Distinct J-protein functions promote amyloid diversity.

<p>(A) Sis1 has five distinct regions denoted using the following notation: DD, dimerization domain; GF, glycine and phenylalanine-rich region; GM, glycine and methionine-rich region; J, J domain; PBDs, peptide-binding domains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref014" target="_blank">14</a>]. (B) Different prions can be selected for or against depending on diverse and sometimes mutually exclusive Sis1 requirements. A construct of Sis1 lacking the GF region (Sis1-ΔGF) maintains all variants of [<i>PSI</i>⁺] but not [<i>RNQ</i>⁺] or [<i>URE3</i>] [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref007" target="_blank">7</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref021" target="_blank">21</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref022" target="_blank">22</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref026" target="_blank">26</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref027" target="_blank">27</a>]. Contrastingly, a construct of Sis1 consisting of only the J domain and GF region (Sis1-J-GF) is sufficient to maintain cell viability and to propagate some variants of [<i>RNQ</i>⁺] and strong but not weak variants of [<i>PSI</i>⁺] [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref021" target="_blank">21</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref022" target="_blank">22</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref044" target="_blank">44</a>], demonstrating the existence of mutually exclusive Sis1 requirements with regard to weak [<i>PSI</i>⁺] and [<i>RNQ</i>⁺]. Finally, either alteration of Sis1 results in the loss of [<i>URE3</i>], but deletion of the J protein Swa2 results in loss of [<i>URE3</i>] alone [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref026" target="_blank">26</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref033" target="_blank">33</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref034" target="_blank">34</a>], demonstrating that novel secondary J-protein requirements exist for some prions. Likewise, but omitted for clarity, the prion [<i>SWI</i>⁺] also exhibits a specific requirement for the J protein Ydj1, whereas all three other prions shown in the figure continue to propagate in a strain lacking Ydj1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref007" target="_blank">7</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref024" target="_blank">24</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref029" target="_blank">29</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref033" target="_blank">33</a>]. This figure is intended to illustrate the possible prions that could propagate in various cells lacking certain chaperone functions. It does not imply that all of these prions and prion variants have been simultaneously observed in a single yeast cell. Although yeast strains can harbor multiple different prions simultaneously, they are often antagonistic toward one another [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref049" target="_blank">49</a>], and weak and strong variants of the same prion are not stable due to competition [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006695#ppat.1006695.ref050" target="_blank">50</a>].</p

Summary statistics of student reactions and self-reported learning following the activity.

<p>Student affective and self-reported learning data were collected and pooled from five courses at three schools. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002351#pbio.1002351.s008" target="_blank">S6 Text</a> for survey questions and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002351#pbio.1002351.s010" target="_blank">S8 Text</a> for additional details regarding data collection and analysis; a representative listing of various student responses for each theme shown is given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002351#pbio.1002351.s010" target="_blank">S8 Text</a>.</p

Case introduction to be read aloud in class to begin the activity.

<p>Students are alerted in advance that this is a true story as part of the initial case study PowerPoint (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002351#pbio.1002351.s001" target="_blank">S1 PowerPoint</a>).</p

Changes in student perceptions about biology.

<p>Student survey data were collected in five independent sections of Introduction to Biology over two years (<i>n</i> = 195). Students were given a nine-question survey (upper panel) immediately before and following the activity. Questions 1–7 were selected for relevance from the CLASS-Bio survey [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002351#pbio.1002351.ref008" target="_blank">8</a>], while questions 8 and 9 were added to address issues that are specifically relevant to the activity but not present in CLASS-Bio. Expert responses are affirmative for questions 1–5 and 9, and negative for questions 6–8. Shifts in student responses were analyzed using a Wilcoxon signed-rank test. Individual test results with <i>p</i>-values less than 0.005 are indicated. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002351#pbio.1002351.s010" target="_blank">S8 Text</a> for additional details regarding data collection and analysis.</p

DryadData

Data used for analysis

Functional Diversification of Hsp40: Distinct J-Protein Functional Requirements for Two Prions Allow for Chaperone-Dependent Prion Selection

<div><p>Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or ‘strains’. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [<i>PSI</i>⁺] are strongly dependent upon overall variant stability. Notably, multiple strong [<i>PSI</i>⁺] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [<i>RNQ</i>⁺] prion propagation. In contrast, weak [<i>PSI</i>⁺] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [<i>RNQ</i>⁺] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [<i>PSI</i>⁺]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [<i>PSI</i>⁺]/[<i>RNQ</i>⁺] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [<i>PSI</i>⁺] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have been maintained in eukaryotic chaperone evolution.</p></div

[<i>RNQ⁺</i>] and weak [<i>PSI⁺</i>]^Sc37 are reciprocally maintained by Sis1ΔG/F and Sis1-121 when assayed simultaneously in the same yeast cells.

