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[SWI], the prion formed by the chromatin remodeling factor Swi1, is highly sensitive to alterations in Hsp70 chaperone system activity.

Hines JK, Li X, Du Z, Higurashi T, Li L, Craig EA - PLoS Genet. (2011)

Bottom Line: In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins.Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state.Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.

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[SWI+] is cured upon deletion of SSE1.[SWI+] cells were transformed with an SSE1-deletion cassette bearing a LEU2 marker and transformants selected on media lacking leucine. (A) sse1-Δ transformants were identified by loss of Sse1 expression, as visualized by SDS-PAGE and immunoblotting using antibody specific for Sse1. Cells which received the marker but maintained Sse1 expression were classified as SSE1 transformants and used as controls in subsequent experiments. Wild-type Sse1 expression is also shown for comparison (SSE1 control) (B and C) Two representative transformants (one sse1-Δ and one SSE1) are shown along with a [SWI+] control strain. (B) sse1-Δ cells regain robust growth on raffinose indicative of [SWI+] loss. To test for [SWI+] maintenance, transformants were streaked onto raffinose- or glucose-based media and growth rates compared to control strains. (C) Loss of prion-specific fluorescent puncta in sse1-Δ cells. The presence or absence of [SWI+] in transformants was also confirmed by subsequently transforming cells with a plasmid expressing Swi1NQ-YFP and scoring for the presence of characteristic punctuate foci. (D) Summary of results for 31 sse1-Δ and 11 SSE1 transformants scored for [SWI+] maintenance using both growth on raffinose and Swi1NQ-YFP aggregation assays. The number of transformants remaining [SWI+] is reported as a fraction of the total examined (Fraction [SWI+]).
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pgen-1001309-g006: [SWI+] is cured upon deletion of SSE1.[SWI+] cells were transformed with an SSE1-deletion cassette bearing a LEU2 marker and transformants selected on media lacking leucine. (A) sse1-Δ transformants were identified by loss of Sse1 expression, as visualized by SDS-PAGE and immunoblotting using antibody specific for Sse1. Cells which received the marker but maintained Sse1 expression were classified as SSE1 transformants and used as controls in subsequent experiments. Wild-type Sse1 expression is also shown for comparison (SSE1 control) (B and C) Two representative transformants (one sse1-Δ and one SSE1) are shown along with a [SWI+] control strain. (B) sse1-Δ cells regain robust growth on raffinose indicative of [SWI+] loss. To test for [SWI+] maintenance, transformants were streaked onto raffinose- or glucose-based media and growth rates compared to control strains. (C) Loss of prion-specific fluorescent puncta in sse1-Δ cells. The presence or absence of [SWI+] in transformants was also confirmed by subsequently transforming cells with a plasmid expressing Swi1NQ-YFP and scoring for the presence of characteristic punctuate foci. (D) Summary of results for 31 sse1-Δ and 11 SSE1 transformants scored for [SWI+] maintenance using both growth on raffinose and Swi1NQ-YFP aggregation assays. The number of transformants remaining [SWI+] is reported as a fraction of the total examined (Fraction [SWI+]).

Mentions: The function of Hsp70 chaperone machinery requires the action of NEFs to stimulate ADP/ATP exchange, and subsequently, peptide release [21]. Consistent with this requirement, one particular NEF of the Hsp110 family, Sse1, has been found to be important for the continued propagation of [URE3] and some weak strains of [PSI+], but not [RNQ+] or strong [PSI+] [43], [49]. To test whether [SWI+] also requires Sse1, we created sse1-Δ strains by transformation of a wild-type [SWI+] strain with an SSE1 deletion cassette bearing the LEU2 selectable marker. The absence of Sse1 expression was verified by immunoblot analysis (Figure 6A). All 31 resulting sse1-Δ transformants became [swi−], as judged by both their ability to grow robustly on raffinose-based media and by the absence of punctate fluorescence (Figure 6B–6D). In contrast, 11 transformants, which obtained the selectable marker but preserved wild-type Sse1 expression, maintained the prion, indicating that like [URE3] and weak [PSI+] strains, [SWI+] is lost upon SSE1 deletion.


