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref>). Transiently elevating the growth temperature from 30°C to 40°C had no effect on viability (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>) or on [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> propagation (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) but induced [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). Notably, both fully red and sectored colonies were observed, indicating that curing happened during both the thermal stress and subsequent recovery (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). Thus, [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> propagation is similarly sensitive to elevated temperature in exponentially growing cultures and in those that have recently exited stationary phase.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.003</object-id><label>Figure 1.</label><caption><title>Thermal stress induces curing through resolution of Sup35 amyloid.</title><p>(<bold>A</bold>) [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> (SLL2606) and [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> (SLL2600) cultures were incubated for 30 min at the indicated temperatures before plating on rich medium at 30°C to analyze curing by colony color phenotype, as described in the text. (<bold>B</bold>) Quantification of [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> (SLL2600) colony color phenotypes following treatment as described in (<bold>A</bold>). Colonies were scored as completely [<italic>psi</italic><sup>−</sup>] (black), or sectored (partially [<italic>psi</italic><sup>−</sup>], white). Data represent averages; error bars represent standard deviations; n = 3. (<bold>C</bold>) Semi-native lysates of [<italic>psi</italic><sup>−</sup>] (SLL2119), [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> (SLL2600), and [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> (SLL2606) cultures were analyzed by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) and immunoblotting for Sup35 after treatment as described in (<bold>A</bold>). (<bold>D</bold>) Sup35 released from amyoid aggregates in a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> strain (SLL2600) following treatment as described in (<bold>A</bold>) and recovery at 30°C in the presence of cycloheximide was determined by treating lysates with 2% SDS at 53°C, followed by SDS-PAGE and quantitative immunoblotting for Sup35. Lines represent medians; boxes represents upper and lower quartiles, and whiskers represent maximum and minimum; n = 5; *p = 0.02, **p = 0.01 by paired t-test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.003">http://dx.doi.org/10.7554/eLife.04288.003</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04288.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Characterization of thermal stress effects.</title><p>(<bold>A</bold>). Exponentially growing [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> cultures (SLL2600) were incubated at 30°C, 37°C, 40°C, or 37°C before 40°C for 30 min and plated to YPD at 30°C to quantify colony forming units. Data represent means; error bars represent standard deviations; n ≥ 3. (<bold>B</bold>) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> cultures (SLL2600) grown exponentially for 2 hr after dilution from a saturated overnight culture (left) or for at least 24 hr (right) were incubated at 40°C for 30 min and allowed to recover for 2 hr at 30°C. Lysates isolated from these cultures were analyzed by SDD-AGE and immunoblotting for Sup35.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.004">http://dx.doi.org/10.7554/eLife.04288.004</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288fs001"/></fig></fig-group></p><p>At the normal growth temperature, large Sup35 aggregates are fragmented into smaller complexes by Hsp104 (<xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref>; <xref ref-type="bibr" rid="bib19">Eaglestone et al., 2000</xref>; <xref ref-type="bibr" rid="bib57">Ness et al., 2002</xref>; <xref ref-type="bibr" rid="bib69">Satpute-Krishnan et al., 2007</xref>; <xref ref-type="bibr" rid="bib38">Kawai-Noma et al., 2009</xref>). In a culture that recently exited stationary phase, the size of SDS-resistant Sup35 aggregates increased, as assessed by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) (<xref ref-type="bibr" rid="bib45">Kryndushkin et al., 2003</xref>), following incubation at 40°C and a 2 hr recovery at 30°C (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>, left) (<xref ref-type="bibr" rid="bib58">Newnam et al., 2011</xref>), consistent with an inhibition of fragmentation (<xref ref-type="bibr" rid="bib58">Newnam et al., 2011</xref>). In contrast, SDS-resistant Sup35 aggregates were immediately reduced in size (<xref ref-type="fig" rid="fig1">Figure 1C</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>) and completely lost after recovery (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>, right) following identical treatment of an exponentially growing [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> strain, a progression suggesting the resolution of existing Sup35 aggregates. To test this possibility, we incubated a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> culture at 40°C, returned the culture to 30°C in the presence of cycloheximide to repress new protein synthesis, and monitored the conversion of existing Sup35 from the amyloid [<italic>PSI</italic><sup><italic>+</italic></sup>] state (i.e. SDS-resistant) to the non-amyloid [<italic>psi</italic><sup>−</sup>] state (i.e. SDS-sensitive) (<xref ref-type="bibr" rid="bib72">Serio et al., 2000</xref>; <xref ref-type="bibr" rid="bib70">Satpute-Krishnan and Serio, 2005</xref>). In a control culture at 30°C, very little pre-existing Sup35 transitioned to an SDS-sensitive state despite the inhibition of new protein synthesis (<xref ref-type="fig" rid="fig1">Figure 1D</xref>), as expected (<xref ref-type="bibr" rid="bib15">DiSalvo et al., 2011</xref>). However, following incubation at 40°C, over 70% of SDS-resistant Sup35 became detergent sensitive during recovery at 30°C (<xref ref-type="fig" rid="fig1">Figure 1D</xref>), indicating disassembly of existing Sup35 amyloid. To determine if the prion curing resulting from this disassembly was mediated by Hsp104, we chemically inhibited this factor with guanidine HCl (GdnHCl) treatment or reduced its dosage by creating a heterozygous disruption in a diploid strain (<xref ref-type="bibr" rid="bib18">Eaglestone et al., 1999</xref>; <xref ref-type="bibr" rid="bib36">Jung and Masison, 2001</xref>; <xref ref-type="bibr" rid="bib26">Grimminger et al., 2004</xref>; <xref ref-type="bibr" rid="bib46">Kummer et al., 2013</xref>; <xref ref-type="bibr" rid="bib81">Tariq et al., 2013</xref>; <xref ref-type="bibr" rid="bib100">Zeymer et al., 2013</xref>), and in both cases, [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing was reduced by more than 50% relative to the wild-type untreated strain (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>). Thus, Hsp104 promotes the disassembly of existing Sup35 amyloid in a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> strain following thermal stress.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.005</object-id><label>Figure 2.</label><caption><title>Curing is mediated by Hsp104 and depends upon propagation efficiency.</title><p>(<bold>A</bold>) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> cultures (SLL2600) were incubated at 40°C for 30 min in the absence (untreated) or presence of guanidine HCl (GdnHCl) and plated on YPD to quantify prion loss by colony color phenotype. Data represent means; error bars represent standard deviations; n = 3; p = 0.0004 by unpaired t-test. (<bold>B</bold>) A WT (<italic>HSP104</italic>/+; SY945) and a heterozygous disruption (<italic>HSP104</italic>/Δ; SY591) [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> diploid strain were incubated at 40°C for 90 min and plated on YPD to quantify prion loss by colony color phenotype. Data represent means; error bars represent standard deviations; n = 3; p < 0.0001 by unpaired t-test. (<bold>C</bold>) [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> strains expressing an extra copy of either WT (SY1646) or G58D (SY1648) Sup35 were incubated at 40°C for 90 min and plated on YPD to quantify prion loss by colony color phenotype. Data represent means; error bars represent standard deviations; n = 4; p < 0.0001 by unpaired t-test. (<bold>D</bold>) A WT (<italic>SUP35</italic>/+; SLL3071) and a heterozygous disruption (<italic>SUP35/</italic>Δ; SY957) diploid [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> strain were incubated at 40°C for 90 min and plated on YPD to quantify prion loss by colony color phenotype. Data represent means; error bars represent standard deviations; n = 3; p < 0.0001 by unpaired t-test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.005">http://dx.doi.org/10.7554/eLife.04288.005</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288f002"/></fig></p><p>At elevated temperature, we noted that the size of SDS-resistant Sup35 aggregates is reduced in a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> strain (<xref ref-type="fig" rid="fig1">Figure 1C</xref>), although curing does not occur (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Because [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> propagates more efficiently than [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup>, the former may be protected from curing at elevated temperature if the rate of Sup35 assembly continued to outpace the rate of its disassembly, a scenario that should be reversed by reducing the efficiency of [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> propagation. To test this idea, we subjected [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> diploid strains heterozygous for either a Sup35 mutant (G58D) or for a Sup35 disruption, which both reduce propagation efficiency (<xref ref-type="bibr" rid="bib13">Derdowski et al., 2010</xref>; <xref ref-type="bibr" rid="bib15">DiSalvo et al., 2011</xref>), to thermal stress. At 30°C, [<italic>PSI</italic><sup><italic>+</italic></sup>] propagation is stable in both of these strains (<xref ref-type="fig" rid="fig2">Figure 2C,D</xref>); however at 40°C, both were now efficiently cured (e.g. ∼100% for WT/<italic>G58D</italic>, ∼60% for <italic>SUP35/</italic>Δ) (<xref ref-type="fig" rid="fig2">Figure 2C,D</xref>). These observations not only provide additional support for Sup35 amyloid disassembly as the mechanism of prion curing in response to thermal stress but also reveal that the inability of chaperones to resolve amyloid in vivo results from both the physical characteristics of these aggregates and cell-based limitations, which are bypassed in the distinct proteostasis niche created at elevated temperature.</p></sec><sec id="s2-2"><title>The asymmetric retention of Hsp104 is required for curing</title><p>Elevated temperature induces protein misfolding, and the cell responds to this stress by elevating the expression of PQC factors (<xref ref-type="bibr" rid="bib56">Morimoto, 2011</xref>). To deconvolute the contributions of each of these events to [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing, we took advantage of the fact that we could modulate the efficiency of curing with variations in temperature. For example, while exposure to 40°C induced quantitative [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing, pretreatment at 37°C prior to exposure to 40°C slightly reduced curing (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>, compare proportion of fully cured colonies), and incubation at 37°C did not induce curing at all (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). This failure to destabilize [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> at 37°C corresponded to an increase in aggregate size (<xref ref-type="fig" rid="fig1">Figure 1C</xref>) and a decrease in Sup35 solubilization (<xref ref-type="fig" rid="fig1">Figure 1D</xref>) relative to growth at 40°C alone, indicating a temperature-dependent modulation of amyloid resolution.</p><p>To determine the molecular basis of these differences in curing efficiency, we first monitored the levels of Sup35, Hsp104, Ssa1/2 (Hsp70), and Sis1 (Hsp40) proteins, which have all been implicated in Sup35 amyloid fragmentation (<xref ref-type="bibr" rid="bib10">Cox, 1965</xref>; <xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref>; <xref ref-type="bibr" rid="bib76">Song et al., 2005</xref>; <xref ref-type="bibr" rid="bib28">Higurashi et al., 2008</xref>; <xref ref-type="bibr" rid="bib85">Tipton et al., 2008</xref>; <xref ref-type="bibr" rid="bib13">Derdowski et al., 2010</xref>). By quantitative immunoblotting, neither Sup35 (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1A</xref>) nor chaperone levels (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1B</xref>) correlated with curing efficiency (<xref ref-type="fig" rid="fig1">Figure 1A</xref>), indicating that [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing could not be explained by simple changes in protein expression. Indeed, the specific overexpression of Hsp104 alone from a galactose-inducible promoter to levels that parallel those achieved during thermal stress (<xref ref-type="fig" rid="fig3">Figure 3A</xref> and <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1C</xref>) induces ∼40% [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1D</xref>, 1.5 gen) in comparison with the ∼95% [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing induced by thermal stress (<xref ref-type="fig" rid="fig1">Figure 1B</xref>) (<xref ref-type="bibr" rid="bib15">DiSalvo et al., 2011</xref>, <xref ref-type="bibr" rid="bib92">Wegrzyn et al., 2001</xref>). Moreover, Hsp104 overexpression alone leads to an increase in the size of SDS-resistant Sup35 aggregates isolated from a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> strain (<xref ref-type="bibr" rid="bib45">Kryndushkin et al., 2003</xref>), as previously reported for [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1E</xref>) (<xref ref-type="bibr" rid="bib45">Kryndushkin et al., 2003</xref>), but this outcome is in obvious contrast to the disassembly of Sup35 amyloid that we observe upon thermal stress (<xref ref-type="fig" rid="fig1">Figure 1C,D</xref>). Thus, thermal stress and chaperone overexpression induce distinct changes in prion propagation.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.006</object-id><label>Figure 3.</label><caption><title>Heat-induced aggregate accumulation but not chaperone levels correlate with temperature.</title><p>(<bold>A</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain (SLL2600) was incubated at 30°C, 37°C, 40°C, or 37°C before 40°C for 30 min, and lysates were prepared and analyzed by SDS-PAGE and quantitative immunoblotting for Hsp104 (black), Ssa1 (gray), and Sis1 (white). Data represent means; error bars represent standard deviations; n ≥ 3. (<bold>B</bold>) Aggregates from lysates of a [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain (SLL2600) following treatment as described in (<bold>A</bold>) were prepared and analyzed by differential centrifugation and Bradford assay. Data represent means; error bars represent standard error; n = 6; *p = 0.0014, **p = 0.0052 by paired t-test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.006">http://dx.doi.org/10.7554/eLife.04288.006</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04288.007</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Effects of thermal stress and Hsp104 on protein accumulation.</title><p>(<bold>A</bold>) Lysates were isolated from [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strains (SLL2600) incubated at 30°C, 37°C, 40°C, or 37°C before 40°C for 30 min and analyzed by SDS-PAGE and quantitative immunoblotting for Sup35. Data represent means; error bars represent standard deviations; n = 4. (<bold>B</bold>) Lysates were isolated from [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strains (SLL2600) incubated at 30°C, 37°C, 40°C, or 37°C before 40°C for 30 min and analyzed by SDS-PAGE and quantitative immunoblotting for Hsp104, Ssa1, Sis1 or phosphoglycerate kinase (PGK) as a loading control (representative blot; see <xref ref-type="fig" rid="fig3">Figure 3A</xref> for quantification). (<bold>C</bold>) Lysates were isolated from a [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain containing a galactose-inducible <italic>HSP104</italic> at the endogenous locus (SY1749) after galactose treatment and Hsp104 protein was quantified by SDS-PAGE and immunoblotting. Data represent means; error bars represent standard deviations; n = 3. (<bold>D</bold>) Galactose-inducible <italic>HSP104</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strains (SY1749) were grown in the presence of galactose for various times and plated on YPD for analysis of [<italic>PSI</italic><sup>+</sup>] phenotype. Data represent means; error bars represent standard deviation; n = 3. (<bold>E</bold>) Lysates isolated from galactose-inducible <italic>HSP104</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> (SY1748) or [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> (SY1749) cultures treated as described in (<bold>D</bold>) were analyzed by SDD-AGE and immunoblotting for Sup35. (<bold>F</bold>) Lysates were isolated from [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> (black) (SLL2606) and [<italic>psi</italic><sup>−</sup>] (white) (SLL2119) strains that were treated as described in (<bold>B</bold>), and heat-induced protein aggregates were quantified following differential centrifugation and Bradford assay. Data represent means; error bars represent standard errors; n ≥ 5. (<bold>G</bold>) Lysates were isolated from a [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain (SLL2600) that was incubated at 30°C or 40°C for 30 min in the absence (untreated) or presence of GdnHCl, and heat-induced protein aggregates were quantified following differential centrifugation and Bradford assay. Data represent means; error bars represent standard error; n = 3.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.007">http://dx.doi.org/10.7554/eLife.04288.007</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288fs002"/></fig></fig-group></p><p>We next assessed the accumulation of misfolded proteins following shifts in temperature to determine if this event correlated with [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing efficiency. By differential centrifugation, protein aggregates accumulated independent of prion status at all elevated temperatures (<xref ref-type="fig" rid="fig3">Figure 3B</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1F</xref>), but in contrast to chaperone expression (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1B</xref>), the severity of this accumulation was impacted by growth temperature. At 37°C, protein aggregation increased by less than 10% in comparison with a culture maintained at 30°C (<xref ref-type="fig" rid="fig3">Figure 3B</xref>, column 1), but in cultures treated at 37°C followed by 40°C or directly at 40°C, this level rose to ∼20% or ∼40%, respectively (<xref ref-type="fig" rid="fig3">Figure 3B</xref>, columns 3 and 2). Thus, the accumulation of protein aggregates (<xref ref-type="fig" rid="fig3">Figure 3B</xref>) correlates directly with curing efficiency at the various temperatures (<xref ref-type="fig" rid="fig1">Figure 1A</xref>).</p><p>We noted, however, that this correlation was not observed for a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> culture treated with GdnHCl during a 40°C incubation, which strongly reduced curing efficiency (<xref ref-type="fig" rid="fig2">Figure 2A</xref>) but did not reduce the accumulation of protein aggregates (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1G</xref>). Nevertheless, numerous studies have reported the localization of chaperones to cytoplasmic quality control foci upon exposure to proteotoxic stresses (<xref ref-type="bibr" rid="bib1">Aguilaniu et al., 2003</xref>; <xref ref-type="bibr" rid="bib21">Erjavec et al., 2007</xref>; <xref ref-type="bibr" rid="bib37">Kaganovich et al., 2008</xref>; <xref ref-type="bibr" rid="bib77">Specht et al., 2011</xref>; <xref ref-type="bibr" rid="bib95">Wolfe et al., 2013</xref>), and GdnHCl blocks the association of Hsp104 with at least one substrate (<xref ref-type="bibr" rid="bib94">Winkler et al., 2012</xref>). To determine if Hsp104 localization to heat-induced aggregates rather than their accumulation per se determined prion-curing efficiency, we replaced endogenous <italic>HSP104</italic> with an <italic>HSP104-GFP</italic> fusion, which supports [<italic>PSI</italic><sup><italic>+</italic></sup>] propagation (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>). At 40°C, this strain exhibited time-dependent [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B</xref>) and accumulated protein aggregates (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1C</xref>) and Hsp104-GFP to wild-type levels, albeit with slightly delayed kinetics (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1D</xref>). At elevated temperatures, we observed an increase Hsp104-interacting proteins as assessed by co-immunocapture (<xref ref-type="fig" rid="fig4">Figure 4A</xref>) and the localization of Hsp104-GFP to cytoplasmic foci (<xref ref-type="fig" rid="fig4">Figure 4B,C</xref>), which also contain the model substrate firefly luciferase-mCherry (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1E</xref>). The amount of co-immunocaptured proteins (<xref ref-type="fig" rid="fig4">Figure 4A</xref> [2.5-fold increase at 37°C and 4.2-fold increase at 40°C relative to 30°C]) and the number and intensity of Hsp104-GFP fluorescent foci (<xref ref-type="fig" rid="fig4">Figure 4C</xref>) corresponded to both the accumulation of heat-induced protein aggregates (<xref ref-type="fig" rid="fig3">Figure 3B</xref>) and the efficiency of curing (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Notably, the Hsp104-GFP fluorescence pattern was unaltered in a non-prion [<italic>psi</italic><sup>−</sup>] strain (<xref ref-type="fig" rid="fig4">Figure 4B</xref>), indicating that Hsp104-GFP was engaged with non-prion substrates. Treatment of a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> culture with GdnHCl during an incubation at 40°C, which strongly reduces Hsp104-GFP association with heat-induced interacting proteins (<xref ref-type="fig" rid="fig4">Figure 4A</xref> [1.7-fold decrease relative to 40°C in the absence of GdnHCl]) and localization to cytoplasmic foci (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1F</xref>), also reduces the efficiency of curing (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1G</xref>). Thus, the specific engagement of Hsp104 with heat-induced aggregates, rather than simply their presence, correlates with curing at elevated temperature.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.008</object-id><label>Figure 4.</label><caption><title>Hsp104 engages heat-induced substrates upon thermal stress.</title><p>(<bold>A</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain with a GFP-tagged endogenous Hsp104 (SY2126) was incubated at 30°C, 37°C, 40°C, or 40°C with GdnHCl for 30 min, and immunocapture in the presence (+) or absence (−) of anti-GFP antibodies (Ab) was performed on native lysates. Proteins were analyzed by SDS-PAGE and general protein staining (Flamingo, top), or immunoblotting for GFP (bottom). (<bold>B</bold>) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> (SY2126) or [<italic>psi</italic><sup>−</sup>] (SY2125) <italic>HSP104GFP</italic> strains were incubated at 30°C, 37°C, 40°C, or 37°C before 40°C for 90 min, and the pattern of Hsp104-GFP fluorescence was examined by microscopy. Scale bar = 1 μm. (<bold>C</bold>) Quantification of Hsp104-GFP fluorescence pattern in [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> (SY2126) cells, treated as described in (<bold>B</bold>): no localization (white); single dot (light gray); faint aggregate (medium gray); bright aggregate (dark gray); multiple bright aggregates (black); n > 25.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.008">http://dx.doi.org/10.7554/eLife.04288.008</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04288.009</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Characterization of HSP104GFP strain.</title><p>(<bold>A</bold>) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> (SLL2600) and [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> (SY2126) strains were grown at 30°C, plated on YPD, and incubated at 30°C for analysis of [<italic>PSI</italic><sup>+</sup>] colony color phenotype. (<bold>B</bold>) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> (SLL2600, gray) and [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> (SY2126, white) strains were incubated at 40°C for the indicated times and plated on YPD at 30°C for analysis of prion curing by colony color phenotype. Data represent means; error bars represent standard deviations; n = 3.(<bold>C</bold>) Lysates were isolated from WT (SLL2600) or <italic>HSP104GFP</italic> (SY2126) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strains that were incubated at 30°C or 40°C for 30 min, and heat-induced protein aggregates were quantified by differential centrifugation and Bradford assay. Data represent means; error bars represent standard error; n = 3. (<bold>D</bold>) Quantitative western blotting for Hsp104 was performed on lysates from [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> (SLL2600) and [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> (SY2126) strains after incubation at 40°C for the indicated times. Data represent means; error bars represent standard deviations; n = 3. (<bold>E</bold>) Hsp104GFP and an mCherry-tagged firefly-luciferase (FFLmCh) reporter were visualized in a [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain (SY2802) by microscopy following incubation at 30°C or after a 30-min recovery from an incubation at 40°C for 90 min (30°C→40°C). Scale bar = 1 μm. (<bold>F</bold>) Hsp104GFP was visualized in a [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain (SY2126) by microscopy after a 90-min recovery from incubation at 40°C for 90 min in the absence (40°C) or presence of GdnHCl added before (GdnHCl→40°C) or after (40°C→GdnHCl) heat treatment. Scale bar = 1 μm. (<bold>G</bold>) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> cultures (SY2126) treated as described in (<bold>F</bold>) were plated on YPD and incubated at 30°C for analysis of [<italic>PSI</italic><sup>+</sup>] colony color phenotype. Data represent means; error bars represent standard deviations; n = 3; *p = 0.0001,**p = 0.0089.