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Small Heat Shock Proteins Potentiate Amyloid Dissolution by Protein Disaggregases from Yeast and Humans

Figure 4

Hsp26 and Hsp42 antagonize Sup35 prion formation in vivo.

(A) Sup35 was overexpressed for 16 h at 30°C in wild-type, Δhsp26, Δhsp42, or Δhsp26Δhsp42 [psi] [RNQ+] cells. Cells were plated on 25% YPD and the proportion of [PSI+] colonies was determined. Compared to wild-type cells there was significantly more [PSI+] induction in Δhsp26 cells (*p = 0.0264, two-tailed Student's t test), Δhsp42 cells (**p = 0.0059, two-tailed Student's t test), or Δhsp26Δhsp42 cells (***p = 0.0002, two-tailed Student's t test). Values represent means±SEM (n = 3). (B) Immunoblots demonstrating that neither Hsp104 nor Hsp70 (3A3 antibody that recognizes yeast Ssa1, Ssa2, Ssa3, and Ssa4 [95]) expression is affected in the Δhsp26, Δhsp42, or Δhsp26Δhsp42 background. Pgk1 is included as a loading control. (C) Sup35 was overexpressed for 16 h at 30°C in [psi] [RNQ+] cells expressing elevated levels of Hsp26, Hsp42, Hsp26 plus Hsp42, or Hsp104. Cells were plated on 25% YPD and the proportion of [PSI+] colonies was determined. Compared to the vector control there was significantly less [PSI+] induction in cells expressing Hsp26 (*p = 0.0253, two-tailed Student's t test), Hsp42 (*p = 0.028, two-tailed Student's t test), Hsp26 and Hsp42 (**p = 0.0021, two-tailed Student's t test), or Hsp104 (***p<0.0001, two-tailed Student's t test). Values represent means±SEM (n = 3). (D) Immunoblots demonstrating that neither Hsp104 nor Hsp70 (3A3 antibody that recognizes yeast Ssa1, Ssa2, Ssa3, and Ssa4 [95]) expression is affected by elevated expression of Hsp26, Hsp42, or Hsp26 and Hsp42. Pgk1 is included as a loading control. (E) NM-YFP was transiently overexpressed for 4 h at 30°C in [psi] [RNQ+] cells expressing elevated levels of Hsp26, Hsp42, or Hsp26 and Hsp42. Cells were processed for fluorescence microscopy. The proportion of cells with NM-YFP foci was then determined. (**p<0.01, ***p<0.001, two-tailed Student's t test). Values represent means±SEM (n = 3). Examples of cells with NM-YFP foci (“dots” and “ribbons” indicated by arrows) and cells with diffuse NM-YFP fluorescence are shown on the right.

Figure 4

doi: https://doi.org/10.1371/journal.pbio.1001346.g004