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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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Overexpression of Sis1 cures [SWI+].(A) Extracts were prepared from either wild-type (WT) or sis1-Δ cells expressing Sis1 from a centromeric plasmid, either under the control of its own (SIS1-Sis1) or the GPD (GPD-Sis1) promoter. The extracts were subjected to immunoblot analysis using either antibody specific for Sis1 or, as a loading control, Ssc1 (control). (A and B) Dashed lines in both figures indicate some sections of the blot or plate have been cropped for clarity. (B) Sis1 expressing cells described in (A) were passaged for one week and then the presence of [SWI+] was assessed by streaking onto raffinose media. Wild-type [SWI+] and [swi−] cells are shown as controls. (C) Loss of [SWI+] from cells shown in (B) was also confirmed by fluorescence analysis following transformation with a plasmid bearing the Swi1NQ-YFP construct.
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pgen-1001309-g003: Overexpression of Sis1 cures [SWI+].(A) Extracts were prepared from either wild-type (WT) or sis1-Δ cells expressing Sis1 from a centromeric plasmid, either under the control of its own (SIS1-Sis1) or the GPD (GPD-Sis1) promoter. The extracts were subjected to immunoblot analysis using either antibody specific for Sis1 or, as a loading control, Ssc1 (control). (A and B) Dashed lines in both figures indicate some sections of the blot or plate have been cropped for clarity. (B) Sis1 expressing cells described in (A) were passaged for one week and then the presence of [SWI+] was assessed by streaking onto raffinose media. Wild-type [SWI+] and [swi−] cells are shown as controls. (C) Loss of [SWI+] from cells shown in (B) was also confirmed by fluorescence analysis following transformation with a plasmid bearing the Swi1NQ-YFP construct.

Mentions: A comparison of the control experiments in Figure 1C and Figure 2B indicates that [SWI+] is more stable in a wild-type background than in the sis1-Δ [TETr-Sis1] strains. To test whether this loss was due to lower than normal Sis1 expression, we asked whether the prion would be stabilized by supplemental expression from a second plasmid. However, we found that the presence of the second Sis1 plasmid exacerbated prion loss (data not shown), suggesting that the instability of [SWI+] might be due to overexpression rather than underexpression. Thus, we tested two sis1-Δ strains, one expressing Sis1 from the native SIS1 promoter and one expressing Sis1 from the stronger GPD promoter, resulting in either normal or approximately two-fold higher Sis1 expression, respectively (Figure 3A). After passage of the strains for one week on glucose-based media, the presence of [SWI+] was assessed by observing the growth of cells on medium containing raffinose as the carbon source. The cells overexpressing Sis1 from the GPD promoter grew more robustly, similar to the control [swi−] cells. Those having normal levels of Sis1 expression grew poorly, similar to the [SWI+] control (Figure 3B), indicating the prion is indeed sensitive to overexpression of Sis1. The maintenance or loss of [SWI+] in these strains was also confirmed using fluorescence analysis (Figure 3C).


[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)

Overexpression of Sis1 cures [SWI+].(A) Extracts were prepared from either wild-type (WT) or sis1-Δ cells expressing Sis1 from a centromeric plasmid, either under the control of its own (SIS1-Sis1) or the GPD (GPD-Sis1) promoter. The extracts were subjected to immunoblot analysis using either antibody specific for Sis1 or, as a loading control, Ssc1 (control). (A and B) Dashed lines in both figures indicate some sections of the blot or plate have been cropped for clarity. (B) Sis1 expressing cells described in (A) were passaged for one week and then the presence of [SWI+] was assessed by streaking onto raffinose media. Wild-type [SWI+] and [swi−] cells are shown as controls. (C) Loss of [SWI+] from cells shown in (B) was also confirmed by fluorescence analysis following transformation with a plasmid bearing the Swi1NQ-YFP construct.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040656&req=5

pgen-1001309-g003: Overexpression of Sis1 cures [SWI+].(A) Extracts were prepared from either wild-type (WT) or sis1-Δ cells expressing Sis1 from a centromeric plasmid, either under the control of its own (SIS1-Sis1) or the GPD (GPD-Sis1) promoter. The extracts were subjected to immunoblot analysis using either antibody specific for Sis1 or, as a loading control, Ssc1 (control). (A and B) Dashed lines in both figures indicate some sections of the blot or plate have been cropped for clarity. (B) Sis1 expressing cells described in (A) were passaged for one week and then the presence of [SWI+] was assessed by streaking onto raffinose media. Wild-type [SWI+] and [swi−] cells are shown as controls. (C) Loss of [SWI+] from cells shown in (B) was also confirmed by fluorescence analysis following transformation with a plasmid bearing the Swi1NQ-YFP construct.
Mentions: A comparison of the control experiments in Figure 1C and Figure 2B indicates that [SWI+] is more stable in a wild-type background than in the sis1-Δ [TETr-Sis1] strains. To test whether this loss was due to lower than normal Sis1 expression, we asked whether the prion would be stabilized by supplemental expression from a second plasmid. However, we found that the presence of the second Sis1 plasmid exacerbated prion loss (data not shown), suggesting that the instability of [SWI+] might be due to overexpression rather than underexpression. Thus, we tested two sis1-Δ strains, one expressing Sis1 from the native SIS1 promoter and one expressing Sis1 from the stronger GPD promoter, resulting in either normal or approximately two-fold higher Sis1 expression, respectively (Figure 3A). After passage of the strains for one week on glucose-based media, the presence of [SWI+] was assessed by observing the growth of cells on medium containing raffinose as the carbon source. The cells overexpressing Sis1 from the GPD promoter grew more robustly, similar to the control [swi−] cells. Those having normal levels of Sis1 expression grew poorly, similar to the [SWI+] control (Figure 3B), indicating the prion is indeed sensitive to overexpression of Sis1. The maintenance or loss of [SWI+] in these strains was also confirmed using fluorescence analysis (Figure 3C).

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