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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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Determination of [SWI+] seed number using a propagon counting assay.[SWI+] cells were grown in the presence or absence of 4mM GdnHCl and plated onto glucose-based media at time intervals at the indicated number of cell generations to obtain single colonies. (A) The presence of [SWI+] in the isolated colonies (n = 24) from each time point was assessed by transformation with a plasmid carrying the Swi1NQ-YFP fusion. Resulting transformants (n≥10 for each colony) were observed under the microscope, scoring for punctuate [SWI+] or diffuse [swi−] fluorescence. The presence of [SWI+] in any transformant indicates the presence of the prion in the original colony. A representative example of cells from a colony collected before (0g) and 13 generations after GdnHCl addition (13g) is shown. (B) The presence or absence of [SWI+] was also tested by assessing growth on raffinose-based medium. 24 individual colonies were also assayed before (0g) and 13 generations after (13g) the addition of GdnHCl (Figure S1A). 10-fold serial dilutions of one representative isolate from each condition were spotted onto raffinose and glucose-based medium. (C) Data obtained from cells grown in the presence (diamonds) or absence (circles) of 4mM GdnHCl, as described in A, were plotted. Solid bold line represents the best-fit curve for [SWI+] using the model of Cox et al. 2003 [29], 45 seeds/cell. Colored dotted lines represent reference curves previously published for other prions using this same model (left to right): Magenta, [URE3], 20 seeds/cell; Orange, weak [PSI+], 60 seeds/cell; Green, [RNQ+], 100 seeds/cell; Blue, strong [PSI+], 260 seeds/cell [15], [24], [39], [41].
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pgen-1001309-g001: Determination of [SWI+] seed number using a propagon counting assay.[SWI+] cells were grown in the presence or absence of 4mM GdnHCl and plated onto glucose-based media at time intervals at the indicated number of cell generations to obtain single colonies. (A) The presence of [SWI+] in the isolated colonies (n = 24) from each time point was assessed by transformation with a plasmid carrying the Swi1NQ-YFP fusion. Resulting transformants (n≥10 for each colony) were observed under the microscope, scoring for punctuate [SWI+] or diffuse [swi−] fluorescence. The presence of [SWI+] in any transformant indicates the presence of the prion in the original colony. A representative example of cells from a colony collected before (0g) and 13 generations after GdnHCl addition (13g) is shown. (B) The presence or absence of [SWI+] was also tested by assessing growth on raffinose-based medium. 24 individual colonies were also assayed before (0g) and 13 generations after (13g) the addition of GdnHCl (Figure S1A). 10-fold serial dilutions of one representative isolate from each condition were spotted onto raffinose and glucose-based medium. (C) Data obtained from cells grown in the presence (diamonds) or absence (circles) of 4mM GdnHCl, as described in A, were plotted. Solid bold line represents the best-fit curve for [SWI+] using the model of Cox et al. 2003 [29], 45 seeds/cell. Colored dotted lines represent reference curves previously published for other prions using this same model (left to right): Magenta, [URE3], 20 seeds/cell; Orange, weak [PSI+], 60 seeds/cell; Green, [RNQ+], 100 seeds/cell; Blue, strong [PSI+], 260 seeds/cell [15], [24], [39], [41].

Mentions: As our first step in the characterization of [SWI+] and its dependence on molecular chaperones for its propagation, we determined its seed number. We employed an established method, a ‘propagon counting assay’, which is based on monitoring prion-loss upon inactivation of Hsp104 activity [39]. The Hsp104 inhibitor GdnHCl was used [40], as [SWI+], like other yeast prions, requires Hsp104 activity for seed generation [16]. At various times after treatment of a culture of [SWI+] cells with GdnHCl, aliquots of cells were plated onto glucose-based medium. To determine the percentage of cells having lost the prion at each time point, cells from a minimum of 24 individual colonies were transformed with a plasmid containing the Asn- and Gln-rich segment of Swi1, which contains the prion-forming domains, fused to YFP (Swi1NQ-YFP). Resulting transformants were observed under the microscope and scored for Swi1 aggregation based on punctuate or diffuse fluorescence, indicative of presence or absence of the prion, respectively (Figure 1A) [16]. As expected, [SWI+] was lost from the population with time; after six generations, approximately 50% of the population was [swi−] (Figure 1C). 13 generations after addition of GdnHCl, none of the colonies tested positive for the prion. No prion loss was observed for the control culture to which no GdnHCl was added. To confirm that the microscopic analysis provided an accurate indicator of the presence or absence of the prion, we also tested the growth of cells on medium containing raffinose as the carbon source. Growth of [SWI+], but not [swi−], cells is greatly impaired on such medium because raffinose transport into cells is reduced when activity of the SWI/SNF complex is impaired [16]. All 24 isolates taken from a culture prior to addition of GdnHCl grew poorly on raffinose-based medium; all 24 isolates taken from a culture 13 generations after GdnHCl addition grew well, confirming loss of the prion (Figure 1B and Figure S1).


