Heat shock protein 104 (Hsp104)-mediated curing of [ PSI+] yeast prions depends on both [ PSI+] conformation and the properties of the Hsp104 homologs

Abstract

Prions arise from proteins that have two possible conformations: properly folded and non-infectious or misfolded and infectious. The [PSI⁺] yeast prion, which is the misfolded and self-propagating form of the translation termination factor eRF3 (Sup35), can be cured of its infectious conformation by overexpression of Hsp104, which helps dissolve the prion seeds. This dissolution depends on the trimming activity of Hsp104, which reduces the size of the prion seeds without increasing their number. To further understand the relationship between trimming and curing, trimming was followed by measuring the loss of GFP-labeled Sup35 foci from both strong and weak [PSI⁺] variants; the former variant has more seeds and less soluble Sup35 than the latter. Overexpression of Saccharomyces cerevisiae Hsp104 (Sc-Hsp104) trimmed the weak [PSI⁺] variants much faster than the strong variants and cured the weak variants an order of magnitude faster than the strong variants. Overexpression of the fungal Hsp104 homologs from Schizosaccharomyces pombe (Sp-Hsp104) or Candida albicans (Ca-Hsp104) also trimmed and cured the weak variants, but interestingly, it neither trimmed nor cured the strong variants. These results show that, because Sc-Hsp104 has greater trimming activity than either Ca-Hsp104 or Sp-Hsp104, it cures both the weak and strong variants, whereas Ca-Hsp104 and Sp-Hsp104 only cure the weak variants. Therefore, curing by Hsp104 overexpression depends on both the trimming ability of the fungal Hsp104 homolog and the strength of the [PSI⁺] variant: the greater the trimming activity of the Hsp104 homolog and the weaker the variant, the greater the curing.

Keywords: Hsp104; amyloid; molecular chaperone; prion; protein folding; yeast.

Figure 1.

Rate of curing and trimming of prion seeds by Sc-Hsp104 is dependent on the [PSI⁺] variant. A, curing of [PSI⁺] by Sc-Hsp104 overexpression was measured in different [PSI⁺] variants. Sc-Hsp104 was expressed from the Gal1 promoter, and the extent of curing was measured at the indicated times by the red/white colony assay. The following variants were used: L1758[PSI⁺], L2888[PSI⁺], 1074[PSI⁺]^DM, 779-6A[PSI⁺]^DM, SY80[PSI⁺], and L1762[PSI⁺]. B, fluorescence imaging of yeast expressing GFP-labeled Sup35 (NGMC) in [PSI⁺] yeast overexpressing Sc-Hsp104. Images are of either the weak L2888[PSI⁺] or the strong SY80 [PSI⁺] variants. Images were taken after overnight incubation in raffinose medium (T = 0), 2 h after addition of galactose medium to induce Sc-Hsp104 expression, or 2 h in galactose medium and 1 h in 5 mm guanidine to stress the cells.

Figure 2.

Curing of different [PSI⁺] variants was measured upon overexpression of different Hsp104 fragments. A–C, curing of different [PSI⁺] variants was measured in yeast overexpressing Sc-Hsp104ΔC7 or Sc-Hsp104ΔC24 from the GAL1 promoter. The dashed lines are the curing curves from Fig. 1A obtained by overexpressing full-length Sc-Hsp104. D, Sc-Hsp104ΔC52 was overexpressed in different [PSI⁺] variants. Curing was measured at the indicated times using the red/white colony assay. E, 5-fluoroorotic acid (5-FOA) shuffle experiment using 1408 yeast in which the pJ312 plasmid, which encodes Sc-Hsp104 under the control of the S. cerevisiae HSP104 promoter on a URA3-based centromeric plasmid, was shuffled with empty vector or plasmids expressing full-length Sc-Hsp104 or the indicated C-terminal truncations of Sc-Hsp104. The full-length and fragments of Sc-Hsp104 were under the control of the Sc-HSP104 promoter. Only constructs that support [PSI⁺] propagation grow on plates without adenine.

Figure 3.