<p>(A and B) A <i>sis1</i>-Δ strain maintaining both [<i>RNQ</i>⁺] and [<i>PSI</i>⁺]^Sc37 by expression of Sis1 from a <i>URA3</i>-marked plasmid ([<i>SIS1</i>-Sis1, <i>URA3</i>]) was transformed by plasmids expressing Sis1ΔG/F, Sis1-121 or Hdj1 and subjected to plasmid shuffling by growth on 5-FOA containing media. The maintenance of [<i>PSI</i>⁺]^Sc37 was assayed by colony color on rich media whereas the maintenance of [<i>RNQ</i>⁺] was assayed by subsequent transformation of each shuffled strain by a Rnq1-GFP reporter plasmid ([<i>CUP1</i>-Rnq1-GFP]) followed by fluorescence microscopy analysis. (A) Color phenotype assays for [<i>PSI</i>⁺]^Sc37 or fluorescence patterns indicative of [<i>RNQ</i>⁺] maintenance (punctate) or loss (diffuse) are shown for representative transformants (<i>n</i>≥6) for each construct following loss of the [<i>SIS1</i>-Sis1, <i>URA3</i>] plasmid. Cells transformed with full-length Sis1 (<i>CEN</i>-Sis1) are included as a positive control for prion maintenance throughout the shuffling and prion-detection procedures. Parental [<i>PSI</i>⁺]^Sc37 cells (parent) and cells cured by growth in the presence of GdnHCl (parent cured) are included as positive and negative controls for colony color. (B) Maintenance or loss of each prion in the cells from (A) was independently confirmed by semi-denaturing detergent agarose gel electrophoresis (SDDAGE). Detergent resistant Sup35 aggregates indicative of the presence of [<i>PSI</i>⁺] (<i>upper image</i>) or detergent resistant Rnq1 aggregates indicative of the presence of [<i>RNQ</i>⁺] (<i>lower image</i>) were resolved by SDDAGE and visualized by immunoblot analysis using antibodies specific for Sup35 or Rnq1, respectively. Control [<i>PRION</i>⁺] and [<i>prion</i>⁻] cells were included for comparison in each case.</p

[<i>PSI</i>⁺] requirements for Sis1 are prion-variant dependent.

<p>(A and B) Sis1-plasmid shuffling experiments to examine the potential effects of Sis1 domain deletions on [<i>PSI</i>⁺] propagation. [<i>PSI</i>⁺] bearing cells were transformed by plasmids expressing Sis1 truncations or deletions and subjected to plasmid shuffling. Color phenotype assays are shown for representative transformants (<i>n</i>≥6 for each plasmid) following loss of the [<i>SIS1</i>-Sis1, <i>URA3</i>] plasmid and bearing either the strong [<i>PSI⁺</i>] variant [<i>PSI⁺</i>]^Sc4 (A), or the weak [<i>PSI⁺</i>] variant [<i>PSI⁺</i>]^Sc37 (B). Parental [<i>PSI</i>⁺] cells for each variant (parent) and cells cured by growth in the presence of GdnHCl (cured) are included as positive and negative controls for colony color. Cells expressing full-length Sis1 (Sis1) from a plasmid were used as a positive control for the stability of the prion throughout the plasmid-shuffling procedure. For clarity, images taken from different parts of the same plate have been arranged in columns. (C) Sis1 protein expression levels in cells containing the weak [<i>PSI⁺</i>] variant [<i>PSI⁺</i>]^Sc37 in the W303 genetic background. Cell extracts from isolates in panel B were subjected to immunoblot analysis using antibody specific for Sis1. A band cross-reacting with the Sis1 antibody is shown as a loading control. Dotted lines separate lanes taken from different parts of the same gel. (D) Maintenance or loss of [<i>PSI</i>⁺] in cells shown in (A) ([<i>PSI⁺</i>]^Sc4, <i>left</i>) and (B) ([<i>PSI⁺</i>]^Sc37, <i>right</i>) was also confirmed by semi-denaturing detergent agarose gel electrophoresis (SDDAGE). Detergent resistant Sup35 aggregates indicative of the presence of [<i>PSI</i>⁺] were resolved by SDDAGE and visualized by immunoblot analysis using an antibody specific for Sup35. Control [<i>PSI</i>⁺] and [<i>psi</i>⁻] cells for each variant were included for comparison.</p

[<i>PSI</i>⁺] requirements for Sis1 are strongly dependent upon overall variant strength.

<p>(A) [<i>PSI</i>⁺] cells of the W303 genetic background bearing either strong [<i>PSI</i>⁺] variants ([<i>PSI</i>⁺]^STR, [<i>PSI</i>⁺]^VH, and [<i>PSI</i>⁺]^93S) or the weak [<i>PSI</i>⁺] variant [<i>PSI</i>⁺]^VL were transformed by plasmids expressing Sis1 truncations or deletions and subjected to plasmid shuffling. Color phenotype assays are shown for representative transformants (<i>n</i>≥6 for each plasmid) following loss of the [<i>SIS1</i>-Sis1, <i>URA3</i>] plasmid. Parental [<i>PSI</i>⁺] cells for each variant (parent) and cells cured by growth in the presence of GdnHCl (cured) are included as positive and negative controls for colony color. Cells expressing full-length Sis1 (Sis1) from a plasmid were used as a positive control for the stability of the prion throughout the plasmid-shuffling procedure. For clarity, images taken from different parts of the same plate have been arranged in columns. (B) Maintenance or loss of [<i>PSI</i>⁺] in cells shown in (A) was also confirmed by semi-denaturing detergent agarose gel electrophoresis (SDDAGE). Detergent resistant Sup35 aggregates indicative of the presence of [<i>PSI</i>⁺] were resolved by SDDAGE and visualized by immunoblot analysis using an antibody specific for Sup35. Control [<i>PSI</i>⁺] and [<i>psi</i>⁻] cells for each variant were included for comparison.</p

Constructs of Sis1 used in plasmid-shuffling experiments.

<p>(<i>left</i>) Gene structure diagrams of Sis1 and Hdj1 (<i>DNAJB1</i>) expression constructs. Gene regions are denoted using the following notation: <i>J</i>, J-domain; <i>G/F</i>, glycine/phenylalanine-rich region; <i>G/M</i>, glycine/methionine-rich region; <i>CTD1/2</i>, C-terminal peptide-binding domains I and II; <i>DD</i>, dimerization domains. Dashed lines indicate where a region had been deleted; gray bars represent a C-terminal tag. (<i>right</i>) A summary of the genetic interactions either previously known or uncovered in this investigation. Prion maintenance and loss are represented by plus and minus signs, respectively; <i>n.v.</i> indicates that the construct does not support cell viability.</p