Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation

Abstract

Hsp100 and Hsp70 chaperones in bacteria, yeast, and plants cooperate to reactivate aggregated proteins. Disaggregation relies on Hsp70 function and on ATP-dependent threading of aggregated polypeptides through the pore of the Hsp100 AAA(+) hexamer. In yeast, both chaperones also promote propagation of prions by fibril fragmentation, but their functional interplay is controversial. Here, we demonstrate that Hsp70 chaperones were essential for species-specific targeting of their Hsp100 partner chaperones ClpB and Hsp104, respectively, to heat-induced protein aggregates in vivo. Hsp70 inactivation in yeast also abrogated Hsp104 targeting to almost all prions tested and reduced fibril mobility, which indicates that fibril fragmentation by Hsp104 requires Hsp70. The Sup35 prion was unique in allowing Hsp70-independent association of Hsp104 via its N-terminal domain, which, however, was nonproductive. Hsp104 overproduction even outcompeted Hsp70 for Sup35 prion binding, which explains why this condition prevented Sup35 fragmentation and caused prion curing. Our findings indicate a conserved mechanism of Hsp70-Hsp100 cooperation at the surface of protein aggregates and prion fibrils.

Figure 1.

Hsp70 chaperones target ClpB/Hsp104 to heat-induced protein aggregates. (A) E. coli wild-type (dnaK+), dnaK103, and ΔclpB cells expressing ClpB-YFP or DnaK-YFP were grown at 30°C and shifted to 45°C. DnaK-V436F is deficient in substrate interaction. The occurrence of foci indicate chaperone binding to protein aggregates. The percentage of 200 cells harboring foci and the percentage of polar fluorescence intensity was quantified (n = 20). Bars, 1 µm. (B) S. cerevisiae cells harboring a single wild-type ssa1 copy or the ssa1-45(ts) allele and expressing mCitrine-Luciferase and Hsp104-CFP were grown at 25°C and shifted to 37°C. Next, cells were shifted to 45°C to induce protein aggregation. The percentage of colocalization of mCitrine-Luciferase and Hsp104-CFP foci at 45°C was quantified in both strains (n = 100). The broken lines indicate the borders of respective yeast cells. Bars, 2 µm.

Figure 2.

The ClpB/Hsp104 M domain mediates Hsp70-dependent targeting to protein aggregates. (A) E. coli ΔclpB cells expressing either ClpB-, Hsp104-, or hybrid HBH-YFP, harboring the ClpB M domain, were grown at 30°C and shifted to 45°C. The percentage of cells showing foci was determined (n = 200). The degree of polar fluorescence intensity was quantified (percentage of total fluorescence, n = 20). Bars, 1 µm. (B) E. coli ΔclpB cells expressing Luciferase and the indicated YFP fusion proteins were grown at 30°C and shifted to 45°C. The recovery of Luciferase activity after heat shock was determined. Standard deviations are given (error bars). wt, ClpB.

Figure 3.

Ssa1 targets Hsp104 to Rnq1 prion fibrils. (A) S. cerevisiae SSA1 and ssa1-45(ts) cells expressing RNQ-mCherry and Hsp104-CFP were grown at 25°C and shifted to 37°C. Binding of Hsp104 to Rnq1 prion fibrils was solely observed at 25°C and 37°C in the SSA1 strain and at 25°C in the ssa1-45(ts) strain. The frequency of colocalization (%) is given (n = 100). (B) ssa1-45(ts) cells were grown at 25°C, and cycloheximide (CHX) was added to stop protein synthesis. Next, cells were shifted to 37°C and incubated further for 60 min at either 25°C or 37°C. At each step, the localizations of Rnq1-mCherry and Hsp104-CFP were analyzed. The reestablishment of Rnq1-mCherry and Hsp104-CFP colocalization during a recovery phase at 25°C indicates that Ssa1-45 activity is restored, enabling it to target Hsp104-CFP to Rnq1-mCherry foci. The frequency of colocalization (%) is given (n = 100). (C) Association of Ssa1 and Ssa1-45 with Rnq1 fibers was monitored by immunofluorescence in SSA1 and ssa1-45(ts) cells at 25°C and 37°C. In SSA1 cells, Ssa1 colocalizes with Rnq1-GFP foci at 25°C and at 37°C. In ssa1-45(ts) cells, efficient binding of Ssa1-45 to Rnq1-GFP foci was only observed at 25°C. The frequency of colocalization (%) is given (n = 100). The broken lines indicate the borders of respective yeast cells. Bars, 2 µm.

Figure 4.

Inactivation of Ssa1 inhibits Rnq1p fiber fragmentation by Hsp104. (A) Rnq1-GFP foci become immobile upon Ssa1 inactivation. FLIP measurements of Rnq1-GFP were performed in SSA1 and ssa1-45(ts) cells at 25°C and after temperature upshift to 37°C. For comparison, the mobility of diffuse Rnq1-GFP was determined in [pin⁻] cells. Mobility of Rnq1-GFP was also monitored in SSA1 cells at 37°C after addition of GdnHCl, causing Hsp104 inactivation. n = 25 cells were analyzed for each condition. Standard errors (error bars) and a photo bleaching control are given. (B) Inactivation of Ssa1 or addition of GdnHCl causes an increase in Rnq1-GFP foci size. SSA1 and ssa1-45(ts) cells were shifted from 25°C to 37°C, and fluorescence intensity of Rnq1-GFP foci was determined before (0 min) and after temperature upshift (90 min). Standard deviations are given (error bars).

