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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+] requires a function of the C-terminal domains of Ydj1.Plasmids expressing J-protein constructs were used to transform a strain expressing Ydj1 from a centromeric plasmid carrying the URA3 genetic marker (ydj1-Δ [YDJ1-Ydj1, URA3] [SWI+]) for plasmid-shuffling experiments (far right column). Following counter-selection against the Ydj1 plasmid by growth on 5-fluoro-orotic acid (5-FOA), individual transformants were then passaged once on selective media, and retransformed with the Swi1NQ-YFP plasmid for [SWI+] scoring by fluorescence analysis. Results are expressed as the fraction of the original transformants that remained [SWI+] over the total number examined (Fraction [SWI+]). In experiments where [SWI+] loss was observed, J-protein-bearing plasmids were also transformed into a wild-type [SWI+] strain to control for potential prion-loss due to protein overexpression (second column from right). For completeness and clarity, results for empty vector and GPD-Apj1 shown in Figure 4 are also included here. Gene domain structure elements are described in Figure 4. The construct S/Y consists of the J-domain and Gly/Phe-rich regions of Sis1 (residues 1–112) and the C-terminal Zn2+- and peptide-binding regions of Ydj1 (residues 93–409). Ydj11–134 contains the J-domain and adjacent Gly/Phe-rich region. The construct Y/A is a chimera substituting the C-terminal Zn2+- and peptide-binding regions of Ydj1 (residues 114–409) with those of Apj1 (residues 162–529). Ydj1H34Q contains a single amino acid alteration (His34→Q) in the J-domain (asterisk) which renders it unable to stimulate Hsp70 ATPase activity [48]. A full list of plasmids used can be found in Table 1. N.T. = Not Tested.
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pgen-1001309-g005: [SWI+] requires a function of the C-terminal domains of Ydj1.Plasmids expressing J-protein constructs were used to transform a strain expressing Ydj1 from a centromeric plasmid carrying the URA3 genetic marker (ydj1-Δ [YDJ1-Ydj1, URA3] [SWI+]) for plasmid-shuffling experiments (far right column). Following counter-selection against the Ydj1 plasmid by growth on 5-fluoro-orotic acid (5-FOA), individual transformants were then passaged once on selective media, and retransformed with the Swi1NQ-YFP plasmid for [SWI+] scoring by fluorescence analysis. Results are expressed as the fraction of the original transformants that remained [SWI+] over the total number examined (Fraction [SWI+]). In experiments where [SWI+] loss was observed, J-protein-bearing plasmids were also transformed into a wild-type [SWI+] strain to control for potential prion-loss due to protein overexpression (second column from right). For completeness and clarity, results for empty vector and GPD-Apj1 shown in Figure 4 are also included here. Gene domain structure elements are described in Figure 4. The construct S/Y consists of the J-domain and Gly/Phe-rich regions of Sis1 (residues 1–112) and the C-terminal Zn2+- and peptide-binding regions of Ydj1 (residues 93–409). Ydj11–134 contains the J-domain and adjacent Gly/Phe-rich region. The construct Y/A is a chimera substituting the C-terminal Zn2+- and peptide-binding regions of Ydj1 (residues 114–409) with those of Apj1 (residues 162–529). Ydj1H34Q contains a single amino acid alteration (His34→Q) in the J-domain (asterisk) which renders it unable to stimulate Hsp70 ATPase activity [48]. A full list of plasmids used can be found in Table 1. N.T. = Not Tested.

Mentions: The plasmid-shuffling system described above also opened up an avenue to test the possibility that overexpression of other J-proteins might be able to substitute for full-length Ydj1 in [SWI+] propagation. As a control to test this system, ydj1-Δ [YDJ1-Ydj1, URA3] was transformed with a second plasmid, either one carrying a second YDJ1 gene, or one lacking an insert, thus serving as a vector control. As expected, after counter-selection against the URA3-based Ydj1-expressing plasmid, none of the 26 vector transformants tested were [SWI+] (Figure 5). However, 37 of 42 isolates having the Ydj1-expressing plasmid tested positive for [SWI+]. It is likely that the loss of the prion in a small portion of these transformants was due to the increased level of Ydj1 during the time that the cells carried two YDJ1 genes, because modest overexpression of J-domains can result in [SWI+] loss, as discussed above. Therefore, we included in our analysis a wild-type strain expressing Ydj1 from the endogenous gene, as a control for possible prion loss due to increased J-protein function during the construction of these strains, rather than a lack of Ydj1 function in the ydj1-Δ test strain. Wild-type control strains carrying either the vector or YDJ1 on the plasmid sustained some loss of [SWI+], with 4 of 46 and 3 of 24 transformants being [swi−], respectively. However, the stability was sufficient to allow use of this system to test the effectiveness of J-protein constructs to substitute for Ydj1 in [SWI+] propagation.