[SWI], the prion formed by the chromatin remodeling factor Swi1, is highly sensitive to alterations in Hsp70 chaperone system activity.

Hines JK, Li X, Du Z, Higurashi T, Li L, Craig EA - PLoS Genet. (2011)

[SWI+] is cured upon deletion of SSE1.[SWI+] cells were transformed with an SSE1-deletion cassette bearing a LEU2 marker and transformants selected on media lacking leucine. (A) sse1-Δ transformants were identified by loss of Sse1 expression, as visualized by SDS-PAGE and immunoblotting using antibody specific for Sse1. Cells which received the marker but maintained Sse1 expression were classified as SSE1 transformants and used as controls in subsequent experiments. Wild-type Sse1 expression is also shown for comparison (SSE1 control) (B and C) Two representative transformants (one sse1-Δ and one SSE1) are shown along with a [SWI+] control strain. (B) sse1-Δ cells regain robust growth on raffinose indicative of [SWI+] loss. To test for [SWI+] maintenance, transformants were streaked onto raffinose- or glucose-based media and growth rates compared to control strains. (C) Loss of prion-specific fluorescent puncta in sse1-Δ cells. The presence or absence of [SWI+] in transformants was also confirmed by subsequently transforming cells with a plasmid expressing Swi1NQ-YFP and scoring for the presence of characteristic punctuate foci. (D) Summary of results for 31 sse1-Δ and 11 SSE1 transformants scored for [SWI+] maintenance using both growth on raffinose and Swi1NQ-YFP aggregation assays. The number of transformants remaining [SWI+] is reported as a fraction of the total examined (Fraction [SWI+]).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040656&req=5

pgen-1001309-g006: [SWI+] is cured upon deletion of SSE1.[SWI+] cells were transformed with an SSE1-deletion cassette bearing a LEU2 marker and transformants selected on media lacking leucine. (A) sse1-Δ transformants were identified by loss of Sse1 expression, as visualized by SDS-PAGE and immunoblotting using antibody specific for Sse1. Cells which received the marker but maintained Sse1 expression were classified as SSE1 transformants and used as controls in subsequent experiments. Wild-type Sse1 expression is also shown for comparison (SSE1 control) (B and C) Two representative transformants (one sse1-Δ and one SSE1) are shown along with a [SWI+] control strain. (B) sse1-Δ cells regain robust growth on raffinose indicative of [SWI+] loss. To test for [SWI+] maintenance, transformants were streaked onto raffinose- or glucose-based media and growth rates compared to control strains. (C) Loss of prion-specific fluorescent puncta in sse1-Δ cells. The presence or absence of [SWI+] in transformants was also confirmed by subsequently transforming cells with a plasmid expressing Swi1NQ-YFP and scoring for the presence of characteristic punctuate foci. (D) Summary of results for 31 sse1-Δ and 11 SSE1 transformants scored for [SWI+] maintenance using both growth on raffinose and Swi1NQ-YFP aggregation assays. The number of transformants remaining [SWI+] is reported as a fraction of the total examined (Fraction [SWI+]).
Mentions: The function of Hsp70 chaperone machinery requires the action of NEFs to stimulate ADP/ATP exchange, and subsequently, peptide release [21]. Consistent with this requirement, one particular NEF of the Hsp110 family, Sse1, has been found to be important for the continued propagation of [URE3] and some weak strains of [PSI+], but not [RNQ+] or strong [PSI+] [43], [49]. To test whether [SWI+] also requires Sse1, we created sse1-Δ strains by transformation of a wild-type [SWI+] strain with an SSE1 deletion cassette bearing the LEU2 selectable marker. The absence of Sse1 expression was verified by immunoblot analysis (Figure 6A). All 31 resulting sse1-Δ transformants became [swi−], as judged by both their ability to grow robustly on raffinose-based media and by the absence of punctate fluorescence (Figure 6B–6D). In contrast, 11 transformants, which obtained the selectable marker but preserved wild-type Sse1 expression, maintained the prion, indicating that like [URE3] and weak [PSI+] strains, [SWI+] is lost upon SSE1 deletion.

Bottom Line: In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins.Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state.Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.

Show MeSH
Related in: MedlinePlus