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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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Sis1 is required for [SWI+] propagation.(A) Western blot showing doxycycline-dependent Sis1 repression. sis1-Δ [TETr-Sis1] cells in liquid culture were harvested before (−Dox., 0g) or the indicated number of generations after the addition of the drug doxycycline (+Dox.). Cell extracts were subjected to immunoblot analysis using either antibody specific for Sis1 or, as a loading control, Ssc1 (control). (B) Time course of [SWI+] loss upon repression of Sis1 expression. [SWI+] sis1-Δ [TETr-Sis1] cells were harvested after the indicated number of generations of growth in the presence (diamonds) or absence (squares) of doxycycline and plated onto glucose-based media. Following transformation of cells from individual colonies with a plasmid bearing Swi1NQ-YFP, the fraction of cells that were [SWI+] was determined by subsequent examination of Swi1 aggregation based on fluorescence (right) and plotted (left). Points represent the average of three independent experiments. Error bars represent ± σ deviation. The solid line represents a best-fit line through the averaged data. (C) Loss of [SWI+] following Sis1 depletion was also confirmed by testing for the restoration of robust growth on raffinose-based media. Serial dilutions of one representative isolate are shown above from a culture before (0g) or 21generations after addition of doxycycline (21g). A total of 24 individual colonies from each of these two time-points were assayed (Figure S1A).
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pgen-1001309-g002: Sis1 is required for [SWI+] propagation.(A) Western blot showing doxycycline-dependent Sis1 repression. sis1-Δ [TETr-Sis1] cells in liquid culture were harvested before (−Dox., 0g) or the indicated number of generations after the addition of the drug doxycycline (+Dox.). Cell extracts were subjected to immunoblot analysis using either antibody specific for Sis1 or, as a loading control, Ssc1 (control). (B) Time course of [SWI+] loss upon repression of Sis1 expression. [SWI+] sis1-Δ [TETr-Sis1] cells were harvested after the indicated number of generations of growth in the presence (diamonds) or absence (squares) of doxycycline and plated onto glucose-based media. Following transformation of cells from individual colonies with a plasmid bearing Swi1NQ-YFP, the fraction of cells that were [SWI+] was determined by subsequent examination of Swi1 aggregation based on fluorescence (right) and plotted (left). Points represent the average of three independent experiments. Error bars represent ± σ deviation. The solid line represents a best-fit line through the averaged data. (C) Loss of [SWI+] following Sis1 depletion was also confirmed by testing for the restoration of robust growth on raffinose-based media. Serial dilutions of one representative isolate are shown above from a culture before (0g) or 21generations after addition of doxycycline (21g). A total of 24 individual colonies from each of these two time-points were assayed (Figure S1A).

Mentions: Sis1 is required for the propagation of at least three prions, [PSI+], [URE3], and [RNQ+] [24], [25], [33]. Therefore, we next tested the dependence of [SWI+] on this J-protein. Because Sis1 is an essential protein, we utilized a system having SIS1 under the control of the tetR promoter (TETr). This system, which, upon addition of the drug doxycycline, allows repression of Sis1 synthesis to a minimal level required for cell growth (Figure 2A), was previously used to analyze the role of Sis1 in the maintenance of other prions [23]–[25]. As a control, samples of cells cultured in the absence of drug were collected at time intervals and plated onto glucose-based media. As discussed above, the status of [SWI+] was assessed after subsequent transformation of resulting individual colonies (n = 24) with a plasmid expressing Swi1NQ-YFP. sis1-Δ [TETr-Sis1] cells initially maintained [SWI+] at a high frequency, but showed gradual loss of the prion (Figure 2B,) as approximately 40% of the cells became [swi−] over 23 cell generations of cell culture in the absence of drug.


[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)

Sis1 is required for [SWI+] propagation.(A) Western blot showing doxycycline-dependent Sis1 repression. sis1-Δ [TETr-Sis1] cells in liquid culture were harvested before (−Dox., 0g) or the indicated number of generations after the addition of the drug doxycycline (+Dox.). Cell extracts were subjected to immunoblot analysis using either antibody specific for Sis1 or, as a loading control, Ssc1 (control). (B) Time course of [SWI+] loss upon repression of Sis1 expression. [SWI+] sis1-Δ [TETr-Sis1] cells were harvested after the indicated number of generations of growth in the presence (diamonds) or absence (squares) of doxycycline and plated onto glucose-based media. Following transformation of cells from individual colonies with a plasmid bearing Swi1NQ-YFP, the fraction of cells that were [SWI+] was determined by subsequent examination of Swi1 aggregation based on fluorescence (right) and plotted (left). Points represent the average of three independent experiments. Error bars represent ± σ deviation. The solid line represents a best-fit line through the averaged data. (C) Loss of [SWI+] following Sis1 depletion was also confirmed by testing for the restoration of robust growth on raffinose-based media. Serial dilutions of one representative isolate are shown above from a culture before (0g) or 21generations after addition of doxycycline (21g). A total of 24 individual colonies from each of these two time-points were assayed (Figure S1A).
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Related In: Results  -  Collection

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

pgen-1001309-g002: Sis1 is required for [SWI+] propagation.(A) Western blot showing doxycycline-dependent Sis1 repression. sis1-Δ [TETr-Sis1] cells in liquid culture were harvested before (−Dox., 0g) or the indicated number of generations after the addition of the drug doxycycline (+Dox.). Cell extracts were subjected to immunoblot analysis using either antibody specific for Sis1 or, as a loading control, Ssc1 (control). (B) Time course of [SWI+] loss upon repression of Sis1 expression. [SWI+] sis1-Δ [TETr-Sis1] cells were harvested after the indicated number of generations of growth in the presence (diamonds) or absence (squares) of doxycycline and plated onto glucose-based media. Following transformation of cells from individual colonies with a plasmid bearing Swi1NQ-YFP, the fraction of cells that were [SWI+] was determined by subsequent examination of Swi1 aggregation based on fluorescence (right) and plotted (left). Points represent the average of three independent experiments. Error bars represent ± σ deviation. The solid line represents a best-fit line through the averaged data. (C) Loss of [SWI+] following Sis1 depletion was also confirmed by testing for the restoration of robust growth on raffinose-based media. Serial dilutions of one representative isolate are shown above from a culture before (0g) or 21generations after addition of doxycycline (21g). A total of 24 individual colonies from each of these two time-points were assayed (Figure S1A).
Mentions: Sis1 is required for the propagation of at least three prions, [PSI+], [URE3], and [RNQ+] [24], [25], [33]. Therefore, we next tested the dependence of [SWI+] on this J-protein. Because Sis1 is an essential protein, we utilized a system having SIS1 under the control of the tetR promoter (TETr). This system, which, upon addition of the drug doxycycline, allows repression of Sis1 synthesis to a minimal level required for cell growth (Figure 2A), was previously used to analyze the role of Sis1 in the maintenance of other prions [23]–[25]. As a control, samples of cells cultured in the absence of drug were collected at time intervals and plated onto glucose-based media. As discussed above, the status of [SWI+] was assessed after subsequent transformation of resulting individual colonies (n = 24) with a plasmid expressing Swi1NQ-YFP. sis1-Δ [TETr-Sis1] cells initially maintained [SWI+] at a high frequency, but showed gradual loss of the prion (Figure 2B,) as approximately 40% of the cells became [swi−] over 23 cell generations of cell culture in the absence of drug.

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