[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)

Determination of [SWI+] seed number using a propagon counting assay.[SWI+] cells were grown in the presence or absence of 4mM GdnHCl and plated onto glucose-based media at time intervals at the indicated number of cell generations to obtain single colonies. (A) The presence of [SWI+] in the isolated colonies (n = 24) from each time point was assessed by transformation with a plasmid carrying the Swi1NQ-YFP fusion. Resulting transformants (n≥10 for each colony) were observed under the microscope, scoring for punctuate [SWI+] or diffuse [swi−] fluorescence. The presence of [SWI+] in any transformant indicates the presence of the prion in the original colony. A representative example of cells from a colony collected before (0g) and 13 generations after GdnHCl addition (13g) is shown. (B) The presence or absence of [SWI+] was also tested by assessing growth on raffinose-based medium. 24 individual colonies were also assayed before (0g) and 13 generations after (13g) the addition of GdnHCl (Figure S1A). 10-fold serial dilutions of one representative isolate from each condition were spotted onto raffinose and glucose-based medium. (C) Data obtained from cells grown in the presence (diamonds) or absence (circles) of 4mM GdnHCl, as described in A, were plotted. Solid bold line represents the best-fit curve for [SWI+] using the model of Cox et al. 2003 [29], 45 seeds/cell. Colored dotted lines represent reference curves previously published for other prions using this same model (left to right): Magenta, [URE3], 20 seeds/cell; Orange, weak [PSI+], 60 seeds/cell; Green, [RNQ+], 100 seeds/cell; Blue, strong [PSI+], 260 seeds/cell [15], [24], [39], [41].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040656&req=5

pgen-1001309-g001: Determination of [SWI+] seed number using a propagon counting assay.[SWI+] cells were grown in the presence or absence of 4mM GdnHCl and plated onto glucose-based media at time intervals at the indicated number of cell generations to obtain single colonies. (A) The presence of [SWI+] in the isolated colonies (n = 24) from each time point was assessed by transformation with a plasmid carrying the Swi1NQ-YFP fusion. Resulting transformants (n≥10 for each colony) were observed under the microscope, scoring for punctuate [SWI+] or diffuse [swi−] fluorescence. The presence of [SWI+] in any transformant indicates the presence of the prion in the original colony. A representative example of cells from a colony collected before (0g) and 13 generations after GdnHCl addition (13g) is shown. (B) The presence or absence of [SWI+] was also tested by assessing growth on raffinose-based medium. 24 individual colonies were also assayed before (0g) and 13 generations after (13g) the addition of GdnHCl (Figure S1A). 10-fold serial dilutions of one representative isolate from each condition were spotted onto raffinose and glucose-based medium. (C) Data obtained from cells grown in the presence (diamonds) or absence (circles) of 4mM GdnHCl, as described in A, were plotted. Solid bold line represents the best-fit curve for [SWI+] using the model of Cox et al. 2003 [29], 45 seeds/cell. Colored dotted lines represent reference curves previously published for other prions using this same model (left to right): Magenta, [URE3], 20 seeds/cell; Orange, weak [PSI+], 60 seeds/cell; Green, [RNQ+], 100 seeds/cell; Blue, strong [PSI+], 260 seeds/cell [15], [24], [39], [41].
Mentions: As our first step in the characterization of [SWI+] and its dependence on molecular chaperones for its propagation, we determined its seed number. We employed an established method, a ‘propagon counting assay’, which is based on monitoring prion-loss upon inactivation of Hsp104 activity [39]. The Hsp104 inhibitor GdnHCl was used [40], as [SWI+], like other yeast prions, requires Hsp104 activity for seed generation [16]. At various times after treatment of a culture of [SWI+] cells with GdnHCl, aliquots of cells were plated onto glucose-based medium. To determine the percentage of cells having lost the prion at each time point, cells from a minimum of 24 individual colonies were transformed with a plasmid containing the Asn- and Gln-rich segment of Swi1, which contains the prion-forming domains, fused to YFP (Swi1NQ-YFP). Resulting transformants were observed under the microscope and scored for Swi1 aggregation based on punctuate or diffuse fluorescence, indicative of presence or absence of the prion, respectively (Figure 1A) [16]. As expected, [SWI+] was lost from the population with time; after six generations, approximately 50% of the population was [swi−] (Figure 1C). 13 generations after addition of GdnHCl, none of the colonies tested positive for the prion. No prion loss was observed for the control culture to which no GdnHCl was added. To confirm that the microscopic analysis provided an accurate indicator of the presence or absence of the prion, we also tested the growth of cells on medium containing raffinose as the carbon source. Growth of [SWI+], but not [swi−], cells is greatly impaired on such medium because raffinose transport into cells is reduced when activity of the SWI/SNF complex is impaired [16]. All 24 isolates taken from a culture prior to addition of GdnHCl grew poorly on raffinose-based medium; all 24 isolates taken from a culture 13 generations after GdnHCl addition grew well, confirming loss of the prion (Figure 1B and Figure S1).

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