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.009">http://dx.doi.org/10.7554/eLife.04288.009</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288fs003"/></fig></fig-group></p><p>How does this chaperone engagement with heat-induced aggregates lead to the resolution of Sup35 amyloid? One possibility is that the asymmetric localization of Hsp104, resulting from its engagement with heat-induced protein aggregates (<xref ref-type="bibr" rid="bib21">Erjavec et al., 2007</xref>), increases its accumulation in a subpopulation of cells beyond that which can be achieved by its transcriptional up-regulation. To test this possibility, we first monitored the partitioning of Hsp104-GFP during cell division following incubation at various temperatures using microfluidics and fluorescence microscopy. Starting with budded cells, mother cells accumulated ∼60% of Hsp104-GFP following the completion of cell division at 30°C (<xref ref-type="fig" rid="fig5">Figure 5A</xref>, gray), which is comparable to the accumulation of untagged GFP expressed from the same promoter (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1A</xref>) and thus likely reflects the volume differences between mother and daughter cells. This baseline asymmetry progressively increased as the temperature was increased to 37°C (∼65% retention), 37°C followed by 40°C (∼73% retention), and finally 40°C (∼75% retention; <xref ref-type="fig" rid="fig5">Figure 5A</xref>, gray). Notably, both Ssa1-GFP and Sis1-GFP fusions also localized to cytoplasmic, and, in the case of Sis1, nuclear foci (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1B,C</xref>), but neither was asymmetrically retained following incubation at 40°C (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1D,E</xref>), although their levels were elevated relative to 30°C (<xref ref-type="table" rid="tbl1">Table 1</xref>) due to their enhanced expression (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). Thus, curing efficiency (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>) correlates directly with the asymmetric retention of Hsp104 in cells at elevated temperature.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.010</object-id><label>Figure 5.</label><caption><title>Curing results from the asymmetric localization of Hsp104 following thermal stress.</title><p>(<bold>A</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> culture (SY2126) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 30°C, 37°C, 40°C, or 37°C before 40°C. Fluorescence intensity in daughter and mother cells was quantified at the first cell division in cells that were budded (gray) or unbudded (orange) after thermal stress. Lines represent medians; boxes represent upper and lower quartiles, and whiskers represent maximum and minimum. All pairwise comparisons are significantly distinct, with a p < 0.015, except where indicated (N.S.), by unpaired t-test; n ≥ 10. (<bold>B</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> WT (SY2126, gray) or <italic>BNI1</italic> deletion strain (Δ<italic>bni1</italic>) (SY2486, green) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C. Fluorescence intensity in daughter and mother cells was quantified at the first cell division. Lines represent medians; boxes represent upper and lower quartiles; and whiskers represent maximum and minimum; n ≥ 14; p = 0.0075 by unpaired t-test. (<bold>C</bold>) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> WT (SLL2600) or Δ<italic>bni1</italic> strains (SY1888), treated as described in (<bold>B</bold>), were plated on YPD to analyze curing by colony color phenotype. Data represent means; error bars represent standard deviations; n = 3; p < 0.0001 by unpaired t-test. (<bold>D</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> strain (SY2126) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C and with GdnHCl added before or after the 40°C incubation. Fluorescence intensity in daughter and mother cells was quantified at the first cell division. Lines represent medians; boxes represent upper and lower quartiles; and whiskers represent maximum and minimum; n > 11; *p = 0.0003, **p = 0.0026 by unpaired t-test. (<bold>E</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain (SLL2600) was incubated at 40°C for 30 min and plated on rich medium. Mother and daughter pairs were separated by micromanipulation and allowed to form colonies, which were then dispersed to YPD for analysis of curing by colony color phenotype. n = 15. (<bold>F</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> culture (SY2126) was incubated at 30°C (dotted) or at 40°C for 30 min and allowed to recover for 30 min at 30°C (solid) before analysis of GFP fluorescence intensity by flow cytometry. Based on these intensities, cells were sorted into four fractions (orange, blue, purple, red) by FACS. (<bold>G</bold>) Cells collected in (<bold>F</bold>) were plated on YPD to analyze curing by colony color phenotype. Data represent means; error bars represent standard deviations; n = 2; *p = 0.02 by paired t-test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.010">http://dx.doi.org/10.7554/eLife.04288.010</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04288.011</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Characterization of chaperone asymmetric retention following thermal stress.</title><p>(<bold>A</bold>) A [<italic>psi</italic><sup>−</sup>] strain expressing heat-inducible untagged GFP (SY2091) was imaged over time in a microfluidics chamber at 30°C after 30 min incubation at 40°C (red) or 30°C (gray). Fluorescence intensity in daughter and mother cells was quantified at the first cell division in budded cells. Lines represent medians, boxes represent upper and lower quartiles, and whiskers represent maximum and minimum; n ≥ 11. (<bold>B</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain expressing a GFP-tagged endogenous Ssa1 and DsRedNLS (SY2659) was imaged after a 90 min incubation at 30°C, 37°C, 40°C, or 37°C before 40°C. Scale bar = 2 μm. (<bold>C</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain expressing a GFP-tagged endogenous Sis1 and DsRedNLS (SY2485) was imaged after a 90-min incubation at 30°C, 37°C, 40°C, or 37°C before 40°C. Scale bar = 2 μm. (<bold>D</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>SSA1GFP</italic> culture (SY2658) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C (red) or 30°C (gray). Fluorescence intensity in daughter and mother cells was quantified at the first cell division in budded cells. Lines represent medians, boxes represent upper and lower quartiles, and whiskers represent maximum and minimum; n > 15. (<bold>E</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>SIS1GFP</italic> culture (SY2447) was imaged over time in a microfluidics chamber at 30°C after a 30 min incubation at 40°C (red) or 30°C (gray). Fluorescence intensity in daughter and mother cells was quantified at the first cell division in budded cells. Lines represent medians, boxes represent upper and lower quartiles, and whiskers represent maximum and minimum; n ≥ 7. (<bold>F</bold>) Quantitative immunoblotting for Hsp104 was performed on lysates from WT (SLL2600) or <italic>Δbni1</italic> (SY1888) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> cultures treated at 30°C (black) and 40°C (white) for 30 min following SDS-PAGE. Data represent means; error bars represent standard deviations; n = 3. (<bold>G</bold>) Lysates were isolated from WT (SLL2600) or <italic>Δbni1</italic> (SY1888) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strains that were incubated at 30°C or 40°C for 30 min, and heat-induced protein aggregates were analyzed by differential centrifugation and Bradford assay. Data represent means; error bars represent standard error; n = 3.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.011">http://dx.doi.org/10.7554/eLife.04288.011</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288fs004"/></fig></fig-group><table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.012</object-id><label>Table 1.</label><caption><p>Relative fluorescence intensity in mother cells</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.012">http://dx.doi.org/10.7554/eLife.04288.012</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center">Treatment (°C)</th><th align="center">Hsp104 (Relative to 30°C)</th><th align="center">Ssa1 (Relative to 30°C)</th><th align="center">Sis1 (Relative to 30°C)</th></tr></thead><tbody><tr><td align="center">30°→30°</td><td align="center">1 ± 0.1 (24)</td><td align="center">1 ± 0.2 (29)</td><td align="center">1 ± 0.1 (18)</td></tr><tr><td align="center">30°→37°</td><td align="center">1.6 ± 0.2 (11)</td><td/><td/></tr><tr><td align="center">30°→40°</td><td align="center">3.5 ± 0.6 (52)</td><td align="center">2.7 ± 0.5 (18)</td><td align="center">1.5 ± 0.1 (7)</td></tr><tr><td align="center">37°→40°</td><td align="center">3.4 ± 0.4 (46)</td><td/><td/></tr></tbody></table><table-wrap-foot><fn><p>[<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic>(SY2126), <italic>SSA1GFP</italic> (SY2658), or <italic>SIS1GFP</italic> (SY2447) cultures were treated at indicated temperatures and were imaged over time at 30°C using microfluidics and fluorescence microscopy. Average fluorescence intensity in mother cells with indicated standard deviations (±), which originated from budded cells at the time of thermal stress, was measured at the first cell division. Number of cells analyzed is indicated in parentheses. p values are <0.001 for all comparisons to 30°C treatment.</p></fn></table-wrap-foot></table-wrap></p><p>To determine if this correlation was a requirement, we next disrupted the asymmetric retention of Hsp104 and determined its effects on curing. Disruption of the formin <italic>BNI1</italic> (<xref ref-type="bibr" rid="bib44">Kohno et al., 1996</xref>) did not alter Hsp104 expression levels or the accumulation of protein aggregates at 30°C and 40°C relative to a wild-type strain, (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1F,G</xref>) but, Hsp104 asymmetric retention was reduced (<xref ref-type="fig" rid="fig5">Figure 5B</xref>, green), as expected (<xref ref-type="bibr" rid="bib49">Liu et al., 2010</xref>). Strikingly, curing was dramatically suppressed from ∼80% for a wild-type strain to ∼10% in the Δ<italic>bni1</italic> strain (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). Likewise, GdnHCl treatment before thermal stress, which blocked both Hsp104 engagement with heat-induced aggregates (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1F</xref>) and curing at elevated temperature (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1G</xref>), also reduced Hsp104-GFP asymmetric retention following exposure to 40°C (<xref ref-type="fig" rid="fig5">Figure 5D</xref>). Thus, the asymmetric retention of Hsp104 is required for curing.</p><p>Our single-cell analyses of Hsp104-GFP partitioning indicated that a relatively minor change in chaperone retention from 65% to 75%, which corresponded to a 2.2-fold increase in accumulation based on fluorescence intensity (compare 37°C–40°C, <xref ref-type="table" rid="tbl1">Table 1</xref>, <xref ref-type="fig" rid="fig5">Figure 5A</xref>), correlated with a quantitative switch from prion stability to curing (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>), suggesting the existence of a biological threshold in this range. To determine directly if cells accumulating Hsp104-GFP corresponded to those cured of [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup>, we incubated a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> culture at 40°C and then isolated single unbudded cells on rich solid medium at 30°C. Following budding and cell division, mother and daughter cells were separated by micromanipulation and grown into colonies, which were then dispersed on rich solid medium to quantify prion retention. Mother cells, which experienced the elevated temperature and accumulated Hsp104 (<xref ref-type="fig" rid="fig5">Figure 5A</xref>), were more likely to be cured than their daughters (<xref ref-type="fig" rid="fig5">Figure 5E</xref>, note most data points fall below the diagonal), as predicted by our hypothesis. To more quantitatively correlate Hsp104-GFP accumulation with curing efficiency, we analyzed the distribution of Hsp104-GFP in a population of cells by flow cytometry. At 30°C, Hsp104-GFP fluorescence was distributed normally in the population (<xref ref-type="fig" rid="fig5">Figure 5F</xref>, dotted). Following incubation at 40°C, Hsp104-GFP fluorescence intensity in the population increased and its distribution was heterogeneous (<xref ref-type="fig" rid="fig5">Figure 5F</xref>, solid). When these subpopulations were separated by FACS and analyzed for colony-based phenotype, the efficiency of curing correlated directly with the accumulation of Hsp104-GFP (<xref ref-type="fig" rid="fig5">Figure 5F,G</xref>). Together, these observations indicate that cells exposed to elevated temperature accumulate heat-induced protein aggregates, asymmetrically retain Hsp104 in a manner that is proportional to these substrates, and ultimately cure [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup>.</p><p>But, is Hsp104 enzymatic activity required for this curing, or is its asymmetric localization alone sufficient? As noted above, when cells are treated with GdnHCl before thermal stress, Hsp104 localization to cytoplasmic foci and asymmetric retention are both reduced (<xref ref-type="fig" rid="fig5">Figure 5D</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1F</xref>). However, we reasoned the Hsp104 association with its substrates would be dynamic and modulated by its ATPase cycle. Indeed, blocking the ATPase activity of Hsp104 after thermal stress with a 90-min treatment with GdnHCl failed to reduce Hsp104-GFP localization to cytoplasmic foci (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1F</xref>) or its asymmetric retention (<xref ref-type="fig" rid="fig5">Figure 5D</xref>), presumably because the chaperone bound to heat-induced substrates but was unable to release them once inhibited with GdnHCl. Despite the asymmetric localization of Hsp104-GFP under these conditions, [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing was reduced by nearly 50% (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1G</xref>). Thus, both Hsp104 asymmetric localization and activity are required to induce [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing following thermal stress.</p></sec><sec id="s2-3"><title>Cell-cycle stage and substrate-chaperone dynamics impact amyloid resolution</title><p>The distribution of Hsp104-GFP in a population of [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> cells that had been exposed to 40°C was very complex in contrast to the normal distribution of Hsp104-GFP at 30°C (<xref ref-type="fig" rid="fig5">Figure 5F</xref>), suggesting that subpopulations of cells were differentially retaining the chaperone. One source of heterogeneity in the population was cell-cycle stage, as our experiments used asynchronous cultures (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). To determine if cell-cycle stage at the time of thermal stress impacted Hsp104 partitioning and explained this heterogeneity, we arrested cells in G1 with α-factor or at the G2/M transition with nocodazole (<xref ref-type="bibr" rid="bib2">Amon, 2002</xref>), exposed these cultures to 40°C incubation, and analyzed them by flow cytometry. Treatment with α-factor (<xref ref-type="fig" rid="fig6">Figure 6B</xref>) and nocodazole (<xref ref-type="fig" rid="fig6">Figure 6C</xref>) efficiently synchronized cultures at the non-budded or large-budded stages, respectively, and did not alter Hsp104 protein levels or localization relative to the asynchronous culture at 30°C (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1A,B</xref>). At 40°C, Hsp104-GFP protein levels increased to similar extents in the asynchronous and arrested cultures (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1A</xref>), and its localization to cytoplasmic foci was similar in all cases (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1B</xref>). By flow cytometry, the distribution of Hsp104-GFP in the α-factor arrested culture remained normal (<xref ref-type="fig" rid="fig6">Figure 6D</xref>), but in the nocodazole-arrested culture, this distribution became bimodal (<xref ref-type="fig" rid="fig6">Figure 6E</xref>), indicating that Hsp104-GFP asymmetry is established immediately, even before cell division.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.013</object-id><label>Figure 6.</label><caption><title>Efficient curing occurs in late cell-cycle staged cells following thermal stress.</title><p>(<bold>A</bold>) Single cells from an asynchronous WT [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> culture (SLL2600) were scored for morphology following bright-field imaging by microscopy: unbudded (black), tiny bud (dark gray), small bud (gray), medium bud (light gray), large bud (white). n = 153. (<bold>B</bold>) α-factor-arrested cultures were analyzed as in (<bold>A</bold>) over time after release. n ≥ 250. (<bold>C</bold>) Nocodazole-arrested cultures were analyzed as in (<bold>A</bold>) over time after release. n ≥ 175. (<bold>D</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> strain (SY2126) released from α-factor arrest was incubated at 40°C (solid black lines) for 30 min before analysis by flow cytometry. 100,000 cells were analyzed per sample. (<bold>E</bold>) A [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> strain (SY2126) released from nocodazole arrest was incubated at at 40°C (black lines) for 30 min before analysis by flow cytometry. 100,000 cells were analyzed per sample. (<bold>F</bold>) α-factor-arrested cultures (SLL2600) were incubated at 40°C for 30 min immediately or 30 min after release, and curing was quantified by colony color phenotype after plating on YPD at 30°C. Data represent means; error bars represent standard deviations; n = 3; p = 0.0255 by unpaired t-test. (<bold>G</bold>) Nocodazole-arrested cultures (SLL2600) were incubated at 40°C for 30 min immediately or 30 min after release, and curing was quantified colony color phenotype after plating on YPD at 30°C. Data represent means; error bars represent standard deviations; n = 3; p = 0.0263 by unpaired t-test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.013">http://dx.doi.org/10.7554/eLife.04288.013</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04288.014</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Characterization of chaperone accumulation and engagement in arrested cultures.</title><p>(<bold>A</bold>) Lysates were isolated from asynchronous, α-factor-arrested, or nocodazole-arrested [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> cultures (SLL2600) following incubation at 30°C (black) or 40°C (white) for 30 min, and the levels of Hsp104 were determined by quantitative immunoblotting following SDS-PAGE. Data represent means; error bars represent standard deviations; n = 3. (<bold>B</bold>) Asynchronous, α-factor-arrested, and nocodazole-arrested [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> (SY2126) cultures were treated for 90 min at 30°C or 40°C and imaged by microscopy. Scale bar = 2 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.014">http://dx.doi.org/10.7554/eLife.04288.014</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288fs005"/></fig></fig-group></p><p>We next assessed the impact of cell-cycle stage on [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing at elevated temperature. Arrest, without exposure to elevated temperature, did not induce curing (<xref ref-type="fig" rid="fig6">Figure 6F,G</xref>). In α-factor arrested cells, exposure to 40°C at release inefficiently cured [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> (∼15%; <xref ref-type="fig" rid="fig6">Figure 6F</xref>), but in nocodazole-arrested cells, curing was nearly quantitative (∼90%; <xref ref-type="fig" rid="fig6">Figure 6G</xref>) consistent with the asymmetric localization of Hsp104-GFP in the latter but not the former case (<xref ref-type="fig" rid="fig6">Figure 6D,E</xref>). These observations suggest that cells at the end of the cell cycle are more sensitive to curing at elevated temperature than those at the beginning of the cell cycle. To test this idea, we released cultures from arrest and, after 30 min of growth at 30°C, exposed them to 40°C. For the culture originally arrested with α-factor, sensitivity to curing at elevated temperature increased (<xref ref-type="fig" rid="fig6">Figure 6F</xref>) as cells progressed into the late stages of the cell cycle (<xref ref-type="fig" rid="fig6">Figure 6B</xref>), and for the culture originally arrested with nocodazole, this sensitivity declined (<xref ref-type="fig" rid="fig6">Figure 6G</xref>) with cell-cycle progression (<xref ref-type="fig" rid="fig6">Figure 6C</xref>). Thus, curing occurs most efficiently when cells at a late stage of the cell cycle are exposed to elevated temperature.</p><p>Our earlier experiments linked curing to the asymmetric retention of Hsp104 at elevated temperature (<xref ref-type="fig" rid="fig5">Figure 5</xref>). To determine if cell-cycle stage impacts this asymmetry, we analyzed Hsp104-GFP distribution in mother–daughter pairs resulting from the growth and division of unbudded cells isolated from asynchronous cultures that were exposed to elevated temperatures. In comparison with budded cells, Hsp104-GFP retention was significantly reduced at all temperatures when unbudded cells were exposed to elevated temperature, but the magnitude of the effect was most severe for conditions that induced curing (30°C→40°C and 37°C→40°C; <xref ref-type="fig" rid="fig5">Figure 5A</xref>, orange), indicating a cell-cycle stage dependence on Hsp104-GFP retention at elevated temperature.</p><p>Because cell-cycle stage did not obviously alter the engagement of Hsp104-GFP with protein aggregates accumulating at elevated temperature (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1B</xref>), the more efficient partitioning of Hsp104-GFP and the reduced curing in unbudded cells could reflect the resolution of heat-induced protein aggregates and thereby the release of Hsp104-GFP during the extended time before cell division in comparison with budded cells. Indeed, nearly 100% of cells contained Hsp104-GFP foci immediately after thermal stress (<xref ref-type="fig" rid="fig7">Figure 7A</xref>) but only ∼80% still contained foci when cell division re-initiated ∼150 min after incubation at 40°C (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). Thus, the relative timing of substrate release and cell division could contribute to Hsp104 asymmetric retention and thereby curing. Consistent with this idea, 60% of unbudded cells, which are inefficiently cured (<xref ref-type="fig" rid="fig6">Figure 6F</xref>), resolved Hsp104-GFP foci prior to cell division (<xref ref-type="fig" rid="fig7">Figure 7B</xref> [165–210 min], <xref ref-type="fig" rid="fig7">Figure 7C</xref>), allowing the partitioning of the chaperone (<xref ref-type="fig" rid="fig5 fig7">Figures 7B and 5A</xref>, orange). In budded cells, which are efficiently cured (<xref ref-type="fig" rid="fig6">Figure 6G</xref>), only ∼8% of cells had resolved heat-induced Hsp104-GFP foci by the time the cell divided (<xref ref-type="fig" rid="fig7">Figure 7B</xref> [105 min], <xref ref-type="fig" rid="fig7">Figure 7C</xref>), leading to the asymmetric retention of Hsp104-GFP (<xref ref-type="fig" rid="fig5 fig7">Figures 7B and 5A</xref>, gray). Together, these observations indicate that Hsp104 is retained in cells exposed to elevated temperature if it is unable to resolve its heat-induced substrates prior to cell division. Because sensitivity to curing at elevated temperature correlated with cell-cycle stage (<xref ref-type="fig" rid="fig6">Figure 6F,G</xref>) and Hsp104-GFP localization to these cytoplasmic foci (<xref ref-type="fig" rid="fig5">Figure 5</xref>), substrate–chaperone dynamics must create a temporal limitation on proteostasis capacity.<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.015</object-id><label>Figure 7.</label><caption><title>Substrate–chaperone engagement must exceed time to cell division to induce curing.