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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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Alterations that impair Sse1 NEF function also impair [SWI+] curing.(A) Domain structure of Sse1. Sse1 shares domain homology with Hsp70s, possessing both an N-terminal ATP-binding domain (NTD) and a C-terminal peptide-binding domain (PBD). Single amino acid alterations tested in this study are indicated by asterisks. (B and C) [SWI+] cells were transformed with either empty vector, or vectors expressing the indicated variants of Sse1 (wild-type Sse1, WT Sse1; Sse1K69Q, K69Q; Sse1G233D, G233D; Sse1Δ1–396, Δ1–396, Sse1Δ394–693, Δ394–693) from the TEF promoter. Analysis of one representative transformant of each variant is shown. (B) Transformants were streaked onto raffinose- and glucose-based media along with [SWI+] and [swi−] control strains for scoring. (C) Transformants and [SWI+] and [swi−] control strains were transformed with vector expressing Swi1NQ-YFP. [SWI+] maintenance was assessed by fluorescence analysis. A representative image of the fluorescence pattern observed in the majority of transformants from each vector and [SWI+] or [swi−] control strains is shown. (D) For each Sse1 variant tested and vector control, results are presented as the fraction of the original transformants which remained [SWI+] over the total number examined (Fraction [SWI+]). For each variant listed, the presence (+) or absence (−) of previously determined nucleotide exchange factor activity (NEF Activity) is shown [51], [52].
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pgen-1001309-g008: Alterations that impair Sse1 NEF function also impair [SWI+] curing.(A) Domain structure of Sse1. Sse1 shares domain homology with Hsp70s, possessing both an N-terminal ATP-binding domain (NTD) and a C-terminal peptide-binding domain (PBD). Single amino acid alterations tested in this study are indicated by asterisks. (B and C) [SWI+] cells were transformed with either empty vector, or vectors expressing the indicated variants of Sse1 (wild-type Sse1, WT Sse1; Sse1K69Q, K69Q; Sse1G233D, G233D; Sse1Δ1–396, Δ1–396, Sse1Δ394–693, Δ394–693) from the TEF promoter. Analysis of one representative transformant of each variant is shown. (B) Transformants were streaked onto raffinose- and glucose-based media along with [SWI+] and [swi−] control strains for scoring. (C) Transformants and [SWI+] and [swi−] control strains were transformed with vector expressing Swi1NQ-YFP. [SWI+] maintenance was assessed by fluorescence analysis. A representative image of the fluorescence pattern observed in the majority of transformants from each vector and [SWI+] or [swi−] control strains is shown. (D) For each Sse1 variant tested and vector control, results are presented as the fraction of the original transformants which remained [SWI+] over the total number examined (Fraction [SWI+]). For each variant listed, the presence (+) or absence (−) of previously determined nucleotide exchange factor activity (NEF Activity) is shown [51], [52].

Mentions: Although Sse1 has NEF activity [51]–[53], the fact that it has a domain structure similar to that of Hsp70s suggests that it might have additional functions as well. For example, Sse1 is known to interact with client proteins, though the relationship between client protein binding and NEF activity is less clear [54], [55]. To assess whether loss of [SWI+] caused by Sse1 overexpression was due to increased NEF activity we took advantage of previously characterized SSE1 mutants. After transformation of plasmids carrying the mutant genes into a wild-type [SWI+] strain, transformants were tested for prion maintenance, using both the raffinose growth assay and visualization of Swi1 distribution. First, we tested whether expression of either the N-terminal ATP-binding domain or the C-terminal putative peptide-binding domain of Sse1 is sufficient to effect prion loss by expressing either Sse1Δ394–693 or Sse1Δ1–396. In both cases, all of the 60 transformants tested positive for [SWI+], indicating a requirement of both domains for prion curing (Figure 8). We then tested two point mutations encoding single amino acid alterations within the N-terminal domain: (1) G233→D in the ATP binding site, which impairs, both in vitro and in vivo, Sse1's NEF activity; (2) K69→Q in a site predicted to be required for ATP hydrolysis, which has no measureable effect on Sse1 NEF function [51], [52]. 57 out of 60 transformants overfexpressing Sse1K69Q lost [SWI+], while the prion was stable in those expressing Sse1G233D. These data are consistent with the hypothesis that Sse1 acts as a nucleotide exchange factor for Hsp70 in [SWI+] curing, rather than performing another uncharacterized function.


