Solubility of Hsp26 in Response to Heat Shock
To identify substrates and cofactors of Hsp104-mediated protein disaggregation in yeast, we analyzed proteins that accumulate in aggregates after heat shock and depend on Hsp104 for resolubilization. Using two-dimensional PAGE, we compared proteins from the soluble and the insoluble fractions of wild-type (WT) and Δhsp104 cells. Proteins were examined: 1. before a non-lethal heat shock, 2. immediately thereafter and 3. after recovery ( and data not shown). Several proteins became insoluble after heat shock and were resolubilized in WT but not in Δhsp104 cells. These proteins were identified by mass spectrometry. Among them, Hsp26 was very abundant and was identified with high confidence (supplemental Table IV).
Two-dimensional PAGE analysis of soluble and insoluble proteins after recovery from heat shock
The results were confirmed by Western blotting using antibodies against Hsp26 (). Heat shock induced the expression of Hsp26 severalfold over untreated levels. Immediately after the heat treatment, most Hsp26 had become insoluble in both the WT and Δhsp104 cells. After recovery at 25 °C for 1 h, the majority of Hsp26 was resolubilized in WT cells but remained insoluble in Δhsp104 cells.
Solubility of Hsp26 and Hsp42 after heat shock in WT and Δhsp104 cells
We next tested by Western blot whether the solubility of Hsp42, the other sHsp in yeast, might also depend on the presence of Hsp104 after heat shock. Approximately 50% of Hsp42 was detected in the insoluble fraction regardless of previous heat treatment (). Upon heat treatment Hsp42 levels increased severalfold, but its solubility in WT and Δhsp104 cells remained similar.
In the accompanying paper, Haslbeck et al
) also observed that Hsp26 is the major protein found in the insoluble fraction of yeast cells after heat shock. These observations reveal a functional interaction between Hsp26 (but not necessarily Hsp42) and Hsp104, wherein Hsp26 is sequestered into the insoluble fraction after heat shock and is resolubilized in an Hsp104-dependent manner during recovery.
Thermotolerance of Mutants
To understand the role of sHsps in thermotolerance, we tested the recovery of sHsp deletion strains after a severe heat shock. Previous work demonstrated that neither Hsp26 nor Hsp42 was required for thermotolerance (survival after a severe heat shock) (7
). In agreement, we saw no thermotolerance defect in Δhsp26
cells (). Even the Δhsp26
double mutant had wild-type levels of thermotolerance. However, Δhsp104
cells have a 5-fold higher rate of survival than Δhsp104
double deletions after a short (10 min) heat shock at 50 °C. The synthetic effect of the deletions of Hsp104 and Hsp26 in our experiments suggests a functional link between these chaperones in response to heat shock and the ensuing protein disaggregation. Surprisingly, the deletion of Hsp42 in Δhsp104
cells does not show this effect, revealing a non-overlapping function of Hsp26 and Hsp42 in thermotolerance.
Thermotolerance of various mutants
In Vivo Effect of Hsp26 Deletion on Hsp104-dependent Reactivation
We hypothesized that Hsp26 binds unfolded proteins after heat shock and thereby enables Hsp104/Ssa1/Ydj1 to resolubilize aggregates more efficiently. To test this, we investigated the Hsp104-dependent disaggregation of a temperature-sensitive protein whose functional state is readily quantified (a previously described luciferase variant (12
)). Each strain constitutively expressing luciferase was pretreated at 37 °C for 30 min and then given a sublethal heat shock at 46 °C for 30 min (12
). Protein synthesis was inhibited by the addition of cycloheximide after the heat shock to focus on the reactivation of luciferase synthesized prior to heat shock.
The Hsp26 deletion did not affect the activity of luciferase at 25 °C or during the conditioning pretreatment at 37 °C. The sublethal heat shock reduced the luciferase activity to <5% in mid-log phase cells (). Cells deleted for Hsp104 did not recover luciferase activity, regardless of the presence of sHsps ( and data not shown).
In vivo reactivation of aggregated luciferase
Cells deleted for Hsp26 reactivated luciferase to a similar extent to WT when grown in mid-log phase. In the stationary phase however, Δhsp26 cells reactivated luciferase at a significantly slower rate than WT cells. The increased dependence of stationary phase cells on Hsp26 for disaggregation is also consistent with the role of sHsps in senescence (21
In Vitro Effect of Hsp26 on Hsp104-dependent Reactivation
To investigate mechanistic aspects of the cooperation between Hsp104/Ssa1/Ydj1 and Hsp26 we used purified proteins in aggregation and disaggregation assays. The Hsp104-dependent return of Hsp26 to the soluble state after heat shock likely reflects its ability to capture denatured proteins and keep them in a refolding competent state.
FFL aggregates were prepared by heating FFL at 45 °C in the absence and presence of various concentrations of Hsp26. Heat not only causes the aggregation of FFL but it also activates Hsp26 by disassembling the oligomers (9
). Moreover, heat-aggregated FFL on its own is a poor substrate for Hsp104 (19
), so we used it to test the effects of Hsp26 (). Disaggregation reactions were initiated by diluting FFL aggregates 10-fold into reaction mixtures containing no chaperones, Hsp104 alone, Ssa1 and Ydj1, or all three chaperones.