</title><p>(<bold>A</bold>) The number of [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> (SY2126) cells containing fluorescent foci was quantified in cultures recovering at 30°C over time following a 90 min incubation at 40°C (white). Colony forming units in these cultures were quantified by plating (black). Data represent means; error bars represent standard deviations; n = 3. (<bold>B</bold>) [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> cells (SY2126) treated for 30 min at 40°C and imaged over time in a microfluidics chamber are shown. Cells that were budded at the time of thermal stress are outlined in white, while unbudded cells are outlined in orange. Solid lines mark mothers, and dotted lines mark daughters. Scale bar = 1 µm. (<bold>C</bold>) A [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> <italic>HSP104GFP</italic> strain (SY2126) was imaged over time in a microfluidics at 30°C after a 30 min incubation at 40°C chamber. Budded or unbudded cells were scored at the first cell division for the presence or absence of fluorescent aggregates. Data represent means; error bars represent standard deviations; n = 3; p = 0.0005 by unpaired t-test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.015">http://dx.doi.org/10.7554/eLife.04288.015</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288f007"/></fig></p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>In <italic>Saccharomyces cerevisiae</italic>, expression of the molecular chaperone Hsp104, even at its low basal level, reduces organismal fitness at the normal growth temperature; however, survival at elevated temperatures is absolutely dependent on Hsp104, whose expression is induced to high levels by heat shock (<xref ref-type="bibr" rid="bib68">Sanchez et al., 1992</xref>; <xref ref-type="bibr" rid="bib22">Escusa-Toret et al., 2013</xref>). Thus, cell-based limitations must finely tune proteostasis capacity not only to control protein misfolding induced by stress but also to allow normal protein folding in the absence of these challenges (<xref ref-type="bibr" rid="bib55">Morimoto, 2008</xref>). Using the yeast prion [<italic>PSI</italic><sup><italic>+</italic></sup>] as a model to understand the in vivo interactions between amyloid and PQC pathways, we have uncovered one such pathway. While [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> is mitotically stable at the normal growth temperature (∼3% loss) (<xref ref-type="bibr" rid="bib14">Derkatch et al., 1996</xref>), a transient sub-lethal thermal stress induces quantitative curing (<xref ref-type="fig" rid="fig1">Figure 1</xref> and <xref ref-type="fig" rid="fig8">Figure 8</xref>) through the disassembly of existing Sup35 amyloid by Hsp104 (<xref ref-type="fig" rid="fig1 fig2 fig8">Figures 1, 2 and 8</xref>). Our studies indicate that the increase in Hsp104 expression at elevated temperature alone is not sufficient to induce Sup35 amyloid resolution and [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing (<xref ref-type="fig" rid="fig1 fig3">Figures 1, 3</xref>). Rather, Hsp104 must engage heat-induced protein aggregates for a period that exceeds the time to the next cell division (<xref ref-type="fig" rid="fig7 fig8">Figures 7, 8</xref>). As a result, Hsp104 is asymmetrically localized to the cells that experienced the thermal stress (<xref ref-type="fig" rid="fig5 fig8">Figures 5, 8</xref>), and this increase in chaperone accumulation, along with its activity, promotes curing in the same cells (<xref ref-type="fig" rid="fig5 fig8">Figures 5, 8</xref>). Thus, chaperone spatial engagement, substrate processing dynamics, and partitioning during cell division represent cell based limitations on proteostasis capacity.<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.016</object-id><label>Figure 8.</label><caption><title>Model for Sup35 amyloid resolubilization and curing upon thermal stress.</title><p>Upon thermal stress, cellular proteins (green) misfold and aggregate, leading to the induction and recruitment of Hsp104 (barrel). If thermal stress occurs in unbudded cells (1), these aggregates are resolved prior to cell division, allowing the partitioning of Hsp104 to both mother (black) and daughter (gray) cells (left). If thermal stress occurs in budded cells (2), heat-induced aggregates persist upon cell division (3), leading to the asymmetric retention of Hsp104 in mother cells. Both heat-induced aggregates (green) and Sup35 amyloid (blue corkscrews) are resolved in cells accumulating high levels of Hsp104, leading to curing (red, 4).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.016">http://dx.doi.org/10.7554/eLife.04288.016</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04288f008"/></fig></p><p>Metazoans lack an Hsp104 homolog (<xref ref-type="bibr" rid="bib86">Torrente and Shorter, 2013</xref>), but disaggregase activity has also recently been linked to a multi-component system in yeast comprised of Hsp110, Hsp70, and Hsp40, and this activity is conserved in the <italic>C. elegans</italic> and human homologs of these chaperones (<xref ref-type="bibr" rid="bib73">Shorter, 2011</xref>; <xref ref-type="bibr" rid="bib66">Rampelt et al., 2012</xref>; <xref ref-type="bibr" rid="bib53">Mattoo et al., 2013</xref>). This system is largely ineffective in the disaggregation of amyloid in vitro (<xref ref-type="bibr" rid="bib73">Shorter, 2011</xref>) but can promote the slow disassembly of amyloid from fiber ends in the presence of small heat shock proteins, such as Hsp26 and Hsp42 from yeast or HspB5 from humans (<xref ref-type="bibr" rid="bib17">Duennwald et al., 2012</xref>). Like Hsp104 in yeast, Hsp110 localizes to foci containing misfolded protein in human cells following thermal stress (<xref ref-type="bibr" rid="bib66">Rampelt et al., 2012</xref>) and interacts with protein amyloids in vivo (<xref ref-type="bibr" rid="bib31">Ishihara et al., 2003</xref>; <xref ref-type="bibr" rid="bib91">Wang et al., 2009</xref>; <xref ref-type="bibr" rid="bib59">Olzscha et al., 2011</xref>), raising the possibility that Hsp110 engagement with stress-induced substrates could also promote its activity toward amyloidogenic substrates in vivo.</p><p>The spatial engagement of PQC factors, including both chaperones and components of the ubiquitin–proteasome system, is a newly appreciated consequence of their function in vivo<italic>.</italic> Numerous cytoplasmic foci arise in response to stressors including heat, aging, oxidation, and/or proteasome inhibition. These foci include aggresomes, the insoluble protein deposit (IPOD), the juxtanuclear quality control compartment (JUNQ), StiF-inducible foci (StiF), and Q-bodies, the latter of which form under the mild thermal stress conditions employed in our studies (<xref ref-type="bibr" rid="bib34">Johnston et al., 1998</xref>; <xref ref-type="bibr" rid="bib21">Erjavec et al., 2007</xref>; <xref ref-type="bibr" rid="bib37">Kaganovich et al., 2008</xref>; <xref ref-type="bibr" rid="bib49">Liu et al., 2010</xref>; <xref ref-type="bibr" rid="bib77">Specht et al., 2011</xref>; <xref ref-type="bibr" rid="bib51">Malinovska et al., 2012</xref>; <xref ref-type="bibr" rid="bib93">Weisberg et al., 2012</xref>; <xref ref-type="bibr" rid="bib22">Escusa-Toret et al., 2013</xref>; <xref ref-type="bibr" rid="bib95">Wolfe et al., 2013</xref>). While the relationship of each of these foci to one another is currently unclear, they are all defined by the co-localization of misfolded and/or aggregation-prone proteins with PQC factors, some of which can be found in more than one of type of focus. The PQC factors that localize to these foci, such as Hsp104, clearly promote survival under stress (<xref ref-type="bibr" rid="bib68">Sanchez et al., 1992</xref>; <xref ref-type="bibr" rid="bib22">Escusa-Toret et al., 2013</xref>), but whether their localization into cytoplasmic foci specifically altered proteostasis capacity had not been previously established. Our studies indicate that the engagement of Hsp104 with heat-induced misfolded protein aggregates enhances proteostasis capacity by increasing the accumulation of this factor beyond the level attainable by changes in gene expression (<xref ref-type="fig" rid="fig5">Figure 5</xref>) and thereby permitting the disassembly of existing Sup35 amyloid (<xref ref-type="fig" rid="fig1 fig5">Figures 1,5</xref>).</p><p>While our studies indicate that chaperone partitioning imposes a limitation on proteostasis capacity, other aspects of this process may be more relevant to this upper boundary in post-mitotic cells, such as neurons. Indeed, our observations reveal other cell-based limitations beyond chaperone partitioning. For example, in contrast to the proteostasis enhancement we observe following thermal stress in yeast, previous studies have linked the accumulation of protein aggregates to reduced proteostasis capacity in vivo (<xref ref-type="bibr" rid="bib4">Broadley and Hartl, 2009</xref>). In these cases, protein aggregates, including those resulting from oxidative damage with age or proteotoxic stresses, have been linked to reduced replicative lifespan, (<xref ref-type="bibr" rid="bib1">Aguilaniu et al., 2003</xref>; <xref ref-type="bibr" rid="bib27">Hernebring et al., 2006</xref>; <xref ref-type="bibr" rid="bib67">Rujano et al., 2006</xref>; <xref ref-type="bibr" rid="bib21">Erjavec et al., 2007</xref>, <xref ref-type="bibr" rid="bib20">2008</xref>; <xref ref-type="bibr" rid="bib84">Tessarz et al., 2009</xref>; <xref ref-type="bibr" rid="bib42">Knorre et al., 2010</xref>; <xref ref-type="bibr" rid="bib49">Liu et al., 2010</xref>; <xref ref-type="bibr" rid="bib89">Unal et al., 2011</xref>; <xref ref-type="bibr" rid="bib101">Zhou et al., 2011</xref>; <xref ref-type="bibr" rid="bib78">Spokoini et al., 2012</xref>) and, the presence of protein amyloids, such as polyglutamine-expanded proteins and other yeast prions, promote the misfolding of metastable proteins, interfere with proteolysis, reduce protein synthesis, inhibit endocytosis, and disrupt prion propagation through the sequestration of chaperones (<xref ref-type="bibr" rid="bib24">Gidalevitz et al., 2006</xref>; <xref ref-type="bibr" rid="bib54">Meriin et al., 2003</xref>; <xref ref-type="bibr" rid="bib41">Kirstein-Miles and Morimoto, 2013</xref>; <xref ref-type="bibr" rid="bib60">Park et al., 2013</xref>; <xref ref-type="bibr" rid="bib98">Yang et al., 2013</xref>; <xref ref-type="bibr" rid="bib99">Yu et al., 2014</xref>). A comparison of these studies with our work suggests that the dynamics of chaperone engagement with distinct substrates, rather than simply their presence, correlates with the impact of these interactions on proteostasis capacity. In the studies resulting in chaperone sequestration, proteotoxicity correlates with imbalances in the system imposed by harsh conditions and/or the unnaturally high expression of amyloidogenic proteins. In contrast, we detect no differences in Hsp104-GFP localization in [<italic>PSI</italic><sup><italic>+</italic></sup>] and [<italic>psi</italic><sup>−</sup>] strains expressing Sup35 at its endogenous level (<xref ref-type="fig" rid="fig4">Figure 4A</xref>, 30°C), and Sup35 amyloid can clearly be resolved at these native stoichiometries when the system is elevated to a distinct but accessible proteostatic niche (<xref ref-type="fig" rid="fig1">Figure 1D</xref>). However, paralleling the studies in metazoans, [<italic>PSI</italic><sup><italic>+</italic></sup>] can transition from a benign to a toxic state upon Sup35 overexpression (<xref ref-type="bibr" rid="bib83">Ter-Avanesyan et al., 1993</xref>), a condition that also induces Hsp70 co-localization (<xref ref-type="bibr" rid="bib94">Winkler et al., 2012</xref>). Thus, proteostasis capacity appears to be finely tuned to maintain a natively expressed proteome.</p><p>Additional evidence of the importance of this balance can be gleaned by a comparison of chaperone overexpression in different cellular contexts. For example, overexpression of Hsp104 alone, to the same level achieved here through asymmetric retention of this factor (<xref ref-type="table" rid="tbl1">Table 1</xref> and <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1C</xref>), also induces curing (<xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref>; <xref ref-type="bibr" rid="bib92">Wegrzyn et al., 2001</xref>). While it has been suggested that Hsp104 overexpression dissolves Sup35 amyloid in vivo, this interpretation is complicated by a lack of temporal resolution and the ability to monitor existing protein (<xref ref-type="bibr" rid="bib61">Park et al., 2014</xref>) and is inconsistent with the increase in the size of SDS-resistant Sup35 aggregates under these conditions (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1D</xref>) (<xref ref-type="bibr" rid="bib45">Kryndushkin et al., 2003</xref>). An alternative model, which is consistent with this biochemical evidence of Hsp104 inhibition, suggests that upon its overexpression, Hsp104 aberrantly and non-productively co-localizes with Sup35 amyloid (<xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref>; <xref ref-type="bibr" rid="bib45">Kryndushkin et al., 2003</xref>; <xref ref-type="bibr" rid="bib3">Bagriantsev et al., 2008</xref>; <xref ref-type="bibr" rid="bib94">Winkler et al., 2012</xref>). In contrast, our studies indicate that Hsp104 overexpression within the context of a thermal stress transitions Sup35 amyloid from outside the buffering capacity of the proteostasis network to within its sphere of protection. Notably, Hsp104 co-localization with Sup35 amyloid varies based on its mode of overexpression (i.e. individual vs network up-regulation), again implicating substrate–chaperone dynamics, rather than simply chaperone availability, in proteostasis capacity. Intriguingly, this interplay is distinct for individual PQC factors within the same cell, as thermal stress induces localization of Hsp104, Ssa1, and Sis1 to cytoplasmic foci, but only Hsp104 is asymmetrically retained upon cell division (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1D, E</xref>), suggesting an additional point of proteostasis regulation.</p><p>Beyond these cell-based limitations on proteostasis capacity, our studies have deconvoluted the contributions of distinct physical characteristics of amyloid variants to their ability to exceed the PQC buffering capacity in vivo. Intriguingly, we find that [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup>, the more thermodynamically stable but less efficiently amplified variant of Sup35 amyloid (<xref ref-type="bibr" rid="bib80">Tanaka et al., 2006</xref>), is susceptible to curing at elevated temperature, while the less thermodynamically stable and more efficiently amplified [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> variant is not (<xref ref-type="fig" rid="fig1">Figure 1</xref>), indicating that amyloid amplification rather than stability imposes the primary limitation on amyloid clearance. Consistent with this idea, reducing [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> amplification by either expressing a Sup35 mutant or decreasing the expression of wild-type Sup35 promotes curing at elevated temperature (<xref ref-type="fig" rid="fig2">Figure 2</xref>). Thus, manipulations that have minor effects on the dynamics of existing amyloid are also sufficient to move this alternative protein-folding pathway within the buffering capacity of the proteostasis network.</p><p>Together, our observations suggest an alternative to the view that the physical characteristics of amyloid complexes alone preclude their accessibility to the cell's natural defenses against protein misfolding. Rather, the dynamics and balance of the system as a whole, including both protein-based and cell-based contributors, create not only a niche that allows amyloid to arise and persist but also another that promotes amyloid clearance. Our studies, therefore, raise the possibility that the proteostasis limitations that allow the accumulation of chronically misfolded proteins may be distinct in a native context and under conditions of their overexpression. Within this framework, our studies provide a proof-of-principle example to support the idea that proteostasis regulators, which are aimed at transitioning proteostasis landscapes to new thresholds, may be the most effective interventions into amyloidosis (<xref ref-type="bibr" rid="bib48">Lindquist and Kelly, 2011</xref>).</p></sec><sec sec-type="materials|methods" id="s4"><title>Materials and methods</title><sec id="s4-1"><title>Plasmid and strain construction</title><p>All plasmids used in this study were previously reported (<xref ref-type="table" rid="tbl2">Table 2</xref>) except for SB1013 (pRS306P<sub>GPD</sub>-FFL-mCherry), which contains firefly luciferase as an <italic>Xba1/BamHI</italic> fragment and mCherry as a <italic>BamHI/XhoI</italic> fragment, separated by a three-repeat glycine–serine linker. The ORFs were amplified by PCR using primers 5XbaI firefly/3BamHI firefly and 5BamHIGS3mCherry/3XhoImCherry, respectively (<xref ref-type="table" rid="tbl3">Table 3</xref>) and confirmed by sequencing. All strains of <italic>Saccharomyces cerevisiae</italic> used in this study are derivatives of 74-D694 (<xref ref-type="table" rid="tbl4">Table 4</xref>) (<xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref>). A WT [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> diploid strain (SY945) was generated by mating SY2600 with SLL3252 (<xref ref-type="table" rid="tbl4">Table 4</xref>). The diploid state was confirmed by sporulation. SY591, a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> strain containing a heterozygous deletion of <italic>HSP104</italic>, was created by transformation of a <italic>Pvu</italic>I-<italic>Bam</italic>HI fragment of pYABL5 (a kind gift of S. Lindquist) into SY945 and selection on medium lacking leucine. Disruptions were verified by PCR and 2:2 marker segregation upon sporulation and dissection. SY957, a [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> diploid strain containing a heterozygous disruption of <italic>SUP35</italic>, was created by transforming a PCR-generated cassette using pFA6a-KanMX4 as a template with primers SD27 and SD28 (<xref ref-type="table" rid="tbl3">Table 3</xref>) into SLL3071 (<xref ref-type="table" rid="tbl4">Table 4</xref>). Integration was confirmed by PCR using primers Psup352/PTEFCH and Sup35 3′chk/pFa6 test (<xref ref-type="table" rid="tbl3">Table 3</xref>). The galactose-inducible <italic>HSP104</italic> strains were made by integrating <italic>BstBI</italic>-linearized SB630 (<xref ref-type="table" rid="tbl2">Table 2</xref>) into SY197 (<xref ref-type="table" rid="tbl4">Table 4</xref>) and selecting transformants on medium lacking uracil. Galactose-inducible expression of Hsp104 was confirmed by western blotting. [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> and [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> were then cytoduced into this strain from SY1698 and SY1699, respectively, to create SY1748 and SY1749 (<xref ref-type="table" rid="tbl4">Table 4</xref>), respectively (<xref ref-type="bibr" rid="bib9">Conde and Fink, 1976</xref>). Cytoductants were selected by growth on synthetic medium containing glycerol and lacking uracil and by colony color on YPD. The <italic>HSP104-GFP</italic> [<italic>psi</italic><sup>−</sup>] strain (SY2125) was created by transforming a PCR-generated cassette using pFA6a-GFP(S65T)-KanMX6 as a template with primers HSP104-GFP F-A and HSP104-GFP R-A (<xref ref-type="table" rid="tbl3">Table 3</xref>) into WT [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> strains and selection on medium containing 300 μg/ml G418. Integration was confirmed by PCR using primers Hsp104for/GFP-R and pFa6 test/Hsp104 3 flank R (<xref ref-type="table" rid="tbl3">Table 3</xref>), and expression was confirmed by fluorescence microscopy. These strains were cured of the prion by growth on YPD plates containing 3 mM GdnHCl (<xref ref-type="bibr" rid="bib87">Tuite et al., 1981</xref>). The [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> variant (SY2126) was generated by mating SY2125 to a WT [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain (SLL2600) and sporulation. Tetrads were dissected to recover haploids, and <italic>HSP104-GFP</italic> isolates were verified by G418 resistance, fluorescence microscopy, and quantitative immunoblotting for Hsp104. The heat inducible GFP strain (SY2091) was generated by transformation of a WT [<italic>psi</italic><sup>−</sup>] strain (SLL2119) with <italic>Bsu</italic>36I-digested SB849 (<xref ref-type="table" rid="tbl2">Table 2</xref>). Expression was confirmed by fluorescence microscopy. <italic>SSA1-GFP</italic> (SY2658) and <italic>SIS1-GFP</italic> (SY2447) [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strains were created by transforming PCR-generated cassettes using pFA6a-GFP(S65T)-KanMX6 as a template with primers GFP-GS-Ssa1-F/GFP-Ssa1-R or Sis1-GFP-F GS/Sis1-GFP-R (<xref ref-type="table" rid="tbl3">Table 3</xref>), respectively, into WT [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> strains and selection on medium containing 300 μg/ml G418. Expression was confirmed by fluorescence microscopy and quantitative immunoblotting for Ssa1/2 and Sis1, respectively. These strains were cured of the prion by growth on YPD plates containing 3 mM GdnHCl, mitochondrial loss was induced by growth in 25 μg/ml ethidium bromide, and [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> was transferred to them by cytoduction (<xref ref-type="bibr" rid="bib10">Cox, 1965</xref>), using SY1699 as a donor strain. Cytoductants were selected by growth on glycerol medium and 300 μg/ml G418. <italic>SSA1-GFP</italic> and <italic>SIS1-GFP</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strains containing a nuclear-localized fluorescent reporter protein (DsRed-NLS, SY2659, and SY485, respectively) were generated by transforming SY2658 or SY2447 with <italic>Bsu</italic>36I-digested SB503 (<xref ref-type="table" rid="tbl2">Table 2</xref>). Expression was confirmed by fluorescence microscopy. The Δ<italic>bni1</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain (SY1888) was created by transforming a PCR-generated cassette using pFA6a-KanMX4 as a template with primers AD-BNI1-f and AD-BNI1-r (<xref ref-type="table" rid="tbl3">Table 3</xref>) into a [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> diploid (SY782, a cross between SY2600 and SY86, <xref ref-type="table" rid="tbl4">Table 4</xref>). Transformants were selected on medium containing 300 μg/ml G418 and verified by PCR using primers AD-BNI1-fseq/PTEFCH and AD-BNI1-rseq/pFa6 test (<xref ref-type="table" rid="tbl3">Table 3</xref>). The haploid Δ<italic>bni1</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain was then generated by sporulation and tetrad dissection and verified by G418 resistance. The <italic>HSP104-GFP</italic> Δ<italic>bni1</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> strain (SY2486) was created by transforming SY2126 with a PCR-generated cassette using pFA6a-hphMX4 as a template with primers AD-BNI1-f and AD-BNI1-r (<xref ref-type="table" rid="tbl3">Table 3</xref>). Transformants were confirmed by PCR using primers AD-BNI1-fseq/PTEFCH and AD-BNI1-rseq/pFa6 test (<xref ref-type="table" rid="tbl4">Table 4</xref>) and growth on YPD plates containing 300 μg/ml hygromycin B.<table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.017</object-id><label>Table 2.</label><caption><p>Plasmids</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.017">http://dx.doi.org/10.7554/eLife.04288.017</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Name</th><th>Description</th></tr></thead><tbody><tr><td>SB20</td><td>pRS306-P<sub>Sup35</sub>N(GS)<sub>3</sub>sGFP(GS)<sub>3</sub>MC</td></tr><tr><td>SB503</td><td>pRS304-P<sub><italic>GPD</italic></sub>GST-DsRED-NLS</td></tr><tr><td>SB630</td><td>pRS306-P<sub><italic>GAL</italic></sub>Hsp104</td></tr><tr><td>SB657</td><td>pRS306-P<sub><italic>tetO2</italic></sub>Sup35</td></tr><tr><td>SB658</td><td>pRS306-P<sub><italic>tetO2</italic></sub>Sup35(G58D)</td></tr><tr><td>SB849</td><td>pRS306-P<sub><italic>HSE</italic></sub>GFP</td></tr><tr><td>SB1013</td><td>pRS306-P<sub><italic>GPD</italic></sub>FFL-mCherry</td></tr></tbody></table></table-wrap><table-wrap id="tbl3" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.018</object-id><label>Table 3.</label><caption><p>Primers</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.018">http://dx.doi.org/10.7554/eLife.04288.018</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Name</th><th>Sequence</th></tr></thead><tbody><tr><td>5XbaI firefly</td><td>5′-TCTAGAATGGAAGATGCCAAAAACATTAAG-3′</td></tr><tr><td>3BamHI firefly</td><td>5′-GGATCCACCTTGAGACTGTGGTTGGAAAC-3′</td></tr><tr><td>5BamHI GS3mCherry</td><td>5′-GGATCCGGTAGTGGTAGTGGTAGTATGGTGAGCAAGGG CGAGGAG-3′</td></tr><tr><td>3XhoI mCherry</td><td>5′-CTCGAGTTACTTGTACAGCTCGTCCATGCCG-3′</td></tr><tr><td>SD27</td><td>5′-ACTTGCTCGGAATAACATCTATATCTGCCCACTAGCAACA CAGCTGAAGCTTCGTACGC-3′</td></tr><tr><td>SD28</td><td>5′-GGTATTATTGTGTTTGCATTTACTTATGTTTGCAAGAAATG CATAGGCCACTAGTGGATCTG-3′</td></tr><tr><td>Psup352</td><td>5′-GAGATGCTCATCAAGGG-3′</td></tr><tr><td>PTEFCH</td><td>5′-GCACGTCAAGACTGTCAAGG-3′</td></tr><tr><td>Sup35 3′chk</td><td>5′-TATTTACGAAGGAGACCCGGAG-3′</td></tr><tr><td>pFa6 test</td><td>5′-TGCCCAGATGCGAAGTTAAGTG-3′</td></tr><tr><td>HSP104-GFP F-A</td><td>5′-CGATAATGAGGACAGTATGGAAATTGATGATGACCTA GATCGGATCCCCGGGTTAATTAA-3′</td></tr><tr><td>Hsp104-GFP R-A</td><td>5′-TATTATATTACTGATTCTTGTTCGAAAGTTTTTAAAAATC GAATTCGAGCTCGTTTAAAC-3′</td></tr><tr><td>Hsp104for</td><td>5′-GGCACATCCTGATGTTTTGA-3′</td></tr><tr><td>GFP-R</td><td>5′-CCTTCACCCTCTCCACTGACAG-3′</td></tr><tr><td>Hsp104 3 flank R</td><td>5′-CCGTATTCTAATAATGGACCAATC-3′</td></tr><tr><td>GFP-GS-Ssa1-F</td><td>5′-AGCTCCAGAGGCTGAAGGTCCAACCGTTGAAGAAGTTG ATGGTTCTGGTTCTGGTTCTCGGATCCCCGGGTTAATTAA-3′</td></tr><tr><td>GFP-Ssa1-R</td><td>5′-ACCCAGATCATTAAAAGACATTTTCGTTATTATCAATTGC GAATTCGAGCTCGTTTAAAC-3′</td></tr><tr><td>Sis1-GFP-F GS</td><td>5′-ACTAAACGACGCTCAAAAACGTGCTATAGATGAAAATTT TGGTTCTGGTTCTGGTTCTCGGATCCCCGGGTTAATTAA-3′</td></tr><tr><td>Sis1-GFP-R</td><td>5′-ATTTATTTGAGTTTATAATTATATTTGCTTAGGATTACTAG AATTCGAGCTCGTTTAAAC-3′</td></tr><tr><td>AD-BNI1-f</td><td>5′-ATGTTGAAGAATTCAGGCTCCAAACATTCGAACTCAAAG GCAGCTGAAGCTTCGTACGC-3′</td></tr><tr><td>AD-BNI1-r</td><td>5′-TTATTTGAAACTTAGCCTGTTACCTGTCCTAGCCTCACCT GCATAGGCCACTAGTGGATCTG-3′</td></tr><tr><td>AD-BNI1-fseq</td><td>5′-GACATCGGTTAGAGGAAG-3′</td></tr><tr><td>AD-BNI1-rseq</td><td>5′-CACTGTGCTTGTCACTTA-3′</td></tr></tbody></table></table-wrap><table-wrap id="tbl4" position="float"><object-id pub-id-type="doi">10.7554/eLife.04288.019</object-id><label>Table 4.