Figure 5.

GdnHCl inhibits Hsp104 binding to Rnq1 fibrils. S. cerevisiae SSA1 cells expressing Rnq1-mCherry or Rnq1-GFP were treated for 0, 90, and 360 min with 3 mM GdnHCl. Colocalization of Rnq1 foci with Hsp104-CFP (A) or SSA1 (B) was analyzed. SSA1 was detected by immunofluorescence. Colocalization of Hsp104-CFP and Rnq1-mCherry is strongly reduced upon prolonged GdnHCl treatment. Addition of GdnHCl does not affect Ssa1 association with Rnq1-GFP fibrils. The frequency (%) of colocalization is given (n = 100). The broken lines indicate the borders of respective yeast cells. Bars, 2 µm.

Figure 6.

Ssa1 modulates the interaction of Hsp104 with NM-YFP fibers. (A) S. cerevisiae SSA1 and ssa1-45(ts) cells expressing NM-YFP and Hsp104-CFP were grown at 25°C and shifted to 37°C. NM-YFP foci colocalize with Hsp104-CFP in SSA1 cells and in ssa1-45(ts) cells at 25°C and 27°C. The frequency of colocalization (%) is given (n = 100). The broken lines indicate the borders of respective yeast cells. Bars, 2 µm. (B and C) Hsp104-CFP and NM-YFP become immobilized upon Ssa1-45 inactivation. FLIP experiments of Hsp104-CFP (B) and NM-YFP (C) were performed in SSA1 and ssa1-45(ts) cells at 25°C and 37°C. For comparison, the motilities of diffuse Hsp104-CFP located outside the foci (cytosol) or of diffuse NM-YFP in [psi⁻] cells were determined. The mobility of NM-YFP was also monitored in SSA1 cells at 37°C after addition of GdnHCl. n = 25 cells analyzed for each condition. Standard errors and photo bleaching controls are given (error bars). (D) Inactivation of Ssa1 or addition of GdnHCl causes an increase in NM-YFP foci size. SSA1 and ssa1-45(ts) cells were shifted from 25°C to 37°C, and the fluorescence intensities of NM-YFP foci were determined before (0 min) and after temperature upshift (90 min). Standard deviations are given (error bars).

Figure 7.

The N domain of Hsp104 mediates binding to NM-YFP fibers and enables high Hsp104 levels to outcompete Ssa1 for NM-YFP association. (A) ΔN-Hsp104-CFP binding to NM-YFP fibers is dependent on SSA1. S. cerevisiae SSA1 and ssa1-45(ts) cells expressing ΔN-Hsp104-CFP and NM-YFP were grown at 25°C and shifted to 37°C. Binding of ΔN-Hsp104-CFP to NM-YFP foci was analyzed by immunofluorescence. Binding of ΔN-Hsp104 with NM-YFP foci was only observed at 25°C and 37°C in SSA1 cells and at 25°C in ssa1-45(ts) cells. The frequency of colocalization (%) is given (n = 100). (B) Hsp104 but not ΔN-Hsp104 competes with Ssa1 for binding to NM-YFP fibers. Ssa localization was determined by immunofluorescence in SSA1 cells overexpressing Hsp104/ΔN-Hsp104 and NM-YFP. The colocalization frequency (%) of Ssa1 and NM-YFP foci was determined (n = 200). The broken lines indicate the borders of respective yeast cells. vc, vector control. (C) FLIP experiments of NM-YFP were performed at 25°C in SSA1 cells overexpressing ΔN-Hsp104 or Hsp104. NM-YFP foci showed reduced mobility in the presence of high levels of Hsp104. n = 25 cells were analyzed for each condition. Standard errors are given (error bars). vc, vector control.

Figure 8.

Inhibition of Hsp90 activity and deletion of sti1Δ prevents outcompetition of Ssa1 by high Hsp104 levels. (A and B) Ssa localization was determined by immunofluorescence in SSA1 pdr5Δ cells or SSA1 sti1Δ overexpressing Hsp104. SSA1 pdr5Δ cells were analyzed in the absence or presence of the Hsp90 inhibitor Radicicol or DMSO (control). Colocalization frequencies with NM-YFP foci were determined (n = 100). The broken lines indicate the borders of respective yeast cells. vc, vector control. Bars, 2 µm.

Figure 9.

Ssa1 has a general role in the targeting of Hsp104 to prion fibrils. S. cerevisiae SSA1 and ssa1-45(ts) cells expressing Hsp104-CFP and YFP fusion to the prion domains of Mot3 (A), Lsm4 (B), or Nrp1 (C) were grown at 25°C and shifted to 37°C. Binding of Hsp104 to the individual prion domains was observed at 25°C and 37°C for the SSA1 cells and solely at 25°C for ssa1-45(ts) cells. The frequency of colocalization (%) is given (n = 60). The broken lines indicate the borders of respective yeast cells. Bars, 2 µm.

Figure 10.

Conserved cooperation between Hsp70 and ClpB/Hsp104 in protein disaggregation and prion fragmentation. Hsp70 chaperones recruit ClpB/Hsp104 to both stress-induced amorphous protein aggregates and prion fibrils. Hsp70-mediated targeting involves the ClpB/Hsp104 M domain. ClpB/Hsp104 threading activity extracts misfolded polypeptides from protein aggregates for subsequent refolding. The same activity leads to prion fiber severing and the generation of oligomeric propagons. High Hsp104 levels bind via N domains to Sup35 fibrils, thereby preventing Hsp70 association and fiber fragmentation.