[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+] requires a function of the C-terminal domains of Ydj1.Plasmids expressing J-protein constructs were used to transform a strain expressing Ydj1 from a centromeric plasmid carrying the URA3 genetic marker (ydj1-Δ [YDJ1-Ydj1, URA3] [SWI+]) for plasmid-shuffling experiments (far right column). Following counter-selection against the Ydj1 plasmid by growth on 5-fluoro-orotic acid (5-FOA), individual transformants were then passaged once on selective media, and retransformed with the Swi1NQ-YFP plasmid for [SWI+] scoring by fluorescence analysis. Results are expressed as the fraction of the original transformants that remained [SWI+] over the total number examined (Fraction [SWI+]). In experiments where [SWI+] loss was observed, J-protein-bearing plasmids were also transformed into a wild-type [SWI+] strain to control for potential prion-loss due to protein overexpression (second column from right). For completeness and clarity, results for empty vector and GPD-Apj1 shown in Figure 4 are also included here. Gene domain structure elements are described in Figure 4. The construct S/Y consists of the J-domain and Gly/Phe-rich regions of Sis1 (residues 1–112) and the C-terminal Zn2+- and peptide-binding regions of Ydj1 (residues 93–409). Ydj11–134 contains the J-domain and adjacent Gly/Phe-rich region. The construct Y/A is a chimera substituting the C-terminal Zn2+- and peptide-binding regions of Ydj1 (residues 114–409) with those of Apj1 (residues 162–529). Ydj1H34Q contains a single amino acid alteration (His34→Q) in the J-domain (asterisk) which renders it unable to stimulate Hsp70 ATPase activity [48]. A full list of plasmids used can be found in Table 1. N.T. = Not Tested.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001309-g005: [SWI+] requires a function of the C-terminal domains of Ydj1.Plasmids expressing J-protein constructs were used to transform a strain expressing Ydj1 from a centromeric plasmid carrying the URA3 genetic marker (ydj1-Δ [YDJ1-Ydj1, URA3] [SWI+]) for plasmid-shuffling experiments (far right column). Following counter-selection against the Ydj1 plasmid by growth on 5-fluoro-orotic acid (5-FOA), individual transformants were then passaged once on selective media, and retransformed with the Swi1NQ-YFP plasmid for [SWI+] scoring by fluorescence analysis. Results are expressed as the fraction of the original transformants that remained [SWI+] over the total number examined (Fraction [SWI+]). In experiments where [SWI+] loss was observed, J-protein-bearing plasmids were also transformed into a wild-type [SWI+] strain to control for potential prion-loss due to protein overexpression (second column from right). For completeness and clarity, results for empty vector and GPD-Apj1 shown in Figure 4 are also included here. Gene domain structure elements are described in Figure 4. The construct S/Y consists of the J-domain and Gly/Phe-rich regions of Sis1 (residues 1–112) and the C-terminal Zn2+- and peptide-binding regions of Ydj1 (residues 93–409). Ydj11–134 contains the J-domain and adjacent Gly/Phe-rich region. The construct Y/A is a chimera substituting the C-terminal Zn2+- and peptide-binding regions of Ydj1 (residues 114–409) with those of Apj1 (residues 162–529). Ydj1H34Q contains a single amino acid alteration (His34→Q) in the J-domain (asterisk) which renders it unable to stimulate Hsp70 ATPase activity [48]. A full list of plasmids used can be found in Table 1. N.T. = Not Tested.
Mentions: The plasmid-shuffling system described above also opened up an avenue to test the possibility that overexpression of other J-proteins might be able to substitute for full-length Ydj1 in [SWI+] propagation. As a control to test this system, ydj1-Δ [YDJ1-Ydj1, URA3] was transformed with a second plasmid, either one carrying a second YDJ1 gene, or one lacking an insert, thus serving as a vector control. As expected, after counter-selection against the URA3-based Ydj1-expressing plasmid, none of the 26 vector transformants tested were [SWI+] (Figure 5). However, 37 of 42 isolates having the Ydj1-expressing plasmid tested positive for [SWI+]. It is likely that the loss of the prion in a small portion of these transformants was due to the increased level of Ydj1 during the time that the cells carried two YDJ1 genes, because modest overexpression of J-domains can result in [SWI+] loss, as discussed above. Therefore, we included in our analysis a wild-type strain expressing Ydj1 from the endogenous gene, as a control for possible prion loss due to increased J-protein function during the construction of these strains, rather than a lack of Ydj1 function in the ydj1-Δ test strain. Wild-type control strains carrying either the vector or YDJ1 on the plasmid sustained some loss of [SWI+], with 4 of 46 and 3 of 24 transformants being [swi−], respectively. However, the stability was sufficient to allow use of this system to test the effectiveness of J-protein constructs to substitute for Ydj1 in [SWI+] propagation.

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