</label><caption><p>Yeast strains</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04288.019">http://dx.doi.org/10.7554/eLife.04288.019</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center">Strain</th><th align="center">Genotype</th><th align="center">Plasmids integrated</th><th align="center">Reference</th><th align="center">Figure</th></tr></thead><tbody><tr><td align="center">SLL2119</td><td align="center"><italic>MATa</italic> [<italic>psi</italic><sup>−</sup>] <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3, 112</italic></td><td align="center">-</td><td align="center"><xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref></td><td align="center">1c, 3Sf</td></tr><tr><td align="center">SLL2600</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3, 112</italic></td><td align="center">-</td><td align="center"><xref ref-type="bibr" rid="bib14">Derkatch et al., 1996</xref></td><td align="center">1, 2a, 3, 5ce, 6abcfg, 1S, 3Sabg, 4Sabcd, 5Sfg, 6Sa</td></tr><tr><td align="center">SLL2606</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3, 112</italic></td><td align="center">-</td><td align="center"><xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref></td><td align="center">1ac, 3Sf</td></tr><tr><td align="center">SLL3071</td><td align="center"><italic>MATa/α</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> <italic>ade1-14/ade1-14 his3Δ200/his3Δ200 trp1-289/ trp1-289 ura3-52/ura3-52 leu2-3112/ leu2-3112</italic></td><td align="center">-</td><td align="center"><xref ref-type="bibr" rid="bib15">DiSalvo et al., 2011</xref></td><td align="center">2d</td></tr><tr><td align="center">SLL3252</td><td align="center"><italic>MATa</italic> [<italic>psi</italic><sup>−</sup>] <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3, 112</italic></td><td align="center">-</td><td align="center"><xref ref-type="bibr" rid="bib7">Chernoff et al., 1995</xref></td><td align="center">‘Materials and methods’</td></tr><tr><td align="center">SY86</td><td align="center"><italic>MATα</italic> [<italic>psi</italic><sup>−</sup>] <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3, 112 sup35::N(GS)</italic><sub><italic>3</italic></sub><italic>GFP(GS)</italic><sub><italic>3</italic></sub><italic>MC</italic></td><td align="center">SB20</td><td align="center"><xref ref-type="bibr" rid="bib13">Derdowski et al., 2010</xref></td><td align="center">‘Materials and methods’</td></tr><tr><td align="center">SY197</td><td align="center"><italic>MATa</italic> [<italic>psi</italic><sup>−</sup>] <italic>ade1-14 his3-11,-15 trp1-1 ura3-1 leu2-3112 can1-100</italic></td><td align="center">-</td><td align="center">J Weissman (YJW513)</td><td align="center">‘Materials and methods’</td></tr><tr><td align="center">SY591</td><td align="center"><italic>MATa/α</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14/ade1-14 his3Δ200/his3Δ200 TRP/ trp1-289 ura3-52/ura3-52 leu2-3112/ leu2-3112 HSP104/hsp104::LEU2</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">2b</td></tr><tr><td align="center">SY782</td><td align="center"><italic>MATa/α</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14/ade1-14 his3Δ200/his3Δ200 trp1-289/ trp1-289 ura3-52/ura3-52 leu2-3112/ leu2-3112 SUP35/sup35::N(GS)</italic><sub><italic>3</italic></sub><italic>GFP(GS)</italic><sub><italic>3</italic></sub><italic>MC</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">‘Materials and methods’</td></tr><tr><td align="center">SY945</td><td align="center"><italic>MATa/α</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14/ade1-14 his3Δ200/his3Δ200 trp1-289/ trp1-289 ura3-52/ura3-52 leu2-3112/ leu2-3112</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">2b</td></tr><tr><td align="center">SY957</td><td align="center"><italic>MATa/α</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> <italic>ade1-14/ade1-14 his3Δ200/his3Δ200 trp1-289/ trp1-289 ura3-52/ura3-52 leu2-3112/ leu2-3112 SUP35/sup35::KANMX4</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">2d</td></tr><tr><td align="center">SY1646</td><td align="center"><italic>MATa/α</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> <italic>ade1-14/ade1-14 his3Δ200/his3Δ200 trp1-289/ trp1-289 ura3-52/ura3-52::URA3::P</italic><sub><italic>tetO2</italic></sub><italic>SUP35 leu2-3112/ leu2-3112 SUP35/sup35::KANMX4</italic></td><td align="center">SB657</td><td align="center"><xref ref-type="bibr" rid="bib15">DiSalvo et al., 2011</xref></td><td align="center">2c</td></tr><tr><td align="center">SY1648</td><td align="center"><italic>MATa/α</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> <italic>ade1-14/ade1-14 his3Δ200/his3Δ200 trp1-289/ trp1-289 ura3-52/ura3-52::URA3::P</italic><sub><italic>tetO2</italic></sub><italic>SUP35(G58D) leu2-3112/ leu2-3112 SUP35/sup35::KANMX4</italic></td><td align="center">SB658</td><td align="center"><xref ref-type="bibr" rid="bib15">DiSalvo et al., 2011</xref></td><td align="center">2c</td></tr><tr><td align="center">SY1698</td><td align="center"><italic>MATα</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> <italic>ade1-14 his3Δ200 ura3-52 leu2-3 kar1-d15 ConR CyhR</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">‘Materials and methods’</td></tr><tr><td align="center">SY1699</td><td align="center"><italic>MATα</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 ura3-52 leu2-3 kar1-d15 ConR CyhR</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">‘Materials and methods’</td></tr><tr><td align="center">SY1748</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Strong</sup> <italic>ade1-14 his3-11,-15 trp1-1 ura3-1::URA3::P</italic><sub><italic>GAL</italic></sub><italic>HSP104 leu2-3112 can1-100</italic></td><td align="center">SB630</td><td align="center">This study</td><td align="center">3Se</td></tr><tr><td align="center">SY1749</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3-11,-15 trp1-1 ura3-1::URA3::P</italic><sub><italic>GAL</italic></sub><italic>HSP104 leu2-3112 can1-100</italic></td><td align="center">SB630</td><td align="center">This study</td><td align="center">3Scde</td></tr><tr><td align="center">SY1888</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3, 112 Δbni1::KANMX4</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">5c, 5Sfg</td></tr><tr><td align="center">SY2091</td><td align="center"><italic>MATa</italic> [<italic>psi</italic><sup>−</sup>] <italic>ade1-14 his3Δ200 trp1-289 ura3-52::URA::P</italic><sub><italic>HSE</italic></sub><italic>GFP leu2-3, 112</italic></td><td align="center">SB849</td><td align="center">This study</td><td align="center">5Sa</td></tr><tr><td align="center">SY2125</td><td align="center"><italic>MATα</italic> [<italic>psi</italic><sup>−</sup>] <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3112 HSP104GFP::KANMX6</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">4b</td></tr><tr><td align="center">SY2126</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3112 HSP104GFP::KANMX6</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">4, 5abdfg, 6de, 7, 4Sabcdfg, 6Sb</td></tr><tr><td align="center">SY2447</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3112 SIS1GFP::KANMX6</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">5Se</td></tr><tr><td align="center">SY2485</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 trp1-289::TRP::P</italic><sub><italic>GPD</italic></sub><italic>GST-DsRed-NLS ura3-52 leu2-3112 SIS1GFP::KANMX6</italic></td><td align="center">SB503</td><td align="center">This study</td><td align="center">5Sc</td></tr><tr><td align="center">SY2486</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3, 112 HSP104GFP::KANMX6 Δbni1::hphMX4</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">5b</td></tr><tr><td align="center">SY2658</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 trp1-289 ura3-52 leu2-3112 SSA1GFP::KANMX6</italic></td><td align="center">-</td><td align="center">This study</td><td align="center">5Sd</td></tr><tr><td align="center">SY2659</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 trp1-289::TRP::P</italic><sub><italic>GPD</italic></sub><italic>GST-DsRed-NLS ura3-52 leu2-3112 SSA1GFP::KANMX6</italic></td><td align="center">SB503</td><td align="center">This study</td><td align="center">5Sb</td></tr><tr><td align="center">SY2802</td><td align="center"><italic>MATa</italic> [<italic>PSI</italic><sup>+</sup>]<sup>Weak</sup> <italic>ade1-14 his3Δ200 trp1-289 ura3-52::URA::P</italic><sub><italic>GPD</italic></sub><italic>Firefly-mCherry leu2-3112 HSP104GFP::KANMX6</italic></td><td align="center">SB1013</td><td align="center">This study</td><td align="center">4Se</td></tr></tbody></table></table-wrap></p></sec><sec id="s4-2"><title>Growth conditions and phenotypic analysis</title><p>Unless otherwise specified, yeast cultures were grown in rich YPD medium supplemented with 0.3 mM adenine. Cultures were maintained at an OD<sub>600</sub> of less that 0.5 at 30°C for at least 10 doublings to ensure exponential growth. Where indicated, cultures were then transferred to 37°C or 40°C for the specified period. Pretreatment of cultures at 37°C prior to shift to 40°C was for 30 min. To analyze colony color phenotype, aliquots of cultures were diluted in H<sub>2</sub>O as needed to ensure well-separated single colonies upon plating to solid YPD medium. After growth at 30°C, each colony was scored based on colony color phenotype: fully cured (completely red, [<italic>psi</italic><sup>−</sup>]), sectored (part red and part white), or [<italic>PSI</italic><sup>+</sup>] (completely white). Unless otherwise indicated, fully cured and sectored colonies were combined in the ‘cured’ category. For all colony counting assays, at least 150 colonies were counted for each experimental condition/timepoint. For the galactose-inducible Hsp104 experiments, cells were grown in rich YP medium containing 3% raffinose supplemented with 3% galactose during induction. α-factor and nocodazole arrests were performed in YPD liquid medium containing final concentrations of 5 μg/ml α-factor or 15 μg/ml nocodazole, respectively, for ∼2 hr. Following confirmation of arrest based on cell morphology by bright-field microscopy, cultures were washed three times with medium containing 1 mM DMSO followed by one wash in YPD before resuspension for indicated manipulation. GdnHCl treatment was performed at 3 mM final concentration in liquid YPD, and for experiments involving recovery, cultures were washed three times with medium before resuspension in YPD for indicated manipulation.</p></sec><sec id="s4-3"><title>Protein analysis</title><p>SDS-PAGE and quantitative immunoblotting were performed as previously described (<xref ref-type="bibr" rid="bib64">Pezza et al., 2009</xref>). Anti-Ssa1/2 rabbit serum was provided by E. Craig (U Wisconsin—Madison), and anti-Sis1 rabbit serum was provided by M. Tuite (U Kent, Canterbury, UK). Semi-native agarose gel electrophoresis (SDD-AGE) was performed as previously described (<xref ref-type="bibr" rid="bib45">Kryndushkin et al., 2003</xref>). The cycloheximide SDS-sensitivity assay was performed as previously described (<xref ref-type="bibr" rid="bib15">DiSalvo et al., 2011</xref>) with the following modifications: 1) cultures were treated at the various experimental temperatures for 30 min prior to the addition of cycloheximide to allow for the induction of chaperone proteins, and 2) after cycloheximide treatment, cultures were incubated with shaking at 30°C for 2 hr before lysis and analysis. For the aggregation analysis, native lysates were prepared as described previously (<xref ref-type="bibr" rid="bib45">Kryndushkin et al., 2003</xref>). Lysates were pre-cleared for 1 min at 500×<italic>g</italic> and total protein content was quantified using the BioRad Bradford assay in triplicate. Lysates were subjected to 15,000×g centrifugation for 15 min, and pellets were washed with 10 mM sodium phosphate buffer (pH7.5) containing 2% NP-40 before being resuspended in 10 mM sodium phosphate buffer (pH7.5) and quantified again in triplicate using the Bradford assay. For the Hsp104 immunocapture, native lysates were prepared at 4°C in IP buffer (50 mM HEPES–NaOH (pH 7.5), 150 mM NaCl, 10 mM MgCl<sub>2</sub>, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, and protease inhibitors (2 mM PMSF, 5 µg/ml pepstatin, complete protease inhibitor tablets (Pierce, Rockford, IL), protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO)). Lysates were pre-cleared for 1 min at 500×<italic>g</italic>, and then incubated for 1 hr with Protein G magnetic beads (NEB, Ipswich, MA) with nutation. Immunocapture was performed using Protein G magnetic beads and anti-GFP mouse monoclonal antibody (Roche, Switzerland). Beads were washed 4× with IP buffer and 1× with 50 mM HEPES–NaOH (pH 7.5), and protein was eluted by boiling in SDS sample buffer. Co-captured proteins were resolved by SDS-PAGE and analyzed by gel staining with Flamingo (Bio-Rad, Hercules, CA) and fluorescent scanning on a Typhoon imager (GE Lifesciences, Marlborough, MA) according to the manufacturer's instructions or by western blot for GFP.</p></sec><sec id="s4-4"><title>Imaging and microfluidics</title><p>Static images were obtained on a Zeiss Axio Imager M2 fluorescence light microscope equipped with a 100× objective. Confocal images were obtained on a Zeiss LSM 510 Meta confocal microscope using a 100× objective. Microfluidics experiments were performed on a Zeiss Axio Observer Z1 using a CellAsics microfluidics plate with temperature controls and media flow of 2 psi on a Y0C4 yeast perfusion plate (channel size 3.5–5 μm). Imaging was performed in complete minimal medium supplemented with 2% glucose and 2.5 mM adenine. Fluorescence intensity was analyzed using the Zen software package (Zeiss, Germany).</p></sec><sec id="s4-5"><title>Flow Cytometry</title><p>Flow cytometry and cell sorting was performed on a BD FACSAria fluorescence-activated cell sorter using a 488 nm laser and a FITC-A filter to measure GFP fluorescence intensity in single cells. Data were obtained at least in triplicate with representative spectra shown. Data were analyzed using the FlowJo software package (TreeStar Inc., Ashland, OR).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Jeff Laney, members of the Serio and Laney laboratories, and Ulrich Hartl for helpful discussions and comments on the manuscript, and E Craig and M Tuite for antisera. This work was funded by awards from the NIH to TRS (R01 GM069802001), CLK (F31 AG034754), and CRL (F31 GM099383).</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>CLK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>MLH, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>CRL, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con4"><p>TRS, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aguilaniu</surname><given-names>H</given-names></name><name><surname>Gustafsson</surname><given-names>L</given-names></name><name><surname>Rigoulet</surname><given-names>M</given-names></name><name><surname>Nyström</surname><given-names>T</given-names></name></person-group><year>2003</year><article-title>Asymmetric inheritance of oxidatively damaged proteins during cytokinesis</article-title><source>Science</source><volume>299</volume><fpage>1751</fpage><lpage>1753</lpage><pub-id pub-id-type="doi">10.1126/science.1080418</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Amon</surname><given-names>A</given-names></name></person-group><year>2002</year><article-title>Synchronization procedures</article-title><source>Methods in Enzymology</source><volume>351</volume><fpage>457</fpage><lpage>467</lpage><pub-id pub-id-type="doi">10.1016/S0076-6879(02)51864-4</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bagriantsev</surname><given-names>SN</given-names></name><name><surname>Gracheva</surname><given-names>EO</given-names></name><name><surname>Richmond</surname><given-names>JE</given-names></name><name><surname>Liebman</surname><given-names>SW</given-names></name></person-group><year>2008</year><article-title>Variant-specific [<italic>PSI</italic><sup><italic>+</italic></sup>] infection is transmitted by Sup35 polymers within [<italic>PSI</italic><sup><italic>+</italic></sup>] aggregates with heterogeneous protein composition</article-title><source>Molecular Biology of the Cell</source><volume>19</volume><fpage>2433</fpage><lpage>2443</lpage><pub-id pub-id-type="doi">10.1091/mbc.E08-01-0078</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Broadley</surname><given-names>SA</given-names></name><name><surname>Hartl</surname><given-names>FU</given-names></name></person-group><year>2009</year><article-title>The role of molecular chaperones in human misfolding diseases</article-title><source>FEBS Letters</source><volume>583</volume><fpage>2647</fpage><lpage>2653</lpage><pub-id pub-id-type="doi">10.1016/j.febslet.2009.04.029</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carmichael</surname><given-names>J</given-names></name><name><surname>Chatellier</surname><given-names>J</given-names></name><name><surname>Woolfson</surname><given-names>A</given-names></name><name><surname>Milstein</surname><given-names>C</given-names></name><name><surname>Fersht</surname><given-names>AR</given-names></name><name><surname>Rubinsztein</surname><given-names>DC</given-names></name></person-group><year>2000</year><article-title>Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington's disease</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>97</volume><fpage>9701</fpage><lpage>9705</lpage><pub-id pub-id-type="doi">10.1073/pnas.170280697</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chai</surname><given-names>Y</given-names></name><name><surname>Koppenhafer</surname><given-names>SL</given-names></name><name><surname>Bonini</surname><given-names>NM</given-names></name><name><surname>Paulson</surname><given-names>HL</given-names></name></person-group><year>1999</year><article-title>Analysis of the role of heat shock protein (Hsp) molecular chaperones in polyglutamine disease</article-title><source>The Journal of Neuroscience</source><volume>19</volume><fpage>10338</fpage><lpage>10347</lpage></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chernoff</surname><given-names>YO</given-names></name><name><surname>Lindquist</surname><given-names>SL</given-names></name><name><surname>Ono</surname><given-names>B</given-names></name><name><surname>Inge-Vechtomov</surname><given-names>SG</given-names></name><name><surname>Liebman</surname><given-names>SW</given-names></name></person-group><year>1995</year><article-title>Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [<italic>PSI</italic><sup><italic>+</italic></sup>]</article-title><source>Science</source><volume>268</volume><fpage>880</fpage><lpage>884</lpage><pub-id pub-id-type="doi">10.1126/science.7754373</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chiti</surname><given-names>F</given-names></name><name><surname>Dobson</surname><given-names>CM</given-names></name></person-group><year>2006</year><article-title>Protein misfolding, functional amyloid, and human disease</article-title><source>Annual Review of Biochemistry</source><volume>75</volume><fpage>333</fpage><lpage>366</lpage><pub-id pub-id-type="doi">10.1146/annurev.biochem.75.101304.123901</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Conde</surname><given-names>J</given-names></name><name><surname>Fink</surname><given-names>GR</given-names></name></person-group><year>1976</year><article-title>A mutant of <italic>Saccharomyces cerevisiae</italic> defective for nuclear fusion</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>73</volume><fpage>3651</fpage><lpage>3655</lpage><pub-id pub-id-type="doi">10.1073/pnas.73.10.3651</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cox</surname><given-names>B</given-names></name></person-group><year>1965</year><article-title>[<italic>PSI</italic>], a cytoplasmic suppressor of super-suppression in yeast</article-title><source>Heredity</source><volume>20</volume><fpage>505</fpage><lpage>521</lpage><pub-id pub-id-type="doi">10.1038/hdy.1965.65</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cox</surname><given-names>BS</given-names></name><name><surname>Tuite</surname><given-names>MF</given-names></name><name><surname>Mclaughlin</surname><given-names>CS</given-names></name></person-group><year>1988</year><article-title>The psi factor of yeast: a problem in inheritance</article-title><source>Yeast</source><volume>4</volume><fpage>159</fpage><lpage>178</lpage><pub-id pub-id-type="doi">10.1002/yea.320040302</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cushman-Nick</surname><given-names>M</given-names></name><name><surname>Bonini</surname><given-names>NM</given-names></name><name><surname>Shorter</surname><given-names>J</given-names></name></person-group><year>2013</year><article-title>Hsp104 suppresses polyglutamine-induced degeneration post onset in a <italic>drosophila</italic> Mjd/Sca3 model</article-title><source>PLOS Genetics</source><volume>9</volume><fpage>e1003781</fpage><pub-id pub-id-type="doi">10.1371/journal.pgen.1003781</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Derdowski</surname><given-names>A</given-names></name><name><surname>Sindi</surname><given-names>SS</given-names></name><name><surname>Klaips</surname><given-names>CL</given-names></name><name><surname>DiSalvo</surname><given-names>S</given-names></name><name><surname>Serio</surname><given-names>TR</given-names></name></person-group><year>2010</year><article-title>A size threshold limits prion transmission and establishes phenotypic diversity</article-title><source>Science</source><volume>330</volume><fpage>680</fpage><lpage>683</lpage><pub-id pub-id-type="doi">10.1126/science.1197785</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Derkatch</surname><given-names>IL</given-names></name><name><surname>Chernoff</surname><given-names>YO</given-names></name><name><surname>Kushnirov</surname><given-names>VV</given-names></name><name><surname>Inge-Vechtomov</surname><given-names>SG</given-names></name><name><surname>Liebman</surname><given-names>SW</given-names></name></person-group><year>1996</year><article-title>Genesis and variability of [<italic>PSI</italic>] prion factors in <italic>Saccharomyces cerevisiae</italic></article-title><source>Genetics</source><volume>144</volume><fpage>1375</fpage><lpage>1386</lpage></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>DiSalvo</surname><given-names>S</given-names></name><name><surname>Derdowski</surname><given-names>A</given-names></name><name><surname>Pezza</surname><given-names>JA</given-names></name><name><surname>Serio</surname><given-names>TR</given-names></name></person-group><year>2011</year><article-title>Dominant prion mutants induce curing through pathways that promote chaperone-mediated disaggregation</article-title><source>Nature Structural & Molecular Biology</source><volume>18</volume><fpage>486</fpage><lpage>492</lpage><pub-id pub-id-type="doi">10.1038/nsmb.2031</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Douglas</surname><given-names>PM</given-names></name><name><surname>Summers</surname><given-names>DW</given-names></name><name><surname>Ren</surname><given-names>HY</given-names></name><name><surname>Cyr</surname><given-names>DM</given-names></name></person-group><year>2009</year><article-title>Reciprocal efficiency of Rnq1 and polyglutamine detoxification in the cytosol and Nucleus</article-title><source>Molecular Biology of the Cell</source><volume>20</volume><fpage>4162</fpage><lpage>4173</lpage><pub-id pub-id-type="doi">10.1091/mbc.E09-02-0170</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Duennwald</surname><given-names>ML</given-names></name><name><surname>Echeverria</surname><given-names>A</given-names></name><name><surname>Shorter</surname><given-names>J</given-names></name></person-group><year>2012</year><article-title>Small heat shock proteins potentiate amyloid dissolution by protein disaggregases from yeast and humans</article-title><source>PLOS Biology</source><volume>10</volume><fpage>e1001346</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.1001346</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eaglestone</surname><given-names>SS</given-names></name><name><surname>Cox</surname><given-names>BS</given-names></name><name><surname>Tuite</surname><given-names>MF</given-names></name></person-group><year>1999</year><article-title>Translation termination efficiency can be regulated in <italic>Saccharomyces cerevisiae</italic> by environmental stress through a prion-mediated mechanism</article-title><source>The EMBO Journal</source><volume>18</volume><fpage>1974</fpage><lpage>1981</lpage><pub-id pub-id-type="doi">10.1093/emboj/18.7.1974</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eaglestone</surname><given-names>SS</given-names></name><name><surname>Ruddock</surname><given-names>LW</given-names></name><name><surname>Cox</surname><given-names>BS</given-names></name><name><surname>Tuite</surname><given-names>MF</given-names></name></person-group><year>2000</year><article-title>Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [<italic>PSI</italic><sup><italic>+</italic></sup>] of <italic>Saccharomyces cerevisiae</italic></article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>97</volume><fpage>240</fpage><lpage>244</lpage><pub-id pub-id-type="doi">10.1073/pnas.97.1.240</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Erjavec</surname><given-names>N</given-names></name><name><surname>Cvijovic</surname><given-names>M</given-names></name><name><surname>Klipp</surname><given-names>E</given-names></name><name><surname>Nyström</surname><given-names>T</given-names></name></person-group><year>2008</year><article-title>Selective benefits of damage partitioning in unicellular systems and its effects on aging</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>105</volume><fpage>18764</fpage><lpage>18769</lpage><pub-id pub-id-type="doi">10.1073/pnas.0804550105</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Erjavec</surname><given-names>N</given-names></name><name><surname>Larsson</surname><given-names>L</given-names></name><name><surname>Grantham</surname><given-names>J</given-names></name><name><surname>Nyström</surname><given-names>T</given-names></name></person-group><year>2007</year><article-title>Accelerated aging and failure to segregate damaged proteins in Sir2 mutants can be suppressed by overproducing the protein aggregation-remodeling factor