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

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

[<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

Hdj1, the human homolog of Sis1, maintains strong but not weak [<i>PSI⁺</i>] variants in two yeast genetic backgrounds.

<p>(A) <i>sis1-Δ</i> cells from two backgrounds (W303 and 74D-694) bearing each of the six variants of [<i>PSI</i>⁺] examined in this study were transformed by a plasmid expressing Hdj1 from a constitutive promoter (<i>p424</i>-<i>GPD</i>-Hdj1) and subjected to plasmid shuffling to remove the covering Sis1-expression plasmid (<i>p316-SIS1-Sis1</i>). Color phenotype assays (<i>left</i>) are shown for representative transformants (<i>n</i>≥6 for each strain) following loss of the [<i>SIS1</i>-Sis1, <i>URA3</i>] plasmid. Maintenance or loss of each [<i>PSI</i>⁺] variant in these cells was also confirmed by semi-denaturing detergent agarose gel electrophoresis (SDDAGE) (<i>right</i>). 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 were included for comparison. (B) Lysates of all cells from (A) were resolved by SDS-PAGE and visualized by immunoblotting with Hdj1-specific antibodies that do not cross-react with Sis1. A lysate of a strain expressing only full-length Sis1 is included as a control. A band cross-reacting with the Hdj1 antibody in all lanes is shown as a loading control (<i>black arrow</i>).</p

Sis1's J-domain and G/M region are sufficient to support cell viability and weak [<i>PSI</i>⁺] propagation.

<p>(A) [<i>PSI</i>⁺]^Sc37 cells of the 74D-694 genetic background were transformed by plasmids expressing Sis1 truncations and subjected to plasmid shuffling. Color phenotype assays are shown for representative transformants following loss of the [<i>SIS1</i>-Sis1, <i>URA3</i>] plasmid. Cells expressing full-length Sis1 (<i>CEN</i>-Sis1) are used as a positive control for both colony color and the stability of the prion throughout the plasmid-shuffling procedure, whereas cells from each transformant were also cured by growth in the presence of GdnHCl and are included as negative controls for colony color. (B) Maintenance of [<i>PSI</i>⁺]^Sc37 in cells shown in (B) 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>⁺]^Sc37 and [<i>psi</i>⁻] cells were included for comparison. (C) The expression of Sis1 constructs, and the absence of wild-type Sis1 expression, in the cells from (A) was determined by subjecting cell lysates to SDS-PAGE followed by immunoblot analysis with Sis1-specific antibodies. Dotted lines separate lanes taken from different parts of the same gel. A band corresponding to Sis1-206ΔG/F* in lane two is clearly visible whereas a faint band corresponding to the approximate size of Sis1-171ΔG/F* is present in the right-most lane (<i>black arrow</i>). A band cross-reacting with the Sis1 antibody is shown as a loading control.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p