Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [PSI+] prion formation

Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in btn2 hsp42 sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an in vivo sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [PSI+] prion, which is the amyloid form of Sup35. [PSI+] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in btn2 hsp42 mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation

Yeast protein kinase A isoforms: a means of encoding specificity in the response to diverse stress conditions?

Eukaryotic cells have developed a complex circuitry of signalling molecules which monitor changes in their intra- and extracellular environments. One of the most widely studied signalling pathways is the highly conserved cyclic AMP (cAMP)/protein kinase A (PKA) pathway, which is a major glucose sensing circuit in the yeast Saccharomyces cerevisiae. PKA activity regulates diverse targets in yeast, positively activating the processes that are associated with rapid cell growth (e.g., fermentative metabolism, ribosome biogenesis and cell division) and negatively regulating the processes that are associated with slow growth, such as respiratory growth, carbohydrate storage and entry into stationary phase. As in higher eukaryotes, yeast has evolved complexity at the level of the PKA catalytic subunit, and Saccharomyces cerevisiae expresses three isoforms, denoted Tpk1-3. Despite evidence for isoform differences in multiple biological processes, the molecular basis of PKA signalling specificity remains poorly defined, and many studies continue to assume redundancy with regards to PKA-mediated regulation. PKA has canonically been shown to play a key role in fine-tuning the cellular response to diverse stressors; however, recent studies have now begun to interrogate the requirement for individual PKA catalytic isoforms in coordinating distinct steps in stress response pathways. In this review, we discuss the known non-redundant functions of the Tpk catalytic subunits and the evolving picture of how these isoforms establish specificity in the response to different stress conditions

Isoform-specific sequestration of protein kinase A fine-tunes intracellular signaling during heat stress

Protein kinase A (PKA) is a conserved kinase crucial for fundamental biological processes linked to growth, development and metabolism. The PKA catalytic subunit is expressed as multiple isoforms in diverse eukaryotes; however, their contribution to ensuring signaling specificity in response to environmental cues remains poorly defined. Catalytic subunit activity is classically moderated via interaction with an inhibitory regulatory subunit. Here, a quantitative mass spectrometry approach was used to examine heat stress-induced changes in the binding of yeast Tpk1-3 catalytic subunits to the Bcy1 regulatory subunit. We show that Tpk3 is not regulated by Bcy1 binding but instead, is deactivated upon heat stress via reversible sequestration into cytoplasmic granules. These ‘Tpk3 granules’ are enriched for multiple PKA substrates involved in various metabolic processes, with the Hsp42 sequestrase required for their formation. Hence, regulated sequestration of Tpk3 provides a mechanism to control isoform-specific kinase signaling activity during stress conditions

Hsp104 is required for oxidative stress tolerance in sequestrase mutants.

A. Representative epifluorescent microscopic images are shown from strains expressing Btn2-GFP or Hsp42-GFP and Hsp104-RFP. Strains were grown to exponential phase and left untreated or treated with with 0.8 mM hydrogen peroxide for one hour. Charts show the percentage of cells that contain 0, 1–3, or >3 Btn2, Hsp42 or Hsp104 puncta per cell scored in 300 cells for each strain. Significance is shown comparing stressed and unstressed strains; *** p B. Quantification is for the co-localisation (%) of Btn2 or Hsp42 puncta with Hsp104 puncta from three biological replicates. Error bars denote SD and significance is shown compared with the untreated strains, * p C. Overexpression of Hsp104 improves the hydrogen peroxide sensitivity of btn2 hsp42 mutants. The wild-type and btn2 hsp42 mutant strains containing vector control or expressing HSP104 under the control of the constitutive TDH3 promoter were grown to exponential phase and the A600 adjusted to 1, 0.1, 0.01 or 0.001 before spotting onto SD plates containing the indicated concentrations of hydrogen peroxide. Representative images are shown from repeat experiments D. Protein aggregates were isolated from the wild-type and btn2 hsp42 mutant strains containing vector control or overexpressing HSP104 and analysed by SDS-PAGE and silver staining. Western blot analysis of the same strains probed with α-Hsp104 or α-Pgk1.</p

Sup35 localizes with Btn2 and Hsp42 and oxidant-induced [<i>PSI</i>⁺] prion formation is increased in mutants lacking <i>BTN2</i> and <i>HSP42</i>.

A. Representative epifluorescent microscopic images are shown from strains expressing Btn2-GFP or Hsp42-GFP and NM-RFP. Strains were grown to exponential phase and left untreated or treated with with 0.8 mM hydrogen peroxide for one hour. Sup35-NM-RFP was induced for two hours under the control of the inducible GAL1 promoter. White arrows indicate examples of colocalization. B. Charts show the percentage of cells that contain 0, 1–3, or >3 Sup35 puncta per cell scored in 300 cells for each strain. Significance is shown comparing stressed and unstressed strains; *** p C. Quantification is shown for the co-localisation of Btn2 or Hsp42 (%) with Sup35 puncta from three biological replicates. Error bars denote SD. D. [PSI+] prion formation was quantified in the wild-type, btn2, hsp42 and btn2 hsp42 mutant strains during non-stress and oxidative stress conditions. Data shown are the means of at least three independent biological repeat experiments expressed as the number of colonies per 105 viable cells. Error bars denote standard deviation. Significance is shown using a one-way ANOVA test; *** p E. Western blot analysis of the same strains as for A. probed with α-Sup35 or α-Pgk1 as a loading control. F. [PSI+] prion formation was quantified in the wild-type and indicated mutant strains containing the Sup35NM-GFP plasmid following 20 hours of copper induction. Data shown are the means of at least three independent biological repeat experiments expressed as the number of colonies per 104 viable cells. Error bars denote standard deviation; * marks statistical significance at p<0.01 (one-way ANOVA). G. Western blot analysis of the same strains as for C. probed with α-Sup35 or α-Pgk1.</p

Non-amyloid aggregate formation underlies the increased Sup35 aggregation observed in <i>btn2</i> hsp42 mutants.