Hsp104p</article-title><source>Genes & Development</source><volume>21</volume><fpage>2410</fpage><lpage>2421</lpage><pub-id pub-id-type="doi">10.1101/gad.439307</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Escusa-Toret</surname><given-names>S</given-names></name><name><surname>Vonk</surname><given-names>WI</given-names></name><name><surname>Frydman</surname><given-names>J</given-names></name></person-group><year>2013</year><article-title>Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress</article-title><source>Nature Cell Biology</source><volume>15</volume><fpage>1231</fpage><lpage>1243</lpage><pub-id pub-id-type="doi">10.1038/ncb2838</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gasch</surname><given-names>AP</given-names></name><name><surname>Spellman</surname><given-names>PT</given-names></name><name><surname>Kao</surname><given-names>CM</given-names></name><name><surname>Carmel-Harel</surname><given-names>O</given-names></name><name><surname>Eisen</surname><given-names>MB</given-names></name><name><surname>Storz</surname><given-names>G</given-names></name><name><surname>Botstein</surname><given-names>D</given-names></name><name><surname>Brown</surname><given-names>PO</given-names></name></person-group><year>2000</year><article-title>Genomic expression programs in the response of yeast cells to environmental changes</article-title><source>Molecular Biology of the Cell</source><volume>11</volume><fpage>4241</fpage><lpage>4257</lpage><pub-id pub-id-type="doi">10.1091/mbc.11.12.4241</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gidalevitz</surname><given-names>T</given-names></name><name><surname>Ben-Zvi</surname><given-names>A</given-names></name><name><surname>Ho</surname><given-names>KH</given-names></name><name><surname>Brignull</surname><given-names>HR</given-names></name><name><surname>Morimoto</surname><given-names>RI</given-names></name></person-group><year>2006</year><article-title>Progressive disruption of cellular protein folding in models of polyglutamine diseases</article-title><source>Science</source><volume>311</volume><fpage>1471</fpage><lpage>1474</lpage><pub-id pub-id-type="doi">10.1126/science.1124514</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Glover</surname><given-names>JR</given-names></name><name><surname>Kowal</surname><given-names>AS</given-names></name><name><surname>Schirmer</surname><given-names>EC</given-names></name><name><surname>Patino</surname><given-names>MM</given-names></name><name><surname>Liu</surname><given-names>JJ</given-names></name><name><surname>Lindquist</surname><given-names>S</given-names></name></person-group><year>1997</year><article-title>Self-seeded fibers formed by Sup35, the protein determinant of [<italic>PSI</italic>+], a heritable prion-like factor of <italic>S. cerevisiae</italic></article-title><source>Cell</source><volume>89</volume><fpage>811</fpage><lpage>819</lpage><pub-id pub-id-type="doi">10.1016/S0092-8674(00)80264-0</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Grimminger</surname><given-names>V</given-names></name><name><surname>Richter</surname><given-names>K</given-names></name><name><surname>Imhof</surname><given-names>A</given-names></name><name><surname>Buchner</surname><given-names>J</given-names></name><name><surname>Walter</surname><given-names>S</given-names></name></person-group><year>2004</year><article-title>The prion curing agent guanidinium chloride specifically inhibits ATP hydrolysis by Hsp104</article-title><source>The Journal of Biological Chemistry</source><volume>279</volume><fpage>7378</fpage><lpage>7383</lpage><pub-id pub-id-type="doi">10.1074/jbc.M312403200</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hernebring</surname><given-names>M</given-names></name><name><surname>Brolen</surname><given-names>G</given-names></name><name><surname>Aguilaniu</surname><given-names>H</given-names></name><name><surname>Semb</surname><given-names>H</given-names></name><name><surname>Nyström</surname><given-names>T</given-names></name></person-group><year>2006</year><article-title>Elimination of damaged proteins during differentiation of embryonic stem cells</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>103</volume><fpage>7700</fpage><lpage>7705</lpage><pub-id pub-id-type="doi">10.1073/pnas.0510944103</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Higurashi</surname><given-names>T</given-names></name><name><surname>Hines</surname><given-names>JK</given-names></name><name><surname>Sahi</surname><given-names>C</given-names></name><name><surname>Aron</surname><given-names>R</given-names></name><name><surname>Craig</surname><given-names>EA</given-names></name></person-group><year>2008</year><article-title>Specificity of the J-protein Sis1 in the propagation of 3 yeast prions</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>105</volume><fpage>16596</fpage><lpage>16601</lpage><pub-id pub-id-type="doi">10.1073/pnas.0808934105</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Holmes</surname><given-names>WM</given-names></name><name><surname>Klaips</surname><given-names>CL</given-names></name><name><surname>Serio</surname><given-names>TR</given-names></name></person-group><year>2014</year><article-title>Defining the limits: protein aggregation and toxicity in vivo</article-title><source>Critical Reviews in Biochemistry and Molecular Biology</source><volume>49</volume><fpage>294</fpage><lpage>303</lpage><pub-id pub-id-type="doi">10.3109/10409238.2014.914151</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Inoue</surname><given-names>Y</given-names></name><name><surname>Taguchi</surname><given-names>H</given-names></name><name><surname>Kishimoto</surname><given-names>A</given-names></name><name><surname>Yoshida</surname><given-names>M</given-names></name></person-group><year>2004</year><article-title>Hsp104 binds to yeast Sup35 prion fiber but needs other factor(s) to sever it</article-title><source>The Journal of Biological Chemistry</source><volume>279</volume><fpage>52319</fpage><lpage>52323</lpage><pub-id pub-id-type="doi">10.1074/jbc.M408159200</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ishihara</surname><given-names>K</given-names></name><name><surname>Yamagishi</surname><given-names>N</given-names></name><name><surname>Saito</surname><given-names>Y</given-names></name><name><surname>Adachi</surname><given-names>H</given-names></name><name><surname>Kobayashi</surname><given-names>Y</given-names></name><name><surname>Sobue</surname><given-names>G</given-names></name><name><surname>Ohtsuka</surname><given-names>K</given-names></name><name><surname>Hatayama</surname><given-names>T</given-names></name></person-group><year>2003</year><article-title>Hsp105alpha suppresses the aggregation of truncated androgen receptor with expanded CAG repeats and cell toxicity</article-title><source>The Journal of Biological Chemistry</source><volume>278</volume><fpage>25143</fpage><lpage>25150</lpage><pub-id pub-id-type="doi">10.1074/jbc.M302975200</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jahn</surname><given-names>TR</given-names></name><name><surname>Radford</surname><given-names>SE</given-names></name></person-group><year>2008</year><article-title>Folding versus aggregation: polypeptide conformations on competing pathways</article-title><source>Archives of Biochemistry and Biophysics</source><volume>469</volume><fpage>100</fpage><lpage>117</lpage><pub-id pub-id-type="doi">10.1016/j.abb.2007.05.015</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jana</surname><given-names>NR</given-names></name><name><surname>Tanaka</surname><given-names>M</given-names></name><name><surname>Wang</surname><given-names>Gh</given-names></name><name><surname>Nukina</surname><given-names>N</given-names></name></person-group><year>2000</year><article-title>Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated n-terminal huntingtin: their role in suppression of aggregation and cellular toxicity</article-title><source>Human Molecular Genetics</source><volume>9</volume><fpage>2009</fpage><lpage>2018</lpage><pub-id pub-id-type="doi">10.1093/hmg/9.13.2009</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Johnston</surname><given-names>JA</given-names></name><name><surname>Ward</surname><given-names>CL</given-names></name><name><surname>Kopito</surname><given-names>RR</given-names></name></person-group><year>1998</year><article-title>Aggresomes: a cellular response to misfolded proteins</article-title><source>The Journal of Cell Biology</source><volume>143</volume><fpage>1883</fpage><lpage>1898</lpage><pub-id pub-id-type="doi">10.1083/jcb.143.7.1883</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jung</surname><given-names>G</given-names></name><name><surname>Jones</surname><given-names>G</given-names></name><name><surname>Wegrzyn</surname><given-names>RD</given-names></name><name><surname>Masison</surname><given-names>DC</given-names></name></person-group><year>2000</year><article-title>A role for cytosolic hsp70 in yeast [<italic>PSI</italic><sup><italic>+</italic></sup>] prion propagation and [<italic>PSI</italic><sup><italic>+</italic></sup>] as a cellular stress</article-title><source>Genetics</source><volume>156</volume><fpage>559</fpage><lpage>570</lpage></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jung</surname><given-names>G</given-names></name><name><surname>Masison</surname><given-names>DC</given-names></name></person-group><year>2001</year><article-title>Guanidine hydrochloride inhibits Hsp104 activity in vivo: a possible explanation for its effect in curing yeast prions</article-title><source>Current Microbiology</source><volume>43</volume><fpage>7</fpage><lpage>10</lpage><pub-id pub-id-type="doi">10.1007/s002840010251</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kaganovich</surname><given-names>D</given-names></name><name><surname>Kopito</surname><given-names>R</given-names></name><name><surname>Frydman</surname><given-names>J</given-names></name></person-group><year>2008</year><article-title>Misfolded proteins partition between two distinct quality control compartments</article-title><source>Nature</source><volume>454</volume><fpage>1088</fpage><lpage>1095</lpage><pub-id pub-id-type="doi">10.1038/nature07195</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kawai-Noma</surname><given-names>S</given-names></name><name><surname>Pack</surname><given-names>CG</given-names></name><name><surname>Tsuji</surname><given-names>T</given-names></name><name><surname>Kinjo</surname><given-names>M</given-names></name><name><surname>Taguchi</surname><given-names>H</given-names></name></person-group><year>2009</year><article-title>Single mother-daughter pair analysis to clarify the diffusion properties of yeast prion Sup35 in guanidine-HCl-treated [<italic>PSI</italic>] cells</article-title><source>Genes to Cells</source><volume>14</volume><fpage>1045</fpage><lpage>1054</lpage><pub-id pub-id-type="doi">10.1111/j.1365-2443.2009.01333.x</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kim</surname><given-names>YE</given-names></name><name><surname>Hipp</surname><given-names>MS</given-names></name><name><surname>Bracher</surname><given-names>A</given-names></name><name><surname>Hayer-Hartl</surname><given-names>M</given-names></name><name><surname>Hartl</surname><given-names>FU</given-names></name></person-group><year>2013</year><article-title>Molecular chaperone functions in protein folding and proteostasis</article-title><source>Annual Review of Biochemistry</source><volume>82</volume><fpage>323</fpage><lpage>355</lpage><pub-id pub-id-type="doi">10.1146/annurev-biochem-060208-092442</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>King</surname><given-names>CY</given-names></name><name><surname>Tittmann</surname><given-names>P</given-names></name><name><surname>Gross</surname><given-names>H</given-names></name><name><surname>Gebert</surname><given-names>R</given-names></name><name><surname>Aebi</surname><given-names>M</given-names></name><name><surname>Wüthrich</surname><given-names>K</given-names></name></person-group><year>1997</year><article-title>Prion-inducing domain 2-114 of yeast Sup35 protein transforms <italic>in vitro</italic> into amyloid-like filaments</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>94</volume><fpage>6618</fpage><lpage>6622</lpage><pub-id pub-id-type="doi">10.1073/pnas.94.13.6618</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kirstein-Miles</surname><given-names>J</given-names></name><name><surname>Morimoto</surname><given-names>RI</given-names></name></person-group><year>2013</year><article-title>Ribosome-associated chaperones act as proteostasis sentinels</article-title><source>Cell Cycle</source><volume>12</volume><fpage>2335</fpage><lpage>2336</lpage><pub-id pub-id-type="doi">10.4161/cc.25703</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Knorre</surname><given-names>DA</given-names></name><name><surname>Kulemzina</surname><given-names>IA</given-names></name><name><surname>Sorokin</surname><given-names>MI</given-names></name><name><surname>Kochmak</surname><given-names>SA</given-names></name><name><surname>Bocharova</surname><given-names>NA</given-names></name><name><surname>Sokolov</surname><given-names>SS</given-names></name><name><surname>Severin</surname><given-names>FF</given-names></name></person-group><year>2010</year><article-title>Sir2-dependent daughter-to-mother transport of the damaged proteins in yeast is required to prevent high stress sensitivity of the daughters</article-title><source>Cell Cycle</source><volume>9</volume><fpage>4501</fpage><lpage>4505</lpage><pub-id pub-id-type="doi">10.4161/cc.9.22.13683</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kobayashi</surname><given-names>Y</given-names></name><name><surname>Kume</surname><given-names>A</given-names></name><name><surname>Li</surname><given-names>M</given-names></name><name><surname>Doyu</surname><given-names>M</given-names></name><name><surname>Hata</surname><given-names>M</given-names></name><name><surname>Ohtsuka</surname><given-names>K</given-names></name><name><surname>Sobue</surname><given-names>G</given-names></name></person-group><year>2000</year><article-title>Chaperones Hsp70 and Hsp40 suppress aggregate formation and apoptosis in cultured neuronal cells expressing truncated androgen receptor protein with expanded polyglutamine tract</article-title><source>The Journal of Biological Chemistry</source><volume>275</volume><fpage>8772</fpage><lpage>8778</lpage><pub-id pub-id-type="doi">10.1074/jbc.275.12.8772</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kohno</surname><given-names>H</given-names></name><name><surname>Tanaka</surname><given-names>K</given-names></name><name><surname>Mino</surname><given-names>A</given-names></name><name><surname>Umikawa</surname><given-names>M</given-names></name><name><surname>Imamura</surname><given-names>H</given-names></name><name><surname>Fujiwara</surname><given-names>T</given-names></name><name><surname>Fujita</surname><given-names>Y</given-names></name><name><surname>Hotta</surname><given-names>K</given-names></name><name><surname>Qadota</surname><given-names>H</given-names></name><name><surname>Watanabe</surname><given-names>T</given-names></name><name><surname>Ohya</surname><given-names>Y</given-names></name><name><surname>Takai</surname><given-names>Y</given-names></name></person-group><year>1996</year><article-title>Bni1p implicated in cytoskeletal control is a putative target of Rho1p small Gtp binding protein in <italic>saccharomyces cerevisiae</italic></article-title><source>The EMBO Journal</source><volume>15</volume><fpage>6060</fpage><lpage>6068</lpage></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kryndushkin</surname><given-names>DS</given-names></name><name><surname>Alexandrov</surname><given-names>IM</given-names></name><name><surname>Ter-Avanesyan</surname><given-names>MD</given-names></name><name><surname>Kushnirov</surname><given-names>VV</given-names></name></person-group><year>2003</year><article-title>Yeast [<italic>PSI</italic><sup>+</sup>] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104</article-title><source>The Journal of Biological Chemistry</source><volume>278</volume><fpage>49636</fpage><lpage>49643</lpage><pub-id pub-id-type="doi">10.1074/jbc.M307996200</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kummer</surname><given-names>E</given-names></name><name><surname>Oguchi</surname><given-names>Y</given-names></name><name><surname>Seyffer</surname><given-names>F</given-names></name><name><surname>Bukau</surname><given-names>B</given-names></name><name><surname>Mogk</surname><given-names>A</given-names></name></person-group><year>2013</year><article-title>Mechanism of Hsp104/Clpb inhibition by prion curing Guanidinium Hydrochloride</article-title><source>FEBS Letters</source><volume>587</volume><fpage>810</fpage><lpage>817</lpage><pub-id pub-id-type="doi">10.1016/j.febslet.2013.02.011</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lim</surname><given-names>Y</given-names></name><name><surname>Kehm</surname><given-names>VM</given-names></name><name><surname>Lee</surname><given-names>EB</given-names></name><name><surname>Soper</surname><given-names>JH</given-names></name><name><surname>Li</surname><given-names>C</given-names></name><name><surname>Trojanowski</surname><given-names>JQ</given-names></name><name><surname>Lee</surname><given-names>VM</given-names></name></person-group><year>2011</year><article-title>Alpha-Syn suppression reverses synaptic and memory defects in a mouse model of dementia with Lewy bodies</article-title><source>The Journal of Neuroscience</source><volume>31</volume><fpage>10076</fpage><lpage>10087</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.0618-11.2011</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lindquist</surname><given-names>SL</given-names></name><name><surname>Kelly</surname><given-names>JW</given-names></name></person-group><year>2011</year><article-title>Chemical and biological approaches for adapting proteostasis to ameliorate protein misfolding and aggregation diseases: progress and prognosis</article-title><source>Cold Spring Harbor Perspectives in Biology</source><volume>3</volume><fpage>a004507</fpage><pub-id pub-id-type="doi">10.1101/cshperspect.a004507</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>B</given-names></name><name><surname>Larsson</surname><given-names>L</given-names></name><name><surname>Caballero</surname><given-names>A</given-names></name><name><surname>Hao</surname><given-names>X</given-names></name><name><surname>Oling</surname><given-names>D</given-names></name><name><surname>Grantham</surname><given-names>J</given-names></name><name><surname>Nyström</surname><given-names>T</given-names></name></person-group><year>2010</year><article-title>The polarisome is required for segregation and retrograde transport of protein aggregates</article-title><source>Cell</source><volume>140</volume><fpage>257</fpage><lpage>267</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2009.12.031</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lo Bianco</surname><given-names>C</given-names></name><name><surname>Shorter</surname><given-names>J</given-names></name><name><surname>Regulier</surname><given-names>E</given-names></name><name><surname>Lashuel</surname><given-names>H</given-names></name><name><surname>Iwatsubo</surname><given-names>T</given-names></name><name><surname>Lindquist</surname><given-names>S</given-names></name><name><surname>Aebischer</surname><given-names>P</given-names></name></person-group><year>2008</year><article-title>Hsp104 antagonizes alpha-synuclein aggregation and reduces dopaminergic degeneration in a rat model of Parkinson disease</article-title><source>The Journal of Clinical Investigation</source><volume>118</volume><fpage>3087</fpage><lpage>3097</lpage><pub-id pub-id-type="doi">10.1172/JCI35781</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Malinovska</surname><given-names>L</given-names></name><name><surname>Kroschwald</surname><given-names>S</given-names></name><name><surname>Munder</surname><given-names>MC</given-names></name><name><surname>Richter</surname><given-names>D</given-names></name><name><surname>Alberti</surname><given-names>S</given-names></name></person-group><year>2012</year><article-title>Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates</article-title><source>Molecular Biology of the Cell</source><volume>23</volume><fpage>3041</fpage><lpage>3056</lpage><pub-id pub-id-type="doi">10.1091/mbc.E12-03-0194</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mallucci</surname><given-names>G</given-names></name><name><surname>Dickinson</surname><given-names>A</given-names></name><name><surname>Linehan</surname><given-names>J</given-names></name><name><surname>Klohn</surname><given-names>PC</given-names></name><name><surname>Brandner</surname><given-names>S</given-names></name><name><surname>Collinge</surname><given-names>J</given-names></name></person-group><year>2003</year><article-title>Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis</article-title><source>Science</source><volume>302</volume><fpage>871</fpage><lpage>874</lpage><pub-id pub-id-type="doi">10.1126/science.1090187302/5646/871</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mattoo</surname><given-names>RU</given-names></name><name><surname>Sharma</surname><given-names>SK</given-names></name><name><surname>Priya</surname><given-names>S</given-names></name><name><surname>Finka</surname><given-names>A</given-names></name><name><surname>Goloubinoff</surname><given-names>P</given-names></name></person-group><year>2013</year><article-title>Hsp110 is a bona fide chaperone using ATP to unfold stable misfolded polypeptides and reciprocally collaborate with Hsp70 to solubilize protein aggregates</article-title><source>The Journal of Biological Chemistry</source><volume>288</volume><fpage>21399</fpage><lpage>21411</lpage><pub-id pub-id-type="doi">10.1074/jbc.M113.479253</pub-id></element-citation></ref><ref id="bib54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Meriin</surname><given-names>AB</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Miliaras</surname><given-names>NB</given-names></name><name><surname>Kazantsev</surname><given-names>A</given-names></name><name><surname>Chernoff</surname><given-names>YO</given-names></name><name><surname>McCaffery</surname><given-names>JM</given-names></name><name><surname>Wendland</surname><given-names>B</given-names></name><name><surname>Sherman</surname><given-names>MY</given-names></name></person-group><year>2003</year><article-title>Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis</article-title><source>Molecular and Cellular Biology</source><volume>23</volume><fpage>7554</fpage><lpage>7565</lpage><pub-id pub-id-type="doi">10.1128/MCB.23.21.7554-7565.2003</pub-id></element-citation></ref><ref id="bib55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Morimoto</surname><given-names>RI</given-names></name></person-group><year>2008</year><article-title>Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging</article-title><source>Genes & Development</source><volume>22</volume><fpage>1427</fpage><lpage>1438</lpage><pub-id pub-id-type="doi">10.1101/gad.1657108</pub-id></element-citation></ref><ref id="bib56"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Morimoto</surname><given-names>RI</given-names></name></person-group><year>2011</year><article-title>The heat shock response: systems biology of proteotoxic stress in aging and disease</article-title><source>Cold Spring Harbor Symposia on Quantitative Biology</source><volume>76</volume><fpage>91</fpage><lpage>99</lpage><pub-id pub-id-type="doi">10.1101/sqb.2012.76.010637</pub-id></element-citation></ref><ref id="bib57"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ness</surname><given-names>F</given-names></name><name><surname>Ferreira</surname><given-names>P</given-names></name><name><surname>Cox</surname><given-names>BS</given-names></name><name><surname>Tuite</surname><given-names>MF</given-names></name></person-group><year>2002</year><article-title>Guanidine hydrochloride inhibits the generation of prion “seeds” but not prion protein aggregation in yeast</article-title><source>Molecular and Cellular Biology</source><volume>22</volume><fpage>5593</fpage><lpage>5605</lpage><pub-id pub-id-type="doi">10.1128/MCB.22.15.5593-5605.2002</pub-id></element-citation></ref><ref id="bib58"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Newnam</surname><given-names>GP</given-names></name><name><surname>Birchmore</surname><given-names>JL</given-names></name><name><surname>Chernoff</surname><given-names>YO</given-names></name></person-group><year>2011</year><article-title>Destabilization and recovery of a yeast prion after mild heat shock</article-title><source>Journal of Molecular Biology</source><volume>408</volume><fpage>432</fpage><lpage>448</lpage><pub-id pub-id-type="doi">10.1016/j.jmb.2011.02.034</pub-id></element-citation></ref><ref id="bib59"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Olzscha</surname><given-names>H</given-names></name><name><surname>Schermann</surname><given-names>SM</given-names></name><name><surname>Woerner</surname><given-names>AC</given-names></name><name><surname>Pinkert</surname><given-names>S</given-names></name><name><surname>Hecht</surname><given-names>MH</given-names></name><name><surname>Tartaglia</surname><given-names>GG</given-names></name><name><surname>Vendruscolo</surname><given-names>M</given-names></name><name><surname>Hayer-Hartl</surname><given-names>M</given-names></name><name><surname>Hartl</surname><given-names>FU</given-names></name><name><surname>Vabulas</surname><given-names>RM</given-names></name></person-group><year>2011</year><article-title>Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions</article-title><source>Cell</source><volume>144</volume><fpage>67</fpage><lpage>78</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2010.11.050</pub-id></element-citation></ref><ref