Overexpression of 444B cures weak [PSI⁺] variants but not strong [PSI⁺] variants. A, effect of overexpression of 444B in different [PSI⁺] variants. Yeast in raffinose medium were grown overnight in galactose medium to induce 444B expression. Yeast were plated on ½ YPD medium before (columns I and III) and after induction of 444B (columns II and IV). Note [psi⁻] yeast in the 779-6A and the 74D-694 backgrounds give different red hues on ½ YPD plates. B, curing of different [PSI⁺] variants was measured in yeast overexpressing 444B. 444B was expressed from the GAL1 promoter in different [PSI⁺] variants and plated at the indicated times. Curing of [PSI⁺] by overexpression of 444B was also done in the 1509 [PSI⁺] ^DM yeast. The 1509 yeast was derived by integrating 444B into the HSP104 chromosomal locus of the 1074 yeast. C, overexpression of 444B causes loss of detectable foci in weak but not strong [PSI⁺] variants. Fluorescence images of GFP-labeled Sup35 are of weak L2888 [PSI⁺] variant overexpressing 444B for one generation and strong SY80 [PSI⁺] variant overexpressing 444B for 15 generations. The L2888 [PSI⁺] yeast were imaged both before stress and after stress. D, rate of curing of different [PSI⁺] variants was determined upon overexpression of 444BΔC19. Curing was measured at the indicated times using the red/white colony assay. E, 5-fluoroorotic acid (5-FOA) shuffle experiment using 1408 yeast in which the pJ312 plasmid, which encodes Sc-Hsp104 under the control of the S. cerevisiae HSP104 promoter on a URA3-based centromeric plasmid., was shuffled with plasmids expressing either 444B or 444BΔC19. The 444B and 444BΔC19 were expressed using the Sc-HSP104 promoter. Only constructs that support [PSI⁺] propagation grow on plates without adenine.

Figure 4.

Overexpression of Sp-Hsp104 or Ca-Hsp104 cures weak [PSI⁺] variants but not strong [PSI⁺] variants. A, curing of different [PSI⁺] variants by overexpression of Sp-Hsp104 was measured as a function of generation time. B, curing of different [PSI⁺] variants by overexpression of Ca-Hsp104 was measured as a function of generation time. C, overexpression of either Sp-Hsp104 or Ca-Hsp104 causes loss of detectable foci in the weak L2888 [PSI⁺] variant but not the strong [PSI⁺] SY80 variant. Fluorescence imaging of GFP-labeled Sup35 of weak L2888 [PSI⁺] variant overexpressing either Sp-Hsp104 or Ca-Hsp104 after overnight incubation in raffinose (T = 0) or for one generation in galactose medium is shown. The latter cells were stressed for 1 h in 5 mm guanidine. Top panel, fluorescence imaging of GFP-labeled Sup35 of strong SY80 [PSI⁺] variant overexpressing either Sp-Hsp104 or Ca-Hsp104 after overnight incubation in raffinose (T = 0) or for 10 generations in galactose medium is shown.

Figure 5.

Growth of strong [PSI⁺] variants was measured in yeast overexpressing 444B, Sp-Hsp104, or Ca-Hsp104. A, overexpression of 444B causes a slow growth phenotype in 1762 [PSI⁺] variant. B, overexpression of 444B causes a slow growth phenotype in 779-6A [PSI⁺]^DM variant. Yeast growth in galactose medium as a function of time is shown for [PSI⁺] yeast expressing 444B (closed squares), [PSI⁺] yeast with empty vector (open diamonds), and cured yeast expressing 444B (open triangles). C, overexpression of 444B causes a slow growth phenotype in strong [PSI⁺] variants due to sequestration of Sup35. 444B was overexpressed in either the 779-6A [PSI⁺]^DM variant or the L1762 [PSI⁺] variant. Growth was measured in [PSI⁺] cells expressing 444B containing an empty vector plasmid (solid symbols) or a plasmid expressing the C-terminal fragment of Sup35 (open symbols). D, Sp-Hsp104 was overexpressed in yeast with the 779-6A [PSI⁺]^DM variant (solid diamonds), in cured yeast (open circles), or in yeast expressing the Sup35 C-terminal domain of Sup35 (open diamonds). E, Sp-Hsp104 was overexpressed in yeast with the 1762 [PSI⁺] variant (solid symbols) or in cured yeast (open symbols). F, Ca-Hsp104 was overexpressed in yeast with the 779-6A [PSI⁺]^DM variant (solid triangles) or in cured yeast (open triangles) and in yeast with the 1762 [PSI⁺] variant (solid diamonds) or in cured yeast (open diamonds). Growth was monitored by optical density, and cells were constantly diluted to maintain them in early log phase. The different [PSI⁺] variants were cured in guanidine.

Figure 6.

Low levels of Sc-Hsp104 cures and trims the weak L2888 [PSI⁺] variant but only trims the strong SY80 [PSI⁺] variant. A, GFP-labeled Sup35 was imaged in the weak L2888 [PSI⁺] overexpressing Sc-Hsp104 from the TET-ON promoter after 1 h of Sc-Hsp104 induction by 10 μg/ml doxycycline. B, curing of the weak L2888 [PSI⁺] variant and strong SY80 [PSI⁺] variant was measured as a function of generation time when Sc-Hsp104 is overexpressed from the TET-ON promoter. C, GFP-labeled Sup35 was imaged in the strong SY80 [PSI⁺] variant overexpressing Sc-Hsp104 from the TET-ON promoter. The SY80 [PSI⁺] cells were imaged 12 generations after induction of Sc-Hsp104 overexpression. Imaging was done in both non-stressed and stressed cells.