A. [PSI+] prion formation was quantified in the wild-type, abp1, btn2 hsp42 and btn2 hsp42 abp1 mutant strains during non-stress conditions and following exposure to 0.8 mM hydrogen peroxide for one hour. Data shown are the means of at least three independent biological repeat experiments expressed as the number of colonies per 105 viable cells. Error bars denote standard deviation. Significance is shown using a one-way ANOVA test; * p p B. SDS-resistant Sup35 and Rnq1 aggregates were detected in the wild-type and btn2 hsp42 mutant strains using SDD-AGE. Strains were grown to exponential phase and left untreated (-) or treated with with 0.8 mM hydrogen peroxide (+) for one hour. [psi-] and rnq1 deletion strains are shown for comparison with Rnq1 and [PSI+] and [psi-] strains are shown for comparison with Sup35. Aggregate and monomer (M) forms are indicated. C. Representative epifluorescent microscopic images are shown from strains expressing Sup35-GFP or ΔN-Sup35-GFP. Strains were grown to exponential phase and left untreated (non-stress) or treated with with 0.8 mM hydrogen peroxide for one hour. Charts show the percentage of cells contain 0, 1–3, or >3 Sup35 puncta per cell scored in 300 cells for each strain. Significance: ** p p < 0.001 (Mann–Whiney U-test).</p

Sup35 aggregate formation is increased in sequestrase mutants.

A. Representative epifluorescent microscopic images are shown from strains expressing Sup35-GFP and Rnq1-CFP. Strains were grown to exponential phase and left untreated (non-stress) or treated with with 0.8 mM hydrogen peroxide for one hour. GFP is false coloured magenta and CFP is false coloured cyan. B. Charts show the percentage of cells contain 0, 1–3, or >3 Sup35 or Rnq1 puncta per cell scored in 300 cells for each strain. Significance: ** p p C. Quantification is shown for the co-localisation of Sup35 puncta with Rnq1 puncta with from three biological replicates. Error bars denote SD.</p

Mutants lacking Btn2 and Hsp42 and are sensitive to oxidative stress.

A. The indicated strains were grown to exponential phase and the A600 adjusted to 1, 0.1, 0.01 or 0.001 before spotting onto SD (control) or plates containing hydrogen peroxide (1.0, 1.25 mM). Representative images are shown from repeat experiments B. Btn2 and Hsp42 protein levels are unaffected in response to oxidative stress conditions. Whole cell extracts were prepared from wild-type strains containing Btn2-Myc or Hsp42-Myc grown under non-stress conditions, subjected to a 37oC, 30-minute heat shock or exposed to 0.8 mM hydrogen peroxide for 30, 60 or 90 minutes. Western blots are shown probed with αMyc or α-Pgk1 as a loading control. Asterisks denote a non-specific band recognized by αMyc. Triplicate blots were quantified and Btn2 and Hsp42 protein levels are shown relative to Pgk1. Significance is shown using a one-way ANOVA test, ** p<0.01, *** p<0.001.</p

Protein aggregation is increased in sequestrase mutants.

A. Wild-type and sequestrase mutants were grown to exponential phase and treated with 0.8 mM hydrogen peroxide (+) or left untreated (-) for one hour. Protein extracts were treated with the carbonyl-specific probe, DNPH, and analyzed by Western blot analysis using an antibody against DNPH. Quantitative data is shown as the means of four independent biological repeat experiments (carbonylation relative to Pgk1) ± SD; * pB. Hsp104-RFP was visualized in wild-type and sequestrase mutant cells grown to exponential phase and treated with 0.8 mM hydrogen peroxide or left untreated for one hour. Charts show the percentage of cells contain 0, 1–3, or >3 puncta per cell scored in 300 cells for each strain. Significance is shown compared with the wild-type strain; *** p C. Western blot analysis blot analysis of the same strains probed with antibodies that recognize Hsp104, Rnq1 or Pgk1.</p

Protein sequestration is a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation.

Soluble proteins such as Sup35 that undergo misfolding in response to oxidative damage are normally triaged by Btn2 and Hsp42 to the various protein deposit sites in cells. In the absence of Hsp42 and Btn2, protein sequestration is deficient and non-specific protein aggregates accumulate in cells. These aggregates are targeted by Hsp104 and other chaperones, but non-specific aggregates ultimately overwhelm the protein homeostasis machinery resulting in sensitivity to oxidative stress conditions. De novo prion formation following protein oxidation depends on IPOD localization which acts as a sorting centre determining whether oxidized proteins are cleared via autophagy, or alternatively, form heritable protein aggregates (prions), dependent on Hsp104. Prion formation is increased in btn2 hsp42 mutants in response to ROS exposure independent of IPOD localization.</p