id="bib60"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Park</surname><given-names>SH</given-names></name><name><surname>Kukushkin</surname><given-names>Y</given-names></name><name><surname>Gupta</surname><given-names>R</given-names></name><name><surname>Chen</surname><given-names>T</given-names></name><name><surname>Konagai</surname><given-names>A</given-names></name><name><surname>Hipp</surname><given-names>MS</given-names></name><name><surname>Hayer-Hartl</surname><given-names>M</given-names></name><name><surname>Hartl</surname><given-names>FU</given-names></name></person-group><year>2013</year><article-title>PolyQ proteins interfere with nuclear degradation of cytosolic proteins by sequestering the Sis1p chaperone</article-title><source>Cell</source><volume>154</volume><fpage>134</fpage><lpage>145</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2013.06.003</pub-id></element-citation></ref><ref id="bib61"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Park</surname><given-names>YN</given-names></name><name><surname>Zhao</surname><given-names>X</given-names></name><name><surname>Yim</surname><given-names>YI</given-names></name><name><surname>Todor</surname><given-names>H</given-names></name><name><surname>Ellerbrock</surname><given-names>R</given-names></name><name><surname>Reidy</surname><given-names>M</given-names></name><name><surname>Eisenberg</surname><given-names>E</given-names></name><name><surname>Masison</surname><given-names>DC</given-names></name><name><surname>Greene</surname><given-names>LE</given-names></name></person-group><year>2014</year><article-title>Hsp104 overexpression cures <italic>Saccharomyces cerevisiae</italic> [<italic>PSI</italic><sup><italic>+</italic></sup>] by causing dissolution of the prion seeds</article-title><source>Eukaryotic Cell</source><volume>13</volume><fpage>635</fpage><lpage>647</lpage><pub-id pub-id-type="doi">10.1128/EC.00300-13</pub-id></element-citation></ref><ref id="bib62"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Patino</surname><given-names>MM</given-names></name><name><surname>Liu</surname><given-names>JJ</given-names></name><name><surname>Glover</surname><given-names>JR</given-names></name><name><surname>Lindquist</surname><given-names>S</given-names></name></person-group><year>1996</year><article-title>Support for the prion hypothesis for inheritance of a phenotypic trait in yeast</article-title><source>Science</source><volume>273</volume><fpage>622</fpage><lpage>626</lpage><pub-id pub-id-type="doi">10.1126/science.273.5275.622</pub-id></element-citation></ref><ref id="bib63"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Paushkin</surname><given-names>SV</given-names></name><name><surname>Kushnirov</surname><given-names>VV</given-names></name><name><surname>Smirnov</surname><given-names>VN</given-names></name><name><surname>Ter-Avanesyan</surname><given-names>MD</given-names></name></person-group><year>1996</year><article-title>Propagation of the yeast prion-like [<italic>PSI</italic><sup><italic>+</italic></sup>] determinant is mediated by oligomerization of the Sup35-encoded polypeptide chain release factor</article-title><source>The EMBO Journal</source><volume>15</volume><fpage>3127</fpage><lpage>3134</lpage></element-citation></ref><ref id="bib64"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pezza</surname><given-names>JA</given-names></name><name><surname>Langseth</surname><given-names>SX</given-names></name><name><surname>Raupp Yamamoto</surname><given-names>R</given-names></name><name><surname>Doris</surname><given-names>SM</given-names></name><name><surname>Ulin</surname><given-names>SP</given-names></name><name><surname>Salomon</surname><given-names>AR</given-names></name><name><surname>Serio</surname><given-names>TR</given-names></name></person-group><year>2009</year><article-title>The Nata acetyltransferase couples Sup35 prion complexes to the [<italic>PSI</italic><sup><italic>+</italic></sup>] phenotype</article-title><source>Molecular Biology of the Cell</source><volume>20</volume><fpage>1068</fpage><lpage>1080</lpage><pub-id pub-id-type="doi">10.1091/mbc.E08-04-0436</pub-id></element-citation></ref><ref id="bib65"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Powers</surname><given-names>ET</given-names></name><name><surname>Morimoto</surname><given-names>RI</given-names></name><name><surname>Dillin</surname><given-names>A</given-names></name><name><surname>Kelly</surname><given-names>JW</given-names></name><name><surname>Balch</surname><given-names>WE</given-names></name></person-group><year>2009</year><article-title>Biological and chemical approaches to diseases of proteostasis deficiency</article-title><source>Annual Review of Biochemistry</source><volume>78</volume><fpage>959</fpage><lpage>991</lpage><pub-id pub-id-type="doi">10.1146/annurev.biochem.052308.114844</pub-id></element-citation></ref><ref id="bib66"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rampelt</surname><given-names>H</given-names></name><name><surname>Kirstein-Miles</surname><given-names>J</given-names></name><name><surname>Nillegoda</surname><given-names>NB</given-names></name><name><surname>Chi</surname><given-names>K</given-names></name><name><surname>Scholz</surname><given-names>SR</given-names></name><name><surname>Morimoto</surname><given-names>RI</given-names></name><name><surname>Bukau</surname><given-names>B</given-names></name></person-group><year>2012</year><article-title>Metazoan Hsp70 machines use Hsp110 to power protein disaggregation</article-title><source>The EMBO Journal</source><volume>31</volume><fpage>4221</fpage><lpage>4235</lpage><pub-id pub-id-type="doi">10.1038/emboj.2012.264</pub-id></element-citation></ref><ref id="bib67"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rujano</surname><given-names>MA</given-names></name><name><surname>Bosveld</surname><given-names>F</given-names></name><name><surname>Salomons</surname><given-names>FA</given-names></name><name><surname>Dijk</surname><given-names>F</given-names></name><name><surname>van Waarde</surname><given-names>MA</given-names></name><name><surname>van der Want</surname><given-names>JJ</given-names></name><name><surname>de Vos</surname><given-names>RA</given-names></name><name><surname>Brunt</surname><given-names>ER</given-names></name><name><surname>Sibon</surname><given-names>OC</given-names></name><name><surname>Kampinga</surname><given-names>HH</given-names></name></person-group><year>2006</year><article-title>Polarised asymmetric inheritance of accumulated protein damage in higher eukaryotes</article-title><source>PLOS Biology</source><volume>4</volume><fpage>e417</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.0040417</pub-id></element-citation></ref><ref id="bib68"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sanchez</surname><given-names>Y</given-names></name><name><surname>Taulien</surname><given-names>J</given-names></name><name><surname>Borkovich</surname><given-names>KA</given-names></name><name><surname>Lindquist</surname><given-names>S</given-names></name></person-group><year>1992</year><article-title>Hsp104 is required for tolerance to many forms of stress</article-title><source>The EMBO Journal</source><volume>11</volume><fpage>2357</fpage><lpage>2364</lpage></element-citation></ref><ref id="bib69"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Satpute-Krishnan</surname><given-names>P</given-names></name><name><surname>Langseth</surname><given-names>SX</given-names></name><name><surname>Serio</surname><given-names>TR</given-names></name></person-group><year>2007</year><article-title>Hsp104-dependent remodeling of prion complexes mediates protein-only inheritance</article-title><source>PLOS Biology</source><volume>5</volume><fpage>e24</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.0050024</pub-id></element-citation></ref><ref id="bib70"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Satpute-Krishnan</surname><given-names>P</given-names></name><name><surname>Serio</surname><given-names>TR</given-names></name></person-group><year>2005</year><article-title>Prion protein remodelling confers an immediate phenotypic switch</article-title><source>Nature</source><volume>437</volume><fpage>262</fpage><lpage>265</lpage><pub-id pub-id-type="doi">10.1038/nature03981</pub-id></element-citation></ref><ref id="bib71"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schaffar</surname><given-names>G</given-names></name><name><surname>Breuer</surname><given-names>P</given-names></name><name><surname>Boteva</surname><given-names>R</given-names></name><name><surname>Behrends</surname><given-names>C</given-names></name><name><surname>Tzvetkov</surname><given-names>N</given-names></name><name><surname>Strippel</surname><given-names>N</given-names></name><name><surname>Sakahira</surname><given-names>H</given-names></name><name><surname>Siegers</surname><given-names>K</given-names></name><name><surname>Hayer-Hartl</surname><given-names>M</given-names></name><name><surname>Hartl</surname><given-names>FU</given-names></name></person-group><year>2004</year><article-title>Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation</article-title><source>Molecular Cell</source><volume>15</volume><fpage>95</fpage><lpage>105</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2004.06.029</pub-id></element-citation></ref><ref id="bib72"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Serio</surname><given-names>TR</given-names></name><name><surname>Cashikar</surname><given-names>AG</given-names></name><name><surname>Kowal</surname><given-names>AS</given-names></name><name><surname>Sawicki</surname><given-names>GJ</given-names></name><name><surname>Moslehi</surname><given-names>JJ</given-names></name><name><surname>Serpell</surname><given-names>L</given-names></name><name><surname>Arnsdorf</surname><given-names>MF</given-names></name><name><surname>Lindquist</surname><given-names>SL</given-names></name></person-group><year>2000</year><article-title>Nucleated conformational conversion and the replication of conformational information by a prion determinant</article-title><source>Science</source><volume>289</volume><fpage>1317</fpage><lpage>1321</lpage><pub-id pub-id-type="doi">10.1126/science.289.5483.1317</pub-id></element-citation></ref><ref id="bib73"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shorter</surname><given-names>J</given-names></name></person-group><year>2011</year><article-title>The mammalian disaggregase machinery: Hsp110 synergizes with Hsp70 and Hsp40 to catalyze protein disaggregation and reactivation in a cell-free system</article-title><source>PLOS ONE</source><volume>6</volume><fpage>e26319</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0026319</pub-id></element-citation></ref><ref id="bib74"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shorter</surname><given-names>J</given-names></name><name><surname>Lindquist</surname><given-names>S</given-names></name></person-group><year>2004</year><article-title>Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers</article-title><source>Science</source><volume>304</volume><fpage>1793</fpage><lpage>1797</lpage><pub-id pub-id-type="doi">10.1126/science.1098007</pub-id></element-citation></ref><ref id="bib75"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shorter</surname><given-names>J</given-names></name><name><surname>Lindquist</surname><given-names>S</given-names></name></person-group><year>2008</year><article-title>Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions</article-title><source>The EMBO Journal</source><volume>27</volume><fpage>2712</fpage><lpage>2724</lpage><pub-id pub-id-type="doi">10.1038/emboj.2008.194</pub-id></element-citation></ref><ref id="bib76"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Song</surname><given-names>Y</given-names></name><name><surname>Wu</surname><given-names>YX</given-names></name><name><surname>Jung</surname><given-names>G</given-names></name><name><surname>Tutar</surname><given-names>Y</given-names></name><name><surname>Eisenberg</surname><given-names>E</given-names></name><name><surname>Greene</surname><given-names>LE</given-names></name><name><surname>Masison</surname><given-names>DC</given-names></name></person-group><year>2005</year><article-title>Role for Hsp70 chaperone in <italic>Saccharomyces cerevisiae</italic> prion seed replication</article-title><source>Eukaryotic Cell</source><volume>4</volume><fpage>289</fpage><lpage>297</lpage><pub-id pub-id-type="doi">10.1128/EC.4.2.289-297.2005</pub-id></element-citation></ref><ref id="bib77"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Specht</surname><given-names>S</given-names></name><name><surname>Miller</surname><given-names>SB</given-names></name><name><surname>Mogk</surname><given-names>A</given-names></name><name><surname>Bukau</surname><given-names>B</given-names></name></person-group><year>2011</year><article-title>Hsp42 is required for sequestration of protein aggregates into deposition sites in <italic>saccharomyces cerevisiae</italic></article-title><source>The Journal of Cell Biology</source><volume>195</volume><fpage>617</fpage><lpage>629</lpage><pub-id pub-id-type="doi">10.1083/jcb.201106037</pub-id></element-citation></ref><ref id="bib78"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Spokoini</surname><given-names>R</given-names></name><name><surname>Moldavski</surname><given-names>O</given-names></name><name><surname>Nahmias</surname><given-names>Y</given-names></name><name><surname>England</surname><given-names>JL</given-names></name><name><surname>Schuldiner</surname><given-names>M</given-names></name><name><surname>Kaganovich</surname><given-names>D</given-names></name></person-group><year>2012</year><article-title>Confinement to organelle-associated inclusion structures mediates asymmetric inheritance of aggregated protein in budding yeast</article-title><source>Cell Reports</source><volume>2</volume><fpage>738</fpage><lpage>747</lpage><pub-id pub-id-type="doi">10.1016/j.celrep.2012.08.024</pub-id></element-citation></ref><ref id="bib79"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tam</surname><given-names>S</given-names></name><name><surname>Geller</surname><given-names>R</given-names></name><name><surname>Spiess</surname><given-names>C</given-names></name><name><surname>Frydman</surname><given-names>J</given-names></name></person-group><year>2006</year><article-title>The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions</article-title><source>Nature Cell Biology</source><volume>8</volume><fpage>1155</fpage><lpage>1162</lpage><pub-id pub-id-type="doi">10.1038/ncb1477</pub-id></element-citation></ref><ref id="bib80"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tanaka</surname><given-names>M</given-names></name><name><surname>Collins</surname><given-names>SR</given-names></name><name><surname>Toyama</surname><given-names>BH</given-names></name><name><surname>Weissman</surname><given-names>JS</given-names></name></person-group><year>2006</year><article-title>The physical basis of how prion conformations determine strain phenotypes</article-title><source>Nature</source><volume>442</volume><fpage>585</fpage><lpage>589</lpage><pub-id pub-id-type="doi">10.1038/nature04922</pub-id></element-citation></ref><ref id="bib81"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tariq</surname><given-names>M</given-names></name><name><surname>Wegrzyn</surname><given-names>R</given-names></name><name><surname>Anwar</surname><given-names>S</given-names></name><name><surname>Bukau</surname><given-names>B</given-names></name><name><surname>Paro</surname><given-names>R</given-names></name></person-group><year>2013</year><article-title><italic>Drosophila</italic> GAGA factor polyglutamine domains exhibit prion-like behavior</article-title><source>BMC Genomics</source><volume>14</volume><fpage>374</fpage><pub-id pub-id-type="doi">10.1186/1471-2164-14-374</pub-id></element-citation></ref><ref id="bib82"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taylor</surname><given-names>RC</given-names></name><name><surname>Dillin</surname><given-names>A</given-names></name></person-group><year>2011</year><article-title>Aging as an event of proteostasis collapse</article-title><source>Cold Spring Harbor Perspectives in Biology</source><volume>3</volume><fpage>a004440</fpage><pub-id pub-id-type="doi">10.1101/cshperspect.a004440</pub-id></element-citation></ref><ref id="bib83"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ter-Avanesyan</surname><given-names>MD</given-names></name><name><surname>Kushnirov</surname><given-names>VV</given-names></name><name><surname>Dagkesamanskaya</surname><given-names>AR</given-names></name><name><surname>Didichenko</surname><given-names>SA</given-names></name><name><surname>Chernoff</surname><given-names>YO</given-names></name><name><surname>Inge-Vechtomov</surname><given-names>SG</given-names></name><name><surname>Smirnov</surname><given-names>VN</given-names></name></person-group><year>1993</year><article-title>Deletion analysis of the Sup35 gene of the yeast <italic>Saccharomyces cerevisiae</italic> reveals two non-overlapping functional regions in the encoded protein</article-title><source>Molecular Microbiology</source><volume>7</volume><fpage>683</fpage><lpage>692</lpage><pub-id pub-id-type="doi">10.1111/j.1365-2958.1993.tb01159.x</pub-id></element-citation></ref><ref id="bib84"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tessarz</surname><given-names>P</given-names></name><name><surname>Schwarz</surname><given-names>M</given-names></name><name><surname>Mogk</surname><given-names>A</given-names></name><name><surname>Bukau</surname><given-names>B</given-names></name></person-group><year>2009</year><article-title>The yeast AAA+ chaperone Hsp104 is part of a network that links the actin cytoskeleton with the inheritance of damaged proteins</article-title><source>Molecular and Cellular Biology</source><volume>29</volume><fpage>3738</fpage><lpage>3745</lpage><pub-id pub-id-type="doi">10.1128/MCB.00201-09</pub-id></element-citation></ref><ref id="bib85"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tipton</surname><given-names>KA</given-names></name><name><surname>Verges</surname><given-names>KJ</given-names></name><name><surname>Weissman</surname><given-names>JS</given-names></name></person-group><year>2008</year><article-title>In vivo monitoring of the prion replication cycle reveals a critical role for Sis1 in delivering substrates to Hsp104</article-title><source>Molecular Cell</source><volume>32</volume><fpage>584</fpage><lpage>591</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2008.11.003</pub-id></element-citation></ref><ref id="bib86"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Torrente</surname><given-names>MP</given-names></name><name><surname>Shorter</surname><given-names>J</given-names></name></person-group><year>2013</year><article-title>The metazoan protein disaggregase and amyloid depolymerase system: Hsp110, Hsp70, Hsp40, and small heat shock proteins</article-title><source>Prion</source><volume>7</volume><fpage>457</fpage><lpage>463</lpage><pub-id pub-id-type="doi">10.4161/pri.27531</pub-id></element-citation></ref><ref id="bib87"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tuite</surname><given-names>MF</given-names></name><name><surname>Mundy</surname><given-names>CR</given-names></name><name><surname>Cox</surname><given-names>BS</given-names></name></person-group><year>1981</year><article-title>Agents that cause a high frequency of genetic change from [<italic>PSI</italic><sup>+</sup>] to [<italic>psi</italic><sup>-</sup>] in <italic>Saccharomyces cerevisiae</italic></article-title><source>Genetics</source><volume>98</volume><fpage>691</fpage><lpage>711</lpage></element-citation></ref><ref id="bib88"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tuite</surname><given-names>MF</given-names></name><name><surname>Serio</surname><given-names>TR</given-names></name></person-group><year>2010</year><article-title>The prion hypothesis: from biological anomaly to basic regulatory mechanism</article-title><source>Nature Reviews Molecular Cell Biology</source><volume>11</volume><fpage>823</fpage><lpage>833</lpage><pub-id pub-id-type="doi">10.1038/nrm3007</pub-id></element-citation></ref><ref id="bib89"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Unal</surname><given-names>E</given-names></name><name><surname>Kinde</surname><given-names>B</given-names></name><name><surname>Amon</surname><given-names>A</given-names></name></person-group><year>2011</year><article-title>Gametogenesis eliminates age-induced cellular damage and resets life span in yeast</article-title><source>Science</source><volume>332</volume><fpage>1554</fpage><lpage>1557</lpage><pub-id pub-id-type="doi">10.1126/science.1204349</pub-id></element-citation></ref><ref id="bib90"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Voisine</surname><given-names>C</given-names></name><name><surname>Pedersen</surname><given-names>JS</given-names></name><name><surname>Morimoto</surname><given-names>RI</given-names></name></person-group><year>2010</year><article-title>Chaperone networks: tipping the balance in protein folding diseases</article-title><source>Neurobiology of Disease</source><volume>40</volume><fpage>12</fpage><lpage>20</lpage><pub-id pub-id-type="doi">10.1016/j.nbd.2010.05.007</pub-id></element-citation></ref><ref id="bib91"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Farr</surname><given-names>GW</given-names></name><name><surname>Zeiss</surname><given-names>CJ</given-names></name><name><surname>Rodriguez-Gil</surname><given-names>DJ</given-names></name><name><surname>Wilson</surname><given-names>JH</given-names></name><name><surname>Furtak</surname><given-names>K</given-names></name><name><surname>Rutkowski</surname><given-names>DT</given-names></name><name><surname>Kaufman</surname><given-names>RJ</given-names></name><name><surname>Ruse</surname><given-names>CI</given-names></name><name><surname>Yates</surname><given-names>JR</given-names><suffix>III</suffix></name><name><surname>Perrin</surname><given-names>S</given-names></name><name><surname>Feany</surname><given-names>MB</given-names></name><name><surname>Horwich</surname><given-names>AL</given-names></name></person-group><year>2009</year><article-title>Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>106</volume><fpage>1392</fpage><lpage>1397</lpage><pub-id pub-id-type="doi">10.1073/pnas.0813045106</pub-id></element-citation></ref><ref id="bib92"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wegrzyn</surname><given-names>RD</given-names></name><name><surname>Bapat</surname><given-names>K</given-names></name><name><surname>Newnam</surname><given-names>GP</given-names></name><name><surname>Zink</surname><given-names>AD</given-names></name><name><surname>Chernoff</surname><given-names>YO</given-names></name></person-group><year>2001</year><article-title>Mechanism of prion loss after Hsp104 inactivation in yeast</article-title><source>Molecular and Cellular Biology</source><volume>21</volume><fpage>4656</fpage><lpage>4669</lpage><pub-id pub-id-type="doi">10.1128/MCB.21.14.4656-4669.2001</pub-id></element-citation></ref><ref id="bib93"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Weisberg</surname><given-names>SJ</given-names></name><name><surname>Lyakhovetsky</surname><given-names>R</given-names></name><name><surname>Werdiger</surname><given-names>AC</given-names></name><name><surname>Gitler</surname><given-names>AD</given-names></name><name><surname>Soen</surname><given-names>Y</given-names></name><name><surname>Kaganovich</surname><given-names>D</given-names></name></person-group><year>2012</year><article-title>Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>109</volume><fpage>15811</fpage><lpage>15816</lpage><pub-id pub-id-type="doi">10.1073/pnas.1205829109</pub-id></element-citation></ref><ref id="bib94"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Winkler</surname><given-names>J</given-names></name><name><surname>Tyedmers</surname><given-names>J</given-names></name><name><surname>Bukau</surname><given-names>B</given-names></name><name><surname>Mogk</surname><given-names>A</given-names></name></person-group><year>2012</year><article-title>Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation</article-title><source>The Journal of Cell Biology</source><volume>198</volume><fpage>387</fpage><lpage>404</lpage><pub-id pub-id-type="doi">10.1083/jcb.201201074</pub-id></element-citation></ref><ref id="bib95"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wolfe</surname><given-names>KJ</given-names></name><name><surname>Ren</surname><given-names>HY</given-names></name><name><surname>Trepte</surname><given-names>P</given-names></name><name><surname>Cyr</surname><given-names>DM</given-names></name></person-group><year>2013</year><article-title>The Hsp70/90 cochaperone, Sti1, suppresses proteotoxicity by regulating spatial quality control of amyloid-like proteins</article-title><source>Molecular Biology of the Cell</source><volume>24</volume><fpage>3588</fpage><lpage>3602</lpage><pub-id pub-id-type="doi">10.1091/mbc.E13-06-0315</pub-id></element-citation></ref><ref id="bib96"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wolff</surname><given-names>S</given-names></name><name><surname>Weissman</surname><given-names>JS</given-names></name><name><surname>Dillin</surname><given-names>A</given-names></name></person-group><year>2014</year><article-title>Differential scales of protein quality control</article-title><source>Cell</source><volume>157</volume><fpage>52</fpage><lpage>64</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2014.03.007</pub-id></element-citation></ref><ref id="bib97"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yamamoto</surname><given-names>A</given-names></name><name><surname>Lucas</surname><given-names>JJ</given-names></name><name><surname>Hen</surname><given-names>R</given-names></name></person-group><year>2000</year><article-title>Reversal of neuropathology and motor dysfunction in a conditional model of huntington's disease</article-title><source>Cell</source><volume>101</volume><fpage>57</fpage><lpage>66</lpage><pub-id pub-id-type="doi">10.1016/S0092-8674(00)80623-6</pub-id></element-citation></ref><ref id="bib98"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>Z</given-names></name><name><surname>Hong</surname><given-names>JY</given-names></name><name><surname>Derkatch</surname><given-names>IL</given-names></name><name><surname>Liebman</surname><given-names>SW</given-names></name></person-group><year>2013</year><article-title>Heterologous gln/asn-rich proteins impede the propagation of yeast prions by altering chaperone availability</article-title><source>PLOS Genetics</source><volume>9</volume><fpage>e1003236</fpage><pub-id pub-id-type="doi">10.1371/journal.pgen.1003236</pub-id></element-citation></ref><ref id="bib99"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname><given-names>A</given-names></name><name><surname>Shibata</surname><given-names>Y</given-names></name><name><surname>Shah</surname><given-names>B</given-names></name><name><surname>Calamini</surname><given-names>B</given-names></name><name><surname>Lo</surname><given-names>DC</given-names></name><name><surname>Morimoto</surname><given-names>RI</given-names></name></person-group><year>2014</year><article-title>Protein aggregation can inhibit clathrin-mediated endocytosis by chaperone competition</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>111</volume><fpage>E1481</fpage><lpage>E1490</lpage><pub-id pub-id-type="doi">10.1073/pnas.1321811111</pub-id></element-citation></ref><ref id="bib100"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zeymer</surname><given-names>C</given-names></name><name><surname>Werbeck</surname><given-names>ND</given-names></name><name><surname>Schlichting</surname><given-names>I</given-names></name><name><surname>Reinstein</surname><given-names>J</given-names></name></person-group><year>2013</year><article-title>The molecular mechanism of Hsp100 chaperone inhibition by the prion curing agent guanidinium chloride</article-title><source>The Journal of Biological Chemistry</source><volume>288</volume><fpage>7065</fpage><lpage>7076</lpage><pub-id pub-id-type="doi">10.1074/jbc.M112.432583</pub-id></element-citation></ref><ref id="bib101"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhou</surname><given-names>C</given-names></name><name><surname>Slaughter</surname><given-names>BD</given-names></name><name><surname>Unruh</surname><given-names>JR</given-names></name><name><surname>Eldakak</surname><given-names>A</given-names></name><name><surname>Rubinstein</surname><given-names>B</given-names></name><name><surname>Li</surname><given-names>R</given-names></name></person-group><year>2011</year><article-title>Motility and segregation of Hsp104-associated protein aggregates in budding yeast</article-title><source>Cell</source><volume>147</volume><fpage>1186</fpage><lpage>1196</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2011.11.002</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04288.020</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Kelly</surname><given-names>Jeffery W</given-names></name><role>Reviewing editor</role></contrib><aff><institution>Scripps Research Institute</institution>, <country>United States</country></aff></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Spatial Quality Control Bypasses Cell-Based Limitations on Proteostasis To Promote Prion Curing” for consideration at <italic>eLife.