Hsp26 facilitates reactivation of aggregated FFL by the Hsp104/Ssa1/Ydj1 chaperone machinery
When Hsp26 was present during the heat denaturation of FFL, it improved resolubilization in a dose-dependent manner (). Hsp104/Ssa1/Ydj1-mediated FFL reactivation was ~20-fold more efficient when FFL aggregates were prepared in the presence of Hsp26 than in its absence. Replacing Hsp26 with another protein such as bovine serum albumin did not influence FFL reactivation (data not shown). When Hsp26 was added after the aggregation of FFL it did not facilitate disaggregation by Hsp104 (FFL activity was not detectable in all samples; data not shown) indicating that formation of the Hsp26-FFL co-complex was essential for reactivation. These observations provide the first direct evidence for cooperation between Hsp26 and Hsp104/Ssa1/Ydj1 chaperones in the reactivation of aggregated proteins.
Increasing FFL concentration with a constant Hsp26 concentration drastically reduced the efficiency of reactivation (). These results suggest that a Hsp26 oligomer:FFL ratio of 2:5 was optimal for FFL as a substrate under these conditions. Our results argue that Hsp26 facilitates protein disaggregation by the Hsp104/Ssa1/Ydj1 machinery in a concentration-dependent manner. Importantly, Hsp26 can only perform its solubilizing function when it co-aggregates with the protein substrate and not after substrate aggregation.
Physical Nature of Hsp26-FFL Interactions
The simplest explanation for the relative inefficiency of FFL reactivation at low Hsp26:FFL ratios, is that Hsp26 was unable to capture all of the FFL. To investigate this we studied the physical nature of Hsp26-FFL complexes by dynamic light scattering ( and supplemental Table V).
Complex formation between Hsp26 and FFL monitored by dynamic light scattering
In the absence of a misfolded client protein, Hsp26 is an oligomer of ~18 nm in diameter () and has been shown to disassemble at 45 °C (9
). FFL aggregated into very large particles when heated to 45 °C without Hsp26 (>300 nm, data not shown). However, when the two proteins are mixed at a ~4:1 or a ~1:2 ratio (Hsp26 oligomer:FFL) and heated to 45 °C no large aggregates were observed. Instead, the measured particle diameter was ~33 or ~66 nm respectively.
Thus, Hsp26 is able to capture all of the FFL even when Hsp26:FFL ratios were low. However, complexes formed with high Hsp26 concentrations lead to the formation of smaller particles. These are readily resolubilized by Hsp104/Ssa1/Ydj1. At lower Hsp26 concentrations, larger particles are formed, which are more resilient to resolubilization.
sHsps Modulate Polyglutamine Aggregation and Toxicity
We next tested whether the function of Hsp26 and the Hsp104/Ssa1/Ydj1 disaggregation machinery applies to proteins involved in human protein misfolding disease. Previously, our laboratory and others demonstrated that yeast serves as a useful model for studying human neurodegenerative diseases involving protein aggregation such as Huntington disease (24
) and Parkinson disease (26
). We tested whether deletion or overexpression of Hsp26, Hsp42, Hsp104, or various combinations thereof, would affect the toxicity conferred by expanded polyglutamine region (72Q) of human huntingtin. Yeast cells expressing a normal polyglutamine stretch (25Q) under identical conditions were used as a control.
Deletions of Hsp26 or Hsp42 had almost no effect on the toxicity of 72Q (supplemental ). This is not surprising given that both sHsps are normally expressed at very low levels in yeast. Overexpressing Hsp104, Hsp26, or Hsp42 reduced 72Q-induced toxicity (). However, only overexpressing combinations of Hsp104 together with Hsp26 or Hsp42 strongly reduced the toxicity of 72Q (). Importantly, the overexpression of these genes does not change the status of the [RNQ+] prion (data not shown), which is crucial for the aggregation and toxicity of 72Q in yeast (25
). Notably, the toxicity of α-synuclein in yeast (26
) could not be suppressed by the over-expression of Hsp104 and Hsp26 or Hsp42 (supplemental ). Thus the ability of these chaperones to suppress toxicity depends on the nature of the aggregated protein.
sHsps and polyglutamine toxicity and aggregation
To test whether the chaperone-mediated reduction of poly-glutamine-induced toxicity correlates with protein disaggregation activity, we analyzed the polyglutamine aggregation in filter-trap assays (). The combinations of Hsp104 with Hsp26 or Hsp42 markedly reduced 72Q aggregation. The over-expression of the chaperones was confirmed by Western blotting (). Notably, the reduction in 72Q aggregation and toxicity was achieved without a reduction of its expression level as demonstrated by the dot blot ().
In summary, these experiments show that sHsps antagonize the toxicity of a polyglutamine protein in yeast. This reduction of toxicity correlates with the solubilization of the polyglutamine aggregates.