[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)

Alterations that impair Sse1 NEF function also impair [SWI+] curing.(A) Domain structure of Sse1. Sse1 shares domain homology with Hsp70s, possessing both an N-terminal ATP-binding domain (NTD) and a C-terminal peptide-binding domain (PBD). Single amino acid alterations tested in this study are indicated by asterisks. (B and C) [SWI+] cells were transformed with either empty vector, or vectors expressing the indicated variants of Sse1 (wild-type Sse1, WT Sse1; Sse1K69Q, K69Q; Sse1G233D, G233D; Sse1Δ1–396, Δ1–396, Sse1Δ394–693, Δ394–693) from the TEF promoter. Analysis of one representative transformant of each variant is shown. (B) Transformants were streaked onto raffinose- and glucose-based media along with [SWI+] and [swi−] control strains for scoring. (C) Transformants and [SWI+] and [swi−] control strains were transformed with vector expressing Swi1NQ-YFP. [SWI+] maintenance was assessed by fluorescence analysis. A representative image of the fluorescence pattern observed in the majority of transformants from each vector and [SWI+] or [swi−] control strains is shown. (D) For each Sse1 variant tested and vector control, results are presented as the fraction of the original transformants which remained [SWI+] over the total number examined (Fraction [SWI+]). For each variant listed, the presence (+) or absence (−) of previously determined nucleotide exchange factor activity (NEF Activity) is shown [51], [52].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040656&req=5

pgen-1001309-g008: Alterations that impair Sse1 NEF function also impair [SWI+] curing.(A) Domain structure of Sse1. Sse1 shares domain homology with Hsp70s, possessing both an N-terminal ATP-binding domain (NTD) and a C-terminal peptide-binding domain (PBD). Single amino acid alterations tested in this study are indicated by asterisks. (B and C) [SWI+] cells were transformed with either empty vector, or vectors expressing the indicated variants of Sse1 (wild-type Sse1, WT Sse1; Sse1K69Q, K69Q; Sse1G233D, G233D; Sse1Δ1–396, Δ1–396, Sse1Δ394–693, Δ394–693) from the TEF promoter. Analysis of one representative transformant of each variant is shown. (B) Transformants were streaked onto raffinose- and glucose-based media along with [SWI+] and [swi−] control strains for scoring. (C) Transformants and [SWI+] and [swi−] control strains were transformed with vector expressing Swi1NQ-YFP. [SWI+] maintenance was assessed by fluorescence analysis. A representative image of the fluorescence pattern observed in the majority of transformants from each vector and [SWI+] or [swi−] control strains is shown. (D) For each Sse1 variant tested and vector control, results are presented as the fraction of the original transformants which remained [SWI+] over the total number examined (Fraction [SWI+]). For each variant listed, the presence (+) or absence (−) of previously determined nucleotide exchange factor activity (NEF Activity) is shown [51], [52].
Mentions: Although Sse1 has NEF activity [51]–[53], the fact that it has a domain structure similar to that of Hsp70s suggests that it might have additional functions as well. For example, Sse1 is known to interact with client proteins, though the relationship between client protein binding and NEF activity is less clear [54], [55]. To assess whether loss of [SWI+] caused by Sse1 overexpression was due to increased NEF activity we took advantage of previously characterized SSE1 mutants. After transformation of plasmids carrying the mutant genes into a wild-type [SWI+] strain, transformants were tested for prion maintenance, using both the raffinose growth assay and visualization of Swi1 distribution. First, we tested whether expression of either the N-terminal ATP-binding domain or the C-terminal putative peptide-binding domain of Sse1 is sufficient to effect prion loss by expressing either Sse1Δ394–693 or Sse1Δ1–396. In both cases, all of the 60 transformants tested positive for [SWI+], indicating a requirement of both domains for prion curing (Figure 8). We then tested two point mutations encoding single amino acid alterations within the N-terminal domain: (1) G233→D in the ATP binding site, which impairs, both in vitro and in vivo, Sse1's NEF activity; (2) K69→Q in a site predicted to be required for ATP hydrolysis, which has no measureable effect on Sse1 NEF function [51], [52]. 57 out of 60 transformants overfexpressing Sse1K69Q lost [SWI+], while the prion was stable in those expressing Sse1G233D. These data are consistent with the hypothesis that Sse1 acts as a nucleotide exchange factor for Hsp70 in [SWI+] curing, rather than performing another uncharacterized function.

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