</italic> Your article has been favorably evaluated by Randy Schekman (Senior editor), Jeffery W Kelly (Reviewing editor), and 3 reviewers.</p><p>The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>Serio and co-workers have submitted a well written and reasonably convincing manuscript demonstrating that spatial temporal retention of Hsp104 by [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> cells induces curing through the disassembly of existing Sup35 amyloid by Hsp104. Serio et al. showed that Hsp104 must engage heat-induced protein aggregates for a period that exceeds the time to the next cell division. As a result, Hsp104 is asymmetrically localized to the cells that experienced the thermal stress (<xref ref-type="fig" rid="fig4 fig7">Figures 4, 7</xref>), and this increase in chaperone accumulation promotes [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing in the same cells (<xref ref-type="fig" rid="fig4 fig7">Figures 4, 7</xref>). This study provides evidence that the engagement of Hsp104 with heat-induced misfolded Sup35 aggregates enhances proteostasis capacity by increasing the accumulation of this factor, perhaps beyond the level attainable by changes in gene expression (<xref ref-type="fig" rid="fig4">Figure 4</xref>), thereby enabling the disassembly of existing Sup35 amyloid (<xref ref-type="fig" rid="fig1 fig4">Figure 1, 4</xref>), restoring homeostasis to the mother cell. While the spatial engagement of proteostasis network components is not new, there are enough novel attributes to this work to render it an interesting <italic>eLife</italic> paper.</p><p>Please carefully consider the three thoughtful reviews pasted below when revising your manuscript. The points common to at least two reviews include:</p><p>1) It is important for the model presented in this work that Hsp104 interacts specifically with aggregated proteins, and that this interaction is dependent on the enzymatic activity of Hsp104. Is it possible to demonstrate this specific interaction, e.g. by co-immunoprecipitation? How does the sequestration of Hsp104 by heat-induced aggregates affect its activity? Is it solely the consequence of achieving high local concentrations?</p><p>2) A broader context here would be useful as a number of laboratories have made observations on Q-bodies (<xref ref-type="bibr" rid="bib22">Escusa-Toret et al., 2013</xref>), iPOD, JUNQ (<xref ref-type="bibr" rid="bib37">Kaganovich et. al., 2008</xref>), aggresomes (<xref ref-type="bibr" rid="bib34">Johnston et. al., 1998</xref>), and spatial sequestration and symmetrical inheritance in yeast (<xref ref-type="bibr" rid="bib76">Song et al., 2014</xref>) have demonstrated that aggregates have spatial restrictions and numerous papers have shown that multiple chaperones are associated with these aggregate structures using a wide range of proteomic and co-localization methods, often with substantial consequences on cellular activity. Some of these would seem to have opposing outcomes, which then poses questions how the mother-daughter cell relationship of yeast relates to metazoan cell division, and broader relevance to neurons being post-mitotic. Integrating these observations into this work without dramatically expanding the Discussion would be useful.</p><p>3) The use of Sup35 as a client and the yeast cellular environment while highly relevant on its own, may not offer broadly relevant concepts than can be extrapolated to metazoans. For example, Hsp104 as a disaggregase activity is restricted to prokaryotes, plants, and yeast whereas a corresponding class of activity in metazoans appears to be a reconfiguration of the Hsp70 and J-domain apparatus by Hsp110, leading to questions whether these concepts on Hsp104 are specific to yeast (which is still interesting) or can help us to understand the larger questions posed by the authors on the upper limit of the cellular environment. Some brief comments addressing this issue will allow the reader to comprehend the bigger picture.</p><p><italic>Reviewer #1</italic>:</p><p>Protein aggregation is linked to many degenerative diseases, either through loss of function of the aggregated protein, or through a novel gain of toxic function of the aggregate. It is therefore of high importance to identify pathways that can prevent or reverse protein aggregation. The submitted manuscript investigates the relationship between protein aggregation and the aggregate-remodeling factor Hsp104.</p><p>The authors report the seemingly paradoxical observation that incubation of cells at an elevated temperature of 40<sup>°</sup>C strongly reduces aggregation of the model substrate Sup35 in its prion form [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup>. Heat shock treatment reduces the size of the aggregates and increases the amount of soluble Sup35 after a recovery period. This process is dependent on the activity of Hsp104. The authors convincingly demonstrate that heat-induced aggregates of proteins other than Sup35 asymmetrically retain Hsp104 during cell division, in a process that is dependent of the enzymatic activity of Hsp104, and thereby increase Hsp104 concentration in mother cells to a level that allows Sup35 disaggregation and prion curing during recovery from heat stress.</p><p>The concept that the (temporary) sequestration of important proteostasis factors into an aggregate can be beneficial instead of detrimental is novel and exciting, and provides insight into an unanticipated role of asymmetric cell division in protein aggregate management. The paper is clearly written and the main conclusions are generally well supported by data.</p><p>There are however a few points the authors should address:</p><p>1) It is important for the model presented in this work that Hsp104 interacts specifically with aggregated proteins, and that this interaction is dependent on the enzymatic activity of Hsp104. Is it possible to demonstrate this specific interaction, e.g. by co-immunoprecipitation?</p><p>2) The use of nocodazole in <xref ref-type="fig" rid="fig5">Figure 5</xref> seems problematic as it may influence several other pathways that are involved in the formation and degradation of aggregates. Since the cell cycle analysis is not contributing critically to the overall model, it might be better to remove this section, or move it to the supplement.</p><p>3) Some of the colors in <xref ref-type="fig" rid="fig4">Figure 4</xref> are hard to distinguish (especially dark blue and black in 4d) and are not explained in the panels.</p><p><italic>Reviewer #2</italic>:</p><p>Klaips et. al. (Serio) have explored the mechanism of curing of Sup35 prion aggregates upon exposure of yeast to transient thermal stress to test a hypothesis that the cellular environment sets limits on protein quality control (PQC). The authors show that exposure of yeast (S. cerevisiae) at specific stages of the cell cycle to short periods of thermal stress leads to the disassembly of Sup35 amyloid, and propose that this is due to the spatial sequestration of Hsp104 in the mother cell rather than the result of overall elevated levels of Hsp104 or other chaperones. The sequestration of Hsp104 is correlated with the accumulation of heat-induced, non-prion aggregates upon thermal stress and the data suggest that the disassembly of Sup35 is more a function of amplification efficiency than thermodynamic stability. FACS experiments coupled with the use of two different cell-cycle inhibitors demonstrated that prion curing was highly dependent upon the cell-cycle stage.</p><p>Overall, the experiments are well done, the results are intriguing and the paper is nicely presented. Some of the conclusions are correlative rather than causal, thus limiting the overall enthusiasm. Also, the use of Sup35 as a probe and the yeast cellular environment while highly relevant on its own, may not offer broadly relevant concepts than can be extrapolated to metazoans. For example, Hsp104 as a disaggregase activity is restricted to prokaryotes, plants, and yeast whereas a corresponding class of activity in metazoans appears to be a reconfiguration of the Hsp70 and J-domain apparatus by Hsp110, leading to questions whether these concepts on Hsp104 are specific to yeast (which is still interesting) or can help us to understand the larger questions posed by the authors on the upper limit of the cellular environment. How does the sequestration of Hsp104 by heat-induced aggregates affect its activity? Is it solely the consequence of achieving high local concentrations? An argument is made that it is the asymmetrical partitioning of Hsp104 by its association with thermal aggregates that leads to a sufficiently high concentration of Hsp104 to resolve Sup35 amyloids. What is this concentration, what is the nature and basis of the interaction? Will elevated levels of Hsp104 achieved using conditional systems or shield-based metastable DHFR achieve the same outcome? Finally, the effects of different temperature conditions are intriguing. What is the nature of the biophysical transformation of Sup35 in 30C cells compared to those exposed to 40C beyond that rather vague terminology of shift from SDS-resistant to SDS-sensitive? What is special about 40C? Is this a useful tool to understand more completely the cellular environment?</p><p>A broader context here would be useful as a number of laboratories have made observations on Q-bodies (<xref ref-type="bibr" rid="bib22">Escusa-Toret et al., 2013</xref>), iPOD, JUNQ (<xref ref-type="bibr" rid="bib37">Kaganovich et. al., 2008</xref>), aggresomes (<xref ref-type="bibr" rid="bib34">Johnston et. al., 1998</xref>), and spatial sequestration and symmetrical inheritance in yeast (<xref ref-type="bibr" rid="bib76">Song et al., 2014</xref>) have demonstrated that aggregates have spatial restrictions and numerous papers have shown that multiple chaperones are associated with these aggregate structures using a wide range of proteomic and co-localization methods, often with substantial consequences on cellular activity. Some of these would seem to have opposing outcomes, which then poses questions how the mother-daughter cell relationship of yeast relates to metazoan cell division, and broader relevance to neurons being post-mitotic.</p><p>Additional comments:</p><p>1) Evidence for involvement of Hsp104 in Sup35 amyloid disassembly comes both from the literature and new data presented here, that (i) the GdnHCl curing of [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> and a ∼50% decrease in curing efficiency was observed in a heterozygous deletion of Hsp104 (<xref ref-type="fig" rid="fig2">Figure 2a, b</xref>), (ii) Hsp104 was asymmetric localized upon thermal stress (<xref ref-type="fig" rid="fig4 fig6">Figure 4, 6</xref>) and (iii) no significant changes were observed for Hsp104, Ssa1 and Sis1 chaperone expression levels upon heat stress (<xref ref-type="fig" rid="fig3">Figure 3</xref>). This establishes necessity, but not sufficiency and therefore does not rule out other 'factor(s)' induced upon heat stress that could contribute to amyloid disassembly.</p><p>These arguments can be strengthened by: (a) demonstrating whether the expression of other chaperone candidates (ie., other Hsp70 and Hsp40 isoforms) are affected by these thermal stress conditions. For example, there could be small changes in the expression of a number of chaperones that affects the cellular environment and enables amyloid resolubilization. Examples of candidates to test include chaperones examined in studies by <xref ref-type="bibr" rid="bib7">Chernoff et. al., 1999</xref>; <xref ref-type="bibr" rid="bib75">Shorter and Lindquist, 2008</xref>; <xref ref-type="bibr" rid="bib17">Duennwald et. al., 2012</xref> and <xref ref-type="bibr" rid="bib94">Winkler et. al., 2012</xref>, and (b) showing the over-expression data, currently not shown (p.18) that establishes that the o/e of Hsp104 alone, to the same level achieved in this study through its asymmetric retention, suffices to induce curing.</p><p>2) The authors' evidence for the formation of heat-induced, non-prion aggregates that accumulate upon heat stress and sequester Hsp104 in the mother cell rests on SDD-AGE (<xref ref-type="fig" rid="fig1">Figure 1c</xref>), differential centrifugation (<xref ref-type="fig" rid="fig3">Figure 3b</xref>) and that formation of aggregates correlates with other Hsp104 specific observations.</p><p>The protocol used for differential centrifugation is not sufficiently well described. It appears that the aggregates were quantified using a Bradford assay after pelleting in a tabletop centrifuge and a mild detergent wash. If the aggregates were not denatured before quantification, the presence of large particles may cause artifacts while making colorimetric measurements in a spectrophotometer. Since the quantification of these aggregates is an important aspect of this story, the authors should quantify aggregate formation with more vigor, using alternative procedures such as sucrose-density gradients and ultra-centrifugation to separate aggregates from other cellular debris. More central, the differential accumulation of aggregates in mother vs. daughter cells should be quantified and compared to the values on the differential retention of Hsp104.</p><p>3) An important conclusion is that the heat-induced aggregates directly retain Hsp104, but there is no evidence for the direct interaction between these two factors, other than strong correlation. This would be greatly strengthened by an experiment that isolates these aggregates (see point 2 above) and demonstrates the presence of Hsp104 bound to these aggregates, using a digest and mass spectrometric quantification, for example. Alternatively, the authors could perform a co-IP experiment if they had more information on one of the proteins that is a constituent of these heat-induced aggregates.</p><p>4) Have the authors ruled out an effect on protein synthesis? Heat shock, in particular, is well known to inhibit protein synthesis, which could affect the flux, and shift equilibria in the proper cellular environment towards dissociation?</p><p>5) A point is regarding the relevance of the conclusions the authors make on the existence of cell-based limitations that preclude amyloid resolubilization in vivo to disease conditions. Since it is clear from their data that the cell-cycle stage is an important determinant that affects the success of resolubilization in vivo, this would not influence our understanding of the PQC in non-dividing neuronal cells. The authors should place their results in the broader context of protein misfolding diseases in the discussion.</p><p>6) Another point in the discussion on spatial engagement of PQC factors and effects on proteostasis capacity is a recent observation (<xref ref-type="bibr" rid="bib99">Yu et. al., 2014</xref>) on aggregate-associated sequestration of Hsc70 leading to down regulation of clathrin-mediated endocytosis. Does this suggest that the interactions of chaperones with aggregates could have different outcomes?</p><p><italic>Reviewer #3:</italic></p><p>In this study, Klaips et al. perform an elegant set of studies, which demonstrate that Hsp104 dissolves Sup35 prions that encode weak [PSI+] at elevated temperatures in yeast. Interestingly, this curing process depends upon an unprecedented spatiotemporal mechanism. Thus, asymmetric Hsp104 retention by heat induced, non-prion aggregates in late cell cycle-stage cells, leads to high enough Hsp104 levels to promote dissolution of Sup35 prions encoding weak [PSI+]. Upon recognizing this potential mechanism, the authors then cleverly tweak the system to enable curing of Sup35 prions that encode strong [PSI+]. Although Sup35 prion dissolution by Hsp104 is not very surprising per se, the spatiotemporal mechanism revealed by these studies is entirely novel and should interest a broad audience. Indeed, this paper will raise awareness that spatiotemporal mechanisms can have a profound impact on proteostasis and amyloid resolution. In my view, the study is high quality and is carefully conducted and controlled, and I do not have any issues with the experiments or data as presented. A limitation is that several conclusions are based upon correlative evidence, but it has already been shown in vitro that Hsp104 can rapidly dissolve Sup35 prions. My only minor objections concern several statements in the Introduction that are incorrect or misleading:</p><p>1) 'but a direct demonstration of amyloid resolubilization in vivo has yet to be reported in any system'. Serio and colleagues have themselves already convincingly demonstrated amyloid resolublization by Hsp104 in vivo in their 2011 NSMB paper (<xref ref-type="bibr" rid="bib15">DiSalvo et al., 2011</xref>). <xref ref-type="bibr" rid="bib61">Park et al. 2014</xref> also provide compelling evidence that Hsp104 promotes amyloid solubilization in vivo. Moreover, there are numerous examples of amyloid clearance in conditional animal models of neurodegenerative disease models (e.g. Yamamoto et al. Cell 2000; Lim et al., J. Neuorosci. 2011). Hence, this statement is incorrect and misleading.</p><p>2) 'Rather, the effects of chaperone overexpression have been shown in some cases to be independent of their catalytic function (<xref ref-type="bibr" rid="bib5">Carmichael et al., 2000</xref>, <xref ref-type="bibr" rid="bib6">Chai et al., 1999</xref>, <xref ref-type="bibr" rid="bib33">Jana et al., 2000</xref>, <xref ref-type="bibr" rid="bib61">Park et al., 2014</xref>)'. This statement is a little misleading as in several cases the effect has been shown to depend on catalytic function (e.g. <xref ref-type="bibr" rid="bib12">Cushman-Nick et al. 2013</xref> PLoS Genet.)</p><p>3) 'Extracts from <italic>C. elegans</italic> and mammalian tissues and cell lines similarly promote amyloid solubilization (Cohen et al., 53 2006, Murray et al., 2010).' This statement should also be revised since Murray et al. subsequently published (Protein Sci. 2013 Nov;22(11):1531-41.) that 'our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity.'</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04288.021</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) It is important for the model presented in this work that Hsp104 interacts specifically with aggregated proteins, and that this interaction is dependent on the enzymatic activity of Hsp104. Is it possible to demonstrate this specific interaction, e.g. by co-immunoprecipitation</italic>?</p><p>We agree with the reviewers that the interaction of Hsp104 with heat-induced aggregates is an essential component of our model. To address this point, we have added two new pieces of data to the manuscript. First, we demonstrate that Hsp104 co-localizes with a model substrate (firefly luciferase-mCherry), which denatures upon thermal stress and is reactivated in an Hsp104-dependent manner (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1e</xref>; Parsell et al. 1994). Second, we demonstrate by immunocapture of Hsp104-GFP, SDS-PAGE and general protein staining, that Hsp104-GFP associates with a larger number of proteins upon thermal stress (<xref ref-type="fig" rid="fig4">Figure 4a</xref>). Importantly, the number of co-captured proteins increases with the severity of the stress (i.e. 40°C > 37°C) and decreases with GdnHCl treatment (<xref ref-type="fig" rid="fig4">Figure 4a</xref>), which blocks Hsp104 activity. Thus, the engagement of Hsp104-GFP with heat-induced substrates parallels the efficiency of [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing, which is increased at 40°C relative to 37°C (<xref ref-type="fig" rid="fig1">Figure 1a, b</xref>) and is inhibited by treatment with GdnHCl (<xref ref-type="fig" rid="fig2">Figure 2a</xref>), providing additional support for our model of prion curing.</p><p><italic>How does the sequestration of Hsp104 by heat-induced aggregates affect its activity? Is it solely the consequence of achieving high local concentrations</italic>?</p><p>Our studies together indicate that both the high local concentration of Hsp104 and its activity are required for [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing. To summarize here, curing correlates only with the highest levels of Hsp104 accumulation (37°C → 40°C and 40°C; <xref ref-type="fig" rid="fig1">Figure 1a, b</xref> and <xref ref-type="fig" rid="fig5">Figure 5a</xref>) and is blocked by treatments that reduce Hsp104 asymmetric accumulation (GdnHCl treatment [<xref ref-type="fig" rid="fig2 fig5">Figure 2a, 5d</xref>] and disruption of <italic>BNI1</italic> [<xref ref-type="fig" rid="fig5">Figure 5b, c</xref>]). In addition to these observations, we now include an additional experiment, in which cells are treated with GdnHCl after the thermal stress (<xref ref-type="fig" rid="fig5s1">Figure 4—figure supplement 1f, g</xref> and <xref ref-type="fig" rid="fig5">Figure 5d</xref>). Under these conditions, Hsp104-GFP still localizes to heat-induced aggregates (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1f</xref>) and is asymmetrically retained (<xref ref-type="fig" rid="fig5">Figure 5d</xref>), but [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing is reduced (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1g</xref>), indicating that both Hsp104-GFP localization and activity are required for curing.</p><p>Prolonged overexpression of Hsp104 from a galactose-inducible promoter alone has been previously reported to induce prion curing of both [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> (<xref ref-type="bibr" rid="bib7">Chernoff <italic>et al.</italic> 1995</xref>) and [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> (<xref ref-type="bibr" rid="bib92">Wegrzyn <italic>et al.</italic> 2001</xref>). We have also previously shown that lower-level overexpression (∼5-fold) is insufficient to cure [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> (<xref ref-type="bibr" rid="bib15">DiSalvo et al. 2011</xref>). Using short induction of a galactose-inducible Hsp104 construct that transiently raises Hsp104 accumulation to the level observed with thermal stress (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1c</xref>), we now demonstrate that [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing does occur, but at a lower frequency than with thermal stress (<xref ref-type="fig" rid="fig1">Figure 1b</xref> and <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1d</xref>). Studies from the Ter-Avanesyan lab revealed that Sup35 SDS-resistant aggregates isolated from a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> strain increase in size upon Hsp104 overexpression (<xref ref-type="bibr" rid="bib45">Kryndushkin <italic>et al.</italic> 2003</xref>). We have confirmed this result and further demonstrated that the same holds true for SDS-resistant aggregates isolated from a [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> strain (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1e</xref>). This observation is in contrast to our analysis of Sup35 SDS-resistant aggregates following thermal stress. Under thermal stress, both [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> and [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> aggregates decrease in size (<xref ref-type="fig" rid="fig1">Figure 1c</xref>), and in the latter case, are disassembled to release soluble Sup35 (<xref ref-type="fig" rid="fig1">Figure 1d</xref>). As we address in the Discussion, these observations together indicate that the mechanisms of curing by specific overexpression of Hsp104 and by thermal stress are distinct.</p><p><italic>2) A broader context here would be useful as a number of laboratories have made observations on Q-bodies (</italic><xref ref-type="bibr" rid="bib22"><italic>Escusa-Torete et. al., 2013</italic></xref><italic>), iPOD, JUNQ (</italic><xref ref-type="bibr" rid="bib37"><italic>Kaganovich et. al., 2008</italic></xref><italic>), aggresomes (</italic><xref ref-type="bibr" rid="bib34"><italic>Johnston et. al., 1998</italic></xref><italic>), and spatial sequestration and symmetrical inheritance in yeast (</italic><xref ref-type="bibr" rid="bib76"><italic>Song et al., 2014</italic></xref><italic>) have demonstrated that aggregates have spatial restrictions and numerous papers have shown that multiple chaperones are associated with these aggregate structures using a wide range of proteomic and co-localization methods, often with substantial consequences on cellular activity. Some of these would seem to have opposing outcomes, which then poses questions how the mother-daughter cell relationship of yeast relates to metazoan cell division, and broader relevance to neurons being post-mitotic. Integrating these observations into this work without dramatically expanding the Discussion would be useful</italic>.</p><p>We thank the reviewers for these helpful suggestions. We have reworked the Discussion to more clearly link our studies to the literature available on spatial quality control and to address the distinctions between our observations of enhanced proteostasis capacity and those in the literature demonstrating reduced proteostasis capacity. We believe that these distinct outcomes arise from imbalance between chaperones and substrates created by significant perturbations of the cellular environment beyond what can be achieved naturally.</p><p>While the limitations on proteostasis capacity imposed by chaperone partitioning are clearly not applicable to post-mitotic neurons, we believe that the issues raised by a comparison of our work with those previous studies highlight the importance of system balance, particularly the role of chaperone-substrate dynamics in determining proteostasis capacity. These latter points are likely to be independent of cell-type and are clarified in the revised Discussion.</p><p><italic>3) The use of Sup35 as a client and the yeast cellular environment while highly relevant on its own, may not offer broadly relevant concepts than can be extrapolated to metazoans. For example, Hsp104 as a disaggregase activity is restricted to prokaryotes, plants, and yeast whereas a corresponding class of activity in metazoans appears to be a reconfiguration of the Hsp70 and J-domain apparatus by Hsp110, leading to questions whether these concepts on Hsp104 are specific to yeast (which is still interesting) or can help us to understand the larger questions posed by the authors on the upper limit of the cellular environment. Some brief comments addressing this issue will allow the reader to comprehend the bigger picture</italic>.</p><p>The reviewer raises an important point that we failed to address in the original submission. We have now included a paragraph on mammalian disaggregase activity in the Discussion, including the similarities and distinctions with the yeast system.</p><p>Reviewer #1:</p><p><italic>1) It is important for the model presented in this work that Hsp104 interacts specifically with aggregated proteins, and that this interaction is dependent on the enzymatic activity of Hsp104</italic>. <italic>Is it possible to demonstrate this specific interaction, e.g. by co-immunoprecipitation?</italic></p><p>Please see the response to point 1 under “Response to points common to at least two reviews” above.</p><p><italic>2) The use of nocodazole in</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref> <italic>seems problematic as it may influence several other pathways that are involved in the formation and degradation of aggregates. Since the cell cycle analysis is not contributing critically to the overall model, it might be better to remove this section, or move it to the supplement</italic>.</p><p>We were also concerned about off-pathway effects of nocodazole treatment, which prompted us to assess both Hsp104 expression and localization in the presence of this treatment (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>), and to further test the suggestions raised by these arrest experiments using single-cell analyses in asynchronous cultures (<xref ref-type="fig" rid="fig7">Figure 7b, c</xref>). We found no deviations from unarrested cells in either case. We believe that the cell-cycle dependence of the curing effect (<xref ref-type="fig" rid="fig6">Figure 6</xref>) is an important aspect of the model because it identifies the importance of heat-induced aggregate resolution relative to cell division as a key component of [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing. Both reviewer 2 and reviewer 3 concur with the importance of this point in their reviews, and we have therefore retained the figure in the main figures of the revised manuscript.</p><p><italic>3) Some of the colors in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref> <italic>are hard to distinguish (especially dark blue and black in 4d) and are not explained in the panels</italic>.</p><p>We have changed the colors in panel <xref ref-type="fig" rid="fig4">Figure 4d</xref> (<xref ref-type="fig" rid="fig5">Figure 5b</xref> in the revised manuscript) to green to increase contrast and have updated the figure legend to reflect this change.</p><p>Reviewer #2:</p><p><italic>Some of the conclusions are correlative rather than causal, thus limiting the overall enthusiasm</italic>.</p><p>We have addressed the specific comments of this reviewer below.</p><p><italic>Also, the use of Sup35 as a probe and the yeast cellular environment while highly relevant on its own, may not offer broadly relevant concepts than can be extrapolated to metazoans. For example, Hsp104 as a disaggregase activity is restricted to prokaryotes, plants, and yeast whereas a corresponding class of activity in metazoans appears to be a reconfiguration of the Hsp70 and J-domain apparatus by Hsp110, leading to questions whether these concepts on Hsp104 are specific to yeast (which is still interesting) or can help us to understand the larger questions posed by the authors on the upper limit of the cellular environment</italic>.</p><p>Please see the response to point 4 under “Response to points common to at least two reviews” above.</p><p><italic>How does the sequestration of Hsp104 by heat-induced aggregates affect its activity</italic>? <italic>Is it solely the consequence of achieving high local concentrations?</italic></p><p>Please see the response to point 2 under “Response to points common to at least two reviews” above.</p><p><italic>An argument is made that it is the asymmetrical partitioning of Hsp104 by its association with thermal aggregates that leads to a sufficiently high concentration of Hsp104 to resolve Sup35 amyloids. What is this concentration, what is the nature and basis of the interaction</italic>?</p><p>On the basis of our microfluidic analyses, curing of [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> occurs when Hsp104 levels are increased ∼3.5-fold by thermal stress, relative to 30°C (<xref ref-type="table" rid="tbl1">Table 1</xref>). The interaction of Hsp104 with heat-induced substrates, as assessed by localization (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>) and immunocapture (<xref ref-type="fig" rid="fig4">Figure 4a</xref>), requires its enzymatic activity (i.e. is blocked by GdnHCl treatment). These points have been clarified in the text with the addition of new data as detailed above.</p><p><italic>Will elevated levels of Hsp104 achieved using conditional systems or shield-based metastable DHFR achieve the same outcome</italic>?</p><p>Please see response to point 2 under “Response to points common to at least two reviews” above for a response to the question on specific elevation of Hsp104 levels.</p><p>For model substrates, we have not attempted this precise experiment; however, in our other recently published work (<xref ref-type="bibr" rid="bib29">Holmes <italic>et al.</italic> 2014</xref>), we note that the appearance of misfolded proteins in a [<italic>PSI</italic><sup><italic>+</italic></sup>] yeast strain disrupted for the N-terminal acetyltransferase NatA leads to a decrease in the size of SDS-resistant Sup35 aggregates, but this effect is not sufficient to induce prion curing. Under these conditions, Hsp104 expression is elevated ∼3-fold, but we see no evidence of asymmetric localization by microscopy presumably due to the continued misfolding of newly-made proteins in both mother and daughter cells. Thus, both elevation of Hsp104 and its asymmetric localization are required for [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing, as noted above in point 2 under “Response to points common to at least two reviews.”</p><p><italic>Finally, the effects of different temperature conditions are intriguing. What is the nature of the biophysical transformation of Sup35 in 30C cells compared to those exposed to 40C beyond that rather vague terminology of shift from SDS-resistant to SDS-sensitive</italic>?</p><p>We have previously demonstrated that the prion domain Sup35 transitions from an SDS-sensitive form in its monomeric state to an SDS-resistant form in its amyloid state both <italic>in vitro</italic> (<xref ref-type="bibr" rid="bib72">Serio <italic>et al.</italic> 2000</xref>) and <italic>in vivo</italic> (<xref ref-type="bibr" rid="bib70">Satpute-Krishnan and Serio, 2005</xref>). We have clarified this point in the text.</p><p><italic>What is special about 40C? Is this a useful tool to understand more completely the cellular environment</italic>?</p><p>Based on our studies, 40°C-treated cells accumulate more heat-induced aggregates than cells treated at 37°C (<xref ref-type="fig" rid="fig3">Figure 3b</xref>). This increase in accumulation correlates with an increase in the Hsp104 interactome (<xref ref-type="fig" rid="fig4">Figure 4a</xref>), an increase in asymmetric localization of Hsp104 (<xref ref-type="fig" rid="fig4 fig5">Figure 4b, 5a</xref>) and an increase in [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing (<xref ref-type="fig" rid="fig1">Figure 1a, b</xref>). Thus, the more severe thermal stress creates a unique proteostatic niche that allows Hsp104 to resolve amyloid <italic>in vivo</italic>. We have stressed these points in the revised manuscript both with the additional of new data and with modifications to the text.</p><p><italic>A broader context here would be useful as a number of laboratories have made observations on Q-bodies (</italic><xref ref-type="bibr" rid="bib22"><italic>Escusa-Torete et. al., 2013</italic></xref><italic>), iPOD, JUNQ (</italic><xref ref-type="bibr" rid="bib37"><italic>Kaganovich et. al., 2008</italic></xref><italic>), aggresomes (</italic><xref ref-type="bibr" rid="bib34"><italic>Johnston et. al., 1998</italic></xref><italic>), and spatial sequestration and symmetrical inheritance in yeast (</italic><xref ref-type="bibr" rid="bib76"><italic>Song et al., 2014</italic></xref><italic>) have demonstrated that aggregates have spatial restrictions and numerous papers have shown that multiple chaperones are associated with these aggregate structures using a wide range of proteomic and co-localization methods, often with substantial consequences on cellular activity. Some of these would seem to have opposing outcomes, which then poses questions how the mother-daughter cell relationship of yeast relates to metazoan cell division, and broader relevance to neurons being post-mitotic</italic>.</p><p>Please see the response to point 3 under “Response to points common to at least two reviews” above.</p><p><italic>Additional comments</italic>:</p><p><italic>1) Evidence for involvement of Hsp104 in Sup35 amyloid disassembly comes both from the literature and new data presented here, that (i) the GdnHCl curing of [PSI+]weak and a ∼50% decrease in curing efficiency was observed in a heterozygous deletion of Hsp104 (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2a, b</italic></xref><italic>), (ii) Hsp104 was asymmetric localized upon thermal stress (</italic><xref ref-type="fig" rid="fig4 fig6"><italic>Figure 4, 6</italic></xref><italic>) and (iii) no significant changes were observed for Hsp104, Ssa1 and Sis1 chaperone expression levels upon heat stress (</italic><xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref><italic>). This establishes necessity, but not sufficiency and therefore does not rule out other 'factor(s)' induced upon heat stress that could contribute to amyloid disassembly</italic>.</p><p><italic>These arguments can be strengthened by: (a) demonstrating whether the expression of other chaperone candidates (ie., other Hsp70 and Hsp40 isoforms) are affected by these thermal stress conditions. For example, there could be small changes in the expression of a number of chaperones that affects the cellular environment and enables amyloid resolubilization. Examples of candidates to test include chaperones examined in studies by</italic> <xref ref-type="bibr" rid="bib7"><italic>Chernoff et. al., 1999</italic></xref><italic>;</italic> <xref ref-type="bibr" rid="bib75"><italic>Shorter and Lindquist, 2008</italic></xref><italic>;</italic> <xref ref-type="bibr" rid="bib17"><italic>Duennwald et. al., 2012</italic></xref> <italic>and</italic> <xref ref-type="bibr" rid="bib94"><italic>Winkler et. al., 2012</italic></xref><italic>, and (b) showing the over-expression data, currently not shown (p.18) that establishes that the o/e of Hsp104 alone, to the same level achieved in this study through its asymmetric retention, suffices to induce curing</italic>.</p><p>We completely agree that our studies demonstrate necessity of Hsp104 asymmetric localization and activity for [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> curing. We believe that establishing these points represents a significant advance in the field because up until this point, the engagement of chaperones with substrates has been correlated with reduced, not enhanced, activity. As we address in the Discussion, we believe that our system, which retains all factors within their native balance, is a major reason for this distinction.</p><p>We also agree that there are likely to be other factors that are necessary to promote Sup35 amyloid disassembly in response to thermal stress. In this manuscript, we focused the assessment of this possibility on factors that are known to be required for Hsp104-mediated fragmentation of Sup35 amyloid <italic>in vivo</italic> (Ssa1 and Sis1). We find no evidence for asymmetric localization of these factors (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1d, e</xref>).</p><p>With regard to curing by overexpression of Hsp104 alone, we have previously published this result for [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> (<xref ref-type="bibr" rid="bib15">Disalvo et al. 2011</xref>). For [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup>, please see the response to point 2 under “Response to points common to at least two reviews” above.</p><p><italic>2) The authors' evidence for the formation of heat-induced, non-prion aggregates that accumulate upon heat stress and sequester Hsp104 in the mother cell rests on SDD-AGE (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1c</italic></xref><italic>), differential centrifugation (</italic><xref ref-type="fig" rid="fig3"><italic>Figure 3b</italic></xref><italic>) and that formation of aggregates correlates with other Hsp104 specific observations</italic>.</p><p><italic>The protocol used for differential centrifugation is not sufficiently well described. It appears that the aggregates were quantified using a Bradford assay after pelleting in a tabletop centrifuge and a mild detergent wash. If the aggregates were not denatured before quantification, the presence of large particles may cause artifacts while making colorimetric measurements in a spectrophotometer. Since the quantification of these aggregates is an important aspect of this story, the authors should quantify aggregate formation with more vigor, using alternative procedures such as sucrose-density gradients and ultra-centrifugation to separate aggregates from other cellular debris. More central, the differential accumulation of aggregates in mother vs. daughter cells should be quantified and compared to the values on the differential retention of Hsp104</italic>.</p><p>We have clarified our protocol for quantifying aggregates. While we did not denature the proteins before Bradford analysis, our protocol does include a preclear step to remove cellular debris. In lieu of additional characterization of aggregates on their own, we have instead included a more rigorous assessment of the Hsp104 interactome by co-immunocapture, SDS-PAGE and general protein staining. This experiment leads to the same conclusion: Hsp104 interacts with more proteins following a 40°C thermal stress than following a 37°C thermal stress (<xref ref-type="fig" rid="fig4">Figure 4a</xref>).</p><p>We have not quantified the differential retention of aggregates following thermal stress because Hsp104GFP has been previously demonstrated to co-localize with model proteins (VHL, ubc9ts), which misfold upon thermal stress (<xref ref-type="bibr" rid="bib37">Kaganovich <italic>et al.</italic> 2008</xref>), and with oxidatively damaged proteins, which are asymmetrically retained in mother cells (<xref ref-type="bibr" rid="bib21">Erjavec et al. 2007</xref>). However, we now show that the asymmetrically retained Hsp104GFP foci co-localize with a model substrate firefly luciferase-mCherry upon thermal stress (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1e</xref>).</p><p><italic>3) An important conclusion is that the heat-induced aggregates directly retain Hsp104, but there is no evidence for the direct interaction between these two factors, other than strong correlation. This would be greatly strengthened by an experiment that isolates these aggregates (see point 2 above) and demonstrates the presence of Hsp104 bound to these aggregates, using a digest and mass spectrometric quantification, for example. Alternatively, the authors could perform a co-IP experiment if they had more information on one of the proteins that is a constituent of these heat-induced aggregates</italic>.</p><p>Please see the response to point 1 under “Response to points common to at least two reviews” above.</p><p><italic>4) Have the authors ruled out an effect on protein synthesis? Heat shock, in particular, is well known to inhibit protein synthesis, which could affect the flux, and shift equilibria in the proper cellular environment towards dissociation</italic>?</p><p>There are two pieces of evidence, included in our original submission, that argues against this possibility. First, Sup35 protein levels are unchanged by the thermal stress (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1a</xref>), a point that we now clarify in the text. Second, treatment with cycloheximide alone does not lead to Sup35 solubilization (<xref ref-type="fig" rid="fig1">Figure 1d</xref>), a point that we now highlight in the text.</p><p><italic>5) A point is regarding the relevance of the conclusions the authors make on the existence of cell-based limitations that preclude amyloid resolubilization in vivo to disease conditions. Since it is clear from their data that the cell-cycle stage is an important determinant that affects the success of resolubilization in vivo, this would not influence our understanding of the PQC in non-dividing neuronal cells. The authors should place their results in the broader context of protein misfolding diseases in the discussion</italic>.</p><p>Please see the response to point 3 under “Response to points common to at least two reviews” above.</p><p><italic>6) Another point in the discussion on spatial engagement of PQC factors and effects on proteostasis capacity is a recent observation (</italic><xref ref-type="bibr" rid="bib99"><italic>Yu et. al., 2014</italic></xref><italic>) on aggregate-associated sequestration of Hsc70 leading to down regulation of clathrin-mediated endocytosis. Does this suggest that the interactions of chaperones with aggregates could have different outcomes</italic>?</p><p>The reviewer raises an interesting point. It is certainly possible that sequestration of chaperones can lead to different outcomes in different situations. Another possibility is that the outcomes are dictated by the experimental conditions. Our studies are distinct from those of Yu <italic>et al.</italic> in that the latter study employs aggregation-prone proteins that are expressed to high levels, which could promote imbalance in the system, as we address in the Discussion. We are in the early stages of understanding non-transcriptional mechanisms for regulating chaperone activity in cells, and these distinctions highlight interesting lines of investigation to pursue in the future.</p><p>Reviewer #3:</p><p><italic>1) 'but a direct demonstration of amyloid resolubilization in vivo has yet to be reported in any system'. Serio and colleagues have themselves already convincingly demonstrated amyloid resolublization by Hsp104 in vivo in their 2011 NSMB paper (</italic><xref ref-type="bibr" rid="bib15"><italic>DiSalvo et al., 2011</italic></xref><italic>).</italic> <xref ref-type="bibr" rid="bib61"><italic>Park et al. 2014</italic></xref> <italic>also provide compelling evidence that Hsp104 promotes amyloid solubilization in vivo. Moreover, there are numerous examples of amyloid clearance in conditional animal models of neurodegenerative disease models (e.g. Yamamoto et al. Cell 2000; Lim et al., J. Neuorosci. 2011). Hence, this statement is incorrect and misleading</italic>.</p><p>We apologize for the confusion regarding this sentence. The text in question was meant to refer to studies in which chaperones were specifically overexpressed in amyloid model systems. We do agree with the reviewer that additional literature demonstrating amyloid clearance upon repression of synthesis of the amyloidogenic protein, as well as the effects of dominant-negative mutants are important to consider as well. We have re-written this part of the Introduction to highlight the facts that 1) chaperone overexpression has not been demonstrated to resolve amyloid <italic>in vivo</italic> and 2) amyloid clearance mechanisms must exist <italic>in vivo</italic>, although they are ineffective against continuously expressed, wildtype amyloid proteins.</p><p>With regard to the studies of Park <italic>et al.</italic> 2014, we disagree with the reviewer on the strength of the evidence supporting amyloid resolubilization <italic>in vivo</italic>. First, these studies are conducted under conditions of continued protein synthesis and over extended time frames, and as we have previously demonstrated (Satpute-Krishnan and Serio, 2005); newly-made protein quickly accumulates in a soluble form when Hsp104 activity is inhibited. Second, the interpretation of these microscopy experiments is inconsistent with previous biochemical studies that demonstrate an increase in the size of Sup35 aggregates upon Hsp104 overexpression, which we have reproduced for both the [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Strong</sup> and [<italic>PSI</italic><sup><italic>+</italic></sup>]<sup>Weak</sup> variants (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1e</xref>). We have clarified these points in the Discussion.</p><p><italic>2) 'Rather, the effects of chaperone overexpression have been shown in some cases to be independent of their catalytic function (</italic><xref ref-type="bibr" rid="bib5"><italic>Carmichael et al., 2000</italic></xref><italic>,</italic> <xref ref-type="bibr" rid="bib6"><italic>Chai et al., 1999</italic></xref><italic>,</italic> <xref ref-type="bibr" rid="bib33"><italic>Jana et al., 2000</italic></xref><italic>,</italic> <xref ref-type="bibr" rid="bib61"><italic>Park et al., 2014</italic></xref><italic>)'. This statement is a little misleading as in several cases the effect has been shown to depend on catalytic function (e.g.</italic> <xref ref-type="bibr" rid="bib12"><italic>Cushman-Nick et al. 2013</italic></xref> <italic>PLoS Genet</italic>.<italic>)</italic></p><p>Again, we apologize for the confusion. Our statement was meant to only refer to the citations listed (hence the “some”), but we can see how this narrow discussion of the literature can lead to confusion. Rather than expand to include the Cushman-Nick study, which incidentally shows that overexpression of Hsp104 promotes amyloid formation by MJD rather than clearance of existing amyloid, we have deleted this sentence.</p><p><italic>3) 'Extracts from</italic> C. elegans <italic>and mammalian tissues and cell lines similarly promote amyloid solubilization (Cohen et al., 53 2006, Murray et al., 2010).' This statement should also be revised since Murray et al. subsequently published (Protein Sci. 2013 Nov;22(11):1531-41.) that 'our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity.</italic>'</p><p>We thank the reviewer for pointing us to the Murray et al. 2013 manuscript. We have removed the previous references to disaggregase activity in metazoan extracts.</p></body></sub-article></article> |